WO2017125076A1 - Application of rhodioloside for preparing pharmaceutical product for treating ischemic disease - Google Patents

Application of rhodioloside for preparing pharmaceutical product for treating ischemic disease Download PDF

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Publication number
WO2017125076A1
WO2017125076A1 PCT/CN2017/071947 CN2017071947W WO2017125076A1 WO 2017125076 A1 WO2017125076 A1 WO 2017125076A1 CN 2017071947 W CN2017071947 W CN 2017071947W WO 2017125076 A1 WO2017125076 A1 WO 2017125076A1
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Prior art keywords
salidroside
treating
medicament
diabetic foot
lower limb
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PCT/CN2017/071947
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French (fr)
Chinese (zh)
Inventor
吴寿荣
江崎慧
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重庆大学
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Priority claimed from CN201610039803.7A external-priority patent/CN105535001B/en
Priority claimed from CN201610039248.8A external-priority patent/CN105687216B/en
Application filed by 重庆大学 filed Critical 重庆大学
Publication of WO2017125076A1 publication Critical patent/WO2017125076A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7032—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a polyol, i.e. compounds having two or more free or esterified hydroxy groups, including the hydroxy group involved in the glycosidic linkage, e.g. monoglucosyldiacylglycerides, lactobionic acid, gangliosides

Definitions

  • the invention relates to the use of salidroside in the preparation of a medicament for treating ischemic diseases.
  • the invention relates to the use of salidroside for the preparation of a medicament for the treatment of diabetic foot.
  • the present invention also relates to a specific inhibitor of PHD3, an angiogenesis factor promoter, a vascular smooth muscle cell migration promoter, a skeletal muscle cell migration promoter, and an agent for mature angiogenesis.
  • An ischemic disease is a type of peripheral vascular disease in which tissue cells are insufficiently supplied due to vascular damage, vascular clogging, and the like.
  • Peripheral Artery Disease refers to the blockage of arteries due to atherosclerosis or the like, thereby limiting the blood flow supply to tissues other than the heart.
  • Limb Ischemia is one of the most common peripheral arterial diseases, resulting in insufficient blood supply to the distal (lower extremities) due to vascular occlusion, thrombosis, hyperglycemia, and the like. Since oxygen is transported from red blood cells in the blood to various tissues and organs, the result of insufficient blood supply is caused by hypoxia and lack of nutrients in the lower limbs, which can lead to tissue gangrene, tissue loss and even individual death.
  • first-generation therapeutic drugs As a drug for lower limb ischemic diseases, there are currently first-generation therapeutic drugs and second-generation therapeutic drugs.
  • the first generation of therapeutic drugs utilizes a single angiogenic factor (Vascular Endothelial Growth Factor (VEGF), etc.), but the clinical trial results are not satisfactory, the new blood vessels are immature, there is leakage, and lack of functionality.
  • VEGF Vascular Endothelial Growth Factor
  • the second generation of therapeutic drugs utilizes a combination of multiple angiogenic factors (Fibroblast growth factor 2 (FGF2) and platelet-derived growth factor (PDGF); VEGF and Angiopoietin-1 (ANG1)), although have certain effects
  • FGF2 Fibroblast growth factor 2
  • PDGF platelet-derived growth factor
  • VEGF vascular endothelial growth factor
  • ANG1 Angiopoietin-1
  • Diabetic foot is one of the most common and serious complications of diabetes. Due to unsatisfactory blood glucose control, peripheral vascular disease of the lower extremities leads to insufficient blood supply to the lower extremities and causes hypoxia in the lower extremity tissue. In addition, tissue repair and wound healing ability are significantly reduced under high glucose conditions, and tissue gangrene and tissue loss occur in severe cases (tissue) Loss) or even death; clinically severe patients often require amputation.
  • the ideal treatment for Diabetic Foot is to improve the blood supply status.
  • methods for treating vascular lesions include stenting, bypass, balloon dilatation, etc.
  • it promotes vascular remodeling. It is considered to be the best treatment route.
  • VEGF vascular endothelial growth factor
  • VEGFR VEGF receptor
  • PDGF-BB platelet-derived growth factor-BB
  • Diabetes Mellitus and Ischemic Diseases Molecular Mechanisms of Vascular Repair Dysfunction. Arteriosclerosis, Thrombosis, and Vascular Biology, 2014, 34(6): 1126-1135.
  • salidroside shown as Formula 1
  • salidroside has a good therapeutic effect on the treatment of ischemic diseases of the lower limbs. Further, it has been found that salidroside has an effect of specifically inhibiting PHD3 and promoting skeletal muscle cell migration ability in skeletal muscle. Furthermore, salidroside was also found to promote skeletal muscle angiogenesis factors (VEGF-A, ANG1, FGF2, PDGF-BB, Heme oxygenase-1 (HO-1), hepatocyte growth).
  • VEGF-A skeletal muscle angiogenesis factors
  • ANG1, FGF2 vascular endothelial growth
  • HO-1 Heme oxygenase-1
  • Hepatocyte growth factor HGF
  • nuclear factor-kappa B NF- ⁇ B
  • FGF2 HGF
  • PDGF-BB vascular maturation
  • ANG1 vascular maturation factor 2
  • salidroside has a function of promoting migration of skeletal muscle cells. It has further been found that salidroside has an effect of promoting the formation of mature blood vessels under conditions of ischemia and/or hypoxia. Thus, the present invention has been completed.
  • salidroside having a structural formula as shown in Formula 1
  • the diabetic foot in the present disclosure refers to lower extremity diabetic vascular complications, or diabetes-related lower extremity vascular lesions.
  • salidroside can promote the angiogenic factor VEGF-A in skeletal muscle, Expression of fibroblast growth factor 2 (FGF2), angiopoietin-1 (ANG1), PDGF-BB, and hepatocyte growth factor (HGF), thereby promoting diabetic lower limbs
  • FGF2 fibroblast growth factor 2
  • ANG1 angiopoietin-1
  • PDGF-BB hepatocyte growth factor
  • HGF hepatocyte growth factor
  • the invention relates to the use of salidroside in the preparation of a medicament for treating ischemic diseases. Specifically, it relates to the following scheme.
  • a medicine for treating an ischemic disease of the lower extremities which comprises salidroside as an active ingredient.
  • the inventors of the present invention have found that salidroside has a good therapeutic effect for treating ischemic diseases of the lower limbs.
  • the inventors of the present invention found that salidroside specifically inhibits the expression of PHD3 and has no effect on the expression of other factors of the PHD family.
  • the inventors of the present invention have found that salidroside can promote the expression of angiogenic factors in skeletal muscle cells and secretion from skeletal muscle cells.
  • said angiogenic factor being a HIF-1 ⁇ dependent and HIF-1 ⁇ independent angiogenic factor.
  • the inventors of the present invention found that salidroside can promote HIF-1 ⁇ -dependent and HIF-1 ⁇ -independent angiogenesis factors, and simultaneously regulate various The role of angiogenic factors to achieve the results of promoting mature blood vessel formation.
  • the promoter according to any one of aspects 6 to 8, which is capable of promoting mature blood vessel formation under conditions of ischemia and/or hypoxia.
  • the inventors of the present invention have found that salidroside can promote the number of vascular endothelial cells and vascular smooth muscle cells in the hypoxic-ischemic region under hypoxic conditions, and further promote the lumen of vascular endothelial cells surrounded by vascular smooth muscle cells. The formation of structures (ie mature blood vessels).
  • a medicament for treating a lower limb ischemic disease which is a specific inhibitor of PHD3 of Scheme 5 or an accelerator of any one of Schemes 6-9.
  • the inventors of the present invention have found that the reduction of PHD3 can simultaneously regulate a variety of angiogenic factors, thereby achieving the effect of promoting the formation of mature blood vessels.
  • An agent for skeletal muscle cell migration the promoter being salidroside.
  • the inventors of the present invention have found that salidroside can promote the migration of skeletal muscle cells while promoting the secretion of various angiogenic factors by skeletal muscle cells, thereby achieving the effect of promoting mature blood vessel formation in a wider range.
  • An agent for vascular smooth muscle cell migration comprising a secreted protein produced by using erythroside to stimulate skeletal muscle cells.
  • the inventors of the present invention have found that salidroside promotes the migration of vascular smooth muscle cells by promoting cell communication between skeletal muscle cells and vascular smooth muscle cells, thereby promoting the formation of mature blood vessels.
  • agent for expression and secretion of an angiogenic factor is a medicament for treating an ischemic disease of the lower extremity.
  • angiogenic factor is one or more of VEGF-A, ANG1, FGF2, PDGF-BB, HO-1, HGF, NF- ⁇ B.
  • An enhancer according to claim 25 which promotes angiogenesis and maturation of neovascularization in tissues of lower limb ischemic diseases.
  • the lower limb ischemic diseases in the above-mentioned regimen are not limited, and include diabetes-related vasculitis such as thromboangiitis obliterans, occlusive atherosclerosis, intermittent claudication (IC), Diabetic Foot, and the like.
  • Diabetes-related vasculitis such as thromboangiitis obliterans, occlusive atherosclerosis, intermittent claudication (IC), Diabetic Foot, and the like.
  • Critical Limb Ischemia preferably intermittent claudication, diabetic foot and lower limb ischemic disease, most preferably diabetic foot and lower limb ischemic disease.
  • the present invention relates to the following aspects.
  • a medicine for treating diabetic foot the active ingredient of which is salidroside.
  • the medicament for treating diabetic foot according to claim 1 or 2 which is a skeletal muscle injection.
  • the accelerator according to claim 4 which promotes angiogenesis of skeletal muscle cells Expression and secretion of the child.
  • angiogenic factors are VEGF-A, FGF2, ANG1, PDGF-BB and HGF.
  • angiogenic factor is one or more of VEGF-A, FGF2, PDGF-BB, ANG1 and HGF.
  • a method of treating a diabetic foot wherein the patient is administered with salidroside.
  • a medicament for treating an ischemic disease of the lower extremities which contains salidroside as an active ingredient.
  • the drug promotes blood by inhibiting the expression of PHD3 Tube neonatal factors (VEGF-A, ANG1, FGF2, PDGF-BB, HO-1, HGF, NF- ⁇ B), especially factors that promote key effects on vascular maturation (FGF2, HGF, PDGF-BB, NF- ⁇ B)
  • VEGF-A, ANG1, FGF2, PDGF-BB, HO-1, HGF, NF- ⁇ B especially factors that promote key effects on vascular maturation
  • FGF2, HGF, PDGF-BB, NF- ⁇ B vascular maturation
  • the expression of ANG1) further promotes the migration of skeletal muscle cells and indirectly promotes the migration of vascular smooth muscle cells and improves the hypoxic adaptation, thereby promoting the formation of mature blood vessels in the lesion site and achieving a therapeutic effect of promoting recovery of blood flow in the lower limbs.
  • salidroside for the preparation of a medicament for treating diabetic foot.
  • salidroside can promote the expression and secretion of various angiogenic factors in skeletal muscle under high glucose and hypoxia conditions, such as expression of VEGF-A, FGF2, ANG1, PDGF-BB and HGF. secretion.
  • VEGF-A vascular endothelial growth factor
  • FGF2 FGF2, ANG1, PDGF-BB
  • HGF-BB vascular endothelial growth factor secretion.
  • a medicament for treating diabetic foot which can promote angiogenic factors VEGF-A, FGF2, ANG1, PDGF-BB and HGF in skeletal muscle under high glucose and hypoxia conditions.
  • the expression and secretion promote the formation of the vascular lumen of the lower limbs of diabetic foot patients, and in particular, promote the formation of mature, leak-free and functional blood vessels, thereby enabling the therapeutic effect of diabetic foot.
  • Figure 1A is a graph showing the effect of salidroside on blood flow recovery in a mouse model of lower limb ischemia.
  • Fig. 1B is a graph showing the quantitative results of the blood flow recovery effect of salidroside in a mouse model of lower limb ischemia.
  • Figure 1C is a graph showing the effect of salidroside on blood flow recovery in a mouse model of lower limb ischemia.
  • Fig. 2A is a photograph showing the results of immunohistochemical staining of salidroside for promoting the increase of vascular endothelial cells, vascular smooth muscle cells, and the formation of a luminal structure (i.e., mature blood vessels) surrounded by vascular smooth muscle cells.
  • Fig. 2B is a quantitative diagram showing the results of immunohistochemical staining of salidroside for promoting vascular endothelial cells and vascular smooth muscle cells.
  • Figure 3 A shows the results of specific inhibition of PHD3 by salidroside.
  • Figure 3B shows the effect of salidroside on the expression of angiogenic factors.
  • Figure 3C shows the effect of PHD3 gene silencing on angiogenic factors.
  • Fig. 4 is a graph showing the promoting effect of salidroside on skeletal muscle cell migration.
  • Fig. 5 is a graph showing the promoting effect of conditioned medium on vascular smooth muscle cell migration.
  • Fig. 6 is a graph showing the results of identification of the types of secreted proteins produced by salidroside-stimulated skeletal muscle cells which promote vascular smooth muscle cell migration.
  • Figure 7 is a graph showing the effect of salidroside on skeletal muscle cell apoptosis under hypoxic conditions.
  • Fig. 8(A) is a diagram showing a color photograph in which the effect of salidroside on promoting blood flow recovery in diabetic foot mice is converted into a grayscale image.
  • Fig. 8(B) is a view showing a state in which the blood flow portion in Fig. 8(A) is processed.
  • Fig. 9 is a graph showing the effect of salidroside on promoting blood flow recovery in diabetic foot mice.
  • Fig. 10 is a photograph showing the effects of mature blood vessel formation in the saline control group and the salidroside-treated group, respectively, 21 days after the operation.
  • Fig. 11 is a quantitative histogram showing the effects of salidroside and physiological saline on vascular endothelial cells and vascular smooth muscle cells, respectively.
  • Figure 12 is a bar graph showing the effect of salidroside on angiogenic factors.
  • the lower limb ischemic disease in the present disclosure may be any kind of lower limb ischemic disease, such as thromboangiitis obliterans, obliterative arteriosclerosis, intermittent claudication, Diabetic Foot and the like.
  • Related vascular lesions and malignant lower limb ischemic disease Critical Limb Ischemia.
  • the therapeutic agent for lower limb ischemic diseases in the present disclosure contains salidroside as an active ingredient.
  • the salidroside in the present disclosure is a compound of the formula (Formula 1).
  • the source of salidroside may be extracted from sedum plants such as Rhodiola rosea, Rhodiola rosea, Rhodiola rosea, and Rhodiola sachalinensis, or may be chemically synthesized.
  • the purity of salidroside is not particularly limited, but is preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, still more preferably 98% or more, and most preferably 99.8% or more.
  • concentration at which salidroside is used to treat lower limb ischemic diseases can be set to be 10-500 mg/kg body weight per injection.
  • This dose can be used in a single or divided dose.
  • the number of administrations may be single or multiple, and may be administered daily or intermittently.
  • the degree of the lower limb ischemic disease in the present disclosure is not limited, and may be a pre-stage lesion, a mild, moderate or severe lower limb ischemic disease, or a chronic or acute lower limb ischemic disease.
  • the "hypoxia” or “hypoxia” in the present disclosure is not limited, and may be hypoxia and hypoxia of tissue cells caused by diseases of lower limb ischemic diseases.
  • the low oxygen content means that the oxygen partial pressure is preferably higher than 0 mmHg and 100 mmHg or less, more preferably 10 to 100 mmHg, still more preferably 20 to 100 mmHg, still more preferably 30 to 30.
  • the lower limb ischemic mouse model in the embodiments of the present disclosure is constructed by completely cutting the thigh aorta (ie, a severe lower limb ischemia and hypoxia model), and the mouse used is vascular remodeling and The Balb/c variety with very poor blood flow recovery; and the cell experiments used conditions of severe hypoxia (oxygen concentration below 0.1%) that had a very serious effect on the cells.
  • the effects of the present disclosure described later have a good therapeutic effect on severe ischemic diseases of the lower extremities, and a pre-stage lesion with mild vascular remodeling and lower limb function recovery ability, mild or moderate lower limbs. Ischemic disease can have a better therapeutic effect.
  • the angiogenic factor in the present disclosure is a factor that contributes to the formation of mature blood vessels, including factors contributing to lumen formation (VEGF-A, FGF2, HGF, etc.) and factors contributing to cell maturation (FGF2). HGF, PDGF-BB, NF- ⁇ B, ANG1, etc.), factors (HO-1, etc.) that improve hypoxia tolerance and cytoprotection.
  • Angiogenesis factors include HIF-1 ⁇ -dependent and HIF-1 ⁇ -independent angiogenic factors. HIF-1 ⁇ -dependent angiogenic factors such as VEGF, PDGF-BB and HO-1, while HIF-1 ⁇ -independent angiogenic factors such as FGF2, HGF, NF- ⁇ B and ANG1.
  • Cell communication in the present disclosure is any means of intercellular communication, such as endocrine, paracrine, autocrine, chemical synapse, contact-dependent communication, gap junction Etc., which can mediate cell communication, can be ligands/receptors, small molecule compounds, vesicles, and the like.
  • angiogenic factors involved in the present disclosure are common in mice and mammals including humans, and therefore, based on this specification, The effect of guanosine in a mouse model and the description of the mechanism of action, it will be understood by those skilled in the art that salidroside can also achieve treatment of lower limb ischemic diseases and specifically inhibit PHD3 in mammals including humans.
  • promote the expression and secretion of angiogenic factors promote the migration of skeletal muscle cells and vascular smooth muscle cells, promote the formation of mature blood vessels, and the effects described in the present specification.
  • the present inventors have found that salidroside has a good therapeutic effect on a mouse diabetic foot model.
  • the diabetic foot in the present disclosure refers to lower extremity diabetic vascular complications or diabetes-related lower extremity vascular lesions.
  • the degree of diabetic foot in the present disclosure is not limited, and may be a pre-stage lesion, a mild, moderate or severe diabetic foot.
  • Diabetes in the present disclosure includes type 1 diabetes, type 2 diabetes, and pre-diabetes.
  • the fasting blood glucose of type 1 diabetes and type 2 diabetes is ⁇ 7.0mmol/L
  • the fasting blood glucose of pre-diabetes is greater than 6.1mmol/L and less than 7.0mmol/L.
  • the "high sugar” described in the present specification means high sugar caused by diabetes and diabetes-related vascular disease.
  • hypoxia hereinafter referred to as "anoxia" of tissue cells caused by diabetes and diabetes-related vascular lesions.
  • anoxia hypoxia
  • the oxygen concentration in the low oxygen state Since the oxygen concentration in the tissue will vary depending on the site (including the site in the same tissue), and considering that the oxygen concentration in the tissue is lower than the arterial oxygen concentration, and the arterial oxygen partial pressure is generally considered to be 100 mmHg (less than 40 mmHg).
  • the low oxygen means that the oxygen partial pressure is preferably higher than 0 mmHg and 100 mmHg or less, more preferably 10 to 100 mmHg, still more preferably 20 to 100 mmHg, still more preferably 30 to 100 mmHg. Most preferably, it is a range of 40 to 100 mmHg.
  • diabetic foot mice in the embodiments of the present disclosure The model was constructed by completely severing the thigh aorta (ie, the lower extremity was in a severe hypoxic state), and the fasting blood glucose of the diabetic mouse model used was ⁇ 16.7 mmol/L, which was much higher than the fasting blood glucose of the diabetic standard (ie ⁇ 7.0). Mmmol/L) is a severe diabetic mouse.
  • Mmmol/L is a severe diabetic mouse.
  • the degree of diabetes i.e., the level of blood sugar
  • the degree of diabetes is inversely proportional to the ability of tissue repair, wound healing, etc., and those skilled in the art will appreciate that the effects of the present disclosure described below have a good therapeutic effect on severe diabetic foot, in addition to Early lesions with mild vascular remodeling and lower limb function recovery, mild or moderate diabetic foot can play a better therapeutic effect.
  • a therapeutic drug for diabetic foot contains salidroside as an active ingredient.
  • the salidroside in the present disclosure is a compound of the formula (Formula 1).
  • the source of salidroside may be extracted from sedum plants such as Rhodiola rosea, Rhodiola rosea, Rhodiola rosea, and Rhodiola sachalinensis, or may be chemically synthesized.
  • the purity of salidroside is not particularly limited, but is preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, still more preferably 98% or more, and most preferably 99.8% or more. Regarding the effect of purity, the applicant explains the following.
  • the presently disclosed embodiments of the present disclosure describe the use of salidroside produced by Shanghai Tongtian Biotechnology Co., Ltd. (purity ⁇ 98.0%), and the applicant also uses the salidroside product of the China Food and Drug Administration Research Institute ( The purity is ⁇ 99.8%), and the effects of using these two concentrations of salidroside are the same, and it is understood that the effects of the present disclosure described later are produced by salidroside rather than by an impurity component.
  • concentration at which salidroside is used to treat lower limb ischemic diseases can be set to be 10-500 mg/kg body weight per injection.
  • This dose can be used in a single or divided dose.
  • the number of administrations may be single or multiple, and may be administered daily or intermittently.
  • the angiogenic factor in the present disclosure is a factor that promotes the formation of mature blood vessels, including factors contributing to lumen formation (VEGF-A, FGF2, HGF, etc.) and factors contributing to cell maturation (FGF2). , HGF, PDGF-BB, ANG1, etc.).
  • mice and mammals including humans and their effects are common, and therefore, those skilled in the art should understand that based on the present specification
  • the effect of salidroside and the description of the mechanism of action, the effects of the present disclosure can also achieve the treatment of diabetic foot in mammals including humans, promote the expression and secretion of angiogenic factors under high glucose and hypoxia conditions, and promote The formation of mature blood vessels and the like described in this specification.
  • the therapeutic agent for the lower limb ischemic disease and/or diabetic foot in the present disclosure may also contain one or more excipients.
  • the excipient is not limited, and is, for example, a solvent, an isotonic agent, an excipient, a pH adjuster, an antioxidant, a disintegrant, a flavoring agent, a perfume, a preservative, and the like which are commonly used in the art.
  • solvent examples include distilled water for injection, physiological saline, vegetable oil, alcohol such as propylene glycol, polyethylene glycol, ethanol, and glycerin.
  • isotonic agent examples include an isotonic agent commonly used in the art such as sorbitol, sodium chloride, and glucose.
  • excipient examples include lactose, mannitol, glucose, microcrystalline cellulose, and starch.
  • pH adjuster examples include hydrochloric acid, citric acid, sodium hydroxide, strong potassium oxide, sodium hydrogencarbonate, and disodium hydrogen phosphate.
  • antioxidant examples include sodium sulfite, sodium hydrogen sulfite, and ascorbic acid.
  • potato starch As a disintegrating agent, potato starch is mentioned.
  • sweeteners such as sucrose and monosaccharide syrup.
  • preservatives examples include preservatives commonly used in the art such as parabens, sorbic acid, and salts thereof.
  • the therapeutic agent for lower limb ischemic diseases and/or diabetic foot in the present disclosure may be any one of dosage forms such as an oral solution, a patch, a tablet, a capsule, an injection, etc., preferably an injection, and most preferably a skeletal muscle injection.
  • the inhibitor of the present disclosure is an agent that inhibits the expression level of a gene, and may include an agent that inhibits transcription, translation, protein synthesis, and protein stability.
  • An agent is an agent that promotes the expression or secretion level of a gene, and may include an agent that promotes transcription, translation, protein synthesis, protein stability, and secretion.
  • Balb/c mice (8 weeks, male) were used to remove the left thigh aorta under anesthesia and blood flow was measured using the Laser Doppler Perfusion Imaging System (MOOR INSTRUMENTS Ltd, MOORLDLS2-IR) (Reference) Shourong Wu et al., Prolyl hydroxylase domain-2 silencing induced by hydrodynamic limb vein injection enhances vascular regeneration in sinus sinus sinus sinus sinus sinus method).
  • MOOR INSTRUMENTS Ltd MOORLDLS2-IR
  • the left thigh is operated, and the mouse is in a prone state, and the mouse in the photo of the blood flow diagram described later is The supine state, so the thigh of the operation in the blood flow picture is on the right side of the figure.
  • the salidroside (Shanghai Tongtian Biotechnology Co., Ltd., purity ⁇ 98.0%) was dissolved in PBS, and prepared into a 40 mg/ml stock solution, filtered through a 0.22 ⁇ m filter, and stored at -20 ° C until use.
  • the salidroside stock solution was diluted to 20 mg/ml before injection.
  • the injection dose of the mice was 100 mg/kg, and the injection was started every three days from the first day after the operation, and the injection was divided into three times for each injection, and three injections were separately administered to the left gastrocnemius muscle.
  • Physiological saline was used as a control, and filtration, preservation, and injection were similarly performed.
  • the gray portion reflects the blood flow condition.
  • the mice in the saline control group and the salidroside-treated mice had the same blood flow status.
  • the Laser Doppler Perfusion Imaging System was originally imaged as a color map, which was converted into a grayscale image, and the original The resulting image looks a little different. In the original color map, red indicates that the blood flow is rich, blue indicates no blood flow. Under the gray image, the blood flow recovery cannot be clearly distinguished (ie, the red color in the original color map) Part) and where there is no blood flow (ie, the blue part of the original color map).
  • the inventor of the present application proceeds to the grayscale image based on the color map result.
  • Image processing is performed, and the processed picture is taken as FIG. 1A.
  • the dot pattern indicates where the blood flow is restored, that is, the red portion in the original color map.
  • the mice in the control group and the salidroside-treated group showed black in the left lower limbs (all in the original color map, and in the processed image, the black portion without the dot pattern) That is, there is no blood flow in the left lower limbs of both of them, and it is known that the surgery successfully produces a mouse model of lower limb ischemia.
  • the left and right lower limbs of the salidroside-treated group began to appear gray and black parts (ie, the red part in the original color map and the half-point part in the processed figure), and it was found that obvious blood appeared.
  • the left lower limb of the saline control group was still black (ie, the blue part of the original color map, and the black part without the dot pattern in the processed image), it is known that there is no Signs of blood flow recovery.
  • the left lower limb of the salidroside-treated group reached almost the same blood flow condition as the untreated left lower limb, that is, the blood flow of the left lower limb was fully recovered;
  • the left lower extremity image of the saline control group mice was absent, which was presumed to be due to the lack of blood flow and the lower limb atrophy and tissue necrosis.
  • Fig. 1B The specific quantitative method is: dividing the quantitative value of the ischemic lower limb (left lower limb) (ie, the pixel of the blood flow area) by the quantitative value of the non-ischemic lower limb (right lower limb) of the same mouse, and then each time The ratio of points is averaged and divided by the average of the preoperative ratios for each group. Referring to Fig. 1B based on the statistical results, the blood flow ratio of the control mice and the salidroside-treated mice decreased to a value lower than 0.2 immediately after the operation.
  • the blood flow ratio of the salidroside-treated mice was higher than 0.5; in contrast, the blood flow ratio of the saline control mice was significantly lower than 0.2.
  • the blood flow ratio of the salidroside-treated mice reached 0.8 or higher; in contrast, the blood flow ratio of the control mice remained at a low value of about 0.4.
  • the applicant shows a gray scale map of the blood flow condition detection result (a graph obtained by grading the color map of the blood flow condition detection result) in FIG. 1C.
  • the frozen section was used, and 21 days after the operation, the left gastrocnemius muscle tissue of the mouse was obtained and stored at -80 °C. The tissue was sectioned after freezing.
  • the sectioning procedure was as follows.
  • the embedding agent was embedded in the tissue and sliced on a microtome (manufactured by Leica), and the section thickness was 10 ⁇ m. After the end of the section, the sections were placed in an oven at 37 ° C for 30 min, and blocked in 2.5% bovine serum albumin (BSA) for 30-60 min.
  • BSA bovine serum albumin
  • ⁇ -SMA anti- ⁇ -Smooth Muscle Actin
  • Mouse monoclonal (Clone 1A4, Sigma-Aldrich Cat C6198) (ie, ⁇ -Smooth Muscle-Cy3, antibody dilution ratio 1:100
  • fluorescently labeled secondary antibody against anti-PECAM-1 antibody Goat anti-Rat IgG (H+L) Secondary Antibody, Alexa
  • Thermo Scientific Cat A11006 antibody dilution ratio 1:100
  • vascular endothelial cells i.e., PECAM-1 positive
  • vascular smooth muscle cells i.e., ⁇ -SMA positive
  • overlapping images it was found that the structure of double positive of PECAM-1 and ⁇ -SMA increased, and the luminal structure surrounded by vascular smooth muscle cells surrounded by vascular endothelial cells was formed, which means that abundant mature blood vessels were formed, and physiological saline was injected.
  • the mouse has only a very weak positive signal and does not form a bureaucratic structure.
  • the quantitative results also show that salidroside can significantly induce more vascular endothelial cells and vascular smooth muscle cells under hypoxic conditions.
  • mice can treat the lower limb ischemia of mice, that is, promote the recovery of ischemic lower limb blood flow, which is probably due to the fact that salidroside promotes angiogenesis and mature blood vessel formation in mice.
  • RNA extraction A mouse model of lower limb ischemic disease was established and injected with salidroside according to the above 1. The mice were euthanized on the third day of surgery and the left gastrocnemius muscle tissue was placed in RNAlater Solution. The tissue was divided into two pieces and ground in liquid nitrogen. After being pulverized, TRIZOL (Invitrogen) was added, and 1.5 mL of the enzyme-free EP tube was collected, and RNA was extracted according to the instructions of Trizol. After RNA extraction, the quality and concentration of the extracted RNA were detected by Nanodrop-2000 (Gene Company, Ltd), followed by reverse transcription.
  • Reagent Usage amount 5*gDNA Eraser Buffer 2.0 ⁇ L gDNA Eraser 1.0 ⁇ L Total RNA 1.0 ⁇ g RNase Free DH 2 O Up to 10.0 ⁇ L
  • PHD1 and PHD2 plays a crucial role in the regulation of angiogenesis. According to the results of the applicant's experiments (see Fig. 3A), it was confirmed that salidroside specifically inhibits PHD3 in skeletal muscle, and has no inhibitory effect on PHD1 and PHD2.
  • salidroside promoted the expression of angiogenic factors (VEGF-A, ANG1, FGF2, PDGF-BB, HO-1, HGF, NF- ⁇ B).
  • FGF2, HGF, PDGF-BB, NF- ⁇ B, and ANG1 are known to be factors related to vascular maturation, and thus those skilled in the art can understand that salidroside promotes various angiogenic factors such as VEGF. Up-regulation of the amount can promote lumen formation and promote the maturation of new blood vessels by promoting the up-regulation of FGF2, HGF, PDGF-BB, NF- ⁇ B and ANG1 expression.
  • the applicant further investigated the relationship between the inhibition of PHD3 and the up-regulation of angiogenic factor expression by PHD3 gene silencing experiments.
  • shRNA expression plasmids Two short hairpin RNA (shRNA) expression plasmids, shPHD3-1 and shPHD3-2, were constructed using PHD3 as a target to induce RNA interference.
  • plasmids see: Yang Yang 1 induces transcriptional activity of p73 through cooperation with E2F1, Shourong Wu et. al., Biochemical and Biophysical Research Communications 365 (2008) 75–81; and Synergistic cooperation of MDM2 and E2F1 contributes To TAp73 transcriptional activity, Vivi Kasim et al., Biochemical and Biophysical Research Communications 449 (2014) 319-326).
  • Mouse skeletal muscle cells C2C12 were plated in a 6-well plate at 300,000 cells per well.
  • DMEM basic + 10% fetal bovine serum (FBS) antibiotic-free medium
  • Transfection was also performed according to the instructions of Lipofectamine 2000 (Invitrogen) reagent. 2 ⁇ g of the plasmid (shPHD3-1, shPHD3-2 shRNA expression plasmid) was uniformly mixed with 200 ⁇ L of Opti-MEM medium, and 4 ⁇ L of Lipofectamine 2000 was mixed with 200 ⁇ L of Opti-MEM medium. Allow to stand at room temperature for 5 min. The two mixing systems were mixed together and allowed to stand for 20 min before being added to a six-well plate.
  • DMEM + 10% fetal bovine serum medium DMEM + 10% fetal bovine serum (FBS) + penicillin-streptomycin solution
  • FBS fetal bovine serum
  • the interference sequences used in the RNA interference test are as follows:
  • the hypoxic treatment was carried out by placing a cell culture plate and AnaeroPack. Anaero (Mitshubishi Gas Chemical, Japan) in a special sealed container (standard square sealed container, Mitsubishi Gas Chemical) and placed in an incubator. The oxygen concentration in the sealed container is less than 0.1%.
  • VEGF-A As can be seen from Fig. 3C, the expression levels of VEGF-A, ANG1, FGF2, PDGF-BB, HO-1, HGF, and NF- ⁇ B were significantly up-regulated after silencing the PHD3 gene.
  • the salidroside (Shanghai Tongtian Biotechnology Co., Ltd., purity ⁇ 98.0%) was dissolved in PBS, prepared into a 2 mg/ml stock solution, filtered through a 0.22 ⁇ m filter, and stored at -20 °C.
  • the skeletal muscle cells C2C12 were seeded in a transwell chamber, 4000 cells in each chamber, and after 5 hours, the 24-well plates in the transwell chamber were replaced with control medium (containing 10% fetal bovine serum and Penicillin, Streptomycin). Double-antibody DMEM medium) or contains red
  • the saponin (adding salidroside to the above control medium, the salidroside content was 100 ⁇ g/ml) was cultured under hypoxic conditions. After 18 hours, the transwell chamber was removed, and the unmigrated cells in the transwell chamber were removed. The cells were filved by DAPI and reached the cells on the other side of the chamber and photographed under a fluorescence microscope (6 or more per group) and passed 6 sheets. The number of cells in the photo (ie, the migrated cells) gives the average number of cells in each photo.
  • salidroside significantly promoted the migration of skeletal muscle cells, but did not promote migration to vascular smooth muscle cells.
  • the mouse skeletal muscle cells C2C12 were sown into a 100 mm*20 mm culture dish, 600,000 cells per plate, cultured for 18 hours, washed with PBS, and replaced with a medium containing salidroside (DMEM + 10% FBS+). Penicillin + Streptomycin + 100 ⁇ g / ml salidroside) to stimulate skeletal muscle cells. After 24 hours of treatment, the medium containing salidroside was removed, and the cells were washed with PBS, replaced with DMEM + 10% FBS + Penicillin + Streptomycin medium, and placed in a hypoxic chamber for 24 hours to collect the medium, 3000 rpm / min.
  • DMEM + 10% FBS+ Penicillin + Streptomycin + 100 ⁇ g / ml salidroside
  • the collected culture medium without salidroside was used as a conditioned medium.
  • conditioned medium containing a variety of secreted proteins secreted by skeletal muscle cells stimulated by salidroside
  • the mouse vascular smooth muscle cells MOVAS were prepared as described in 2-3-2. Perform a Transwell chamber experiment.
  • the preparation method was the same as that of the conditioned medium, but without adding salidroside, PBS having the same volume as salidroside was added.
  • the mouse vascular smooth muscle cells were treated as described in 2-3-2.
  • MOVAS performed Transwell chamber experiments.
  • Figure 5 shows that secreted proteins secreted by salidroside-stimulated skeletal muscle cells can Promotes migration of vascular smooth muscle cells. Combined with the above-mentioned medium containing salidroside, there is no effect on the migration of vascular smooth muscle cells. We believe that the migration of vascular smooth muscle cells is promoted in this experiment because skeletal muscle cells are stimulated by salidroside The secreted protein is then secreted into the culture medium, and the secreted protein functions as a cellular communication between skeletal muscle cells and vascular smooth muscle cells. It is also known that migration of vascular smooth muscle cells is very important for the formation of functional mature blood vessels.
  • the vascular smooth muscle cells MOVAS were cultured in the above conditioned medium, and the inhibitor of each angiogenic factor receptor was added to the MOVAS medium to confirm the migration of mouse vascular smooth muscle cells MOVAS.
  • salidroside promotes the migration of vascular smooth muscle cells by promoting cell communication between skeletal muscle cells and vascular smooth muscle cells mediated by FGF2 and PDGF-BB, thereby promoting the formation of mature blood vessels.
  • KGI cell apoptosis PI staining kit Item No.: KGA214
  • Mouse skeletal muscle cells C2C12 were seeded in 6 cm plates, 100,000 cells per plate, washed with PBS 18 hours later, and replaced with medium containing salidroside (DMEM + 10% FBS + Penicillin + Streptomycin + 100 ⁇ g) /ml salidroside). After continuing to culture for 24 hours, the medium was removed and washed with PBS, replaced with a medium containing no salidroside, and cultured under hypoxic conditions for 48 hours. After collecting the cells, apoptosis was detected using apoptotic PI staining kit (KGI, Cat. No. KGA214) and flow cytometry (FACS). Specific steps are as follows:
  • the cells were washed once with 1 x Buffer A (centrifugation 2000 rpm, 5 min), and the cells were collected and the cell concentration was diluted to 1 x 10 6 cells/ml.
  • RNase A was added to a final concentration of 0.25 mg/ml, and reacted at 37 ° C for 30 min.
  • PI positive cells apoptotic cells
  • the inventors confirmed the number of PI positive cells by flow cytometry, and hypoxic conditions did not significantly induce apoptosis of skeletal muscle cells.
  • the apoptosis rate of salidroside under hypoxic conditions was compared with normoxic conditions and hypoxia. There was no significant difference in the rate of apoptosis.
  • salidroside can specifically inhibit the expression of PHD3 in skeletal muscle cells, thereby promoting the expression of various angiogenic factors of skeletal muscle cells under hypoxic conditions, and also improving bones.
  • the ability of muscle cells to migrate, these factors increase the number of angiogenic factors secreted by skeletal muscle cells, and the range of their effects is increased; and PDGF-BB and FGF2 in angiogenic factors secreted by these skeletal muscle cells act through
  • the respective receptors on vascular smooth muscle cells also referred to in the present disclosure as skeletal muscle cells - cellular communication between vascular smooth muscle cells
  • promote the migration of vascular smooth muscle cells which are critical for the formation of mature blood vessels, and ultimately The formation of mature blood vessels, thereby obtaining a good therapeutic effect of lower limb ischemic diseases.
  • mice (6 weeks, male) were purchased (purchased from the Third Military Medical University of the People's Liberation Army) and the blood glucose of the mice was measured one week after returning.
  • the blood sugar was measured after feeding for 4 weeks with the following high-fat diet, and pre-diabetes occurred (pre- After the case of diabetes, streptozotocin was continuously injected through the muscle for five days, and the dose was 50 mg/kg.
  • the blood glucose of the mice was measured after one week of high-fat diet feeding.
  • the blood glucose above 16.7 mmol/L was selected as the next step. experiment.
  • blood glucose of 7 or more is considered to have diabetes, but the model of the present disclosure selects a mouse having blood glucose of 16.7 or more.
  • the left thigh aorta was excised under anesthesia using the above-described diabetic mouse model, and blood flow was measured using a Laser Doppler Perfusion Imaging System.
  • the left thigh is operated, and the mouse is in a prone state, and the mouse in the photo of the blood flow diagram described later is The supine state, so the thigh of the operation in the blood flow picture is shown on the right side of the figure) (refer to the literature Shourong Wu et al., Prolyl hydroxylase domain-2silencing induced by hydrodynamic limb vein injection enhances vascular regeneration in the essence limb ischemia mice into activation of Multiple genes (2015) Curr Gene Ther., 15(3): Method in 313-325).
  • the salidroside (purchased from Shanghai Tongtian Biotechnology Co., Ltd., the sample purity is higher than 98%) was dissolved in phosphate buffer solution (PBS), and prepared into a 40 mg/ml stock solution, which was filtered through a 0.22 ⁇ m filter. Store at -20 °C for later use.
  • PBS phosphate buffer solution
  • the salidroside stock solution was diluted to 20 mg/ml before injection, and the dose of the mouse intramuscular injection was 100 mg/kg.
  • the injection was started every three days from the first day after the operation, and the injection was divided into three times for each injection. Three sites were injected in the left gastrocnemius muscle.
  • Physiological saline was used as a control, and filtration, preservation, and injection were similarly performed.
  • the gray portion reflects the blood flow condition.
  • the mice in the saline control group and the salidroside-treated mice had the same blood flow status.
  • the Laser Doppler Perfusion Imaging System was originally imaged as a color map, which was converted into a grayscale image, and the original The resulting image looks a little different. In the original color map, red indicates that the blood flow is rich, blue indicates no blood flow. Under the gray image, the blood flow recovery cannot be clearly distinguished (ie, the red color in the original color map) Part) and where there is no blood flow (ie, the blue part of the original color map).
  • the inventors of the present invention performed image processing on the grayscale image based on the result of the color map, thereby obtaining FIG. 8(B).
  • the dot pattern indicates where the blood flow is restored, that is, the red portion in the original color map.
  • the left lower limbs of the mice in the control group and the salidroside-treated group showed Black (both in the original color map, there is no dot pattern in the processed image), that is, there is no blood flow in the left lower limbs of both, and it is known that the diabetic foot mouse model is successfully manufactured.
  • the left and right lower limbs of the salidroside-treated group began to appear gray and black parts (ie, the red part in the original color map and the half-point part in the processed figure), and it was found that obvious blood appeared.
  • the left lower limb of the saline control group was still black (ie, the blue part of the original color map, and the black part without the dot pattern in the processed image), it is known that there is no Signs of blood flow recovery.
  • the left lower limb of the salidroside-treated group reached almost the same blood flow condition as the untreated left lower limb, that is, the blood flow of the left lower limb was fully recovered;
  • the left lower extremity image of the saline control group mice was absent, which was presumed to be due to the lack of blood flow and the lower limb atrophy and tissue necrosis.
  • the inventors of the present application quantified the results of the Laser Doppler Perfusion Imaging System.
  • the specific calculation method is: dividing the quantitative value of the ischemic lower limb (left lower limb) by the quantitative value of the non-ischemic lower limb (right lower limb) of the same mouse (ie, the pixel of the blood flow area), and then each time The ratio of points is averaged and divided by the average of the preoperative ratios for each group. Referring to Fig. 9 based on the statistical results, the blood flow ratio of the control group and the salidroside-treated mice decreased to a value lower than 0.3 immediately after the operation.
  • the blood flow ratio of the salidroside-treated mice reached 0.6; in contrast, the blood flow ratio of the saline control mice was significantly lower than 0.4.
  • the blood flow ratio of the salidroside-treated mice reached 0.8; in contrast, the blood flow ratio of the control mice was still close to the value of the third day after surgery.
  • salidroside has excellent blood flow recovery effect on the treatment of diabetic foot mice. Compared with the saline-injected control, the blood flow status of the diabetic foot mice injected with salidroside was significantly restored, and it was found that salidroside had a good effect on the treatment of diabetic foot, and thus it was found that the mature, no Leaky and functional neovascularization.
  • the gastrocnemius muscle tissues of the salidroside-treated group and the saline-treated group were obtained and stored at -80 °C. After the tissue was frozen, it was sliced to obtain an experimental sample.
  • the sectioning procedure was as follows.
  • the embedding agent was embedded in the tissue and sliced on a microtome (manufactured by Leica), and the section thickness was 10 ⁇ M. After the end of the section, the sections were placed in an oven at 37 ° C for 30 min, and blocked in 2.5% bovine serum albumin (BSA) for 30-60 min.
  • BSA bovine serum albumin
  • a fluorescently labeled antibody i.e. ⁇ - Smooth Muscle-Cy3, Mouse monoclonal (Clone 1A4, Sigma-Aldrich Cat C6198
  • PECAM- 1 antibody secondary antibody Goat anti-Rat IgG (H+L) Secondary Antibody, Alex
  • the mixture of secondary antibodies was incubated for 30 min at room temperature.
  • the PBS-T was then washed three times for 5 min each time.
  • detection was performed using a fluorescence microscope (Leica Microsystems, DMI6000B).
  • vascular endothelial cells i.e., PECAM-1 positive
  • vascular smooth muscle cells i.e., ⁇ -SMA positive
  • results further indicate that the injection of salidroside can treat the recovery of blood flow in the ischemic lower limb of diabetic foot, which is presumed to be due to the fact that salidroside promotes angiogenesis and mature blood vessel formation in mice.
  • the inventors of the present application measured the blood glucose of diabetic foot mice.
  • the blood sugar test method is: dripping blood to Vibrant blood glucose test strip insertion Vital blood glucose meter ( Active, Model GU, Roche). use Blood collection pen Lancing Device, Roche) and Single use lancet ( Roche), collecting blood from the tail of diabetic foot mice and adding blood drops The middle of the orange area of the vital blood glucose test strip.
  • the blood glucose level of the salidroside-treated mice was the same as that of the saline control group. It can be seen that local injection of salidroside on the ischemic thigh skeletal muscle of diabetic foot mice did not affect blood glucose levels. That is, it can be seen that the local injection of salidroside in the skeletal muscle of the ischemic site for blood flow recovery of diabetic foot mice and further treatment of diabetic foot is not achieved by adjusting the blood sugar level of the living body.
  • the table shows the mean ⁇ standard deviation, and the p value is calculated by the chi-square test.
  • the salidroside was dissolved in PBS, and a 40 mg/ml stock solution was prepared, filtered through a 0.22 ⁇ m filter, and stored at -20 ° C until use.
  • the salidroside stock solution was diluted to 20 mg/ml before injection.
  • a diabetic foot mouse model was established according to the description in item 1. above, and the obtained salidroside dilution was injected.
  • the dose of intramuscular injection of diabetic mice was 100 mg/kg, which was injected on the first day after surgery. The injection was divided into 3 times and injected into the left gastrocnemius muscle at 3 sites.
  • the instruments in the experiment mainly include: NanoDrop2000 micronucleic acid analyzer (Gene Company). R&D (domestic), PCR instrument (T100 Thermal cycler, Bio-RAD), Erlian (Bio-RAD), Q-PCR (CFX-96 Optical Reaction Module #1845097, Bio-RAD).
  • RNA Reverse Transcription and Real-Time Quantification Kit (TaKaRa RR047A PrimeScript RT reagent Kit with gDNA Eraser), TriZol (Invitrogen).
  • TAKARA-PrimeScriptTM RT reagent Kit with gDNA Eraser (Code No. RR047A)
  • the reagents used in this step and the amounts thereof used are as follows.
  • the reaction process is as follows: 42 ° C 2 min
  • the reverse transcription reaction was carried out using the following reagents in the following amounts.
  • the reaction process is as follows: 37 ° C 15 min
  • the cDNA was diluted 10 times after the cDNA was obtained.
  • the diluted sample was used for the Q-PCR reaction.
  • the reaction system is as follows:
  • the Q-PCR instrument is CFX-96 Optical Reaction Module #1845097 (Bio-RAD).
  • Fig. 12 The experimental results are shown in Fig. 12. As can be seen from Fig. 12, angiogenic factors VEGF-A, FGF2, ANG1 in skeletal muscle after administration of salidroside to the ischemic lower limb of a diabetic model mouse. The expression levels of PDGF-BB and HGF were significantly up-regulated. It is believed that salidroside can promote the expression of various angiogenic factors under high glucose and hypoxia conditions.

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Abstract

The present invention relates to an application of a rhodioloside for preparing a pharmaceutical product for treating an ischemic disease. The invention further relates to an application of the rhodioloside for preparing a pharmaceutical product for treating a diabetic foot disease. In a mouse model for the diabetic foot pathology, the rhodioloside resulted in an exceptional improvement to the expression and secretion of an angiogenic growth factor under a high sugar and low oxygen condition. The invention further provides a pharmaceutical product for treating a lower limb ischemia, wherein the pharmaceutical product contains a rhodioloside as an active ingredient. The invention further provides an exceptional inhibitor for PHD3, wherein the inihibitor is a rhodioloside.

Description

红景天苷在制备治疗缺血性疾病的药物中的应用Application of salidroside in preparing medicine for treating ischemic diseases 技术领域Technical field
本发明涉及红景天苷在制备治疗缺血性疾病的药物中的应用。具体地，本发明涉及红景天苷在制备治疗糖尿病足的药物中的应用。本发明还涉及PHD3的特异性抑制剂、血管新生因子促进剂、血管平滑肌细胞迁移促进剂、骨骼肌细胞迁移促进剂以及成熟血管形成的促进剂。The invention relates to the use of salidroside in the preparation of a medicament for treating ischemic diseases. In particular, the invention relates to the use of salidroside for the preparation of a medicament for the treatment of diabetic foot. The present invention also relates to a specific inhibitor of PHD3, an angiogenesis factor promoter, a vascular smooth muscle cell migration promoter, a skeletal muscle cell migration promoter, and an agent for mature angiogenesis.
背景技术Background technique
缺血性疾病是由于血管损伤、血管堵塞等原因造成组织细胞供血量不足的外周血管疾病的一种。An ischemic disease is a type of peripheral vascular disease in which tissue cells are insufficiently supplied due to vascular damage, vascular clogging, and the like.
外周动脉疾病(Peripheral Artery Disease)是指由于动脉粥样硬化等导致动脉阻塞，从而限制心脏以外的组织的血流供应。下肢缺血性疾病(Limb Ischemia)是一种最常见的外周动脉疾病，由于血管堵塞、血栓、高血糖等原因导致远端(下肢)供血不足。由于氧气是由血液中的红血球运输到各个组织器官，供血不足的结果是引起下肢组织缺氧、缺营养，严重时会导致组织坏疽，组织缺失(tissue loss)甚至个体的死亡。Peripheral Artery Disease refers to the blockage of arteries due to atherosclerosis or the like, thereby limiting the blood flow supply to tissues other than the heart. Limb Ischemia is one of the most common peripheral arterial diseases, resulting in insufficient blood supply to the distal (lower extremities) due to vascular occlusion, thrombosis, hyperglycemia, and the like. Since oxygen is transported from red blood cells in the blood to various tissues and organs, the result of insufficient blood supply is caused by hypoxia and lack of nutrients in the lower limbs, which can lead to tissue gangrene, tissue loss and even individual death.
目前，下肢缺血性疾病尚未有有效的治疗和控制方法，严重时需要对患者进行截肢，给患者带来巨大的痛苦和损失。目前被认为有望起到良好效果的治疗方法是通过促进血管新生，改善远端供血的情况，从而阻止组织继续坏死并起到改善下肢功能的作用。At present, there is no effective treatment and control method for lower limb ischemic diseases. In severe cases, the patient needs to be amputated, causing tremendous pain and loss to the patient. At present, it is considered that a therapeutic effect that is expected to have a good effect is to prevent angiogenesis and improve the blood supply to the distal end, thereby preventing the tissue from continuing to be necrotic and improving the function of the lower limb.
作为下肢缺血性疾病的药物，目前有第一代治疗药物和第二代治疗药物。其中，第一代治疗药物利用的是单个血管新生因子(血管内皮生长因子(Vascular endothelial growth factor，VEGF)等)，但临床试验结果不理想，新生血管不成熟，出现漏的情况，缺乏功能性(参见：Therapeutic angiogenesis for critical limb ischaemia.Nature Reviews Cardiology,2013,10(7):387-96)。治疗结果不理想的原因被认为是由于血管重构是个多因子参与的复杂的过程。第二代治疗药物则利用了多种血管新生因子的组合 (成纤维细胞生长因子2(Fibroblast growth factor 2，FGF2)和血小板衍生因子(Platelet-derived growth factor，PDGF)；VEGF和血管生成素-1(Angiopoietin-1，ANG1))，虽然有一定的效果，然而血管新生因子种类繁多，它们在血管重构的不同的阶段起到不同的作用。因此，存在着血管新生因子种类的选择、组合时的比率、何时给药等难题。另外，目前对下肢缺血性疾病的治疗手段还面临外来血管新生因子的局部化，因此不足以在广泛的缺血缺氧区域内诱导足够的新生成熟血管的难题。As a drug for lower limb ischemic diseases, there are currently first-generation therapeutic drugs and second-generation therapeutic drugs. Among them, the first generation of therapeutic drugs utilizes a single angiogenic factor (Vascular Endothelial Growth Factor (VEGF), etc.), but the clinical trial results are not satisfactory, the new blood vessels are immature, there is leakage, and lack of functionality. (See: Therapeutic angiogenesis for critical limb ischaemia. Nature Reviews Cardiology, 2013, 10(7): 387-96). The reason for the unsatisfactory treatment outcome is believed to be due to the complex process of vascular remodeling involved in multiple factors. The second generation of therapeutic drugs utilizes a combination of multiple angiogenic factors (Fibroblast growth factor 2 (FGF2) and platelet-derived growth factor (PDGF); VEGF and Angiopoietin-1 (ANG1)), although have certain effects However, there are many types of angiogenic factors that play different roles in different stages of vascular remodeling. Therefore, there are problems in the selection of angiogenic factor types, the ratio at the time of combination, and when to administer. In addition, the current treatment of lower limb ischemic diseases is also faced with the localization of foreign angiogenic factors, and thus is not sufficient to induce sufficient new mature blood vessels in a wide area of ischemia and hypoxia.
糖尿病足是糖尿病的最常见、最严重并发症之一。由于血糖控制不理想，导致下肢外周血管病变，从而引起下肢供血不足并导致下肢组织细胞缺氧，加上高糖条件下组织修复、伤口愈合能力明显下降，严重时出现组织坏疽、组织缺失(tissue loss)甚至死亡；临床上严重患者往往需要截肢。Diabetic foot is one of the most common and serious complications of diabetes. Due to unsatisfactory blood glucose control, peripheral vascular disease of the lower extremities leads to insufficient blood supply to the lower extremities and causes hypoxia in the lower extremity tissue. In addition, tissue repair and wound healing ability are significantly reduced under high glucose conditions, and tissue gangrene and tissue loss occur in severe cases (tissue) Loss) or even death; clinically severe patients often require amputation.
糖尿病足(Diabetic Foot)的理想的治疗方法为改善供血状态，目前针对治疗血管病变的方法有利用支架、搭桥、气囊扩张术等；另外，最近，由于其无侵袭性等优势，促进血管重构被认为是最好的治疗途径。The ideal treatment for Diabetic Foot is to improve the blood supply status. At present, methods for treating vascular lesions include stenting, bypass, balloon dilatation, etc. In addition, recently, due to its non-invasive advantages, it promotes vascular remodeling. It is considered to be the best treatment route.
然而，糖尿病病人的血管病变反复率高，且病变部位较广，因此上述利用支架、搭桥或气囊扩张术对糖尿病足不是理想的治疗方法。However, in diabetic patients, the recurrence rate of vascular lesions is high and the lesions are wide. Therefore, the above-mentioned stenting, bypass or balloon dilatation is not an ideal treatment for diabetic foot.
更为重要的是，在高血糖这种独特的病理环境条件下，各种体内机能下降，各种因子的行为和生物调控途径以及反应也与正常条件有所不同。例如，糖尿病人的组织/细胞缺失了细胞对低氧环境的应激能力。高糖环境使血管内皮生长因子(Vascular endothelial growth factor,VEGF)以及VEGF受体(VEGFR)、血小板衍生因子-BB(Platelet-derived growth factor-BB,PDGF-BB)等血管新生因子的表达异常降低、且这些因子对低氧的应激反应也受损害，而这些因子在血管重构中起到重要作用。且也有报道，高糖条件下细胞表面的VEGFR2下降，阻止了VEGF-VEGFR2通路的信号传导，抑制了VEGF所诱导的血管内皮细胞的增殖等血管重构过程，导致糖尿病小鼠对VEGF的灵敏度明显低于正常小鼠，因此利用VEGF诱导下肢血管重构对治疗糖尿病足不是一个理想的治疗方法。More importantly, under the unique pathological environment of hyperglycemia, various in vivo functions decline, and the behavior and biological regulation pathways and responses of various factors are also different from normal conditions. For example, a tissue/cell of a diabetic person lacks the ability of the cell to stress the hypoxic environment. High glucose environment abnormally decreased the expression of angiogenic factors such as vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR) and platelet-derived growth factor-BB (PDGF-BB). These factors also impair the hypoxia stress response, and these factors play an important role in vascular remodeling. It has also been reported that the decrease of VEGFR2 on the cell surface under high glucose conditions prevents the signal transduction of VEGF-VEGFR2 pathway, inhibits the vascular remodeling process of vascular endothelial cells induced by VEGF, and leads to the sensitivity of diabetic mice to VEGF. Lower than normal mice, therefore, the use of VEGF to induce lower limb vascular remodeling is not an ideal treatment for diabetic foot.
现有技术文献 Prior art literature
非专利文献Non-patent literature
Diabetes Mellitus and Ischemic Diseases:Molecular Mechanisms of Vascular Repair Dysfunction.Arteriosclerosis,Thrombosis,and Vascular Biology,2014,34(6):1126-1135.Diabetes Mellitus and Ischemic Diseases: Molecular Mechanisms of Vascular Repair Dysfunction. Arteriosclerosis, Thrombosis, and Vascular Biology, 2014, 34(6): 1126-1135.
发明内容Summary of the invention
发明所要解决的问题Problems to be solved by the invention
基于上述现状，作为解决上述问题的最佳方法，目前迫切需要一种能在广泛的缺血缺氧区域内起作用，并同时调控多种血管新生因子和参与成熟血管重构的多种体内通路的药物。Based on the above situation, as the best way to solve the above problems, there is an urgent need for a variety of in vivo pathways that can function in a wide range of ischemic and hypoxic regions, and simultaneously regulate multiple angiogenic factors and participate in mature vascular remodeling. Drug.
另外，作为解决上述问题的途径，目前迫切需要一种能够在高糖、低氧条件下有效促进血管重构的方法及药物，即迫切需要对于糖尿病足的治疗有良好效果的方法及药物。In addition, as a way to solve the above problems, there is an urgent need for a method and a drug capable of effectively promoting vascular remodeling under conditions of high sugar and hypoxia, that is, a method and a drug which have a good effect on the treatment of diabetic foot.
解决问题的手段Means of solving problems
本发明的发明人等对此进行了深入研究，结果发现，红景天苷(结构式如式1所示)对治疗下肢缺血性疾病有良好的治疗效果。进而，还发现红景天苷具有在骨骼肌内特异性抑制PHD3并促进骨骼肌细胞迁移能力的效果。进而，还发现红景天苷能促进骨骼肌内血管新生因子(VEGF-A、ANG1、FGF2、PDGF-BB、血红素加氧酶-1(Heme oxygenase-1，HO-1)、肝细胞生长因子(Hepatocyte growth factor，HGF)、核因子-kappa B(Nuclear factor-kappa B，NF-κB))、特别是促进对血管成熟起关键作用的因子(FGF2、HGF、PDGF-BB、NF-κB和ANG1)的表达以及分泌的作用。进而还发现，红景天苷具有促进骨骼肌细胞迁移的作用。进而还发现，红景天苷具有在缺血以及/或缺氧条件下促进成熟血管形成的作用。由此，完成了本发明。The inventors of the present invention conducted intensive studies and found that salidroside (shown as Formula 1) has a good therapeutic effect on the treatment of ischemic diseases of the lower limbs. Further, it has been found that salidroside has an effect of specifically inhibiting PHD3 and promoting skeletal muscle cell migration ability in skeletal muscle. Furthermore, salidroside was also found to promote skeletal muscle angiogenesis factors (VEGF-A, ANG1, FGF2, PDGF-BB, Heme oxygenase-1 (HO-1), hepatocyte growth). Hepatocyte growth factor (HGF), nuclear factor-kappa B (NF-κB), and especially factors that promote vascular maturation (FGF2, HGF, PDGF-BB, NF-κB) And the expression of ANG1) and the role of secretion. Further, it has been found that salidroside has a function of promoting migration of skeletal muscle cells. It has further been found that salidroside has an effect of promoting the formation of mature blood vessels under conditions of ischemia and/or hypoxia. Thus, the present invention has been completed.
另外，本申请的发明人发现，红景天苷(结构式如式1所示)对于糖尿病足的治疗有良好的效果。本公开中的糖尿病足是指下肢糖尿病血管并发症、或糖尿病相关的下肢血管病变。Further, the inventors of the present application found that salidroside (having a structural formula as shown in Formula 1) has a good effect on the treatment of diabetic foot. The diabetic foot in the present disclosure refers to lower extremity diabetic vascular complications, or diabetes-related lower extremity vascular lesions.
另外，还发现红景天苷能够促进骨骼肌里的血管新生因子VEGF-A、 成纤维细胞生长因子2(Fibroblast growth factor 2，FGF2)、血管生成素-1(Angiopoietin-1，ANG1)、PDGF-BB和肝细胞生长因子(Hepatocyte growth factor，HGF)的表达，从而促进糖尿病下肢血管病变患者(即糖尿病足患者)的血管管腔的形成，尤其是本发明能够促进成熟、无漏且具有功能的血管的形成。由此，完成了本发明。In addition, it has also been found that salidroside can promote the angiogenic factor VEGF-A in skeletal muscle, Expression of fibroblast growth factor 2 (FGF2), angiopoietin-1 (ANG1), PDGF-BB, and hepatocyte growth factor (HGF), thereby promoting diabetic lower limbs The formation of vascular lumens in patients with vascular disease (i.e., diabetic foot patients), and in particular, the present invention is capable of promoting the formation of mature, leak-free, and functional blood vessels. Thus, the present invention has been completed.
Figure PCTCN2017071947-appb-000001
Figure PCTCN2017071947-appb-000001
本发明涉及红景天苷在制备治疗缺血性疾病的药物中的应用。具体地涉及如下方案。The invention relates to the use of salidroside in the preparation of a medicament for treating ischemic diseases. Specifically, it relates to the following scheme.
1.一种治疗下肢缺血性疾病的药物，所述药物含有红景天苷作为活性成分。本发明的发明人发现，红景天苷对于治疗下肢缺血性疾病起到良好的治疗效果。A medicine for treating an ischemic disease of the lower extremities, which comprises salidroside as an active ingredient. The inventors of the present invention have found that salidroside has a good therapeutic effect for treating ischemic diseases of the lower limbs.
2.根据方案1的治疗下肢缺血性疾病的药物，所述药物为注射剂。2. The medicament for treating an ischemic disease of the lower limb according to item 1, which is an injection.
3.根据方案1或2的治疗下肢缺血性疾病的药物，所述药物为骨骼肌注射剂。3. The medicament for treating an ischemic disease of the lower extremities according to claim 1 or 2, which is a skeletal muscle injection.
4.根据方案1～3中任一项的治疗下肢缺血性疾病的药物，所述药物进一步包含辅料。4. The medicament for treating an ischemic disease of the lower extremities according to any one of items 1 to 3, further comprising an adjuvant.
5.一种PHD3的特异性抑制剂，所述抑制剂含有红景天苷。本发明的发明人发现，红景天苷能特异性抑制PHD3的表达，同时对PHD家族的其他因子的表达没有影响。5. A specific inhibitor of PHD3, the inhibitor comprising salidroside. The inventors of the present invention found that salidroside specifically inhibits the expression of PHD3 and has no effect on the expression of other factors of the PHD family.
6.一种血管新生因子的表达和分泌的促进剂，所述促进剂含有红景天苷。6. An agent for the expression and secretion of an angiogenic factor, the promoter comprising salidroside.
7.根据方案6的促进剂，所述促进剂促进骨骼肌细胞的血管新生因子的表达和分泌。本发明的发明人发现，红景天苷能促进血管新生因子在骨骼肌细胞内的表达以及从骨骼肌细胞里的分泌。7. The accelerator according to aspect 6, which promotes expression and secretion of angiogenic factors of skeletal muscle cells. The inventors of the present invention have found that salidroside can promote the expression of angiogenic factors in skeletal muscle cells and secretion from skeletal muscle cells.
8.根据方案6或7的促进剂，所述血管新生因子为HIF-1α依赖性和HIF-1α非依赖性的血管新生因子。本发明的发明人发现，红景天苷能促进HIF-1α依赖性和HIF-1α非依赖性的血管新生因子，起到同时调控多种 血管新生因子的作用，从而达到促进成熟血管形成的结果。8. The enhancer of claim 6 or 7, said angiogenic factor being a HIF-1α dependent and HIF-1α independent angiogenic factor. The inventors of the present invention found that salidroside can promote HIF-1α-dependent and HIF-1α-independent angiogenesis factors, and simultaneously regulate various The role of angiogenic factors to achieve the results of promoting mature blood vessel formation.
9.根据方案6～8中任一项的促进剂，所述血管新生因子能在缺血以及/或缺氧条件下促进成熟血管形成。本发明的发明人发现，红景天苷能在缺血缺氧条件下促进缺血缺氧区域内血管内皮细胞以及血管平滑肌细胞的数量，进一步能促进由血管平滑肌细胞包围血管内皮细胞的管腔结构(即成熟血管)的形成。9. The promoter according to any one of aspects 6 to 8, which is capable of promoting mature blood vessel formation under conditions of ischemia and/or hypoxia. The inventors of the present invention have found that salidroside can promote the number of vascular endothelial cells and vascular smooth muscle cells in the hypoxic-ischemic region under hypoxic conditions, and further promote the lumen of vascular endothelial cells surrounded by vascular smooth muscle cells. The formation of structures (ie mature blood vessels).
10.一种治疗下肢缺血性疾病的药物，所述药物为方案5的PHD3的特异性抑制剂或方案6～9中任一项的促进剂。本发明的发明人发现，PHD3的降低能起到同时调控多种血管新生因子的作用，从而达到促进成熟血管形成的效果。A medicament for treating a lower limb ischemic disease, which is a specific inhibitor of PHD3 of Scheme 5 or an accelerator of any one of Schemes 6-9. The inventors of the present invention have found that the reduction of PHD3 can simultaneously regulate a variety of angiogenic factors, thereby achieving the effect of promoting the formation of mature blood vessels.
11.一种骨骼肌细胞迁移的促进剂，所述促进剂为红景天苷。本发明的发明人发现，红景天苷在促进骨骼肌细胞分泌各种血管新生因子的同时，能促进骨骼肌细胞的迁移，从而达到在更广泛的范围内促进成熟血管形成的效果。11. An agent for skeletal muscle cell migration, the promoter being salidroside. The inventors of the present invention have found that salidroside can promote the migration of skeletal muscle cells while promoting the secretion of various angiogenic factors by skeletal muscle cells, thereby achieving the effect of promoting mature blood vessel formation in a wider range.
12.一种血管平滑肌细胞迁移的促进剂，所述促进剂含有利用红景天苷刺激骨骼肌细胞而产生的分泌性蛋白。12. An agent for vascular smooth muscle cell migration, the promoter comprising a secreted protein produced by using erythroside to stimulate skeletal muscle cells.
13.根据方案11的骨骼肌细胞迁移的促进剂或方案12的血管平滑肌细胞迁移的促进剂，所述促进剂通过骨骼肌细胞与血管平滑肌细胞之间的细胞通讯促进血管平滑肌细胞的迁移。本发明的发明人发现，红景天苷能通过促进骨骼肌细胞与血管平滑肌细胞之间的细胞通讯，促进血管平滑肌细胞的迁移，从而促进成熟血管的形成。13. The promoter of skeletal muscle cell migration according to protocol 11 or the promoter of vascular smooth muscle cell migration of protocol 12, which promotes migration of vascular smooth muscle cells by cellular communication between skeletal muscle cells and vascular smooth muscle cells. The inventors of the present invention have found that salidroside promotes the migration of vascular smooth muscle cells by promoting cell communication between skeletal muscle cells and vascular smooth muscle cells, thereby promoting the formation of mature blood vessels.
14.根据方案11的骨骼肌细胞迁移的促进剂或根据方案12的血管平滑肌细胞迁移的促进剂，所述促进剂通过促进FGF2介导的骨骼肌细胞与血管平滑肌细胞间的细胞通讯促进血管平滑肌细胞的迁移。14. The promoter of skeletal muscle cell migration according to item 11 or the promoter of vascular smooth muscle cell migration according to protocol 12, which promotes vascular smooth muscle by promoting FGF2-mediated cellular communication between skeletal muscle cells and vascular smooth muscle cells. Migration of cells.
15.根据方案11的骨骼肌细胞迁移的促进剂或根据方案12的血管平滑肌细胞迁移的促进剂，所述促进剂通过PDGF-BB介导的骨骼肌细胞与血管平滑肌细胞间的细胞通讯促进血管平滑肌细胞的迁移。15. The promoter of skeletal muscle cell migration according to item 11 or the promoter of vascular smooth muscle cell migration according to protocol 12, which promotes blood vessels by PDGF-BB-mediated cellular communication between skeletal muscle cells and vascular smooth muscle cells Smooth muscle cell migration.
16.红景天苷在制备治疗下肢缺血性疾病的药物中的用途。16. Use of salidroside for the manufacture of a medicament for the treatment of ischemic diseases of the lower extremities.
17.根据方案16所述的用途，所述治疗下肢缺血性疾病的药物为注射剂。 17. The use according to claim 16, wherein the medicament for treating an ischemic disease of the lower extremity is an injection.
18.根据方案17所述的用途，所述治疗下肢缺血性疾病的药物为骨骼肌注射剂。18. The use according to claim 17, wherein the medicament for treating a lower limb ischemic disease is a skeletal muscle injection.
19.红景天苷在制备PHD3的特异性抑制剂中的用途。19. Use of salidroside for the preparation of a specific inhibitor of PHD3.
20.根据方案19所述的用途，所述PHD3的特异性抑制剂为治疗下肢缺血性疾病的药物。20. The use according to claim 19, wherein the specific inhibitor of PHD3 is a medicament for treating ischemic diseases of the lower extremities.
21.红景天苷在制备血管新生因子的表达和分泌的促进剂中的用途。21. Use of salidroside for the preparation of a promoter for the expression and secretion of angiogenic factors.
22.根据方案21所述的用途，所述血管新生因子的表达和分泌的促进剂为治疗下肢缺血性疾病的药物。22. The use according to claim 21, wherein the agent for expression and secretion of an angiogenic factor is a medicament for treating an ischemic disease of the lower extremity.
23.根据方案21所述的用途，所述血管新生因子的表达和分泌的促进剂促进骨骼肌细胞的血管新生因子的表达和分泌。23. The use according to embodiment 21, wherein the expression and secretion promoting agent of angiogenic factors promotes expression and secretion of angiogenic factors of skeletal muscle cells.
24.根据方案21～23中任一项的促进剂，所述血管新生因子为VEGF-A、ANG1、FGF2、PDGF-BB、HO-1、HGF、NF-κB中的一种或多种。The promoter according to any one of aspects 21 to 23, wherein the angiogenic factor is one or more of VEGF-A, ANG1, FGF2, PDGF-BB, HO-1, HGF, NF-κB.
25.红景天苷在制备成熟血管形成促进剂中的用途。25. Use of salidroside for the preparation of a mature angiogenesis promoter.
26.根据方案25的促进剂，其促进下肢缺血性疾病的组织中的血管新生以及新生血管的成熟。26. An enhancer according to claim 25 which promotes angiogenesis and maturation of neovascularization in tissues of lower limb ischemic diseases.
另外，上述方案中的下肢缺血性疾病没有限制，包括血栓闭塞性脉管炎、闭塞性动脉硬化症、间歇性跛行(Intermittent Claudication，IC)、糖尿病足(Diabetic Foot)等糖尿病相关血管病变以及恶性下肢缺血性疾病(Critical Limb Ischemia,CLI)，优选为间歇性跛行、糖尿病足和下肢缺血性疾病，最优选为糖尿病足和下肢缺血性疾病。In addition, the lower limb ischemic diseases in the above-mentioned regimen are not limited, and include diabetes-related vasculitis such as thromboangiitis obliterans, occlusive atherosclerosis, intermittent claudication (IC), Diabetic Foot, and the like. Critical Limb Ischemia (CLI), preferably intermittent claudication, diabetic foot and lower limb ischemic disease, most preferably diabetic foot and lower limb ischemic disease.
另外，本发明涉及如下方案。Further, the present invention relates to the following aspects.
1.一种治疗糖尿病足的药物，该药物的活性成份为红景天苷。A medicine for treating diabetic foot, the active ingredient of which is salidroside.
2.根据方案1的治疗糖尿病足的药物，所述药物为注射剂。2. The medicament for treating diabetic foot according to item 1, which is an injection.
3.根据方案1或2的治疗糖尿病足的药物，所述药物为骨骼肌注射剂。3. The medicament for treating diabetic foot according to claim 1 or 2, which is a skeletal muscle injection.
4.一种血管新生因子的表达和分泌的促进剂，所述促进剂为红景天苷。4. An agent for expression and secretion of an angiogenic factor, the promoter being salidroside.
5.根据方案4的促进剂，所述促进剂促进骨骼肌细胞的血管新生因 子的表达和分泌。5. The accelerator according to claim 4, which promotes angiogenesis of skeletal muscle cells Expression and secretion of the child.
6.根据方案4或5的促进剂，所述血管新生因子为VEGF-A、FGF2、ANG1、PDGF-BB和HGF。6. The promoter according to embodiment 4 or 5, wherein the angiogenic factors are VEGF-A, FGF2, ANG1, PDGF-BB and HGF.
7.根据方案4-6中任一项的促进剂，所述促进剂在高糖条件下促进血管新生因子的表达。7. The promoter of any of embodiments 4-6, which promotes expression of an angiogenic factor under high glucose conditions.
8.根据方案4-7中任一项的促进剂，所述促进剂在低氧条件下促进血管新生因子的表达。8. The promoter of any of embodiments 4-7, which promotes expression of an angiogenic factor under hypoxic conditions.
9.红景天苷在制备治疗糖尿病足的药物中的应用。9. The use of salidroside in the preparation of a medicament for treating diabetic foot.
10.根据方案9的应用，其中，所述红景天苷为注射剂。10. The use according to item 9, wherein the salidroside is an injection.
11.根据方案9或10的应用，其中，所述红景天苷为骨骼肌肉注射剂。11. The use according to claim 9 or 10, wherein the salidroside is a musculoskeletal injection.
12.红景天苷在制备血管新生因子的表达和分泌的促进剂中的用途。12. Use of salidroside for the preparation of a promoter for the expression and secretion of angiogenic factors.
13.根据方案12所述的用途，所述血管新生因子的表达和分泌的促进剂为治疗糖尿病足的药物。13. The use according to claim 12, wherein the agent for expression and secretion of an angiogenic factor is a drug for treating diabetic foot.
14.根据方案13的用途，所述血管新生因子的表达和分泌的促进剂促进血管新生因子的表达和分泌。14. The use of the angiogenic factor expression and secretion promoter to promote expression and secretion of angiogenic factors according to the use of Scheme 13.
15.根据方案14的用途，所述血管新生因子为VEGF-A、FGF2、PDGF-BB、ANG1和HGF中的一种或多种。15. The use according to variant 14, wherein the angiogenic factor is one or more of VEGF-A, FGF2, PDGF-BB, ANG1 and HGF.
16.根据方案12-15中任一项的用途，所述促进剂在高糖条件下促进血管新生因子的表达。16. The use according to any one of the embodiments 12-15, wherein the promoter promotes expression of an angiogenic factor under high glucose conditions.
17.根据方案12-16中任一项的用途，所述促进剂在低氧条件下促进血管新生因子的表达。17. The use according to any one of the aspects 12-16, wherein the promoter promotes expression of an angiogenic factor under hypoxic conditions.
18.红景天苷在制备高糖条件下成熟血管形成促进剂中的用途。18. Use of salidroside for the preparation of a mature angiogenesis promoter under high glucose conditions.
19.红景天苷在制备高糖、低氧条件下成熟血管形成促进剂中的用途。19. Use of salidroside for the preparation of a mature angiogenesis promoter under conditions of high glucose and hypoxia.
20.一种治疗糖尿病足的方法，其中，对患者进行红景天苷的给药。20. A method of treating a diabetic foot, wherein the patient is administered with salidroside.
21.根据方案20的治疗糖尿病足的方法，该给药方法为肌肉注射。21. The method of treating diabetic foot according to item 20, which is by intramuscular injection.
发明效果Effect of the invention
根据本发明，能够提供一种治疗下肢缺血性疾病的药物，所述药物含有红景天苷作为活性成分。所述药物通过抑制PHD3的表达，从而促进血 管新生因子(VEGF-A、ANG1、FGF2、PDGF-BB、HO-1、HGF、NF-κB)、特别是促进对血管成熟起关键作用的因子(FGF2、HGF、PDGF-BB、NF-κB和ANG1)的表达，进而通过促进骨骼肌细胞迁移以及间接促进血管平滑肌细胞迁移以及改善低氧适应性，从而促进病变部位的成熟血管的形成，实现促进下肢血流的恢复的治疗效果。According to the present invention, it is possible to provide a medicament for treating an ischemic disease of the lower extremities, which contains salidroside as an active ingredient. The drug promotes blood by inhibiting the expression of PHD3 Tube neonatal factors (VEGF-A, ANG1, FGF2, PDGF-BB, HO-1, HGF, NF-κB), especially factors that promote key effects on vascular maturation (FGF2, HGF, PDGF-BB, NF-κB) The expression of ANG1) further promotes the migration of skeletal muscle cells and indirectly promotes the migration of vascular smooth muscle cells and improves the hypoxic adaptation, thereby promoting the formation of mature blood vessels in the lesion site and achieving a therapeutic effect of promoting recovery of blood flow in the lower limbs.
根据本发明，能够提供红景天苷在制备治疗糖尿病足的药物中的应用。所述应用中，红景天苷能够在高糖、低氧条件下促进骨骼肌里的多种血管新生因子的表达和分泌，例如VEGF-A、FGF2、ANG1、PDGF-BB和HGF的表达和分泌。进而，能够促进糖尿病足患者的下肢血管管腔的形成，尤其是能够促进成熟、无漏且具有功能性的血管的形成，从而能够实现糖尿病足的治疗效果。According to the present invention, it is possible to provide the use of salidroside for the preparation of a medicament for treating diabetic foot. In the application, salidroside can promote the expression and secretion of various angiogenic factors in skeletal muscle under high glucose and hypoxia conditions, such as expression of VEGF-A, FGF2, ANG1, PDGF-BB and HGF. secretion. Further, it is possible to promote the formation of the vascular lumen of the lower limbs of the diabetic foot patient, and in particular, it is possible to promote the formation of mature, leak-free and functional blood vessels, and the therapeutic effect of the diabetic foot can be achieved.
另外，根据本发明，能够提供一种治疗糖尿病足的药物，所述药物能够在高糖、低氧条件下促进骨骼肌里的血管新生因子VEGF-A、FGF2、ANG1、PDGF-BB和HGF的表达和分泌，从而促进糖尿病足患者的下肢血管管腔的形成，尤其是能够促进成熟、无漏且具有功能性的血管的形成，从而能够实现糖尿病足的治疗效果。Further, according to the present invention, it is possible to provide a medicament for treating diabetic foot which can promote angiogenic factors VEGF-A, FGF2, ANG1, PDGF-BB and HGF in skeletal muscle under high glucose and hypoxia conditions. The expression and secretion promote the formation of the vascular lumen of the lower limbs of diabetic foot patients, and in particular, promote the formation of mature, leak-free and functional blood vessels, thereby enabling the therapeutic effect of diabetic foot.
另外，根据本发明，能够提供一种治疗糖尿病足的方法，其具有优异的糖尿病足的治疗效果。Further, according to the present invention, it is possible to provide a method for treating a diabetic foot which has an excellent therapeutic effect of a diabetic foot.
附图说明DRAWINGS
图1A表示红景天苷促进下肢缺血模型小鼠的血流恢复作用的图。Figure 1A is a graph showing the effect of salidroside on blood flow recovery in a mouse model of lower limb ischemia.
图1B表示红景天苷促进下肢缺血模型小鼠的血流恢复作用的定量结果图。Fig. 1B is a graph showing the quantitative results of the blood flow recovery effect of salidroside in a mouse model of lower limb ischemia.
图1C表示红景天苷促进下肢缺血模型小鼠的血流恢复作用的图。Figure 1C is a graph showing the effect of salidroside on blood flow recovery in a mouse model of lower limb ischemia.
图2A表示红景天苷促进血管内皮细胞、血管平滑肌细胞的增多以及由血管平滑肌细胞包围血管内皮细胞的管腔结构(即成熟血管)的形成的免疫组织染色结果的照片。Fig. 2A is a photograph showing the results of immunohistochemical staining of salidroside for promoting the increase of vascular endothelial cells, vascular smooth muscle cells, and the formation of a luminal structure (i.e., mature blood vessels) surrounded by vascular smooth muscle cells.
图2B表示红景天苷促进血管内皮细胞以及血管平滑肌细胞增多的免疫组织染色结果的定量图。Fig. 2B is a quantitative diagram showing the results of immunohistochemical staining of salidroside for promoting vascular endothelial cells and vascular smooth muscle cells.
图3 A表示红景天苷对PHD3的特异性抑制作用的结果。 Figure 3 A shows the results of specific inhibition of PHD3 by salidroside.
图3B表示红景天苷对血管新生因子的表达的促进作用。Figure 3B shows the effect of salidroside on the expression of angiogenic factors.
图3C表示PHD3基因沉默对血管新生因子的影响。Figure 3C shows the effect of PHD3 gene silencing on angiogenic factors.
图4是示出红景天苷对骨骼肌细胞迁移的促进作用的图。Fig. 4 is a graph showing the promoting effect of salidroside on skeletal muscle cell migration.
图5是示出条件培养基对于血管平滑肌细胞迁移的促进作用的图。Fig. 5 is a graph showing the promoting effect of conditioned medium on vascular smooth muscle cell migration.
图6是示出促进血管平滑肌细胞迁移的、红景天苷刺激下骨骼肌细胞产生的分泌蛋白的种类的鉴定结果的图。Fig. 6 is a graph showing the results of identification of the types of secreted proteins produced by salidroside-stimulated skeletal muscle cells which promote vascular smooth muscle cell migration.
图7是示出低氧条件下红景天苷对骨骼肌细胞凋亡的影响的图。Figure 7 is a graph showing the effect of salidroside on skeletal muscle cell apoptosis under hypoxic conditions.
图8(A)是示出红景天苷促进糖尿病足小鼠血流恢复的效果的彩色照片被转化为灰度图像后的图。Fig. 8(A) is a diagram showing a color photograph in which the effect of salidroside on promoting blood flow recovery in diabetic foot mice is converted into a grayscale image.
图8(B)是示出对图8(A)中的血流部分进行处理后的图。Fig. 8(B) is a view showing a state in which the blood flow portion in Fig. 8(A) is processed.
图9是示出红景天苷促进糖尿病足小鼠血流恢复的效果的坐标图。Fig. 9 is a graph showing the effect of salidroside on promoting blood flow recovery in diabetic foot mice.
图10是分别示出术后21天生理盐水对照组与红景天苷处理组中成熟血管形成的效果的照片。Fig. 10 is a photograph showing the effects of mature blood vessel formation in the saline control group and the salidroside-treated group, respectively, 21 days after the operation.
图11是分别示出红景天苷和生理盐水对血管内皮细胞以及血管平滑肌细胞的作用效果的定量柱状图。Fig. 11 is a quantitative histogram showing the effects of salidroside and physiological saline on vascular endothelial cells and vascular smooth muscle cells, respectively.
图12是示出红景天苷对血管新生因子影响的柱状图。Figure 12 is a bar graph showing the effect of salidroside on angiogenic factors.
具体实施方式detailed description
(一)下肢缺血性疾病的治疗药物(1) Therapeutic drugs for lower limb ischemic diseases
本公开中的下肢缺血性疾病可以为任何一种下肢缺血性疾病，例如血栓闭塞性脉管炎、闭塞性动脉硬化症、间歇性跛行(Intermittent claudication)、糖尿病足(Diabetic Foot)等糖尿病相关血管病变以及恶性下肢缺血性疾病(Critical Limb Ischemia)。The lower limb ischemic disease in the present disclosure may be any kind of lower limb ischemic disease, such as thromboangiitis obliterans, obliterative arteriosclerosis, intermittent claudication, Diabetic Foot and the like. Related vascular lesions and malignant lower limb ischemic disease (Critical Limb Ischemia).
本公开中的下肢缺血性疾病的治疗药物包含红景天苷作为活性成份。本公开中的红景天苷为结构式如式1的化合物。红景天苷的来源可以是从高山红景天、大花红景天、蔷薇红景天、西藏红景天等景天科植物中提取，也可以是通过化学合成。红景天苷的纯度没有限制，优选为80％以上，更优选为90％以上，进而优选为95％以上，再优选为98％以上，最优选为99.8％以上。关于纯度的影响，申请人说明如下：后述的本公开实施例中记载了使用上海同田生物技术有限公司(纯度≥98.0％)生产的红景天 苷，申请人另外也使用了中国食品药品检定研究院的红景天苷产品(纯度≥99.8％)，且使用这两种浓度的红景天苷效果相同，由此可以理解，后述的本公开效果是由红景天苷所产生的，而不是由杂质成分产生。The therapeutic agent for lower limb ischemic diseases in the present disclosure contains salidroside as an active ingredient. The salidroside in the present disclosure is a compound of the formula (Formula 1). The source of salidroside may be extracted from sedum plants such as Rhodiola rosea, Rhodiola rosea, Rhodiola rosea, and Rhodiola sachalinensis, or may be chemically synthesized. The purity of salidroside is not particularly limited, but is preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, still more preferably 98% or more, and most preferably 99.8% or more. Regarding the influence of purity, the applicant has explained the following: The embodiment of the present disclosure described later describes Rhodiola produced by Shanghai Tongtian Biotechnology Co., Ltd. (purity ≥ 98.0%). Glycoside, the applicant also used the salidroside product of China Food and Drug Administration Institute (purity ≥99.8%), and the effect of using these two concentrations of salidroside is the same, which can be understood, the following The public effect is produced by salidroside rather than by impurity components.
另外，关于使用红景天苷治疗下肢缺血性疾病时的浓度，可以设定为每次注射10-500mg/kg体重。该剂量可以单次或者分成多次使用。给药次数可以单次或多次，可以连续性每天给药或间隔性给药。In addition, regarding the concentration at which salidroside is used to treat lower limb ischemic diseases, it can be set to be 10-500 mg/kg body weight per injection. This dose can be used in a single or divided dose. The number of administrations may be single or multiple, and may be administered daily or intermittently.
本公开中的下肢缺血性疾病的程度没有任何限制，可为前期病变、轻度、中度或严重的下肢缺血性疾病，也可为慢性或急性下肢缺血性疾病。本公开中的“低氧”或“缺氧”没有任何限制，可以是下肢缺血性疾病疾病所导致的组织细胞低氧、缺氧。对于低氧、缺氧状态下的氧浓度没有任何限定。由于组织里的氧浓度会根据部位(包括同一个组织里的部位)而有所不同，并且考虑到组织里的氧气浓度低于动脉氧气浓度、且动脉氧气分压一般认为为100mmHg(低于40mmHg就会使生物体至死)，因此本公开中低氧是指氧气分压优选为高于0mmHg且为100mmHg以下的范围，更优选为10～100mmHg、进一步优选为20～100mmHg、再优选30～100mmHg、最优选40～100mmHg的范围。本领域技术人员应可以理解，本公开实施例中的下肢缺血性小鼠模型是采用完全切断大腿大动脉构建的(即严重下肢缺血缺氧模型)，且采用的小鼠为血管重构和血流恢复能力非常差的Balb/c品种；而细胞实验采用的也是对细胞影响非常严重的严重低氧(氧气浓度低于0.1％)的条件。由此可以理解，后述的本公开的效果除了对严重下肢缺血性疾病具有良好的治疗效果之外，在血管重构和下肢功能恢复能力更强的前期病变、轻度或中度的下肢缺血性疾病能起到更好的治疗效果。The degree of the lower limb ischemic disease in the present disclosure is not limited, and may be a pre-stage lesion, a mild, moderate or severe lower limb ischemic disease, or a chronic or acute lower limb ischemic disease. The "hypoxia" or "hypoxia" in the present disclosure is not limited, and may be hypoxia and hypoxia of tissue cells caused by diseases of lower limb ischemic diseases. There is no limitation on the oxygen concentration in the hypoxic or anoxic state. Since the oxygen concentration in the tissue will vary depending on the site (including the site in the same tissue), and considering that the oxygen concentration in the tissue is lower than the arterial oxygen concentration, and the arterial oxygen partial pressure is generally considered to be 100 mmHg (less than 40 mmHg). In the present disclosure, the low oxygen content means that the oxygen partial pressure is preferably higher than 0 mmHg and 100 mmHg or less, more preferably 10 to 100 mmHg, still more preferably 20 to 100 mmHg, still more preferably 30 to 30. A range of 100 mmHg, most preferably 40 to 100 mmHg. It will be understood by those skilled in the art that the lower limb ischemic mouse model in the embodiments of the present disclosure is constructed by completely cutting the thigh aorta (ie, a severe lower limb ischemia and hypoxia model), and the mouse used is vascular remodeling and The Balb/c variety with very poor blood flow recovery; and the cell experiments used conditions of severe hypoxia (oxygen concentration below 0.1%) that had a very serious effect on the cells. It can be understood from the above that the effects of the present disclosure described later have a good therapeutic effect on severe ischemic diseases of the lower extremities, and a pre-stage lesion with mild vascular remodeling and lower limb function recovery ability, mild or moderate lower limbs. Ischemic disease can have a better therapeutic effect.
本公开中的血管新生因子为对成熟血管的形成起到作用的因子，其中包括对管腔形成起作用的因子(VEGF-A、FGF2、HGF等)、对细胞成熟起作用的因子(FGF2、HGF、PDGF-BB、NF-κB和ANG1等)、起到提高低氧耐受性和细胞保护作用的因子(HO-1等)等。血管新生因子包含HIF-1α依赖性和HIF-1α非依赖性的血管新生因子。HIF-1α依赖性的血管新生因子例如VEGF、PDGF-BB和HO-1，而HIF-1α非依赖性的血管新生因子例如FGF2、HGF、NF-κB和ANG1。 The angiogenic factor in the present disclosure is a factor that contributes to the formation of mature blood vessels, including factors contributing to lumen formation (VEGF-A, FGF2, HGF, etc.) and factors contributing to cell maturation (FGF2). HGF, PDGF-BB, NF-κB, ANG1, etc.), factors (HO-1, etc.) that improve hypoxia tolerance and cytoprotection. Angiogenesis factors include HIF-1α-dependent and HIF-1α-independent angiogenic factors. HIF-1α-dependent angiogenic factors such as VEGF, PDGF-BB and HO-1, while HIF-1α-independent angiogenic factors such as FGF2, HGF, NF-κB and ANG1.
本公开中的细胞通讯为任意一种细胞间交流的方式，例如内分泌(endocrine)、旁分泌(paracrine)、自分泌(autocrine)、化学突触(chemical synapse)、接触性依赖的通讯、间隙连接等，介导细胞通讯的可以为配体/受体、小分子化合物、囊泡等。Cell communication in the present disclosure is any means of intercellular communication, such as endocrine, paracrine, autocrine, chemical synapse, contact-dependent communication, gap junction Etc., which can mediate cell communication, can be ligands/receptors, small molecule compounds, vesicles, and the like.
本领域技术人员应可以理解，本公开所涉及的血管新生因子、它们的调控机制以及它们的作用效果在小鼠和包括人类在内的哺乳动物中是共通的，因此，基于本说明书对红景天苷在小鼠模型中的作用效果以及作用机制的描述，本领域技术人员应可以理解：红景天苷在包括人类在内的哺乳动物体内也能达到治疗下肢缺血性疾病、特异抑制PHD3、促进血管新生因子的表达分泌、促进骨骼肌细胞以及血管平滑肌细胞迁移、促进成熟血管的形成等本说明书所描述的效果。It will be understood by those skilled in the art that the angiogenic factors involved in the present disclosure, their regulatory mechanisms, and their effects are common in mice and mammals including humans, and therefore, based on this specification, The effect of guanosine in a mouse model and the description of the mechanism of action, it will be understood by those skilled in the art that salidroside can also achieve treatment of lower limb ischemic diseases and specifically inhibit PHD3 in mammals including humans. Promote the expression and secretion of angiogenic factors, promote the migration of skeletal muscle cells and vascular smooth muscle cells, promote the formation of mature blood vessels, and the effects described in the present specification.
(二)糖尿病足的治疗药物(B) the treatment of diabetic foot
本公开人发现红景天苷能够对小鼠糖尿病足模型具有良好的治疗效果。另外，本公开中的糖尿病足是指下肢糖尿病血管并发症、或糖尿病相关的下肢血管病变。本公开中的糖尿病足的程度没有任何限制，可为前期病变、轻度、中度或严重的糖尿病足。The present inventors have found that salidroside has a good therapeutic effect on a mouse diabetic foot model. In addition, the diabetic foot in the present disclosure refers to lower extremity diabetic vascular complications or diabetes-related lower extremity vascular lesions. The degree of diabetic foot in the present disclosure is not limited, and may be a pre-stage lesion, a mild, moderate or severe diabetic foot.
本公开中的糖尿病包括1型糖尿病、2型糖尿病以及糖尿病前期。1型糖尿病和2型糖尿病的空腹血糖≥7.0mmol/L，糖尿病前期(pre-diabetes)的空腹血糖大于6.1mmol/L而小于7.0mmol/L。本说明书中记载的“高糖”是指由糖尿病以及糖尿病相关血管病变引起的高糖。Diabetes in the present disclosure includes type 1 diabetes, type 2 diabetes, and pre-diabetes. The fasting blood glucose of type 1 diabetes and type 2 diabetes is ≥7.0mmol/L, and the fasting blood glucose of pre-diabetes is greater than 6.1mmol/L and less than 7.0mmol/L. The "high sugar" described in the present specification means high sugar caused by diabetes and diabetes-related vascular disease.
本说明书中记载的“低氧”是指由糖尿病以及糖尿病相关血管病变引起的组织细胞低氧(以下有时称为“缺氧”)。关于低氧状态下的氧浓度没有任何限定。由于组织里的氧浓度会根据部位(包括同一个组织里的部位)而有所不同，并且考虑到组织里的氧气浓度低于动脉氧气浓度、且动脉氧气分压一般认为为100mmHg(低于40mmHg就会使生物体致死)，因此本公开中低氧是指氧气分压优选为高于0mmHg且为100mmHg以下的范围，更优选为10～100mmHg、进一步优选为20～100mmHg、再优选30～100mmHg、最优选40～100mmHg的范围。The "hypoxia" described in the present specification means hypoxia (hereinafter referred to as "anoxia") of tissue cells caused by diabetes and diabetes-related vascular lesions. There is no limitation on the oxygen concentration in the low oxygen state. Since the oxygen concentration in the tissue will vary depending on the site (including the site in the same tissue), and considering that the oxygen concentration in the tissue is lower than the arterial oxygen concentration, and the arterial oxygen partial pressure is generally considered to be 100 mmHg (less than 40 mmHg). In the present disclosure, the low oxygen means that the oxygen partial pressure is preferably higher than 0 mmHg and 100 mmHg or less, more preferably 10 to 100 mmHg, still more preferably 20 to 100 mmHg, still more preferably 30 to 100 mmHg. Most preferably, it is a range of 40 to 100 mmHg.
另外，本领域技术人员应可以理解，本公开实施例中的糖尿病足小鼠 模型是采用完全切断大腿大动脉构建的(即该下肢处于严重的缺氧状态)，且所用的糖尿病小鼠模型的空腹血糖≥16.7mmol/L，远远高于糖尿病标准的空腹血糖(即≥7.0mmol/L)，是严重的糖尿病小鼠。因此可知，本公开实施例中的糖尿病足小鼠患有严重的糖尿病足。基于糖尿病的程度(即血糖的高低)与组织修复、伤口愈合等能力成反比，本领域技术人员应可以理解，后述的本公开的效果除了对严重糖尿病足具有良好的治疗效果之外，对血管重构和下肢功能恢复能力更强的前期病变、轻度或中度的糖尿病足能起到更好的治疗效果。In addition, those skilled in the art should understand that diabetic foot mice in the embodiments of the present disclosure The model was constructed by completely severing the thigh aorta (ie, the lower extremity was in a severe hypoxic state), and the fasting blood glucose of the diabetic mouse model used was ≥16.7 mmol/L, which was much higher than the fasting blood glucose of the diabetic standard (ie ≥7.0). Mmmol/L) is a severe diabetic mouse. Thus, it can be seen that the diabetic foot mice in the examples of the present disclosure suffer from severe diabetic foot. The degree of diabetes (i.e., the level of blood sugar) is inversely proportional to the ability of tissue repair, wound healing, etc., and those skilled in the art will appreciate that the effects of the present disclosure described below have a good therapeutic effect on severe diabetic foot, in addition to Early lesions with mild vascular remodeling and lower limb function recovery, mild or moderate diabetic foot can play a better therapeutic effect.
本公开中，糖尿病足的治疗药物包含红景天苷作为活性成份。本公开中的红景天苷为结构式如式1的化合物。红景天苷的来源可以是从高山红景天、大花红景天、蔷薇红景天、西藏红景天等景天科植物中提取，也可以是通过化学合成。红景天苷的纯度没有限制，优选为80％以上，更优选为90％以上，进而优选为95％以上，再优选为98％以上，最优选为99.8％以上。关于纯度的影响，申请人说明如下。后述的本公开实施例中记载了使用上海同田生物技术有限公司(纯度≥98.0％)生产的红景天苷，申请人另外也使用了中国食品药品检定研究院的红景天苷产品(纯度≥99.8％)，且使用这两种浓度的红景天苷效果相同，由此可以理解，后述的本公开效果是由红景天苷所产生的，而不是由杂质成分产生。In the present disclosure, a therapeutic drug for diabetic foot contains salidroside as an active ingredient. The salidroside in the present disclosure is a compound of the formula (Formula 1). The source of salidroside may be extracted from sedum plants such as Rhodiola rosea, Rhodiola rosea, Rhodiola rosea, and Rhodiola sachalinensis, or may be chemically synthesized. The purity of salidroside is not particularly limited, but is preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, still more preferably 98% or more, and most preferably 99.8% or more. Regarding the effect of purity, the applicant explains the following. The presently disclosed embodiments of the present disclosure describe the use of salidroside produced by Shanghai Tongtian Biotechnology Co., Ltd. (purity ≥ 98.0%), and the applicant also uses the salidroside product of the China Food and Drug Administration Research Institute ( The purity is ≥99.8%), and the effects of using these two concentrations of salidroside are the same, and it is understood that the effects of the present disclosure described later are produced by salidroside rather than by an impurity component.
另外，关于使用红景天苷治疗下肢缺血性疾病时的浓度，可以设定为每次注射10-500mg/kg体重。该剂量可以单次或者分成多次使用。给药次数可以单次或多次，可以连续性每天给药或间隔性给药。In addition, regarding the concentration at which salidroside is used to treat lower limb ischemic diseases, it can be set to be 10-500 mg/kg body weight per injection. This dose can be used in a single or divided dose. The number of administrations may be single or multiple, and may be administered daily or intermittently.
本公开中的血管新生因子为对促进成熟血管的形成起到作用的因子，其中包括对管腔形成起作用的因子(VEGF-A、FGF2、HGF等)、对细胞成熟起作用的因子(FGF2、HGF、PDGF-BB、ANG1等)等。另外，本领域技术人员应可以理解，这些因子在小鼠和包括人类在内的哺乳动物中的调控机制以及它们的作用效果是共通的，因此，本领域技术人员应可以理解，基于本说明书对红景天苷作用效果以及作用机制的描述，本公开的效果在包括人类在内的哺乳动物体内也能达到治疗糖尿病足、在高糖、低氧条件下促进血管新生因子的表达和分泌、促进成熟血管的形成等本说明书所描述的效果。 The angiogenic factor in the present disclosure is a factor that promotes the formation of mature blood vessels, including factors contributing to lumen formation (VEGF-A, FGF2, HGF, etc.) and factors contributing to cell maturation (FGF2). , HGF, PDGF-BB, ANG1, etc.). In addition, it will be understood by those skilled in the art that the regulatory mechanisms of these factors in mice and mammals including humans and their effects are common, and therefore, those skilled in the art should understand that based on the present specification The effect of salidroside and the description of the mechanism of action, the effects of the present disclosure can also achieve the treatment of diabetic foot in mammals including humans, promote the expression and secretion of angiogenic factors under high glucose and hypoxia conditions, and promote The formation of mature blood vessels and the like described in this specification.
(三)其他添加剂和剂型(3) Other additives and dosage forms
本公开中的下肢缺血性疾病和/或糖尿病足的治疗药物也可以包含一种或多种辅料。辅料没有限定，例如溶剂、等张剂、赋形剂、pH调整剂、抗氧化剂、崩解剂、调味剂、香料、保存剂等本领域常用的辅料。The therapeutic agent for the lower limb ischemic disease and/or diabetic foot in the present disclosure may also contain one or more excipients. The excipient is not limited, and is, for example, a solvent, an isotonic agent, an excipient, a pH adjuster, an antioxidant, a disintegrant, a flavoring agent, a perfume, a preservative, and the like which are commonly used in the art.
作为溶剂可以列举：注射用蒸馏水、生理盐水、植物油，丙二醇、聚乙二醇、乙醇、甘油之类的醇类等。Examples of the solvent include distilled water for injection, physiological saline, vegetable oil, alcohol such as propylene glycol, polyethylene glycol, ethanol, and glycerin.
作为等张剂可以列举：山梨醇、氯化钠、葡萄糖等本领域常用的等张剂。Examples of the isotonic agent include an isotonic agent commonly used in the art such as sorbitol, sodium chloride, and glucose.
作为赋形剂可以列举：乳糖、甘露醇、葡萄糖、微晶纤维素、淀粉等。Examples of the excipient include lactose, mannitol, glucose, microcrystalline cellulose, and starch.
作为pH调整剂可以列举：盐酸、枸橼酸、氢氧化钠、强氧化钾、碳酸氢钠、磷酸氢二钠等。Examples of the pH adjuster include hydrochloric acid, citric acid, sodium hydroxide, strong potassium oxide, sodium hydrogencarbonate, and disodium hydrogen phosphate.
作为抗氧化剂可以列举：亚硫酸钠、亚硫酸氢钠、抗坏血酸等。Examples of the antioxidant include sodium sulfite, sodium hydrogen sulfite, and ascorbic acid.
作为崩解剂可以列举：马铃薯淀粉。As a disintegrating agent, potato starch is mentioned.
作为调味剂可以列举：蔗糖、单糖浆等甜味剂，等。Examples of the flavoring agent include sweeteners such as sucrose and monosaccharide syrup.
作为香料可以列举：薄荷油，橙皮油等。As a fragrance, peppermint oil, orange peel oil, etc. are mentioned.
作为保存剂可以列举：尼泊金类、山梨酸及其盐等本领域常用的保存剂。Examples of the preservative include preservatives commonly used in the art such as parabens, sorbic acid, and salts thereof.
本公开中的下肢缺血性疾病和/或糖尿病足的治疗药物可以为任何一种剂型，例如口服液、贴剂、片剂、胶囊、注射剂等，优选为注射剂，最优选为骨骼肌注射剂。The therapeutic agent for lower limb ischemic diseases and/or diabetic foot in the present disclosure may be any one of dosage forms such as an oral solution, a patch, a tablet, a capsule, an injection, etc., preferably an injection, and most preferably a skeletal muscle injection.
以下举出实施例来具体说明本发明，但本发明并不限于下述实施例。本领域技术人员应可以理解，本说明书中的“促进血管重构”和“促进成熟血管的形成”为促进血管的新生和/或促进新生血管的成熟。本公开中的抑制剂为抑制基因的表达水平的药剂，可包含抑制转录、翻译、蛋白质合成以及蛋白质稳定性的药剂。促进剂为促进基因的表达或分泌水平的药剂，可包含促进转录、翻译、蛋白质合成、蛋白质稳定性以及分泌的药剂。The invention will be specifically described below by way of examples, but the invention is not limited to the examples described below. It will be understood by those skilled in the art that "promoting vascular remodeling" and "promoting the formation of mature blood vessels" in the present specification promotes the angiogenesis and/or promotes the maturation of new blood vessels. The inhibitor of the present disclosure is an agent that inhibits the expression level of a gene, and may include an agent that inhibits transcription, translation, protein synthesis, and protein stability. An agent is an agent that promotes the expression or secretion level of a gene, and may include an agent that promotes transcription, translation, protein synthesis, protein stability, and secretion.
实施例Example
(一)关于下肢缺血性疾病的治疗药物(1) Treatment drugs for lower limb ischemic diseases
1.红景天苷对下肢缺血模型小鼠的血流恢复作用的测定1. Determination of the recovery of blood flow in mice with lower limb ischemia model by salidroside
1-1下肢缺血模型小鼠的建立Establishment of 1-1 lower limb ischemia model mice
使用Balb/c小鼠(8周，雄性)，在麻醉条件下对左侧大腿大动脉进行切除手术，并利用Laser Doppler Perfusion Imaging System(MOOR INSTRUMENTS Ltd,MOORLDLS2-IR)检测血流的情况(参照文献Shourong Wu et al.,Prolyl hydroxylase domain-2 silencing induced by hydrodynamic limb vein injection enhances vascular regeneration in critical limb ischemia mice through activation of multiple genes(2015)Curr Gene Ther.,15(3):313-25.中的方法)。需要说明的是，关于本申请说明书中“左侧”、“右侧”的表述，进行了手术的是左侧大腿，此时小鼠处于俯卧状态，后述的血流图的照片中小鼠处于仰卧状态，所以该血流图照片中手术的大腿在图中右侧。Balb/c mice (8 weeks, male) were used to remove the left thigh aorta under anesthesia and blood flow was measured using the Laser Doppler Perfusion Imaging System (MOOR INSTRUMENTS Ltd, MOORLDLS2-IR) (Reference) Shourong Wu et al., Prolyl hydroxylase domain-2 silencing induced by hydrodynamic limb vein injection enhances vascular regeneration in sinus sinus sinus sinus sinus sinus method). In addition, regarding the expressions of "left side" and "right side" in the specification of the present application, the left thigh is operated, and the mouse is in a prone state, and the mouse in the photo of the blood flow diagram described later is The supine state, so the thigh of the operation in the blood flow picture is on the right side of the figure.
1-2红景天苷的注射1-2 injection of salidroside
将红景天苷(上海同田生物技术有限公司，纯度≥98.0％)用PBS溶解，配成40mg/ml的储存液，用0.22μm滤膜过滤后，于-20℃保存备用。The salidroside (Shanghai Tongtian Biotechnology Co., Ltd., purity ≥98.0%) was dissolved in PBS, and prepared into a 40 mg/ml stock solution, filtered through a 0.22 μm filter, and stored at -20 ° C until use.
注射前将红景天苷储存液稀释至20mg/ml。小鼠注射剂量是100mg/kg，术后第1天开始每三天注射一次，每次注射将注射液分3次，连续往左侧腓肠肌里分三个位点分别注射。The salidroside stock solution was diluted to 20 mg/ml before injection. The injection dose of the mice was 100 mg/kg, and the injection was started every three days from the first day after the operation, and the injection was divided into three times for each injection, and three injections were separately administered to the left gastrocnemius muscle.
使用生理盐水作为对照，同样地过滤、保存和注射。Physiological saline was used as a control, and filtration, preservation, and injection were similarly performed.
1-3观察结果1-3 observation results
利用Laser Doppler Perfusion Imaging System检测术前、刚手术后、术后第3、7、14、21天的血流情况。Blood flow was measured before, immediately after, and on days 3, 7, 14, and 21 after surgery using the Laser Doppler Perfusion Imaging System.
本实验获得了令人惊奇的优异效果。参照图1A，灰色部分反应血流状况。术前，生理盐水对照组小鼠和红景天苷处理组小鼠具有同等的血流状况(需要说明的是，Laser Doppler Perfusion Imaging System原初成像为彩图，转化为灰度图片后，与原初所成的像看起来有些许差异。在原彩图中，红色表示血流丰富，蓝色表示没有血流。在灰度图像下无法较清晰地区分血流恢复的地方(即原彩图中红色部位)和没有血流(即原彩图中蓝色部位)的地方)。针对该问题，本申请发明人依据彩图结果对灰度图进 行了图像处理，并将处理后的图片作为图1A。在图1A中，网点状图案表示血流恢复的地方，即原彩图中的红色部分。刚手术后，对照组小鼠和红景天苷处理组小鼠左侧下肢均显示出黑色(在原彩图中均为蓝色部分，在处理后的图中为没有网点状图案的黑色部分)，即二者左侧下肢均没有血流，可知手术成功地制造出下肢缺血小鼠模型。在术后第3天，红景天苷处理组小鼠左侧下肢开始出现灰色和黑色部分(即原彩图中的红色部分、处理后的图中的网点部分)，可知出现了明显的血流恢复状况；与之相对，生理盐水对照组小鼠左侧下肢仍为黑色(即原彩图中的蓝色部分，在处理后的图中为没有网点状图案的黑色部分)，可知没有出现血流恢复的迹象。术后第21天时，红景天苷处理组小鼠左侧下肢达到了与未处理的左侧下肢几乎同等的血流状况，即左侧下肢的血流得到了较为充分的恢复；与之相对，图中生理盐水对照组小鼠左侧下肢影像缺失，推测是由于没有血流恢复而导致了下肢萎缩和组织坏死。另外从图中也可以看出，随着治疗时间的延长，红景天苷处理组左侧下肢的灰色和黑色部分(即原彩图中的红色部分、处理后的图中的网点部分)越来越达到下肢的远端(即靠近脚趾)部位，可知血流逐渐恢复到距离动脉切除较远的部位。This experiment achieved surprisingly excellent results. Referring to Figure 1A, the gray portion reflects the blood flow condition. Before the operation, the mice in the saline control group and the salidroside-treated mice had the same blood flow status. (It should be noted that the Laser Doppler Perfusion Imaging System was originally imaged as a color map, which was converted into a grayscale image, and the original The resulting image looks a little different. In the original color map, red indicates that the blood flow is rich, blue indicates no blood flow. Under the gray image, the blood flow recovery cannot be clearly distinguished (ie, the red color in the original color map) Part) and where there is no blood flow (ie, the blue part of the original color map). In response to this problem, the inventor of the present application proceeds to the grayscale image based on the color map result. Image processing is performed, and the processed picture is taken as FIG. 1A. In Fig. 1A, the dot pattern indicates where the blood flow is restored, that is, the red portion in the original color map. After the operation, the mice in the control group and the salidroside-treated group showed black in the left lower limbs (all in the original color map, and in the processed image, the black portion without the dot pattern) That is, there is no blood flow in the left lower limbs of both of them, and it is known that the surgery successfully produces a mouse model of lower limb ischemia. On the third day after surgery, the left and right lower limbs of the salidroside-treated group began to appear gray and black parts (ie, the red part in the original color map and the half-point part in the processed figure), and it was found that obvious blood appeared. In contrast, the left lower limb of the saline control group was still black (ie, the blue part of the original color map, and the black part without the dot pattern in the processed image), it is known that there is no Signs of blood flow recovery. On the 21st day after surgery, the left lower limb of the salidroside-treated group reached almost the same blood flow condition as the untreated left lower limb, that is, the blood flow of the left lower limb was fully recovered; In the figure, the left lower extremity image of the saline control group mice was absent, which was presumed to be due to the lack of blood flow and the lower limb atrophy and tissue necrosis. In addition, it can be seen from the figure that the more the treatment time is extended, the more the gray and black parts of the left lower limb of the salidroside treatment group (ie, the red part in the original color map and the halftone part in the processed figure) The more the distal end of the lower limb (ie, near the toe) is reached, the blood flow gradually recovers to a location farther away from the arterial resection.
另外，发明人对Laser Doppler Perfusion Imaging System的结果进行了定量，结果示于图1B。具体定量方法为：将缺血下肢(左侧下肢)的定量值(即血流面积的像素)除以同一只小鼠的非缺血下肢(右侧下肢)的定量值，然后将每一个时间点的该比值平均化并除以各组术前的比值的平均值。参照基于统计结果的图1B可知，刚手术后，对照组小鼠和红景天苷处理组小鼠的血流比值均降为低于0.2的数值。在术后第3天，红景天苷处理组小鼠的血流比值达到高于0.5；与之相对，生理盐水对照组小鼠的血流比值明显低于0.2。术后第21天时，红景天苷处理组小鼠的血流比值达到了0.8以上；与之相对，对照组小鼠的血流比值仍为大约0.4的低值。Further, the inventors quantified the results of the Laser Doppler Perfusion Imaging System, and the results are shown in Fig. 1B. The specific quantitative method is: dividing the quantitative value of the ischemic lower limb (left lower limb) (ie, the pixel of the blood flow area) by the quantitative value of the non-ischemic lower limb (right lower limb) of the same mouse, and then each time The ratio of points is averaged and divided by the average of the preoperative ratios for each group. Referring to Fig. 1B based on the statistical results, the blood flow ratio of the control mice and the salidroside-treated mice decreased to a value lower than 0.2 immediately after the operation. On the third day after surgery, the blood flow ratio of the salidroside-treated mice was higher than 0.5; in contrast, the blood flow ratio of the saline control mice was significantly lower than 0.2. On the 21st day after surgery, the blood flow ratio of the salidroside-treated mice reached 0.8 or higher; in contrast, the blood flow ratio of the control mice remained at a low value of about 0.4.
另外，申请人将血流状况检测结果的灰度图(即将血流状况检测结果的彩色图灰度化后的图)示于图1C中。Further, the applicant shows a gray scale map of the blood flow condition detection result (a graph obtained by grading the color map of the blood flow condition detection result) in FIG. 1C.
2.红景天苷治疗下肢缺血疾病的机理的研究2. Study on the mechanism of salidroside in the treatment of lower limb ischemic diseases
对于这一优异效果，申请人进行了机理方面的研究。 For this excellent effect, the applicant conducted a mechanism study.
2-1.免疫组织染色2-1. Immunohistochemical staining
采用冰冻切片进行，术后21天，取得小鼠的左侧腓肠肌组织后保存在-80℃。待组织冷冻后进行切片。The frozen section was used, and 21 days after the operation, the left gastrocnemius muscle tissue of the mouse was obtained and stored at -80 °C. The tissue was sectioned after freezing.
切片流程如下，包埋剂包埋组织后在切片机(Leica生产)上进行切片，切片厚度是10μm。切片结束后将切片置于37℃烘箱中烘干30min，2.5％牛血清白蛋白(BSA)中封闭30～60min。将组织周围的BSA除去后，用抗PECAM-1(别名：CD31)抗体室温孵育1h(PECAM-1：Purified Rat Anti-Mouse CD31(Clone MEC13.3,BD PharmingenTM Cat 550274)，抗体稀释比例1:50)，然后用含有0.1％吐温的生理盐水(PBS-T)清洗三次，每次5min。进一步将该切片用带有荧光标记的抗α-Smooth Muscle Actin(α-SMA)的抗体Mouse monoclonal(Clone 1A4,Sigma-Aldrich Cat C6198)(即α-Smooth Muscle-Cy3，抗体稀释比例1:100)、和荧光标记的针对抗PECAM-1抗体的二抗(Goat anti-Rat IgG(H+L)Secondary Antibody,Alexa
Figure PCTCN2017071947-appb-000002
488 conjugate(Thermo Scientific Cat A11006，抗体稀释比例1:100))的混合液室温孵育30min。然后PBS-T清洗三次，每次5min。免疫荧光染色结束并用丙三醇封片后，在荧光显微镜(Leica Microsystems，DMI6000B)上面检测。对注射了红景天苷和生理盐水的小鼠确认血管生成及血管成熟情况。结果如图2A所示。另外，对所获得的图片中的被染色部分的面积利用Leica Application Suite Version 4.6软件进行定量，得出PECAM-1阳性和α-SMA阳性的面积。结果如图2B所示。The sectioning procedure was as follows. The embedding agent was embedded in the tissue and sliced on a microtome (manufactured by Leica), and the section thickness was 10 μm. After the end of the section, the sections were placed in an oven at 37 ° C for 30 min, and blocked in 2.5% bovine serum albumin (BSA) for 30-60 min. After removing BSA around the tissue, incubate with anti-PECAM-1 (alias: CD31) antibody for 1 h at room temperature (PECAM-1: Purified Rat Anti-Mouse CD31 (Clone MEC 13.3, BD Pharmingen TM Cat 550274), antibody dilution ratio 1 :50), then washed three times with physiological saline (PBS-T) containing 0.1% Tween for 5 min each time. Further, the section was subjected to a fluorescently labeled anti-α-Smooth Muscle Actin (α-SMA) antibody Mouse monoclonal (Clone 1A4, Sigma-Aldrich Cat C6198) (ie, α-Smooth Muscle-Cy3, antibody dilution ratio 1:100 , and fluorescently labeled secondary antibody against anti-PECAM-1 antibody (Goat anti-Rat IgG (H+L) Secondary Antibody, Alexa
Figure PCTCN2017071947-appb-000002
A mixture of 488 conjugate (Thermo Scientific Cat A11006, antibody dilution ratio 1:100) was incubated for 30 min at room temperature. The PBS-T was then washed three times for 5 min each time. After immunofluorescence staining was completed and mounted with glycerol, it was detected on a fluorescence microscope (Leica Microsystems, DMI 6000B). Angiogenesis and vascular maturation were confirmed in mice injected with salidroside and physiological saline. The result is shown in Figure 2A. In addition, the area of the stained portion in the obtained image was quantified using Leica Application Suite Version 4.6 software to obtain a PECAM-1 positive and α-SMA positive area. The result is shown in Figure 2B.
结果确认，注射红景天苷的小鼠的组织内发现了血管内皮细胞(即PECAM-1阳性)和血管平滑肌细胞(即α-SMA阳性)的细胞增多。通过重叠图片(Merge image)发现PECAM-1和α-SMA双阳性的结构增多，并形成由血管平滑肌细胞包围血管内皮细胞的管腔结构，意味着形成了丰富的成熟血管，而注射生理盐水的小鼠仅有非常微弱的阳性信号，且不形成官腔结构。另外，定量结果也显示了红景天苷能在缺血缺氧条件下显著地诱导更多的血管内皮细胞和血管平滑肌细胞。As a result, it was confirmed that cells of vascular endothelial cells (i.e., PECAM-1 positive) and vascular smooth muscle cells (i.e., α-SMA positive) were increased in tissues of mice injected with salidroside. Through overlapping images (Merge image), it was found that the structure of double positive of PECAM-1 and α-SMA increased, and the luminal structure surrounded by vascular smooth muscle cells surrounded by vascular endothelial cells was formed, which means that abundant mature blood vessels were formed, and physiological saline was injected. The mouse has only a very weak positive signal and does not form a bureaucratic structure. In addition, the quantitative results also show that salidroside can significantly induce more vascular endothelial cells and vascular smooth muscle cells under hypoxic conditions.
该结果表明，注射红景天苷能够治疗小鼠下肢缺血、即促进缺血下肢血流的恢复，其原因很可能是由于红景天苷促进了小鼠的血管新生和成熟血管的形成。 The results indicate that injection of salidroside can treat the lower limb ischemia of mice, that is, promote the recovery of ischemic lower limb blood flow, which is probably due to the fact that salidroside promotes angiogenesis and mature blood vessel formation in mice.
2-2.红景天苷对PHD3的特异性抑制和对血管新生因子的诱导2-2. Specific inhibition of PHD3 by salidroside and induction of angiogenic factors
RNA提取：根据上述1.建立下肢缺血性疾病小鼠模型并注射红景天苷。在手术第三天对小鼠进行安乐死，并取左侧腓肠肌组织置于RNAlater Solution。将组织分成两块在液氮中研磨，待磨成粉末状之后加入TRIZOL(Invitrogen)后收集到1.5mL的无酶EP管中，并根据Trizol的说明书提取RNA。RNA提取之后用Nanodrop-2000(Gene Company,Ltd)检测所提取的RNA的质量和浓度后进行反转录。 RNA extraction : A mouse model of lower limb ischemic disease was established and injected with salidroside according to the above 1. The mice were euthanized on the third day of surgery and the left gastrocnemius muscle tissue was placed in RNAlater Solution. The tissue was divided into two pieces and ground in liquid nitrogen. After being pulverized, TRIZOL (Invitrogen) was added, and 1.5 mL of the enzyme-free EP tube was collected, and RNA was extracted according to the instructions of Trizol. After RNA extraction, the quality and concentration of the extracted RNA were detected by Nanodrop-2000 (Gene Company, Ltd), followed by reverse transcription.
mRNA水平的测定： Determination of mRNA levels :
RT-PCRRT-PCR
TAKARA-PrimeScriptTM RT reagent Kit with gDNA Eraser(Code No.RR047A)TAKARA-PrimeScript TM RT reagent Kit with gDNA Eraser (Code No. RR047A)
(1)(1)
去除基因组DNA反应Genomic DNA reaction removal
试剂 Reagent 使用量Usage amount
5*gDNA Eraser Buffer5*gDNA Eraser Buffer 2.0μL2.0μL
gDNA ErasergDNA Eraser 1.0μL1.0μL
Total RNATotal RNA 1.0μg1.0μg
RNase Free DH2ORNase Free DH 2 O Up to 10.0μLUp to 10.0μL
体系完成之后置于Bio-Rad T100 Thermal cycler中，反应条件如下：After the system was completed, it was placed in a Bio-Rad T100 Thermal cycler with the following reaction conditions:
42℃ 2min42 ° C 2 min
4℃ 。4 ° C.
⑵(2)
反转录反应Reverse transcription reaction
试剂Reagent 使用量Usage amount
PrimeScript RT Enzyme Mix ⅠPrimeScript RT Enzyme Mix I 1.0μL1.0μL
RT Prime Mix*4 RT Prime Mix *4 1.0μL1.0μL
5*PrimeScript Buffer25*PrimeScript Buffer2 4.0μL4.0μL
RNase Free DH2ORNase Free DH 2 O 4.0μL4.0μL
步骤1的反应液Step 1 reaction solution 10.0μL10.0 μL
TotalTotal 20.0μL20.0 μL
体系完成之后置于Bio-Rad T100 Thermal cycler中，反应条件如下：After the system was completed, it was placed in a Bio-Rad T100 Thermal cycler with the following reaction conditions:
37℃ 15min37 ° C 15 min
85℃ 5sec85°C 5sec
4℃4 ° C
⑶得到cDNA后稀释10倍。稀释后的样品用来做定量PCR实验(定量PCR仪：CFX96 Optical Reaction Module#1845097，Bio-Rad)，测定PHD1、PHD2、PHD3、FGF2、HGF、ANG1、VEGF-A、HO-1、NF-κB以及PDGF-BB的基因的表达水平，并用β-Actin的表达量归一化。反应体系如下(3) Dilute 10 times after obtaining cDNA. The diluted sample was used for quantitative PCR experiments (quantitative PCR instrument: CFX96 Optical Reaction Module #1845097, Bio-Rad) to determine PHD1, PHD2, PHD3, FGF2, HGF, ANG1, VEGF-A, HO-1, NF- The expression levels of the genes for κB and PDGF-BB were normalized to the expression levels of β-Actin. The reaction system is as follows
试剂Reagent 用量Dosage
SYBRSYBR 5.0μL5.0μL
PCR Forward Primer(10Μm)PCR Forward Primer (10Μm) 0.4μL0.4μL
PCR Reverse Primer(10Μm)PCR Reverse Primer (10Μm) 0.4μL0.4μL
RT反应液RT reaction solution 2.5μL2.5μL
DH2ODH 2 O 1.7μL1.7μL
TotalTotal 10μL10μL
定量PCR反应程序Quantitative PCR reaction procedure
1. 50.0℃ for 2 min1. 50.0°C for 2 min
2. 95.0℃ for 10 min2. 95.0°C for 10 min
3. 95.0℃ for 15 sec3. 95.0°C for 15 sec
4. 60.0℃ for 35 sec4. 60.0°C for 35 sec
5.GOTO 3.40 more times5.GOTO 3.40 more times
6. 95.0℃ for 15 sec6. 95.0°C for 15 sec
7. 60.0℃ for 1 min7. 60.0°C for 1 min
8.Melt Curve 65.0 to 95.0,increment 0.5℃. 8.Melt Curve 65.0 to 95.0, increment 0.5 °C.
定量PCR相关引物序列Quantitative PCR-related primer sequences
Figure PCTCN2017071947-appb-000003
Figure PCTCN2017071947-appb-000003
测定结果示于图3A～C。The measurement results are shown in Figs. 3A to 3C.
目前已知的是，PHD家族(PHD1、PHD2、PHD3)对血管新生起到至关重要的调控作用。根据本申请人的实验结果(参见图3A)，确认红景天苷在骨骼肌中对PHD3有特异性的抑制作用，对于PHD1和PHD2则没有抑制作用。It is currently known that the PHD family (PHD1, PHD2, PHD3) plays a crucial role in the regulation of angiogenesis. According to the results of the applicant's experiments (see Fig. 3A), it was confirmed that salidroside specifically inhibits PHD3 in skeletal muscle, and has no inhibitory effect on PHD1 and PHD2.
由图3B可知，红景天苷促进了血管新生因子(VEGF-A、ANG1、FGF2、PDGF-BB、HO-1、HGF、NF-κB)的表达。其中，已知FGF2、HGF、PDGF-BB、NF-κB和ANG1为与血管成熟相关的因子，因此本领域技术人员可以理解，红景天苷通过促进VEGF等多种血管新生因子表 达量的上调能够促进管腔形成，通过促进FGF2、HGF、PDGF-BB、NF-κB和ANG1表达量的上调而能够促进新生血管的成熟。As can be seen from FIG. 3B, salidroside promoted the expression of angiogenic factors (VEGF-A, ANG1, FGF2, PDGF-BB, HO-1, HGF, NF-κB). Among them, FGF2, HGF, PDGF-BB, NF-κB, and ANG1 are known to be factors related to vascular maturation, and thus those skilled in the art can understand that salidroside promotes various angiogenic factors such as VEGF. Up-regulation of the amount can promote lumen formation and promote the maturation of new blood vessels by promoting the up-regulation of FGF2, HGF, PDGF-BB, NF-κB and ANG1 expression.
申请人进而通过PHD3基因沉默实验研究了PHD3的抑制与血管新生因子表达量上调间的关系。The applicant further investigated the relationship between the inhibition of PHD3 and the up-regulation of angiogenic factor expression by PHD3 gene silencing experiments.
PHD3基因沉默(RNA干扰)试验：PHD3 gene silencing (RNA interference) assay:
构建2个以PHD3为靶标，表达能诱导RNA干扰的短发夹型RNA(short hairpin RNA,shRNA)表达质粒，即shPHD3-1和shPHD3-2。质粒的制作可参见下述文献：Yin Yang 1 induces transcriptional activity of p73 through cooperation with E2F1,Shourong Wu et.al.,Biochemical and Biophysical Research Communications 365(2008)75–81；以及Synergistic cooperation of MDM2 and E2F1 contributes to TAp73 transcriptional activity,Vivi Kasim et al.,Biochemical and Biophysical Research Communications 449(2014)319–326)。种小鼠骨骼肌细胞C2C12于6孔板中，每孔30万细胞，18小时后，细胞换成不含抗生素的培养基(DMEM basic+10％胎牛血清(FBS))。同时按照Lipofectamine2000(Invitrogen)试剂的指导说明进行转染。2μg质粒(shPHD3-1、shPHD3-2shRNA表达质粒)分别与200μL的Opti-MEM培养基混合均匀，另外取4μL Lipofectamine2000与200μL的Opti-MEM培养基混合均匀。室温静置5min。将两个混合体系混合在一起，静置20min后加入六孔板中。6h后换DMEM+10％胎牛血清培养基培养(DMEM+10％胎牛血清(FBS)+penicillin-streptomycin solution)，20h后更换培养基并在低氧环境下培养12h，之后收集样品进行总RNA提取。Two short hairpin RNA (shRNA) expression plasmids, shPHD3-1 and shPHD3-2, were constructed using PHD3 as a target to induce RNA interference. For the preparation of plasmids, see: Yang Yang 1 induces transcriptional activity of p73 through cooperation with E2F1, Shourong Wu et. al., Biochemical and Biophysical Research Communications 365 (2008) 75–81; and Synergistic cooperation of MDM2 and E2F1 contributes To TAp73 transcriptional activity, Vivi Kasim et al., Biochemical and Biophysical Research Communications 449 (2014) 319-326). Mouse skeletal muscle cells C2C12 were plated in a 6-well plate at 300,000 cells per well. After 18 hours, the cells were replaced with antibiotic-free medium (DMEM basic + 10% fetal bovine serum (FBS)). Transfection was also performed according to the instructions of Lipofectamine 2000 (Invitrogen) reagent. 2 μg of the plasmid (shPHD3-1, shPHD3-2 shRNA expression plasmid) was uniformly mixed with 200 μL of Opti-MEM medium, and 4 μL of Lipofectamine 2000 was mixed with 200 μL of Opti-MEM medium. Allow to stand at room temperature for 5 min. The two mixing systems were mixed together and allowed to stand for 20 min before being added to a six-well plate. After 6 hours, DMEM + 10% fetal bovine serum medium (DMEM + 10% fetal bovine serum (FBS) + penicillin-streptomycin solution) was changed. After 20 hours, the medium was changed and cultured in a hypoxic environment for 12 hours, after which the samples were collected for total. RNA extraction.
RNA干扰试验中使用的干扰用序列如下所示： The interference sequences used in the RNA interference test are as follows:
Figure PCTCN2017071947-appb-000004
Figure PCTCN2017071947-appb-000004
低氧环境下的细胞培养：低氧处理是将细胞培养板和AnaeroPack.Anaero(Mitshubishi Gas Chemical,Japan)放入专用密封容器(标准四角形密封容器，Mitsubishi Gas Chemical)内，放入培养箱。所述密封容器内的氧气浓度低于0.1％。Cell culture in a hypoxic environment: The hypoxic treatment was carried out by placing a cell culture plate and AnaeroPack. Anaero (Mitshubishi Gas Chemical, Japan) in a special sealed container (standard square sealed container, Mitsubishi Gas Chemical) and placed in an incubator. The oxygen concentration in the sealed container is less than 0.1%.
实验结果示于图3C。The experimental results are shown in Figure 3C.
由图3C可知，使PHD3基因沉默后，VEGF-A、ANG1、FGF2、PDGF-BB、HO-1、HGF、NF-κB的表达量均显著上调。As can be seen from Fig. 3C, the expression levels of VEGF-A, ANG1, FGF2, PDGF-BB, HO-1, HGF, and NF-κB were significantly up-regulated after silencing the PHD3 gene.
2-3成熟血管形成的深入研究2-3 in-depth study of mature blood vessel formation
申请人在上述实验的基础上，继续进行了深入研究。为了确认所形成的成熟血管中的细胞的来源，申请人进行了细胞实验。On the basis of the above experiments, the applicant continued to conduct in-depth research. In order to confirm the source of the cells in the mature blood vessels formed, the applicant conducted a cell experiment.
2-3-1细胞实验中所用的红景天苷的制备Preparation of salidroside used in 2-3-1 cell experiment
将红景天苷(上海同田生物技术有限公司，纯度≥98.0％)用PBS溶解，配成2mg/ml的储存液，用0.22μm滤膜过滤后，于-20℃保存。The salidroside (Shanghai Tongtian Biotechnology Co., Ltd., purity ≥98.0%) was dissolved in PBS, prepared into a 2 mg/ml stock solution, filtered through a 0.22 μm filter, and stored at -20 °C.
2-3-1细胞迁移实验2-3-1 cell migration experiment
使用骨骼肌细胞的细胞迁移实验Cell migration assay using skeletal muscle cells
将骨骼肌细胞C2C12种在transwell小室内，每个小室内种4000细胞，5小时后将盛放transwell小室的24孔板内分别换成对照培养基(含有10％胎牛血清和Penicillin、Streptomycin的双抗DMEM培养基)或含有红景 天苷的(在上述对照培养基的基础上添加红景天苷，红景天苷含量是100μg/ml)，置于低氧条件下培养。18小时后取出transwell小室，去除transwell小室内部未迁移的细胞后，用DAPI染色透过滤膜而到达小室另一侧的细胞并在荧光显微镜下拍照(每组6张以上)，并通过数6张照片中的细胞数(即迁移的细胞)得出每张照片中细胞数的平均。The skeletal muscle cells C2C12 were seeded in a transwell chamber, 4000 cells in each chamber, and after 5 hours, the 24-well plates in the transwell chamber were replaced with control medium (containing 10% fetal bovine serum and Penicillin, Streptomycin). Double-antibody DMEM medium) or contains red The saponin (adding salidroside to the above control medium, the salidroside content was 100 μg/ml) was cultured under hypoxic conditions. After 18 hours, the transwell chamber was removed, and the unmigrated cells in the transwell chamber were removed. The cells were filved by DAPI and reached the cells on the other side of the chamber and photographed under a fluorescence microscope (6 or more per group) and passed 6 sheets. The number of cells in the photo (ie, the migrated cells) gives the average number of cells in each photo.
使用血管平滑肌细胞的细胞迁移实验Cell migration assay using vascular smooth muscle cells
除了将细胞由小鼠骨骼肌细胞C2C12变更为小鼠血管平滑肌细胞MOVAS外，与上述“使用骨骼肌细胞的细胞迁移实验”同样进行。Except that the cells were changed from mouse skeletal muscle cell C2C12 to mouse vascular smooth muscle cell MOVAS, the same procedure as described above for " cell migration experiment using skeletal muscle cells " was carried out.
将实验结果示于图4。The experimental results are shown in Fig. 4.
由图4可知，红景天苷显著促进了骨骼肌细胞的迁移，但对血管平滑肌细胞则没有促进迁移的作用。As can be seen from Fig. 4, salidroside significantly promoted the migration of skeletal muscle cells, but did not promote migration to vascular smooth muscle cells.
2-3-3条件培养基对于血管平滑肌迁移的作用Effect of 2-3-3 conditioned medium on vascular smooth muscle migration
条件培养基处理组Conditioned medium treatment group
首先将小鼠骨骼肌细胞C2C12播种到100mm*20mm培养皿内，每个板子60万细胞，培养18小时后用PBS清洗，并更换为含有红景天苷的培养基(DMEM+10％FBS+Penicillin+Streptomycin+100μg/ml红景天苷)，来刺激骨骼肌细胞。处理24小时后，去除含红景天苷的培养基，用PBS清洗细胞，换上DMEM+10％FBS+Penicillin+Streptomycin培养基后放入低氧盒内培养24h,收集培养基，3000rpm/min离心5min，收集上清液，用0.22μm膜滤器进行过滤。将所收集的不含红景天苷的培养基作为条件培养基。用条件培养基(该条件培养基里包含了由骨骼肌细胞受到红景天苷的刺激而分泌的多种分泌性蛋白)按照2-3-2里所述的方法对小鼠血管平滑肌细胞MOVAS进行Transwell小室实验。First, the mouse skeletal muscle cells C2C12 were sown into a 100 mm*20 mm culture dish, 600,000 cells per plate, cultured for 18 hours, washed with PBS, and replaced with a medium containing salidroside (DMEM + 10% FBS+). Penicillin + Streptomycin + 100 μg / ml salidroside) to stimulate skeletal muscle cells. After 24 hours of treatment, the medium containing salidroside was removed, and the cells were washed with PBS, replaced with DMEM + 10% FBS + Penicillin + Streptomycin medium, and placed in a hypoxic chamber for 24 hours to collect the medium, 3000 rpm / min. After centrifugation for 5 min, the supernatant was collected and filtered through a 0.22 μm membrane filter. The collected culture medium without salidroside was used as a conditioned medium. Using conditioned medium (containing a variety of secreted proteins secreted by skeletal muscle cells stimulated by salidroside), the mouse vascular smooth muscle cells MOVAS were prepared as described in 2-3-2. Perform a Transwell chamber experiment.
对照组Control group
与条件培养基的制作方法相同，但不添加红景天苷，而是添加与红景天苷同等体积的PBS。用所获得的对照培养基(该对照培养基里包含了由骨骼肌细胞在未受到的刺激的情况下分泌的分泌性蛋白)按照2-3-2里所述的方法对小鼠血管平滑肌细胞MOVAS进行Transwell小室实验。The preparation method was the same as that of the conditioned medium, but without adding salidroside, PBS having the same volume as salidroside was added. Using the obtained control medium (containing the secreted protein secreted by skeletal muscle cells in the absence of stimulation), the mouse vascular smooth muscle cells were treated as described in 2-3-2. MOVAS performed Transwell chamber experiments.
将条件培养基处理和对照组的实验结果示于图5。The results of the conditioned medium treatment and the control group are shown in Fig. 5.
图5表明，经红景天苷刺激的骨骼肌细胞所分泌的分泌性蛋白质能够 促进血管平滑肌细胞的迁移。结合上述含红景天苷的培养基对于血管平滑肌细胞的迁移没有促进作用的结果，我们认为，本实验中，血管平滑肌细胞的迁移之所以受到促进，是由于骨骼肌细胞受到红景天苷刺激后将分泌性蛋白分泌到培养基中，该分泌性蛋白起到了骨骼肌细胞-血管平滑肌细胞间的细胞通讯的作用，从而实现的。另外已知的是，血管平滑肌细胞的迁移对形成功能性的成熟血管非常重要。Figure 5 shows that secreted proteins secreted by salidroside-stimulated skeletal muscle cells can Promotes migration of vascular smooth muscle cells. Combined with the above-mentioned medium containing salidroside, there is no effect on the migration of vascular smooth muscle cells. We believe that the migration of vascular smooth muscle cells is promoted in this experiment because skeletal muscle cells are stimulated by salidroside The secreted protein is then secreted into the culture medium, and the secreted protein functions as a cellular communication between skeletal muscle cells and vascular smooth muscle cells. It is also known that migration of vascular smooth muscle cells is very important for the formation of functional mature blood vessels.
2-3-4分泌性蛋白的确认Confirmation of 2-3-4 secreted protein
利用上述的条件培养基培养血管平滑肌细胞MOVAS，并在MOVAS的培养基里添加各个血管新生因子受体的抑制剂，对小鼠血管平滑肌细胞MOVAS的迁移情况进行确认。The vascular smooth muscle cells MOVAS were cultured in the above conditioned medium, and the inhibitor of each angiogenic factor receptor was added to the MOVAS medium to confirm the migration of mouse vascular smooth muscle cells MOVAS.
结果示于图6。The results are shown in Figure 6.
由图6可知，红景天苷通过促进骨骼肌细胞与血管平滑肌细胞的由FGF2和PDGF-BB介导的细胞通讯促进血管平滑肌细胞的迁移，从而促进成熟血管的形成。As can be seen from Fig. 6, salidroside promotes the migration of vascular smooth muscle cells by promoting cell communication between skeletal muscle cells and vascular smooth muscle cells mediated by FGF2 and PDGF-BB, thereby promoting the formation of mature blood vessels.
2-3-5红景天苷对于低氧下的骨骼肌细胞的凋亡的影响Effect of 2-3-5 salidroside on apoptosis of skeletal muscle cells under hypoxia
凯基细胞凋亡PI染色试剂盒。货号：KGA214KGI cell apoptosis PI staining kit. Item No.: KGA214
将小鼠骨骼肌细胞C2C12种于6cm板中，每板种细胞10万，18小时后用PBS清洗，并更换为含有红景天苷的培养基(DMEM+10％FBS+Penicillin+Streptomycin+100μg/ml红景天苷)。继续培养24小时后去除培养基并用PBS清洗，换成不含红景天苷的培养基后在低氧条件下培养48小时。收集细胞后用细胞凋亡PI染色试剂盒(凯基，货号：KGA214)和流式细胞仪(FACS)检测细胞凋亡。具体步骤如下：Mouse skeletal muscle cells C2C12 were seeded in 6 cm plates, 100,000 cells per plate, washed with PBS 18 hours later, and replaced with medium containing salidroside (DMEM + 10% FBS + Penicillin + Streptomycin + 100 μg) /ml salidroside). After continuing to culture for 24 hours, the medium was removed and washed with PBS, replaced with a medium containing no salidroside, and cultured under hypoxic conditions for 48 hours. After collecting the cells, apoptosis was detected using apoptotic PI staining kit (KGI, Cat. No. KGA214) and flow cytometry (FACS). Specific steps are as follows:
(1)用1xBuffer A洗涤细胞一次(离心2000rpm,5min),收集细胞并将细胞浓度稀释到1x106细胞/ml。(1) The cells were washed once with 1 x Buffer A (centrifugation 2000 rpm, 5 min), and the cells were collected and the cell concentration was diluted to 1 x 10 6 cells/ml.
(2)细胞加9倍体积的70％乙醇，在-20℃固定12-14小时。(2) The cells were supplemented with 9 volumes of 70% ethanol and fixed at -20 ° C for 12-14 hours.
(3)离心收集细胞后用1xBuffer A洗涤细胞除去乙醇，细胞重悬于500μL Buffer A中。(3) After collecting the cells by centrifugation, the cells were washed with 1 x Buffer A to remove ethanol, and the cells were resuspended in 500 μL of Buffer A.
(4)加入RNase A,使其终浓度为0.25mg/ml，37℃反应30min。(4) RNase A was added to a final concentration of 0.25 mg/ml, and reacted at 37 ° C for 30 min.
(5)加入5μL PI，室温避光染色30min，然后利用流式细胞仪检测PI阳性细胞(凋亡细胞)。 (5) 5 μL of PI was added, stained at room temperature for 30 min in the dark, and then PI positive cells (apoptotic cells) were detected by flow cytometry.
发明人通过流式细胞仪检测PI阳性细胞数确认，低氧条件并未明显诱导骨骼肌细胞的凋亡，低氧条件下使用红景天苷的凋亡率与常氧条件下和低氧对照的凋亡率无显著差异。The inventors confirmed the number of PI positive cells by flow cytometry, and hypoxic conditions did not significantly induce apoptosis of skeletal muscle cells. The apoptosis rate of salidroside under hypoxic conditions was compared with normoxic conditions and hypoxia. There was no significant difference in the rate of apoptosis.
结果示于图7。The results are shown in Figure 7.
2-3-6红景天苷调控血管新生的机理的推测。Speculation of the mechanism by which 2-3-6 salidroside regulates angiogenesis.
通过上述各个实验，可以合理推测，红景天苷通过特异性抑制骨骼肌细胞中的PHD3的表达，从而促进了骨骼肌细胞在低氧条件下的多种血管新生因子的表达，还提高了骨骼肌细胞自身的迁移能力，这些因素使得骨骼肌细胞分泌出的血管新生因子增多，受其影响的范围增广；并且这些骨骼肌细胞分泌出的血管新生因子中的PDGF-BB和FGF2通过作用于血管平滑肌细胞上的各自的受体(本公开中也称为骨骼肌细胞-血管平滑肌细胞间的细胞通讯)而促进了对成熟血管形成起至关重要的作用的血管平滑肌细胞的迁移，最终实现了成熟血管的形成，从而获得了良好的下肢缺血性疾病的治疗效果。Through the above experiments, it can be reasonably speculated that salidroside can specifically inhibit the expression of PHD3 in skeletal muscle cells, thereby promoting the expression of various angiogenic factors of skeletal muscle cells under hypoxic conditions, and also improving bones. The ability of muscle cells to migrate, these factors increase the number of angiogenic factors secreted by skeletal muscle cells, and the range of their effects is increased; and PDGF-BB and FGF2 in angiogenic factors secreted by these skeletal muscle cells act through The respective receptors on vascular smooth muscle cells (also referred to in the present disclosure as skeletal muscle cells - cellular communication between vascular smooth muscle cells) promote the migration of vascular smooth muscle cells, which are critical for the formation of mature blood vessels, and ultimately The formation of mature blood vessels, thereby obtaining a good therapeutic effect of lower limb ischemic diseases.
(二)关于糖尿病足的治疗药物(2) Treatment drugs for diabetic foot
1.糖尿病小鼠模型的建立1. Establishment of a diabetic mouse model
C57BL/6J小鼠(6周，雄性)购买(购自中国人民解放军第三军医大学)回来一周后测量小鼠血糖，用下述高脂饲料喂养4周后测量血糖，出现糖尿病前期(pre-diabetes)的情况后，链脲佐菌素通过肌肉连续注射五天，射量为50mg/kg，继续高脂饲料喂养一周后测量小鼠血糖，血糖高于16.7mmol/L的被选用做下一步实验。在此，需要说明的是，一般血糖为7以上已被认为患有糖尿病，但本公开的模型选定血糖为16.7以上的小鼠。C57BL/6J mice (6 weeks, male) were purchased (purchased from the Third Military Medical University of the People's Liberation Army) and the blood glucose of the mice was measured one week after returning. The blood sugar was measured after feeding for 4 weeks with the following high-fat diet, and pre-diabetes occurred (pre- After the case of diabetes, streptozotocin was continuously injected through the muscle for five days, and the dose was 50 mg/kg. The blood glucose of the mice was measured after one week of high-fat diet feeding. The blood glucose above 16.7 mmol/L was selected as the next step. experiment. Here, it is to be noted that, in general, blood glucose of 7 or more is considered to have diabetes, but the model of the present disclosure selects a mouse having blood glucose of 16.7 or more.
高脂饲料的配方：15％猪油Formula of high fat feed: 15% lard
10％蛋黄10% egg yolk
10％白糖10% white sugar
65％普通饲料65% ordinary feed
其中，普通饲料、蛋黄、猪油、白糖由第三军医大学大坪医院提供，并由第三军医大学大坪医院生产高脂饲料。 Among them, ordinary feed, egg yolk, lard, and sugar were provided by Daping Hospital of the Third Military Medical University, and high-fat feed was produced by Daping Hospital of the Third Military Medical University.
2.红景天苷对糖尿病足的治疗效果2. The therapeutic effect of salidroside on diabetic foot
使用上述糖尿病小鼠模型，在麻醉条件下对左侧大腿大动脉进行切除手术，并利用Laser Doppler Perfusion Imaging System检测血流的情况。需要说明的是，关于本申请说明书中“左侧”、“右侧”的表述，进行了手术的是左侧大腿，此时小鼠处于俯卧状态，后述的血流图的照片中小鼠处于仰卧状态，所以该血流图照片中手术的大腿在图中右侧)(参照文献Shourong Wu et al.,Prolyl hydroxylase domain-2silencing induced by hydrodynamic limb vein injection enhances vascular regeneration in critical limb ischemia mice through activation of multiple genes(2015)Curr Gene Ther.,15(3):313-325中的方法)。The left thigh aorta was excised under anesthesia using the above-described diabetic mouse model, and blood flow was measured using a Laser Doppler Perfusion Imaging System. In addition, regarding the expressions of "left side" and "right side" in the specification of the present application, the left thigh is operated, and the mouse is in a prone state, and the mouse in the photo of the blood flow diagram described later is The supine state, so the thigh of the operation in the blood flow picture is shown on the right side of the figure) (refer to the literature Shourong Wu et al., Prolyl hydroxylase domain-2silencing induced by hydrodynamic limb vein injection enhances vascular regeneration in the essence limb ischemia mice into activation of Multiple genes (2015) Curr Gene Ther., 15(3): Method in 313-325).
将红景天苷(从上海同田生物技术股份有限公司购买，样品纯度高于98％)用磷酸盐缓冲溶液(PBS)溶解，配成40mg/ml的储存液，用0.22μm滤膜过滤后，于-20℃保存备用。The salidroside (purchased from Shanghai Tongtian Biotechnology Co., Ltd., the sample purity is higher than 98%) was dissolved in phosphate buffer solution (PBS), and prepared into a 40 mg/ml stock solution, which was filtered through a 0.22 μm filter. Store at -20 °C for later use.
注射前将红景天苷储存液稀释至20mg/ml，小鼠肌肉注射剂的剂量为100mg/kg，术后第1天开始每三天注射一次，每次注射将注射液分3次，连续往左侧腓肠肌里分三个位点分别注射。The salidroside stock solution was diluted to 20 mg/ml before injection, and the dose of the mouse intramuscular injection was 100 mg/kg. The injection was started every three days from the first day after the operation, and the injection was divided into three times for each injection. Three sites were injected in the left gastrocnemius muscle.
使用生理盐水作为对照，同样地过滤、保存和注射。Physiological saline was used as a control, and filtration, preservation, and injection were similarly performed.
利用Laser Doppler Perfusion Imaging System(MOOR INSTRUMENTS Ltd，MOORLDLS2-IR)检测手术前、刚手术后、术后第3、7、14、21天的血流情况。Blood flow was measured before, immediately after, and on days 3, 7, 14, and 21 after surgery using the Laser Doppler Perfusion Imaging System (MOOR INSTRUMENTS Ltd, MOORLDLS2-IR).
参照图8(A)，灰色部分反应血流状况。术前，生理盐水对照组小鼠和红景天苷处理组小鼠具有同等的血流状况(需要说明的是，Laser Doppler Perfusion Imaging System原初成像为彩图，转化为灰度图片后，与原初所成的像看起来有些许差异。在原彩图中，红色表示血流丰富，蓝色表示没有血流。在灰度图像下无法较清晰地区分血流恢复的地方(即原彩图中红色部位)和没有血流(即原彩图中蓝色部位)的地方)。针对该问题，本申请发明人依据彩图结果对灰度图进行了图像处理，从而得到了图8(B)。在图8(B)中，网点状表示血流恢复的地方，即原彩图中的红色部分。刚手术后，对照组小鼠和红景天苷处理组小鼠左侧下肢均显示 出黑色(在原彩图中均为蓝色部分，在处理后的图中没有网点状图案)，即二者左侧下肢均没有血流，可知手术成功地制造出糖尿病足小鼠模型。在术后第3天，红景天苷处理组小鼠左侧下肢开始出现灰色和黑色部分(即原彩图中的红色部分、处理后的图中的网点部分)，可知出现了明显的血流恢复状况；与之相对，生理盐水对照组小鼠左侧下肢仍为黑色(即原彩图中的蓝色部分，在处理后的图中为没有网点状图案的黑色部分)，可知没有出现血流恢复的迹象。术后第21天时，红景天苷处理组小鼠左侧下肢达到了与未处理的左侧下肢几乎同等的血流状况，即左侧下肢的血流得到了较为充分的恢复；与之相对，图中生理盐水对照组小鼠左侧下肢影像缺失，推测是由于没有血流恢复而导致了下肢萎缩和组织坏死。另外从图中也可以看出，随着治疗时间的延长，红景天苷处理组左侧下肢的灰色和黑色部分(即原彩图中的红色部分、处理后的图中的网点部分)越来越达到下肢的远端(即靠近脚趾)部位，可知血流逐渐恢复到距离动脉切除较远的部位。Referring to Fig. 8(A), the gray portion reflects the blood flow condition. Before the operation, the mice in the saline control group and the salidroside-treated mice had the same blood flow status. (It should be noted that the Laser Doppler Perfusion Imaging System was originally imaged as a color map, which was converted into a grayscale image, and the original The resulting image looks a little different. In the original color map, red indicates that the blood flow is rich, blue indicates no blood flow. Under the gray image, the blood flow recovery cannot be clearly distinguished (ie, the red color in the original color map) Part) and where there is no blood flow (ie, the blue part of the original color map). In response to this problem, the inventors of the present invention performed image processing on the grayscale image based on the result of the color map, thereby obtaining FIG. 8(B). In Fig. 8(B), the dot pattern indicates where the blood flow is restored, that is, the red portion in the original color map. After the operation, the left lower limbs of the mice in the control group and the salidroside-treated group showed Black (both in the original color map, there is no dot pattern in the processed image), that is, there is no blood flow in the left lower limbs of both, and it is known that the diabetic foot mouse model is successfully manufactured. On the third day after surgery, the left and right lower limbs of the salidroside-treated group began to appear gray and black parts (ie, the red part in the original color map and the half-point part in the processed figure), and it was found that obvious blood appeared. In contrast, the left lower limb of the saline control group was still black (ie, the blue part of the original color map, and the black part without the dot pattern in the processed image), it is known that there is no Signs of blood flow recovery. On the 21st day after surgery, the left lower limb of the salidroside-treated group reached almost the same blood flow condition as the untreated left lower limb, that is, the blood flow of the left lower limb was fully recovered; In the figure, the left lower extremity image of the saline control group mice was absent, which was presumed to be due to the lack of blood flow and the lower limb atrophy and tissue necrosis. In addition, it can be seen from the figure that the more the treatment time is extended, the more the gray and black parts of the left lower limb of the salidroside treatment group (ie, the red part in the original color map and the halftone part in the processed figure) The more the distal end of the lower limb (ie, near the toe) is reached, the blood flow gradually recovers to a location farther away from the arterial resection.
为了进一步明确红景天苷处理组的实验效果，本申请发明人对Laser Doppler Perfusion Imaging System的结果进行了定量。具体计算方法为：将缺血下肢(左侧下肢)的定量值除以同一只小鼠的非缺血下肢(右侧下肢)的定量值(即血流面积的像素)，然后将每一个时间点的该比值平均化并除以各组术前的比值的平均值。参照基于统计结果的图9可知，刚手术后，对照组小鼠和红景天苷处理组小鼠的血流比值均降为低于0.3的数值。在术后第3天，红景天苷处理组小鼠的血流比值达到0.6；与之相对，生理盐水对照组小鼠的血流比值明显低于0.4。术后第21天时，红景天苷处理组小鼠的血流比值达到了0.8；与之相对，对照组小鼠的血流比值仍为与术后第3天的值接近的低值。In order to further clarify the experimental effect of the salidroside treatment group, the inventors of the present application quantified the results of the Laser Doppler Perfusion Imaging System. The specific calculation method is: dividing the quantitative value of the ischemic lower limb (left lower limb) by the quantitative value of the non-ischemic lower limb (right lower limb) of the same mouse (ie, the pixel of the blood flow area), and then each time The ratio of points is averaged and divided by the average of the preoperative ratios for each group. Referring to Fig. 9 based on the statistical results, the blood flow ratio of the control group and the salidroside-treated mice decreased to a value lower than 0.3 immediately after the operation. On the third day after surgery, the blood flow ratio of the salidroside-treated mice reached 0.6; in contrast, the blood flow ratio of the saline control mice was significantly lower than 0.4. On the 21st day after surgery, the blood flow ratio of the salidroside-treated mice reached 0.8; in contrast, the blood flow ratio of the control mice was still close to the value of the third day after surgery.
由此可知红景天苷对糖尿病足小鼠的处理实验获得了优异的血流恢复效果。与注射生理盐水的对照相比，注射了红景天苷的糖尿病足小鼠的血流状况显著恢复，可知红景天苷对于糖尿病足的治疗有良好的效果，由此可知产生了成熟、无漏且具有功能性的新生血管。It can be seen that salidroside has excellent blood flow recovery effect on the treatment of diabetic foot mice. Compared with the saline-injected control, the blood flow status of the diabetic foot mice injected with salidroside was significantly restored, and it was found that salidroside had a good effect on the treatment of diabetic foot, and thus it was found that the mature, no Leaky and functional neovascularization.
3.免疫组织染色 3. Immunohistochemical staining
(1)制造糖尿病足模型小鼠组织冰冻切片(1) Tissue frozen sections of mice with diabetic foot model
术后21天，取得红景天苷处理组和生理盐水处理组小鼠的腓肠肌组织后保存在-80℃。待组织冷冻后进行切片，得到实验样本。On the 21st day after surgery, the gastrocnemius muscle tissues of the salidroside-treated group and the saline-treated group were obtained and stored at -80 °C. After the tissue was frozen, it was sliced to obtain an experimental sample.
切片流程如下，包埋剂包埋组织后在切片机(Leica生产)上进行切片，切片厚度是10μM。切片结束后将切片置于37℃烘箱中烘干30min，2.5％牛血清白蛋白(BSA)中封闭30～60min。将组织周围的BSA除去后，用抗PECAM-1(别名：CD31)抗体染色(PECAM-1：Purified Rat Anti-Mouse CD31(Clone MEC13.3,BD PharmingenTM Cat 550274，抗体稀释比例1:50)。室温孵育1h后，用含有0.1％吐温的生理盐水(PBS-T)清洗三次，每次5min。The sectioning procedure was as follows. The embedding agent was embedded in the tissue and sliced on a microtome (manufactured by Leica), and the section thickness was 10 μM. After the end of the section, the sections were placed in an oven at 37 ° C for 30 min, and blocked in 2.5% bovine serum albumin (BSA) for 30-60 min. After removal of BSA around the tissue, staining with anti-PECAM-1 (alias: CD31) antibody (PECAM-1: Purified Rat Anti-Mouse CD31 (Clone MEC 13.3, BD Pharmingen TM Cat 550274, antibody dilution ratio 1:50) After incubating for 1 h at room temperature, it was washed three times with physiological saline (PBS-T) containing 0.1% Tween for 5 min each time.
将带有荧光标记的抗α-Smooth Muscle Actin(α-SMA)的抗体(即α-Smooth Muscle-Cy3,Mouse monoclonal(Clone 1A4,Sigma-Aldrich Cat C6198))和荧光标记的，针对抗PECAM-1抗体的二抗(Goat anti-Rat IgG(H+L)Secondary Antibody,Alexa
Figure PCTCN2017071947-appb-000005
488 conjugate(Thermo Scientific Cat A11006))混合(抗体稀释比例均为1:100)，并将上述用抗PECAM-1抗体孵育并清洗过的切片进一步用上述抗α-SMA和抗PECAM-1抗体的二抗的混合液在室温孵育30min。然后PBS-T清洗三次，每次5min。免疫荧光染色结束并用丙三醇封片后，用荧光显微镜(Leica Microsystems，DMI6000B)进行检测。The anti-α-Smooth Muscle Actin (α- SMA) with a fluorescently labeled antibody (i.e. α - Smooth Muscle-Cy3, Mouse monoclonal (Clone 1A4, Sigma-Aldrich Cat C6198)) and a fluorescent-labeled, directed against PECAM- 1 antibody secondary antibody (Goat anti-Rat IgG (H+L) Secondary Antibody, Alexa
Figure PCTCN2017071947-appb-000005
488 conjugate (Thermo Scientific Cat A11006)) was mixed (antibody dilution ratio was 1:100), and the above-mentioned sections incubated and washed with anti-PECAM-1 antibody were further used with the above anti-α-SMA and anti-PECAM-1 antibodies. The mixture of secondary antibodies was incubated for 30 min at room temperature. The PBS-T was then washed three times for 5 min each time. After immunofluorescence staining was completed and mounted with glycerol, detection was performed using a fluorescence microscope (Leica Microsystems, DMI6000B).
对注射了红景天苷和生理盐水的糖尿病足小鼠分别确认了血管生成及血管成熟状况。结果如图10所示。根据图10所示的结果可知，红景天苷促进了糖尿病足小鼠缺血下肢中的成熟血管的形成。可以确认，注射红景天苷后的小鼠的组织内血管内皮细胞(即PECAM-1阳性)和血管平滑肌细胞(即α-SMA阳性)增多。通过重叠图片(Merge image)发现PECAM-1和α-SMA双阳性的结构增多，并形成了由血管平滑肌细胞包围血管内皮细胞的管腔结构，意味着形成了丰富的成熟血管，而注射生理盐水的小鼠仅有非常微弱的阳性信号，且绝大部分不形成管腔结构。定量结果也显示了红景天苷能显著地诱导更多的血管内皮细胞和血管平滑肌细胞。Angiogenesis and vascular maturation were confirmed in diabetic foot mice injected with salidroside and physiological saline, respectively. The result is shown in Figure 10. According to the results shown in Fig. 10, salidroside promoted the formation of mature blood vessels in the ischemic lower limbs of diabetic foot mice. It was confirmed that vascular endothelial cells (i.e., PECAM-1 positive) and vascular smooth muscle cells (i.e., α-SMA positive) in the tissues of mice after injection of salidroside increased. Through overlapping images (Merge image), it was found that the structure of double positive of PECAM-1 and α-SMA increased, and the luminal structure surrounded by vascular smooth muscle cells surrounded by vascular endothelial cells was formed, which means that abundant mature blood vessels were formed, and saline was injected. The mice have only very weak positive signals, and most of them do not form a luminal structure. The quantitative results also show that salidroside can significantly induce more vascular endothelial cells and vascular smooth muscle cells.
为了进一步明确红景天苷组的实验效果，本申请发明人利用Leica Application Suite Version 4.6软件对所获得的图10的结果进行了定量。结 果如图11所示。In order to further clarify the experimental effect of the salidroside group, the inventors of the present application quantified the obtained results of FIG. 10 using Leica Application Suite Version 4.6 software. Knot As shown in Figure 11.
由该定量结果可以明确地读出，在针对PECAM-1的实验结果中，红景天苷处理后的样本的PECAM-1的阳性面积超过了80000pixel，与之相对，生理盐水处理后的样本的阳性面积仅稍大于20000pixel，是相当低的值。在针对α-SMA的实验结果中，红景天苷处理后的样本的阳性的面积超过了30000pixel，与之相对，生理盐水处理后的样本的阳性面积仅刚超过10000pixel，是相当低的值。From the quantitative results, it can be clearly read out. In the experimental results for PECAM-1, the positive area of PECAM-1 of the sample treated with salidroside exceeds 80,000 pixels, whereas the sample after physiological saline treatment The positive area is only slightly larger than 20,000 pixels, which is a fairly low value. In the experimental results for α-SMA, the positive area of the sample treated with salidroside exceeded 30,000 pixels, whereas the positive area of the sample treated with physiological saline was just over 10,000 pixels, which was a relatively low value.
该结果进一步表明，注射红景天苷能够治疗糖尿病足缺血下肢血流的恢复，推测其原因是由于红景天苷促进了小鼠的血管新生和成熟血管的形成。The results further indicate that the injection of salidroside can treat the recovery of blood flow in the ischemic lower limb of diabetic foot, which is presumed to be due to the fact that salidroside promotes angiogenesis and mature blood vessel formation in mice.
4.红景天苷对糖尿病足小鼠血糖的影响4. Effects of salidroside on blood glucose in diabetic foot mice
为了检测红景天苷对于糖尿病足小鼠血流恢复的影响是否是基于其对生物体血糖水平的影响的结果，本申请的发明人对糖尿病足小鼠的血糖进行了测定。In order to examine whether the effect of salidroside on blood flow recovery in diabetic foot mice is based on the effect on blood glucose levels of the organism, the inventors of the present application measured the blood glucose of diabetic foot mice.
血糖检测方法为：将血滴到
Figure PCTCN2017071947-appb-000006
活力型血糖试纸***
Figure PCTCN2017071947-appb-000007
活力型血糖仪(
Figure PCTCN2017071947-appb-000008
Active，Model GU，Roche)。利用
Figure PCTCN2017071947-appb-000009
采血笔(
Figure PCTCN2017071947-appb-000010
Lancing Device，Roche)和
Figure PCTCN2017071947-appb-000011
一次性使用采血针(
Figure PCTCN2017071947-appb-000012
Roche)，从糖尿病足小鼠尾巴采血，并将血滴添加在
Figure PCTCN2017071947-appb-000013
活力型血糖试纸橙色区域的中间。The blood sugar test method is: dripping blood to
Figure PCTCN2017071947-appb-000006
Vibrant blood glucose test strip insertion
Figure PCTCN2017071947-appb-000007
Vital blood glucose meter (
Figure PCTCN2017071947-appb-000008
Active, Model GU, Roche). use
Figure PCTCN2017071947-appb-000009
Blood collection pen
Figure PCTCN2017071947-appb-000010
Lancing Device, Roche) and
Figure PCTCN2017071947-appb-000011
Single use lancet (
Figure PCTCN2017071947-appb-000012
Roche), collecting blood from the tail of diabetic foot mice and adding blood drops
Figure PCTCN2017071947-appb-000013
The middle of the orange area of the vital blood glucose test strip.
对于生理盐水对照组和红景天苷处理组的小鼠，分别对术前、以及术后第3、7、14和21天的血糖值进行检测。For the saline control group and the salidroside-treated group, blood glucose levels were measured before surgery and on days 3, 7, 14, and 21 after surgery.
由下表1可知，红景天苷处理组小鼠的血糖水平与生理盐水对照组小鼠为同等水平。可知，对糖尿病足小鼠缺血大腿骨骼肌局部注射红景天苷未影响血糖水平。即，可知在缺血部位的骨骼肌局部注射红景天苷对于糖尿病足小鼠的血流恢复状况、进一步地对于糖尿病足的治疗，并不是通过对生物体血糖水平的调节来实现的。As can be seen from Table 1 below, the blood glucose level of the salidroside-treated mice was the same as that of the saline control group. It can be seen that local injection of salidroside on the ischemic thigh skeletal muscle of diabetic foot mice did not affect blood glucose levels. That is, it can be seen that the local injection of salidroside in the skeletal muscle of the ischemic site for blood flow recovery of diabetic foot mice and further treatment of diabetic foot is not achieved by adjusting the blood sugar level of the living body.
表1(单位：mmol/L) Table 1 (unit: mmol / L)
  术前Preoperative 术后3天3 days after surgery 术后7天7 days after surgery 术后14天14 days after surgery 术后21天21 days after surgery
对照Control 22.61±2.6322.61±2.63 19.34±1.5419.34±1.54 24.65±3.4524.65±3.45 20.26±1.7020.26±1.70 19.16±1.9119.16±1.91
红景天苷Salidroside 21.77±4.1821.77±4.18 18.46±1.0018.46±1.00 25.93±4.1425.93±4.14 19.69±1.2319.69±1.23 19.19±1.3019.19±1.30
p值(对照vs红景天苷)p value (control vs salidroside) 0.6130.613 0.3970.397 0.5360.536 0.3360.336 11
表中为平均值±标准偏差，p值由卡方检验计算得到。The table shows the mean ± standard deviation, and the p value is calculated by the chi-square test.
5.糖尿病小鼠模型中红景天苷对血管新生因子的影响5. Effects of salidroside on angiogenesis factors in a diabetic mouse model
(1)红景天苷的注射(1) Injection of salidroside
将红景天苷用PBS溶解，配成40mg/ml的储存液，用0.22μm滤膜过滤后，于-20℃保存备用。The salidroside was dissolved in PBS, and a 40 mg/ml stock solution was prepared, filtered through a 0.22 μm filter, and stored at -20 ° C until use.
注射前将红景天苷储存液稀释至20mg/ml。根据上述项目1.中的记载建立糖尿病足小鼠模型，并注射所得到的红景天苷稀释液。糖尿病小鼠肌肉注射剂的剂量是100mg/kg，术后第一天注射，注射时将注射液分3次，连续往左侧腓肠肌里分3个位点分别注射。The salidroside stock solution was diluted to 20 mg/ml before injection. A diabetic foot mouse model was established according to the description in item 1. above, and the obtained salidroside dilution was injected. The dose of intramuscular injection of diabetic mice was 100 mg/kg, which was injected on the first day after surgery. The injection was divided into 3 times and injected into the left gastrocnemius muscle at 3 sites.
(2)RNA的提取(2) RNA extraction
在小鼠手术第三天将其麻醉后处死，取腓肠肌组织并置于
Figure PCTCN2017071947-appb-000014
Solution。将组织分成两块在液氮中研磨，待磨成粉末状之后加入TRIZOL后收集到1.5mL的无酶EP管中。RNA提取后，用NanoDrop进行RNA定量后，进行反转录PCR(RT-PCR)和定量PCR(Q-PCR)。On the third day of the mouse surgery, the rats were anesthetized and sacrificed. The gastrocnemius muscle tissue was taken and placed.
Figure PCTCN2017071947-appb-000014
Solution. The tissue was divided into two pieces and ground in liquid nitrogen. After being pulverized, TRIZOL was added and 1.5 mL of the enzyme-free EP tube was collected. After RNA extraction, RNA was quantified by NanoDrop, followed by reverse transcription PCR (RT-PCR) and quantitative PCR (Q-PCR).
实验过程中的仪器主要有：NanoDrop2000微量核酸测定仪(Gene Company公司)。研钵(国产)，PCR仪(T100Thermal cycler，Bio-RAD公司)，八连管(Bio-RAD公司)，Q-PCR(CFX-96Optical Reaction Module#1845097，Bio-RAD公司)。The instruments in the experiment mainly include: NanoDrop2000 micronucleic acid analyzer (Gene Company). R&D (domestic), PCR instrument (T100 Thermal cycler, Bio-RAD), Erlian (Bio-RAD), Q-PCR (CFX-96 Optical Reaction Module #1845097, Bio-RAD).
实验过程中主要使用如下试剂：液氮，
Figure PCTCN2017071947-appb-000015
Solution(Ambion公司)，RNA反转录及实时定量试剂盒(TaKaRa RR047A PrimeScript RT reagent Kit with gDNA Eraser)，TriZol(Invitrogen公司)。The following reagents were mainly used in the experiment: liquid nitrogen,
Figure PCTCN2017071947-appb-000015
Solution (Ambion), RNA Reverse Transcription and Real-Time Quantification Kit (TaKaRa RR047A PrimeScript RT reagent Kit with gDNA Eraser), TriZol (Invitrogen).
(3)mRNA水平的测定(3) Determination of mRNA levels
通过RT-PCR，测定表2中记载的各血管新生因子的基因的表达水平， 并用β-Actin的表达量归一化。The expression levels of the genes of each angiogenic factor described in Table 2 were determined by RT-PCR. And normalized by the expression level of β-Actin.
(I)基因组DNA的去除(I) Removal of genomic DNA
使用TAKARA-PrimeScriptTM RT reagent Kit with gDNA Eraser(Code No.RR047A)，该步骤中使用的试剂及其使用量如下所示。Using TAKARA-PrimeScriptTM RT reagent Kit with gDNA Eraser (Code No. RR047A), the reagents used in this step and the amounts thereof used are as follows.
Figure PCTCN2017071947-appb-000016
Figure PCTCN2017071947-appb-000016
加样完成之后置于Bio-RAD T100 Thermal Cycler中。After the addition was completed, it was placed in a Bio-RAD T100 Thermal Cycler.
反应过程如下：42℃ 2minThe reaction process is as follows: 42 ° C 2 min
4℃ 。4 ° C.
(II)反转录反应(II) Reverse transcription reaction
使用如下试剂并按照如下的使用量进行反转录反应。The reverse transcription reaction was carried out using the following reagents in the following amounts.
Figure PCTCN2017071947-appb-000017
Figure PCTCN2017071947-appb-000017
加样完成之后置于Bio-RAD T100 Thermal Cycler中。After the addition was completed, it was placed in a Bio-RAD T100 Thermal Cycler.
反应过程如下：37℃ 15minThe reaction process is as follows: 37 ° C 15 min
85℃ 5sec85°C 5sec
4℃。4 ° C.
得到cDNA后稀释10倍。稀释后的样品用来做Q-PCR反应。反应体系如下：The cDNA was diluted 10 times after the cDNA was obtained. The diluted sample was used for the Q-PCR reaction. The reaction system is as follows:
Figure PCTCN2017071947-appb-000018
Figure PCTCN2017071947-appb-000018
Figure PCTCN2017071947-appb-000019
Figure PCTCN2017071947-appb-000019
(III)Q-PCR反应程序(III) Q-PCR reaction procedure
1 50.0℃ for 2min1 50.0°C for 2min
2 95.0℃ for 10min2 95.0°C for 10min
3 95.0℃ for 15sec3 95.0°C for 15sec
4 60.0℃ for 35sec4 60.0°C for 35sec
5 GOTO 3.40more times5 GOTO 3.40more times
6 95.0℃ for 15sec6 95.0°C for 15sec
7 60.0℃ for 1min7 60.0°C for 1min
8 Melt Curve 65.0 to 95.0,increment 0.5℃.8 Melt Curve 65.0 to 95.0, increment 0.5°C.
Q-PCR仪为CFX-96 Optical Reaction Module#1845097(Bio-RAD公司)。The Q-PCR instrument is CFX-96 Optical Reaction Module #1845097 (Bio-RAD).
Q-PCR相关引物序列Q-PCR related primer sequences
表2Table 2
Figure PCTCN2017071947-appb-000020
Figure PCTCN2017071947-appb-000020
实验结果示于图12。由图12可知，在对糖尿病模型小鼠的缺血下肢进行红景天苷给药后，骨骼肌里的血管新生因子VEGF-A、FGF2、ANG1、 PDGF-BB和HGF的表达量均显著上调。认为红景天苷能够在高糖、低氧条件下促进多种血管新生因子的表达。 The experimental results are shown in Fig. 12. As can be seen from Fig. 12, angiogenic factors VEGF-A, FGF2, ANG1 in skeletal muscle after administration of salidroside to the ischemic lower limb of a diabetic model mouse. The expression levels of PDGF-BB and HGF were significantly up-regulated. It is believed that salidroside can promote the expression of various angiogenic factors under high glucose and hypoxia conditions.

Claims (21)

  1. 红景天苷在制备治疗缺血性疾病的药物中的应用。The use of salidroside in the preparation of a medicament for treating ischemic diseases.
  2. 红景天苷在制备治疗糖尿病足的药物中的应用。The use of salidroside in the preparation of a medicament for treating diabetic foot.
  3. 根据权利要求2的应用，其中，所述红景天苷为注射剂。The use according to claim 2, wherein the salidroside is an injection.
  4. 根据权利要求2或3的应用，其中，所述红景天苷为骨骼肌注射剂。The use according to claim 2 or 3, wherein the salidroside is a skeletal muscle injection.
  5. 根据权利要求2-4中任一项的应用，其中，所述红景天苷在高糖、低氧环境下促进血管新生因子的表达和分泌。The use according to any one of claims 2 to 4, wherein the salidroside promotes expression and secretion of angiogenic factors in a high glucose, hypoxic environment.
  6. 根据权利要求5的应用，其中，所述血管新生因子为VEGF-A、FGF2、ANG1、PDGF-BB和HGF。The use according to claim 5, wherein the angiogenic factors are VEGF-A, FGF2, ANG1, PDGF-BB and HGF.
  7. 一种治疗糖尿病足的方法，其中，对患者进行红景天苷的给药。A method of treating a diabetic foot, wherein the patient is administered with salidroside.
  8. 根据权利要求7的治疗糖尿病足的方法，该给药方法为肌肉注射。A method of treating a diabetic foot according to claim 7, which is intramuscular injection.
  9. 一种治疗糖尿病足的药物，该药物的活性成份为红景天苷。A medicament for treating diabetic foot, the active ingredient of which is salidroside.
  10. 如权利要求9的药物，所述红景天苷为血管新生因子的促进剂，该促进剂在高糖、低氧条件下促进血管新生因子的表达和分泌。The medicament according to claim 9, wherein the salidroside is an enhancer of an angiogenic factor which promotes expression and secretion of an angiogenic factor under conditions of high glucose and hypoxia.
  11. 如权利要求10的药物，其中，所述血管新生因子为VEGF-A、FGF2、ANG1、PDGF-BB和HGF。The medicament according to claim 10, wherein said angiogenic factors are VEGF-A, FGF2, ANG1, PDGF-BB and HGF.
  12. 一种PHD3的特异性抑制剂，所述抑制剂含有红景天苷。A specific inhibitor of PHD3, which contains salidroside.
  13. 一种血管新生因子的表达和分泌的促进剂，所述促进剂含有红景天苷。An accelerator for the expression and secretion of an angiogenic factor, the promoter comprising salidroside.
  14. 一种治疗下肢缺血性疾病的药物，所述药物含有权利要求12的PHD3的特异性抑制剂或权利要求13的促进剂。A medicament for treating a lower limb ischemic disease, the medicament comprising the specific inhibitor of PHD3 of claim 12 or the promoter of claim 13.
  15. 一种治疗下肢缺血性疾病的药物，所述药物含有红景天苷作为活性成分。A medicament for treating a lower limb ischemic disease, the medicament comprising salidroside as an active ingredient.
  16. 根据权利要求14或15的治疗下肢缺血性疾病的药物，所述药物进一步含有药用辅料。A medicament for treating an ischemic disease of the lower extremities according to claim 14 or 15, which further comprises a pharmaceutically acceptable adjuvant.
  17. 一种血管平滑肌细胞迁移的促进剂，所述促进剂含有利用红景天苷刺激骨骼肌细胞而产生的分泌性蛋白。An accelerator for vascular smooth muscle cell migration, the promoter comprising a secreted protein produced by using erythroside to stimulate skeletal muscle cells.
  18. 一种骨骼肌细胞迁移的促进剂，所述促进剂含有红景天苷。 An accelerator for skeletal muscle cell migration, the promoter comprising salidroside.
  19. 红景天苷在制备治疗下肢缺血性疾病的药物中的用途。Use of salidroside for the preparation of a medicament for treating ischemic diseases of the lower limbs.
  20. 红景天苷在制备PHD3的特异性抑制剂中的用途。Use of salidroside for the preparation of specific inhibitors of PHD3.
  21. 红景天苷在制备血管新生因子的表达和分泌的促进剂中的用途。 Use of salidroside in the preparation of a promoter for the expression and secretion of angiogenic factors.
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