CN108969509A - Application of the tyrosol in the drug of preparation treatment diabetic complication diabetes - Google Patents

Abstract

The promotor of expression and the secretion of the application and a kind of angiogenic factor that the invention discloses tyrosols in the drug of preparation treatment diabetic complication diabetes, it is characterised in that: the effective component of the promotor is tyrosol.The promotor promotes the expression and secretion of the angiogenic factor of Skeletal Muscle Cell.The angiogenic factor includes PDGF-BB and VEGF-A.Under the conditions of high sugar, the promotor promotes the expression and secretion of the angiogenic factor of Skeletal Muscle Cell.The proliferation of Skeletal Muscle Cell C2C12 a kind of and the promotor of migration, it is characterised in that: the effective component of the promotor is tyrosol.Under the conditions of high sugar, the promotor promotes the proliferation and migration of Skeletal Muscle Cell C2C12.

Description

Application of the tyrosol in the drug of preparation treatment diabetic complication diabetes

Technical field

The present invention relates to drug fields, and specifically tyrosol is in the drug of preparation treatment diabetic complication diabetes Using.

Background technique

Diabetes are one of the most common of diabetes, most serious complication.Since glycemic control is undesirable, lead to lower limb Peripheral blood vessel lesion so as to cause lower limb blood supply insufficiency and leads to lower limb histocyte anoxic, in addition Gao Tang condition undertissue repairs Multiple, wound-healing abilities are decreased obviously, and it is even dead to occur tissue necrosis, tissue missing (tissue loss) when serious；It is clinical Upper severe patient generally requires amputation.

The ideal treatment method of diabetes (Diabetic Foot) is to improve blood supply state, at present for treatment blood The method of pipe lesion, which has, utilizes bracket, bridging, balloon dilation etc..However, rate is high repeatedly for the vascular lesion of diabetic, and Diseased region is wider, therefore above-mentioned utilization bracket, bridging or balloon dilation are not ideal treatment method to diabetes；And Promote vascular remodeling due to being considered as best therapy approach with advantages such as no invasions.

More importantly under the conditions of this unique pathology environment of hyperglycemia, various internal functions declines, it is various because The behavior of son and bioelectric detecting approach and reaction are also different with normal condition.For example, the tissue/cell of diabetes patient lacks Cell has been lost to the stress ability of low-oxygen environment.High saccharide ring border makes vascular endothelial growth factor (Vascular endothelial Growth factor, VEGF) and vegf receptor (VEGFR), platelet-derived growth factor beta B (Platelet-derived Growth factor-BB, PDGF-BB) etc. angiogenic factors abnormal expression reduce and these angiogenic factors to low The stress reaction of oxygen is also damaged, these angiogenic factors play a significant role in vascular remodeling.Also studies have reported that It points out, the VEGFR2 decline of cell surface, prevents the signal transduction of VEGF-VEGFR2 access under the conditions of high sugar, it is suppressed that The vascular remodelings process such as proliferation for the vascular endothelial cell that VEGF is induced causes diabetic mice obvious to the sensitivity of VEGF Lower than normal mouse, therefore using only VEGF Induction of hindlimb vascular remodeling treatment is not an ideal to treatment diabetes Method.

Summary of the invention

Problem to be solved by the invention

Based on above-mentioned status, as the approach to solve the above problems, there is an urgent need to one kind at present can be in high sugar, hypoxemia item The method and drug of vascular remodeling are effectively facilitated under part, i.e., there is an urgent need to the methods that the treatment for diabetes has good result And drug.

The means solved the problems, such as

Present inventor has carried out research extensively and profoundly, as a result, it has been found that, tyrosol (structural formula is as shown in Equation 1) for The treatment of diabetes has good effect.Diabetes in the present invention refer to lower limb diabetic vascular complications or glycosuria The relevant vascular change at lower extremities of disease.

Still further, it was discovered that tyrosol can promote the expression of Skeletal Muscle Cell medium vessels new life factor Ⅴ EGF-A and PDGF-BB, To promote the formation of the vessel lumen of vasculopathy of lower extremity in diabetes change patient (i.e. patient with diabetic feet), can especially promote It is mature, without the formation for leaking and having functional blood vessel.The present invention is completed as a result,.

The present invention relates to following schemes.

1. a kind of drug for treating diabetes, the active constituent of the drug is tyrosol.

2. according to the drug of the treatment diabetes of scheme 1, the drug is injection.

3. according to the drug of the treatment diabetes of scheme 1 or 2, the drug is skeletal muscle injection agent.

4. the promotor of expression and the secretion of a kind of angiogenic factor, the promotor is tyrosol.

5. the promotor promotes the expression of the angiogenic factor of Skeletal Muscle Cell and divides according to the promotor of scheme 4 It secretes.

6. the angiogenic factor is VEGF-A and PDGF-BB according to the promotor of scheme 4 or 5.

7. the promotor promotes angiogenic factor under the conditions of high sugar according to the promotor of any one of scheme 4-6 Expression.

8. the promotor promotes angiogenic factor under low oxygen conditions according to the promotor of any one of scheme 4-7 Expression.

9. application of the tyrosol in the drug of preparation treatment diabetes.

10. according to the application of scheme 9, wherein the tyrosol is injection.

11. according to the application of scheme 9 or 10, wherein the tyrosol is intramuscular dose.

12. purposes of the tyrosol in the promotor of the expression and secretion that prepare angiogenic factor.

13. purposes according to scheme 12, the promotor of expression and the secretion of the angiogenic factor is treatment sugar Urinate the drug of foot disease.

14. the promotor of expression and the secretion of the angiogenic factor promotes angiogenesis according to the purposes of scheme 13 The expression and secretion of the factor.

15. the angiogenic factor is the one or more of VEGF-A and PDGF-BB according to the purposes of scheme 14.

16. the promotor promotes angiogenic factor under the conditions of high sugar according to the purposes of any one of scheme 12-15 Expression.

17. the promotor promotes angiogenic factor under low oxygen conditions according to the purposes of any one of scheme 12-16 Expression.

18. purposes of tyrosol under the conditions of preparation high sugar in mature vascularization promotor.

19. tyrosol induces the purposes under high sugared hypoxia condition in the promotor of mature vascularization in preparation.

Invention effect

In accordance with the invention it is possible to provide application of the tyrosol in the drug of preparation treatment diabetes.In the application, junket Alcohol can promote the expression and secretion of a variety of angiogenic factors of Skeletal Muscle Cell, such as VEGF-A under high sugar, hypoxia condition With the expression and secretion of PDGF-BB.In turn, the formation that can promote the lower limb vascular lumen of patient with diabetic feet, especially can It is enough to promote formation mature, without leaking and with functional blood vessel, so as to realize the therapeutic effect of diabetes.

In addition, the drug can be high sugared, low in accordance with the invention it is possible to provide a kind of drug for treating diabetes The expression and secretion for promoting the angiogenic factor VEGF-A and PDGF-BB in skeletal muscle under the conditions of oxygen, to promote diabetes The formation of the lower limb vascular lumen of sufficient patient can especially promote formation mature, without leaking and with functional blood vessel, from And it can be realized the therapeutic effect of diabetes.

In addition, according to the present invention, by the new application, realizing the therapeutic effect of excellent diabetes.

Detailed description of the invention

Fig. 1 is the detection of Skeletal Muscle Cell C2C12 PDGF-BB and VEGF-A protein expression level after tyrosol is handled；

Fig. 2 is the secretion result figure (ELISA) of Skeletal Muscle Cell C2C12 PDGF-BB after tyrosol is handled；

Fig. 3 is the secretion result figure (ELISA) of Skeletal Muscle Cell C2C12 VEGF after tyrosol is handled；

Fig. 4 is cell Proliferation influence diagram of the tyrosol to Skeletal Muscle Cell C2C12 under the conditions of high sugar；

Fig. 5 is cell Proliferation quantitative result figure of the tyrosol to Skeletal Muscle Cell C2C12 under the conditions of high sugar；

Fig. 6 is for tyrosol to the result figure of the cell migration of Skeletal Muscle Cell C2C12 under the conditions of high sugar；

Fig. 7 is after tyrosol promotes the photochrome of the effect of diabetes mouse restoration of blood flow to be converted into gray level image Figure；

Fig. 8 is after tyrosol promotes the photochrome of the effect of diabetes mouse restoration of blood flow to be converted into gray level image Figure；

Fig. 9 is the coordinate diagram that tyrosol promotes diabetes mouse restoration of blood flow effect.

Specific embodiment

The inventors discovered that tyrosol can have good therapeutic effect to diabetes mice foot model.In addition, of the invention In diabetes refer to lower limb diabetic vascular complications or the relevant vascular change at lower extremities of diabetes.Sugar in the present invention The degree of urine foot disease does not have any restrictions, can be cercinoma prophase pathologic change, slight, moderate or serious diabetes.

Diabetes in the present invention include type 1 diabetes, diabetes B and prediabetes.Type 1 diabetes and 2 types Fasting blood-glucose >=7.0mmol/L of diabetes, the fasting blood-glucoses of prediabetes (pre-diabetes) be greater than 6.1mmol/L and Less than 7.0mmol/L." the high sugar " recorded in this specification refers to as caused by diabetes and diabetes relevant blood vessel lesion High sugar.

" hypoxemia " recorded in this specification refers to that the tissue as caused by diabetes and diabetes relevant blood vessel lesion is thin Born of the same parents' hypoxemia (hereinafter sometimes referred to " anoxic ").There is no any restriction about the oxygen concentration under hypoxia.Due to the oxygen in tissue Concentration can be different according to position (including the position in the same tissue), and in view of the oxygen concentration in tissue is low In artery oxygen concentration and artery oxygen partial pressure was generally acknowledged that as 100mmHg (organism will be made lethal lower than 40mmHg), because Hypoxemia refers to that oxygen partial pressure is preferably more than 0mmHg and for 100mmHg range below in this present invention, more preferably 10~ The range of 100mmHg, further preferably 20~100mmHg, further preferably 30~100mmHg, most preferably 40~100mmHg.

In addition, those skilled in the art it would be appreciated that, the diabetes mouse model in the embodiment of the present invention be use It completely cuts through (i.e. the lower limb are in serious anaerobic condition) of the building of thigh main artery, and diabetic mouse model used Fasting blood-glucose >=16.7mmol/L, the significantly larger than fasting blood-glucose (i.e. >=7.0mmol/L) of diabetes standard, are serious glycosurias Sick mouse.Thus, it can be known that the diabetes mouse in the embodiment of the present invention suffers from serious diabetes.Journey based on diabetes Degree (i.e. the height of blood glucose) be inversely proportional with abilities such as tissue repair, wound healings, those skilled in the art it would be appreciated that, it is aftermentioned Effect of the invention other than there is good therapeutic effect enough to severe diabetes mellitus, it is extensive to vascular remodeling and lower limb function The stronger cercinoma prophase pathologic change of reactivation power, slight or moderate diabetes can play better therapeutic effect.

In the present invention, the therapeutic agent of diabetes includes tyrosol as active constituent.Tyrosol in the present invention is structure The compound of formula such as formula 1.There is no limit for the purity of tyrosol, and preferably 80% or more, more preferably 90% or more, and then preferably 95% or more, it is further preferably 98% or more, most preferably 99.8% or more.About the influence of purity, applicant is described as follows.Afterwards The tyrosol produced using Shanghai Tongtian Biotechnology Co., Ltd. (purity >=98.0%) is described in the embodiment of the present invention stated, In addition applicant also uses the tyrosol product (purity >=99.5%) of U.S.'s Sigma Aldrich, and use both The tyrosol effect of concentration is identical, it is thus understood that, aftermentioned effect of the present invention is as caused by tyrosol, rather than by impurity Ingredient generates.

In addition, about use tyrosol treat ischemic disease of lower extremity when concentration, can be set as per injection 5- 200mg/kg weight.The dosage can be used for multiple times with single or be divided into.Administration number of times can be single or multiple, can be with continuity Daily administration or intermittent administration.

The therapeutic agent of diabetes in the present invention also may include one or more auxiliary materials.Auxiliary material does not limit, example Such as solvent, isotonic agent, excipient, pH regulator, antioxidant, disintegrating agent, flavoring agent, fragrance, preservative agent are commonly used in the art Auxiliary material.

It can be enumerated as solvent: distilled water for injection, physiological saline, vegetable oil, it is propylene glycol, polyethylene glycol, ethyl alcohol, sweet The alcohols etc. of oil etc.

It can be enumerated as isotonic agent: the isotonic agent commonly used in the art such as sorbierite, sodium chloride, glucose.

It can be enumerated as excipient: lactose, mannitol, glucose, microcrystalline cellulose, starch etc..

It can be enumerated as pH regulator: hydrochloric acid, citric acid, sodium hydroxide, Strong oxdiative potassium, sodium bicarbonate, phosphoric acid hydrogen two Sodium etc..

It can be enumerated as antioxidant: sodium sulfite, sodium hydrogensulfite, ascorbic acid etc..

It can be enumerated as disintegrating agent: potato starch.

It can be enumerated as flavoring agent: the sweeteners such as sucrose, simple syrup, etc..

It can be enumerated as fragrance: peppermint oil, orange oil etc..

It can be enumerated as preservative agent: the preservative agent commonly used in the art such as parabens, sorbic acid and its salt.

The therapeutic agent of diabetes in the present invention can be any dosage form, for example, oral solution, patch, tablet, Capsule, injection etc., preferably injection, most preferably skeletal muscle injection agent.

Promotor is the expression for promoting gene or the medicament of secretion level, may include that transcription, translation, protein is promoted to close At, protein stability and the medicament of secretion.

Angiogenic factor in the present invention is to the factor for promoting the formation of mature blood vessel to play a role, including right The factor (VEGF-A etc.), the factor (PDGF-BB etc.) to work to cell maturation that segment dislocation works etc..In addition, ability Field technique personnel it would be appreciated that, regulatory mechanism in mammal of these factors in mouse and including humans and it Function and effect be it is common, therefore, those skilled in the art it would be appreciated that, based on this specification to tyrosol function and effect And the description of mechanism of action, effect of the invention including humans can also reach treatment diabetes in the mammalian body This specification such as foot, the expression for promoting angiogenic factor under high sugar, hypoxia condition and secretion, the formation for promoting mature blood vessel Described effect.In addition, those skilled in the art it would be appreciated that, " promote vascular remodeling " and " promotion in this specification The formation of mature blood vessel " is the new life for promoting blood vessel and/or the maturation for promoting new vessels.

Below with reference to embodiment, the invention will be further described, but should not be construed the above-mentioned subject area of the present invention only It is limited to following embodiments.Without departing from the idea case in the present invention described above, according to ordinary skill knowledge and used With means, various replacements and change are made, should all include within the scope of the present invention.

Embodiment

1. influence of the tyrosol to Skeletal Muscle Cell angiogenic factor in high sugar system

C2C12, HUVECs and MOVAS cell strain are purchased from American Type Culture Collection (ATCC)。

All cell culture are in Dulbecco ' s modified Eagle medium basic (Gibco, Life Technologies, Grand Island, NY), and 10% fetal calf serum (Biological is added in culture medium Industries,Israel)。

For high sugar experiment, cell with the culture medium culture (high glucose medium) containing 25mM glucose for 24 hours, control be by Cell culture (low sugar culture medium) in 5.5mM dextrose culture-medium.

By Skeletal Muscle Cell C2C12 after tyrosol is handled, the protein expression level of PDGF-BB and VEGF-A albumen is carried out Detection, comprising the following steps:

1) Skeletal Muscle Cell C2C12 is inoculated into the tissue culture plate of 6cm with the quantity of 150,000/well；

2) pass through low sugar (5.5mM), tyrosol (is dissolved in PBS, makes culture medium by high sugar (25mM) and height sugar+tyrosol combination The ultimate density of middle tyrosol is 50 μ g/ml) it handles for 24 hours, it is placed in hypoxemia box (0.1%O₂, Mitsubishi Gas Chemical, Tokyo, Japan) after hypoxemia 12h, collect albumen.Concrete operations are as follows:

1. preparing sample solution

The cell sample handled well is washed with PBS, outwells the egg for after remaining PBS, being added and preparing in tissue culture plate White lysate collects cell sample using cell scraper, and places 30min on ice, and high-speed low temperature centrifugation 20min (12000r, 4 DEG C), draw supernatant, as total protein sample, using BCA working solution (BCA reagent A: BCA reagent B=50:1, Beyotime, China) and after microplate reader (BioTek, USA) measurement protein sample concentration, total protein sample and sample-loading buffer (SDS-PAGE Albumen sample-loading buffer, 5 ×, Beyotime, China) it is mixed according to the ratio of 4:1,5min is boiled at 100 DEG C of metal bath. According to the sample concentration that experiment measures, keeps the applied sample amount of total protein consistent (40 μ g), calculate the loading volume of each sample.

2. the preparation of PAGE gel

A. the glass plate of the drying standby gel of cleaning and wiping, after clamping glass plate using shelf and detect and be not in leakage situation Start glue.

B. according to following 12% separation gel of system configurations lower layer:

Each component and mixing (being eventually adding TEMED and APS) needed for separation gel preparation is added as needed, use liquid relief The mixed liquor of separation gel is quickly added in the gap between glass plate (solution speed in filling is fast) by rifle, to be separated Glue stops filling separation gel when being added to appropriate position, carry out fluid-tight to separation gel using distilled water and flatten separation gel (when fluid-tight It is noted that filling should be slow, it otherwise will appear phenomenon of the separation gel not on a horizontal plane).

C. after separation gel solidifies, distilled water is outwelled, and blotted with filter paper.

D. glue is concentrated in the upper layer for preparing 5%

Each component and mixing needed for concentration glue preparation is added as needed, using liquid-transfering gun quickly by the mixed of separation gel It closes liquid to be added in the gap between glass plate, comb needed for wanting quick insertion corresponding after the completion of filling, and prevents the production of bubble It is raw.

3. loading and electrophoresis

Experimental facilities is assembled, and gel is fixed on the electrode according to operating instruction, is put into electrophoresis tank, takes out 1 × running buffer is added into electrophoresis tank, and is rinsed using syringe to each hole for comb.Use microsyringe Loading standard albumen Marker (PageRuler Prestained Protein Ladder, Thermo Scientific, Cat# 26616) and identical quality is added after protein quantification result calculates before in protein sample, protein sample.

Engaging means power supply, on upper layer, glue compresses protein sample using 60V voltage, when protein sample compression is into a line When, it converts voltage and continues to carry out electrophoresis in separation gel into 120V, wait samples to stop gel electrophoresis close to separation gel bottom, and turn Film.

4. transferring film

A. gel is first taken out, concentration glue part is prescinded, according to the position marker and the size that detect protein band, is retained The separation gel part needed cuts glue and after the completion immerses separation gel in preprepared 1 × Trans Buffer buffer, together When measure glue size, cut pvdf membrane of corresponding size, by film first in methanol solution impregnate to complete to activate, then will Pvdf membrane is transferred in 1 × Trans Buffer buffer solution, balances film in buffer solution.

Testing protein is transferred to total on pvdf membrane from gel and is similar to " sandwich biscuits ", the black transferring film face of clip Filter paper is located at lowest level on pad, is secondly separation gel, pvdf membrane, filter paper is later the white transferring film face of clip, works as whole process By clamp after end of operation, in operation, it is careful not to make between various pieces with the presence of bubble, otherwise gel In albumen can by bubble obstruct so that transferring film is failed, clip will be immersed in 1 × Trans Buffer in entire During migration and protect It holds wet.

Clip is put into slot after the completion of operation, to make clip black to the black side of slot, and white is to red face, otherwise egg It is white to enter in electrophoresis tank rather than on pvdf membrane, entire clip structure is put into electrophoresis tank after the completion of operation, because of transferring film High heat can be generated in the process, is carried out so can place an ice bag on vacant one side of slot and be placed in ice basin, or directly Transferring film under the conditions of 4 DEG C.Power on, selects 200mA electric current, transferring film 120min.

B. after the completion of transferring film, can also be led to by observing albumen marker to determine whether being transferred on pvdf membrane It crosses and Ponceaux dyeing liquor method is added dropwise on film to confirm, if albumen Marker is clear or Ponceaux dyeing liquor dyes Later protein band is clear, then illustrates transferring film success, subsequent step can just be carried out by needing to clean after Ponceaux dyeing.

5. immune response and development

A. it closes: after the completion of transferring film, being marked according to Marker in the front of film, first with TBST rinsing film surface Pvdf membrane taking-up is placed in preparatory prepared 5% protein blocking liquid, in room temperature by the surfactants such as SDS after removing TBST Under the conditions of closing 2 hours is shaken on shaking table at a slow speed.

B. the incubation of primary antibody: being diluted to suitable concentration with TBST buffer for primary antibody, and antibody and film are incubated under the conditions of 4 DEG C It educates overnight.Pvdf membrane is cleaned three times with 1 × TBST after the completion of being incubated for, 10 minutes every time.

C. the incubation of secondary antibody: it will proportionally be diluted to using secondary antibody, be incubated for 90 minutes on shaking table at room temperature.It is incubated for It is cleaned three times with 1 × TBST after the completion, 10 minutes every time.

Antibody	Product number	Maker	Experiment	Dilution
					Goat antirabbit lgG	ZB2301	ZSGB-BIO	Western Blotting	1/10000
Goat anti-mouse lgG	ZB2305	ZSGB-BIO	WesternBlotting	1/10000

6. ECL chemiluminescence chromogenic reaction

A.ECL developing solution (photo-biological Technology Co., Ltd. is preserved in ECL working solution A:ECL working solution B=1:1, Chongqing) matches System: it by reagent A and reagent B according to isometric mixing, is kept in dark place stand-by.

B. pvdf membrane egg front is come into full contact with developing solution and is reacted, put and carry out development imaging in imaging systems.

C. the colour developing situation of protein band, statistics and analysis data are observed.

As shown in Figure 1, (β-actin makees the protein expression result figure of VEGF-A and PDGF-BB as in Skeletal Muscle Cell For internal reference).

For Skeletal Muscle Cell in high sugar system, the expression of PDGF-BB and VEGF-A angiogenic factor albumen is obvious Decline, after tyrosol is added, PDGF-BB and VEGF-A angiogenic factor protein expression is significantly increased；

Therefore, this example demonstrated the expression that tyrosol can remarkably promote Skeletal Muscle Cell C2C12 angiogenic factor. PDGF-BB and VEGF-A are important angiogenic factor, can promote the formation of mature blood vessel, can speculate that tyrosol can promote maturation The formation of blood vessel.

2. tyrosol promotes Skeletal Muscle Cell to secrete PDGF-BB

Using enzyme linked immunosorbent assay (ELISA) kit (mouse PDGF-BB ELISA kit, source leaf biology, Cat#:CK-E94206M), in testing conditions culture medium platelet-derived growth factor-BB (PDGF-BB) amount；

The collection of conditioned medium, comprising the following steps:

1) Skeletal Muscle Cell C2C12 is seeded in the tissue culture plate of 6cm, cell quantity is 150,000/well；

2) the high sugar+tyrosol of C2C12 cell obtained in step 1) is handled for 24 hours, PBS washing, then in hypoxia condition Lower culture is for 24 hours；

3) collection condition culture medium (CM- high sugar+tyrosol), and filtered with 0.22 μm of sterile filter.

4) control uses low sugar (5.5mM) respectively, then high sugar (25mM) processing is cultivated for 24 hours under low oxygen conditions, finally obtained Obtain conditioned medium: CM- low sugar, CM- high sugar.

Wherein: CM is conditioned medium.According to operating instruction, the amount of PDGF-BB in testing conditions culture medium.Such as Fig. 2 institute Show, as the secretion of tyrosol promotion Skeletal Muscle Cell C2C12 angiogenic factor PDGF-BB.**p<0.01.

Skeletal Muscle Cell has the function of paracrine, is secretory important in vivo, in high sugar system, bone The secretion capacity of myocyte's angiogenic factor declines, and tyrosol can effectively improve point of the high sugar system medium vessels new life factor It secretes, promotes angiogenesis.

3. tyrosol promotes Skeletal Muscle Cell secretion of VEGF

Use enzyme linked immunosorbent assay kit (the mouse VEGF ELISA reagent of blood vessel endothelial cell growth factor VEGF Box, NeoBioscience, Cat#:EMC103) low sugar in detection embodiment 2, high sugar, high sugar combines three conditions with tyrosol Cell culture medium in VEGF secretory volume；

The results showed that tyrosol remarkably promotes the secretion of Skeletal Muscle Cell C2C12 angiogenic factor VEGF.

As shown in figure 3, the secretion of as tyrosol promotion Skeletal Muscle Cell C2C12 angiogenic factor VEGF.**p<0.01.

4. the influence that tyrosol is proliferated Skeletal Muscle Cell

Carry out the cell proliferation experiment of Skeletal Muscle Cell C2C12, comprising the following steps:

1) Skeletal Muscle Cell C2C12 is inoculated into six orifice plates, cell quantity is 100,000/well；

2) C2C12 cell low sugar (5.5mM), high sugared (25mM) and sugared (the 25mM)+tyrosol (50 μ g/ml) of height are handled 24h；

3) by above-mentioned three kinds of cells again with the quantity kind of 20,000/well in 48 orifice plates, hypoxemia box culture is put into after pasting wall 6h。

According to the concrete operations of EdU kit, the proliferative conditions of C2C12 cell are detected.

As shown in figure 4, tyrosol processing can remarkably promote the proliferation of Skeletal Muscle Cell C2C12 under the conditions of high sugar.**p< 0.01；To Fig. 4 in embodiment 4, analysis of statistical results is carried out, statistical result shows that high sugared condition significantly reduces Skeletal Muscle Cell The cell Proliferation of C2C12, and tyrosol promotes the proliferation of cell；As shown in figure 5, as tyrosol is thin to skeletal muscle under the conditions of high sugar The cell Proliferation quantitative result of born of the same parents C2C12.

5. the influence that tyrosol migrates Skeletal Muscle Cell

Carry out the Cell migration assay of Skeletal Muscle Cell C2C12, comprising the following steps:

1) Skeletal Muscle Cell C2C12 is inoculated into six orifice plates, cell quantity be 100,000/well, with low sugar (5.5mM), High sugar (25mM) and sugared (the 25mM)+tyrosol (50 μ g/ml) of height are handled for 24 hours；

2) above-mentioned three kinds of cells are inoculated into the cell transwell upper layer with identical quantity to (transwell is small again Room infiltrates in the medium in advance), the culture of hypoxemia box is for 24 hours；

3) cell for washing off the cell transwell upper layer is moved to crystal violet (Beyotime, China) dye The cell of the cell transwell lower layer, observation of taking pictures, and counting statistics moves to the cell number of lower layer from cell upper layer respectively.

The Skeletal Muscle Cell C2C12 migration of result of study discovery tyrosol processing significantly increases.As shown in fig. 6, as tyrosol The migration of Skeletal Muscle Cell C2C12 under the conditions of promoting height sugared.

6. the foundation of diabetic mouse model

C57BL/6J mouse (6 weeks, male) purchase (being purchased from Military Medical Univ No.3, P.L.A) is returned after a week Mouse blood sugar is measured, blood glucose is measured after being fed 4 weeks with following high lipid foods, the feelings of prediabetes (pre-diabetes) occurs After condition, streptozotocin is continuously injected five days by muscle, and the amount of penetrating is 50mg/kg, is continued high lipid food nursing and is measured after a week Mouse blood sugar, blood glucose are used as experiment in next step higher than the selected of 16.7mmol/L.Herein, it should be noted that general blood glucose is 7mmol/L's diabetes, but mouse that the selected blood glucose of model of the invention is 16.7mmol/L or more or more has been thought suffering from.

The formula of high lipid food: 15% lard

10% yolk

10% white sugar

65% normal diet

Wherein, normal diet, yolk, lard, white sugar are provided by great Ping hospital of Third Military Medical University, and by medical university of third army The level ground Xue great hospital produces high lipid food.

7. tyrosol is to the therapeutic effect of diabetes

Using above-mentioned diabetic mouse model, resection operation, and benefit are carried out to left side thigh main artery under anesthesia The case where detecting blood flow with Laser Doppler Perfusion Imaging System.It should be noted that about this Shen Please in specification " left side ", " right side " statement, what is performed the operation is left side thigh, and mouse is in prostrate state at this time, after Mouse is in supine position in the photo for the rheography stated, so the thigh performed the operation in rheography photo right side in figure) (ginseng According to document 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): the method in 313-325).

Tyrosol (is bought, sample purity is higher than 98%) slow with phosphate from Shanghai Tauto Biotechnology Co., Ltd. Solution (PBS) dissolution is rushed, the storing liquid of 20mg/ml is made into, after 0.22 μm of membrane filtration, is saved backup in -20 DEG C.

Tyrosol storing liquid is diluted to 10mg/ml before injection, the dosage of mouse muscle injection is 50mg/kg, the postoperative 1st It starts injection every three days once, and per injection is continuous to infuse respectively toward point three sites in the gastrocnemius of left side by injection point 3 times It penetrates.

Use physiological saline as control, similarly filters, saves and inject.

Using Laser Doppler Perfusion Imaging System (MOOR INSTRUMENTS Ltd, MOORLDLS2-IR) detect operation consent, just operation after, postoperative 21st day blood circumstance.

Referring to Fig. 7, grey parts react blood flow state.It is preoperative, saline control group mouse and tyrosol processing group mouse With same blood flow state (it should be noted that Laser Doppler Perfusion Imaging System originally at As being coloured picture, after being converted into gray scale picture, little bit different is appeared to have with originally imaging.In former coloured picture, red is indicated Blood flow is abundant, and blue indicates no blood flow.Place (the i.e. former coloured picture of restoration of blood flow can not be distinguished more visiblely under gray level image Middle red position) and without blood flow (blue position in i.e. former coloured picture) place).For this problem, present inventor's foundation Coloured picture result has carried out image procossing to grayscale image, to obtain Fig. 8.In fig. 8, net-point shape indicates the ground of restoration of blood flow Side, i.e. RED sector in original coloured picture.Just after operation, lower limb are all shown black on the left of control group mice and tyrosol processing group mouse There there is no lower limb on the left of color (being blue portion in former coloured picture, do not have net-point shape pattern in figure after treatment), i.e. the two Blood flow, it is known that produce to successful surgery diabetes mouse model.At postoperative 21st day, lower limb on the left of tyrosol processing group mouse Reach that almost same blood flow state, the i.e. blood flow of left side lower limb have obtained more adequately extensive with untreated left side lower limb It is multiple；In contrast, in figure on the left of saline control group mouse lower extremity blood flow restore with it is postoperative without obvious gap, thus it is speculated that be due to Lower extremity blood flow is relatively difficult to recover in diabetic mice.In addition from figure it can also be seen that with treatment time extension, at tyrosol The grey and black portions (RED sector in i.e. former coloured picture, the site part in treated figure) of lower limb are more next on the left of reason group More reach distal end (i.e. close to toe) position of lower limb, it is known that blood flow is gradually restored to apart from the farther away position of arterial resection.

In order to further clarify the experiment effect of tyrosol processing group, present inventor is to Laser Doppler The result of Perfusion Imaging System is quantified.Circular are as follows: by ischemic limb (left side lower limb) Quantitative values divided by same mouse non-ischemic limb (right side lower limb) quantitative values (i.e. the pixel of blood flow area), then will The average of relatives at each time point and divided by the average value of the preoperative ratio of each group.Referring to Fig. 9 based on statistical result It is found that the ventilation perfusion ratio of control group mice and tyrosol processing group mouse is reduced to the numerical value lower than 0.35 just after operation.Postoperative At 21 days, the ventilation perfusion ratio of tyrosol processing group mouse has reached 0.9；In contrast, though the ventilation perfusion ratio of control group mice has one Fixed rise, but recovery rate is smaller.In addition, as shown in table 1, before tyrosol administration, the diabetes of all experimental groups are small It has not improved significantly within the blood glucose of mouse 21 days after surgery, therefore, the effect of restoration of blood flow is not due to the improvement of blood glucose environment, But the effect of due to tyrosol itself.

Blood sugar detecting method are as follows: arrive drop of bloodThe insertion of vigor type blood sugar test paper Vigor type blood glucose Instrument (Accu-Active, Model GU, Roche).It utilizes Blood collecting pen (Accu- Lancing Device, Roche) andDisposable lancet (Roche), from The blood sampling of diabetes mousetail, and drop of blood addition is existedThe centre of vigor type blood sugar test paper orange areas.

Table 1 (unit: mmol/L)

	It is preoperative	It is postoperative	Postoperative 21 days
				Control	25.53±1.77	22.80±1.50	24.93±1.47
Tyrosol	26.20±0.7	23.67±0.43	25.07±1.03
				P value	0.610	0.337	0.904

Note: being average value ± standard deviation in table, and p value is obtained by T checking computation.

It can thus be appreciated that tyrosol obtains excellent restoration of blood flow effect to the processing experiment of diabetes mouse.It is given birth to injection The control of reason salt water is compared, and the blood flow state for having injected the diabetes mouse of tyrosol significantly restores, it is known that tyrosol is for glycosuria The treatment of foot disease has good effect, it can thus be appreciated that producing maturation, without leakage and with functional new vessels.

Claims (5)

1. application of the tyrosol in the drug of preparation treatment diabetes.

2. application according to claim 1, wherein the tyrosol is injection.

3. application according to claim 1, wherein the tyrosol is skeletal muscle injection agent.

4. application according to claim 1 or 2, wherein the tyrosol promotes angiogenic factor under high sugar, low-oxygen environment Expression and secretion.

5. application according to claim 3, wherein the angiogenic factor is VEGF-A and PDGF-BB.