WO2023241715A1 - 视黄酸受体激活剂及其组合在哺乳动物再生修复中的应用 - Google Patents

视黄酸受体激活剂及其组合在哺乳动物再生修复中的应用 Download PDF

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WO2023241715A1
WO2023241715A1 PCT/CN2023/100866 CN2023100866W WO2023241715A1 WO 2023241715 A1 WO2023241715 A1 WO 2023241715A1 CN 2023100866 W CN2023100866 W CN 2023100866W WO 2023241715 A1 WO2023241715 A1 WO 2023241715A1
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retinoic acid
regeneration
acid receptor
composition
repair
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PCT/CN2023/100866
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English (en)
French (fr)
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李伟
周琪
何正泉
卢宗宝
袁雪薇
王馨
王帅
王柳
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中国科学院动物研究所
北京干细胞与再生医学研究院
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Publication of WO2023241715A1 publication Critical patent/WO2023241715A1/zh

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    • A—HUMAN NECESSITIES
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    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
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    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/202—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having three or more double bonds, e.g. linolenic
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/42—Oxazoles
    • A61K31/423—Oxazoles condensed with carbocyclic rings
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    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/45—Non condensed piperidines, e.g. piperocaine having oxo groups directly attached to the heterocyclic ring, e.g. cycloheximide
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4741—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having oxygen as a ring hetero atom, e.g. tubocuraran derivatives, noscapine, bicuculline
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    • A61K38/00—Medicinal preparations containing peptides
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    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
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    • A61P17/00—Drugs for dermatological disorders
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    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
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    • A61P25/00—Drugs for disorders of the nervous system
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D211/80—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members
    • C07D211/84—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen directly attached to ring carbon atoms
    • C07D211/86—Oxygen atoms
    • C07D211/88—Oxygen atoms attached in positions 2 and 6, e.g. glutarimide

Definitions

  • the present invention relates to the field of biotechnology, and in particular to a method for promoting mammalian organ regeneration and repair and related applications.
  • Regeneration refers to the repair process in which the whole body or an organ is partially lost due to trauma, and a structure that is the same in form and function as the lost part is grown based on the remaining part. Failure of regeneration can lead to loss of tissue or organ functionality, ultimately leading to various diseases and even death. Different species in nature have different regeneration abilities, which can be divided into: 1. Individual level regeneration. For example, lower plants can regenerate a plant from a single cell, and some higher plants can use roots, stems, leaves and other tissues to regenerate new ones. Plants and lower organisms such as planarians can use any part of the body to regenerate a complete individual; 2.
  • Regeneration from excision, tailed amphibians such as salamanders, geckos and some fish can regenerate tails, limbs and fins.
  • Regeneration at the tissue level such as liver cell proliferation and regeneration after liver resection; 4.
  • Regeneration at the cellular level such as regrowth of neuron axons after rupture.
  • mammals, including humans have greatly lost their ability to regenerate and have almost no ability to regenerate.
  • Regeneration is limited to the fetal period and specific periods such as the liver and skin, and specific tissues or organs. .
  • the general trend is: as the evolutionary level increases, the regeneration ability becomes weaker or even lost.
  • Fibrosis is a non-regenerative way of repairing damage. Promoting the regeneration of damaged tissue (regenerative therapy) can not only control fibrosis, but also restore the original function of the tissue. Therefore, regenerative therapy is the most effective way to prevent and treat fibrosis-related diseases. ideal means.
  • this application provides a small molecule compound with the ability to promote regeneration and repair, and has achieved technological and unexpected results. technical effects.
  • the technical solution of this application is as follows:
  • the present application provides a compound that can promote the regeneration and repair ability of tissues and organs.
  • the compound is a retinoic acid receptor activator.
  • the present application provides the use of the retinoic acid receptor activator or a composition comprising the retinoic acid receptor activator in promoting the regeneration and repair ability of mammalian tissues or complex structures or organs.
  • the present application also provides the use of the retinoic acid receptor activator or the composition containing the retinoic acid receptor activator in the preparation of drugs or reagents for promoting the regeneration and repair ability of mammalian tissues or complex structures or organs. application.
  • the present application also provides the use of the retinoic acid receptor activator or the composition comprising the retinoic acid receptor activator in the preparation of medicaments for the treatment of diseases related to the regeneration and repair of mammalian tissues or complex structures or organs. or application in reagents.
  • the retinoic acid receptor activator provided in this application can be one or more of all-trans retinoic acid and selective retinoic acid receptor gamma agonist.
  • the selective retinoic acid receptor gamma agonist is CD437 and/or BMS961.
  • the regenerative repair described in this application is to promote the regeneration of tissues or complex structures or organs after tissue or organ removal or damage.
  • the tissue described in this application is skin, fat, muscle, bone, hair follicle, blood vessel or nerve.
  • the complex structure described in this application is at least two or more body structures including skin, hair follicles, glands, cartilage, muscles, fat, blood vessels, nerves or limbs.
  • the organ described in this application is lung, liver, heart, pancreatic islet or kidney.
  • the complex structure is an ear, a limb, a finger, an eye or a nose.
  • the regenerative repair described in this application is regeneration after the ear has been removed.
  • the regenerative repair described in this application is to promote regeneration and repair after skin damage, hair regeneration after hair loss, regeneration and repair of cartilage and muscle damage, regeneration of lungs, liver, skin, heart, kidney, muscle fibrosis, and blood vessels, nerves and Regeneration after limb injury.
  • the diseases related to the regeneration and repair of tissues or complex structures or organs described in this application are skin burns, skin trauma, skin burns, hair loss, cartilage and muscle damage, liver fibrosis, pulmonary fibrosis or limb damage.
  • the retinoic acid receptor activator provided in this application is used to promote the regeneration and repair ability of tissues and organs by activating retinoic acid. Preferably, this is achieved through activation of retinoic acid receptor gamma.
  • composition containing the retinoic acid receptor activator includes a protein synthesis inhibitor, all-trans retinoic acid and a BMP activator.
  • the protein synthesis inhibitor in the composition is Narciclasine
  • the BMP activator is BMP signaling agonist sb4.
  • the retinoic acid receptor activator or the composition containing the retinoic acid receptor activator in the method is the retinoic acid receptor activator or the retinoic acid receptor activator provided in this application. agent composition.
  • the retinoic acid receptor activator or the retinoic acid receptor activating agent may be administered to the subject in need by intraperitoneal injection, intravenous injection, intragastric administration, oral administration, or skin application. agent composition.
  • Figure 1B is a schematic diagram of the healing of mouse ear holes with a diameter of 2 mm after treatment with Vehicle and different translation inhibitors Ani and CHX drugs.
  • Figure 1C is a schematic diagram of the closure of a 2 mm diameter mouse ear hole after treatment with Vehicle and different doses of CHX drugs.
  • Figure 1D is a schematic diagram of the closure of mouse ear holes with 2 mm diameter ear punch trauma after treatment with CHX drugs for 30 days.
  • the scale bar is 1 mm.
  • Figure 1F shows the results of HE staining of mouse auricle tissue after treatment with CHX drugs.
  • the scale bar is 1 mm.
  • Figure 1H is a schematic diagram of the HE staining results of mouse auricle tissue after 180 days of ear hole closure after CHX drug treatment.
  • the scale bar is 1 mm.
  • Figure 1I is a schematic diagram of the healing effect of mouse ear holes after 3 weeks of treatment with different administration methods (21 days after injury).
  • Figure 1J shows the healing effect of 2mm ear piercing in Nsun2 knockout mice (21 days after injury).
  • Figure 8B shows the phenomenon of acral induced regeneration after 20, 40, and 120 days of drug treatment. Scale bar is 2mm.
  • Figure 8G shows the Masson staining results of mouse acral regenerated tissue after drug treatment for 21, 50, and 120 days. Scale bar is 2mm, n ⁇ 3, t test.
  • Figure 9B is a schematic diagram of the immunohistochemistry results after NRB treatment.
  • the present application mainly relates to a compound or a composition containing the compound that can promote the regeneration and repair ability of mammalian tissues or complex structures or organs.
  • the compound is a retinoic acid receptor activator.
  • the present application also relates to the use of the compounds in the preparation of medicaments or agents for promoting the regeneration and repair capacity of mammalian tissues or complex structures or organs.
  • the present application also relates to the use of said compounds in the preparation of medicaments or agents for the treatment of diseases associated with the regeneration and repair of mammalian tissues or complex structures or organs.
  • the ability to promote tissue and organ regeneration and repair is achieved through activation of retinoic acid or retinoic acid receptor ⁇ .
  • the retinoic acid receptor activators described in this application include compounds or combinations of compounds that can produce equivalent activating effects on each pathway of retinoic acid receptor RARs, as well as compounds that can activate the pathways of retinoic acid receptor RARs.
  • the RAR ⁇ pathway produces selectively activating compounds or combinations of several compounds.
  • the retinoic acid receptor activator is a selective retinoic acid receptor gamma agonist.
  • the selective retinoic acid receptor gamma agonist is selected from CD437 and/or or BMS961
  • the regenerative repair is to promote the regeneration of tissues or complex structures or organs after removal or damage of mammalian tissues or organs.
  • the tissue is epidermis, dermis, muscle, bone, fat, hair follicles, blood vessels, or nerves.
  • the complex structure is at least two or more body structures including skin, hair follicles, glands, cartilage, muscles, fat, blood vessels, nerves, or limbs.
  • the organ is lung, skin, heart, liver, kidney, stomach, intestine, etc.
  • the complex structures described in this application are body structural parts composed of different tissues or body functional parts capable of completing specific physiological functions or functional activities, such as ears, various organs, limbs, eyes, nose, etc.
  • the complex structure is an ear hole.
  • the regenerative repair is to promote regeneration after the ear is partially removed.
  • the regeneration and repair is to promote the regeneration and repair of burnt skin.
  • the diseases related to the regeneration and repair of tissues and organs include but are not limited to skin burns, fibrosis of organs, muscle/cartilage damage or neurological diseases, etc., preferably skin burns/scalds/wounds, Hair loss, cartilage and muscle damage, pulmonary fibrosis, liver fibrosis, renal fibrosis, myocardial fibrosis, limb trauma or various neurological diseases, etc.
  • composition containing the retinoic acid receptor activator may also include other compounds with regenerative repair capabilities.
  • the Amaryllidaceae plant extract or Amaryllidaceae alkaloid includes Narciclasine or Pancratisatin.
  • the composition includes a protein synthesis inhibitor, a retinoic acid receptor activator, and a BMP activator.
  • the composition includes cycloheximide (CHX), all-trans retinoic acid and BMP signaling agonist sb4. Further, based on the cycloheximide in the composition being 1 part by weight, the all-trans retinoic acid is 0.25-8 parts by weight, and the BMP signaling agonist sb4 is 0.25-4 parts by weight.
  • CHX cycloheximide
  • all-trans retinoic acid is 0.25-8 parts by weight
  • BMP signaling agonist sb4 is 0.25-4 parts by weight.
  • the medicament or agent can be prepared in the following form: the protein synthesis inhibitor or a composition containing the same is mixed with a pharmaceutically acceptable carrier, for example, to obtain an oral preparation, such as a tablet (including sugar-coated tablet, Film-coated tablets, sublingual tablets, orally disintegrating tablets), capsules (including soft capsules, microcapsules), granules, powders, lozenges, syrups, emulsions, suspensions, films (e.g., orally disintegrating films), etc., parenteral preparations such as injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections, infusions), external preparations (e.g., skin preparations, ointments), suppositories (such as rectal suppositories, vaginal suppositories), pills, nasal drops, respiratory preparations (inhalants), eye drops, etc.
  • these formulations can be used as controlled release formulations (eg.,
  • the HE staining picture of mouse auricle tissue in Figure 1F shows that after ear piercing trauma mice were treated with DMSO/CHX (20 mg/kg), focal necrosis was visible in both tissues on the first day after trauma (D1 group). There is diffuse infiltration of inflammatory cells in the tissue, as shown by arrow 1; diffuse infiltration of inflammatory cells can be seen in the dermis of the tissue, as shown by arrow 2; hemosiderin deposition can be seen in some cells in the CHX group, as shown by arrow 3 shown.
  • the HE staining picture of mouse auricle tissue in Figure 1H shows that after 180 days of closure of mouse ear holes treated with CHX (20 mg/kg), the wound part contains hair follicles, glands, cartilage, muscles, blood vessels and other tissues and tissue derivatives. regeneration.
  • Nsun2 knockout has been reported to inhibit translation by regulating tRNA stability, so Nsun2 knockout mice were used as a genetic model to verify the effect of translation inhibition on regeneration.
  • the specific method is: using wild-type mice (WT) and Nsun2 knockout mice (KO) to establish ear piercing trauma mouse models, making 2 mm diameter ear holes (the method is the same as the above embodiment), and observing and measuring respectively after 3 weeks. Ear piercing healing status.
  • the results showed that Nsun2 knockout mice had significantly smaller ear piercing areas than wild-type mice. This also suggests that translation inhibition has a role in regeneration (Fig. 1J).
  • Example 3 Cycloheximide CHX promotes ear piercing regeneration independent of the inhibition of ferroptosis and autophagy.
  • the concentration used is 10-20mg/kg, the administration method is the same as CHX, n ⁇ 8, **p ⁇ 0.01, ***p ⁇ 0.001, ns: no significant difference, t test.
  • Inhibitors of ferroptosis and autophagy respectively cannot promote ear piercing healing like CHX, which shows that CHX's promotion of ear piercing regeneration does not depend on its inhibitory activity on ferroptosis or autophagy.
  • Example 5 Activating RAR ⁇ promotes ear piercing regeneration.
  • Tazarotene (MCE, HY-15388, 20mg/kg) and Bexarotene (MCE, HY-14171, 20mg/kg), retinoic acid receptor agonists; VA (VitaminA, Selleck, S5592, 20mg/kg), vitamin A, Metabolized in vivo to produce RA; Talarozole (MCE, HY-14531, 10mg/kg), inhibits CYP26A1, CYP26B1, and increases endogenous all-trans retinoic acid; AGN 195183 (MCE, HY-16684, 10mg/kg), selective RAR ⁇ Agonist; CD437 (MCE, HY-100532, 10mg/kg) and BMS 961 (Glpbio, GC17382, 10mg/kg), selective RAR ⁇ agonist; GW0742 (MCE, HY-13928, 20mg/mg), activates PPAR ⁇ / ⁇ ; ML385 (MCE,
  • Example 6 Combination of CRB (C: protein synthesis inhibitor CHX; R: RARs activator all-trans retinoic acid (All-trans retinoic acid); B: BMP activator BMP (signaling agonist sb4)) promotes 4mm in mice Ear holes are closed and cuts are regenerated.
  • CRB protein synthesis inhibitor CHX
  • R RARs activator all-trans retinoic acid (All-trans retinoic acid)
  • B BMP activator BMP (signaling agonist sb4)) promotes 4mm in mice Ear holes are closed and cuts are regenerated.
  • mice were anesthetized with 5% chloral hydrate, injected intraperitoneally with 10 mL/kg, and anesthetized according to body weight. The anesthetized mice were bound, and 75% ethanol was used to sterilize the mouse ears and instruments. Taking the center of the mouse's auricle, use an ear hole punch with a diameter of 4 mm to punch holes in the left and right auricles of the mouse respectively.
  • DMSO/CRB DMSO/CRB
  • CB dosage CHX 20mg/kg, full ATRA 20mg/kg, BMP signaling agonist sb4 10-20mg/kg
  • the mice were anesthetized every 7 days, and vernier calipers were used to measure the proximal–distal (DPD) and anterior–posterior (DAP) axes of the mouse’s ear piercings, and the time of the mice’s pierced ears was calculated.
  • Figure 6A shows the closure of mouse ear holes after DMSO/CRB treatment respectively. The results showed that CRB promoted the closure of ear holes with a diameter of 4 mm. After 30 days of administration, the mouse ear holes were completely closed.
  • Figure 6B shows the closure of a 4mm ear hole in mice after 30 days of drug treatment. It can be seen that in mice treated with the drug, the ear hole trauma has been closed.
  • Figure 6C shows the HE staining results and epidermal thickness statistics of mouse auricle tissue after 7 days of drug treatment. Scale bar is 200um. After drug treatment, the auricles of mice had bud base formed and the thickness of the epidermis was significantly reduced. n ⁇ 3, t test.
  • Figure 6D shows the immunofluorescence staining results of ⁇ -SMA in mouse auricle tissue after 7 days of drug treatment. Scale bar is 100um. Seven days after trauma, a large amount of ⁇ -SMA expression appeared in both the control group and the drug-treated group. Compared with the control group, the ⁇ -SMA expression in the drug-treated group was arranged in a linear and orderly manner, while in the control group, it was disorderly accumulated, and the expression pattern was similar to that of the control group. Scars form in a similar way.
  • Figure 6E shows the regeneration of various tissues after drug (CRB) induction for more than 90 days, showing the regeneration of epidermis, dermis, glands, hair follicles, muscles, cartilage, fat, and muscles.
  • CRB drug
  • FIG. 6F Immunofluorescence staining of the vascular marker CD31 further identifies vascular regeneration, and triangles indicate regenerated blood vessels.
  • Example 7 RA reduces scar formation and promotes skin regeneration in mice after burns.
  • mice Use 7-week-old mice, (1) anesthetize the mice with intraperitoneal injection of 0.5% sodium pentobarbital (100 ⁇ l/10g b.w.); (2) shave the back, and disinfect the skin with 75% alcohol. (3) Scald in boiling water for 10-15 seconds. The burnt area is a circle with a diameter of 1.5cm. (4) After burns, the patients were evenly divided into two groups, and the Vehicle group (DMSO) and the RA group (20 mg/kg, dissolved in DMSO) were administered. (5) Detect wound repair status. The experimental results are shown in Figure 7.
  • DMSO Vehicle group
  • RA group 20 mg/kg, dissolved in DMSO
  • mice were anesthetized with 5% chloral hydrate, injected intraperitoneally with 10 mL/kg, and anesthetized according to body weight. The anesthetized mice were bound, and 75% ethanol was used to sterilize the mouse's left upper limb and surgical instruments. In order to accurately define the occurrence of regeneration, the distance from the elbow joint to the radius and ulna is measured, 10 mm is retained, and the rest to the palm, fingers and other segments are removed and modeled (the ulna and radius are two bones of the forearm.
  • FIG. 8A is a schematic diagram of extremity modeling.
  • Figure 8B shows the phenomenon of acral induced regeneration after 20, 40, and 120 days of drug treatment. It can be seen that outgrowth occurs at the amputation site of mice treated with the drug.
  • FIG. 8C shows the statistical results of the extremity outgrowth length. It can be seen that the CR administration group induces extremity regeneration very well, and there is a very significant difference in length from the control group. n ⁇ 3, ***p ⁇ 0.001, t test.
  • Figure 8D shows that after 20 and 40 days of drug CR treatment, computerized tomography (CT) was used to obtain high-definition images of the bone tissue reconstruction process. The CR treatment group showed a good acral bone tissue induction growth process.
  • CT computerized tomography
  • HE section staining found that the combination can promote the regeneration of multiple tissue types at the amputated extremity, such as skin, hair follicles, capillaries, new bone and other tissues
  • Figure 8F “Ep” indicates epidermal regeneration; “CT” indicates connective tissue regeneration; “CV” indicates capillary regeneration; “HF” indicates hair follicle regeneration; “OT” indicates bone tissue regeneration.
  • Figure 8G shows the Masson staining results of the regenerated tissue of the mouse acral tissue after drug treatment for 21, 50, and 120 days. In the control group, the blunted shape of the bone tissue can be observed, which is consistent with the CT results. The bone tissue in the drug treatment group showed a growth trend. What is important is that the experimental group stained and observed muscle tissue regeneration at 120 days, while the control group showed disordered accumulation, and the expression pattern was similar to scar formation. Scale bar is 2mm, n ⁇ 3, t test.
  • Example 9 Combination NRB (N: Narciclasine; R: RARs activator all-trans retinoic acid (All-trans retinoic acid); B: BMP activator BMP (signaling agonist sb4)) promotes 4mm ear diameter in mice The cut hole is regenerated.
  • NRB Narciclasine
  • R RARs activator all-trans retinoic acid (All-trans retinoic acid)
  • B BMP activator BMP (signaling agonist sb4)) promotes 4mm ear diameter in mice The cut hole is regenerated.
  • mice The mouse model construction method is the same as Example 2.
  • DMSO/NRB N-(n-(n-(n-(n-(n-(n-(n-(n-(N-(N-(DPD)) once every 2 days, and the mice were anesthetized every 7 days.
  • DPD proximal–distal
  • DAP anterior–posterior
  • the experimental results are shown in Figures 9A to 9C.
  • Figure 9A shows the healing effect of a 4mm ear piercing in mice after 30 days of NRB treatment, and shows that it has a healing-promoting effect. After 30 days of treatment, the wound in the mouse ear piercing was completely closed, and it was identified as a regeneration event.
  • Figures 9B and 9C show that the HE section staining data well indicates the regenerative structures of cartilage (long black arrow), hair follicles (asterisks), glands/sebaceous glands (triangular arrows), muscles (dotted line selection area) and other structures. .

Abstract

视黄酸受体激活剂或包含视黄酸受体激活剂的组合物在制备用于促进哺乳动物组织或复杂结构或器官再生修复能力的药物或试剂中的应用，视黄酸受体激活剂包括全反式视黄酸、选择性视黄酸受体γ激动剂的一种或两种以上，再生修复为促进组织或器官切除或损伤后的组织或复杂结构或器官的再生。

Description

视黄酸受体激活剂及其组合在哺乳动物再生修复中的应用 技术领域
本发明涉及生物技术领域，具体涉及一种促进哺乳动物器官再生修复的方法及其相关应用。
 背景技术
再生是指生物体的整体或器官发生创伤而部分丢失,在剩余部分的基础上又生长出与丢失部分在形态与功能上相同的结构的修复过程。再生失败会导致组织或器官功能性丧失，最终导致各类疾病甚至死亡。自然界不同物种拥有不同的再生能力，再生能力可以分为：一、个体水平再生，如低等植物能从单个细胞再生出一个植株，一些高等植物可利用根、茎、叶等组织再生出新的植株，低等生物如涡虫等可以利用身体任何部分再生出完整个体；二、割除再生，有尾两栖类如蝾蚺、壁虎及某些鱼类等可以进行断尾再生、肢体再生及鱼鳍再生；三、组织水平再生，如肝切除后可以通过肝细胞增殖再生、皮肤组织的再生；四、细胞水平再生，如神经元轴突断裂重新生长等。然而，相比于植物和低等动物，哺乳动物包括人在内，极大地丧失了再生能力，几乎没有割除再生能力，再生仅局限发生于胎儿时期以及肝脏、皮肤等特定时期、特定组织或器官。总体趋势为：随着进化等级越高，再生能力越弱，甚至丧失。
包括人在内的哺乳动物面临各种损伤的时候，会发生疤痕形成的修复方式，这种方式会直接引起组织器官丧能，如截肢运动丧失等；或纤维化相关疾病，如心血管疾病、退行性神经***疾病、肺部疾病、肝部疾病、糖尿病、皮肤病等。在全世界范围内，组织、器官的损伤导致的疤痕形成是许多疾病致残、致死的主要原因。因此提高哺乳动物原位再生修复能力是理解生命本质及治疗一系列相关疾病的重要手段。
对哺乳动物而言，重度和慢性损伤通常通过形成疤痕而不是组织再生来修复，其特点是用大量***增生和细胞外基质沉积的纤维化组织取代功能性组织。研究表明，纤维化是许多慢性疾病致残、致死的主要原因。许多COVID-19患者在出院后都有炎症后肺纤维化，这极大地影响病人的预后生活质量。目前在临床上，尽管少数药物或细胞治疗手段可以用来缓解特定器官、特定类型的纤维化进程，但无法恢复损伤的组织，并且极度缺乏广泛而有效的抗纤维化手段，因此亟待开发广谱的抗纤维化并促进再生的候选靶点及药物。纤维化是损伤的一种非再生的修复方式，促进损伤组织的再生(再生疗法)不仅可以控制纤维化，还能恢复组织的原有的功能，因此再生疗法是预防和治疗纤维化相关疾病最理想的手段。
肺纤维化是由成纤维细胞增殖及大量细胞外基质堆积并伴炎症损伤、组织结构破坏为特征病理性改变，即是正常的肺泡组织被损坏后经过异常修复导致结构异常(疤痕形成)。肺纤维化会严重影响人体呼吸功能，表现为各种呼吸困难并随着病情、肺部损伤的加重及患者呼吸功能不断恶化。据报道特发性肺纤维化在世界范围内的发病率和死亡率逐年增加，诊断后的平均生存期不到3年，高于大多数肿瘤，因此又被称为一种“类肿瘤疾病”。因此促进损伤组织的再生是治疗和预防肺纤维化相关疾病最根本的手段，具有重要的应用价值。
发明内容
为了实现哺乳动物组织、器官的再生修复，为与之相关疾病的预防和诊疗开发更多技术途径，本申请提供了一种具有促进再生修复能力的小分子化合物，取得了开创性的、预料不到的技术效果。本申请的技术方案如下：
本申请提供了一种可以促进组织器官再生修复能力的化合物，所述化合物为视黄酸受体激活剂。
本申请提供了所述视黄酸受体激活剂或包含所述视黄酸受体激活剂的组合物在促进哺乳动物组织或复杂结构或器官再生修复能力中的应用。
本申请还提供了所述视黄酸受体激活剂或包含所述视黄酸受体激活剂的组合物在制备用于促进哺乳动物组织或复杂结构或器官再生修复能力的药物或试剂中的应用。
本申请还提供了所述视黄酸受体激活剂或包含所述视黄酸受体激活剂的组合物在制备用于治疗与哺乳动物组织或复杂结构或器官的再生修复相关的疾病的药物或试剂中的应用。
进一步的，本申请所提供的所述视黄酸受体激活剂可以全反式视黄酸、选择性视黄酸受体γ激动剂的一种或两种以上。
优选地，所述选择性视黄酸受体γ激动剂为CD437和/或BMS961。
进一步的，本申请中所述再生修复为促进组织或器官切除或损伤后的组织或复杂结构或器官的再生。
优选地，本申请所述组织为皮肤、脂肪、肌肉、骨骼、毛囊、血管或神经。
优选地，本申请所述复杂结构为至少包括皮肤、毛囊、腺体、软骨、肌肉、脂肪、血管、神经或与肢体中的两种以上的机体结构。
优选地，本申请所述器官为肺、肝、心、胰岛或肾。
进一步优选地，所述复杂结构为耳朵、肢体、手指、眼或鼻。
优选地，本申请所述再生修复为耳朵被切除后的再生。
优选地，本申请所述再生修复为促进皮肤损伤后的再生修复，脱发后的毛发再生，软骨肌肉损伤再生修复，肺、肝、皮肤、心、肾、肌肉纤维化的再生以及血管、神经与肢体损伤后的再生。
优选地，本申请所述再生修复为促进烫伤皮肤的再生修复。
优选地，本申请所述与组织或复杂结构或器官的再生修复相关的疾病为皮肤烫伤、皮肤创伤、皮肤烧伤、脱发、软骨肌肉损伤、肝纤维化、肺纤维化或肢体损伤。
本申请所提供的所述视黄酸受体激活剂，在促进组织器官再生修复能力的应用中，是通过对视黄酸的激活实现的。优选地，通过对视黄酸受体γ的激活实现的。
进一步的，本申请提供的包含所述视黄酸受体激活剂的组合物中包括蛋白质合成抑制剂、全反式视黄酸和BMP激活剂。
优选地，所述组合物中的蛋白质合成抑制剂为环己酰亚胺(Cycloheximide(CHX))，BMP激活剂为BMP signaling agonist sb4。
进一步优选地，在所述组合物中，以所述环己酰亚胺为1重量份计，全反式视黄酸为0.25～8重量份，BMP signaling agonist sb4为0.25～4重量份。
另一种优选地，所述组合物中的蛋白质合成抑制剂为水仙环素(Narciclasine)，BMP激活剂为BMP signaling agonist sb4。
进一步优选地，在所述组合物中，以所述水仙环素(Narciclasine)为1重量份计，全反式视黄酸为0.25～10重量份，BMP signaling agonist sb4为2～10重量份。
本申请还提供了所述视黄酸受体激活剂及包含其的组合物的给药方式可以为腹腔注射、静脉注射、灌胃、口服或皮肤涂抹。
本申请还提供了一种促进哺乳动物组织或复杂结构或器官再生修复的方法，其包括向有需要的受试者施用视黄酸受体激活剂或包含视黄酸受体激活剂的组合物。
进一步的，所述方法中的视黄酸受体激活剂或包含视黄酸受体激活剂的组合物即为本申请中所提供的视黄酸受体激活剂或包含视黄酸受体激活剂的组合物。
进一步的，所述方法中，可以以腹腔注射、静脉注射、灌胃、口服或皮肤涂抹等给药方式向有需要的受试者施用视黄酸受体激活剂或包含视黄酸受体激活剂的组合物。
 附图说明
图1A为再生的非洲刺鼠与非再生的小鼠转录组之间的差异基因分析及其功能富集结果。
图1B为Vehicle与不同翻译抑制剂Ani、CHX药物处理后，直径2mm的小鼠耳洞愈合情况示意图。
图1C为经Vehicle与不同剂量的CHX药物处理后，直径2mm的小鼠耳洞闭合情况示意图。
图1D为直径2mm耳洞打孔器创伤的小鼠耳洞经CHX药物处理30天的闭合情况示意图，比例尺为1mm。
图1E为经CHX药物处理后，小鼠耳廓组织HE染色结果，比例尺为200um。
图1F为经CHX药物处理后，小鼠耳廓组织HE染色结果，比例尺为1mm。
图1G为经CHX药物处理7天后，小鼠耳廓组织的KI67免疫组化染色结果示意图，比例尺为100um。
图1H为经CHX药物处理耳洞闭合180天后，小鼠耳廓组织HE染色结果示意图，比例尺为1mm。
图1I为不同给药方式处理3周后小鼠耳洞愈合效果(损伤后21天)示意图。
图1J为Nsun2敲除小鼠2mm耳洞愈合效果(损伤后21天)。
图2A为经DMSO/CHX处理后，直径4mm的小鼠耳洞闭合情况示意图。
图2B为直径4mm耳洞打孔器创伤的小鼠耳洞经CHX处理50天的闭合情况示意图，比例尺为1mm。
图3为蛋白质与RNA合成、铁死亡和自噬的抑制剂处理小鼠耳洞愈合效果(损伤后21天)。
图4为CHX同时干扰发育及低等生物再生相关通路促进耳洞再生效果示意图(损伤后21天)。
图5A为RARs激活剂RA促进耳洞再生效果示意图(损伤后21天)。RA(Retinoic acid，MCE，HY-14649，5/20mg/kg)，激活RARs。n≥6。***p<0.001，t检验。
图5B为RARs激活剂RA促进耳洞再生效果示意图(损伤后21天)。RA(Retinoic acid，MCE，HY-14649，5/20mg/kg)，激活RARs。n≥6。***p<0.001，t检验。
图6A为经Vehicle/CRB处理后，小鼠耳洞的愈合情况示意图。
图6B为小鼠4mm耳洞经Vehicle/CRB处理30天后的愈合效果图。
图6C为经Vehicle/CRB处理后小鼠耳廓组织HE染色图。
图6D为经Vehicle/CRB处理7天后小鼠耳廓组织α-SMA免疫荧光染色图。
图6E为小鼠4mm耳洞经Vehicle/CRB处理大于180天后，小鼠耳廓组织HE染色结果示意图，比例尺为100μm。
图6F免疫荧光染色血管标记物CD31染色结果，比例尺为20μm。
图7为经Vehicle/RA处理后，小鼠烫伤疤痕面积示意图。
图8A为肢端造模的模式图。
图8B显示经药物处理20、40、120天后的肢端诱导再生现象。比例尺为2mm。
图8C为肢端outgrowth长度的统计结果。n≥3，***p<0.001，t检验。
图8D显示经药物CR处理20天和40天后，利用电子计算机断层扫描(CT)获取骨组织重建的过程高清图。
图8E为药物CR处理30后骨组织的再生结果。
图8F为HE染色发现该组合可以促进截肢肢端多组织类型的再生。
图8G显示经药物处理21、50、120天后，小鼠肢端再生组织Masson染色结果。比例尺为2mm，n≥3，t检验。
图9A为经Vehicle/NRB处理后，小鼠4mm耳洞的愈合情况示意图。
图9B为经NRB处理后的免疫组化结果示意图。
图9C为经NRB处理后的，HE切片染色结果示意图。
 具体实施方式
以下通过具体实施例来详细阐述和说明本发明的实施方式，但以下内容不应理解为对本发明作任何限制。
本申请主要涉及一种能够促进哺乳动物组织或复杂结构或器官再生修复能力的化合物或包含所述化合物的组合物，具体的，所述化合物为视黄酸受体激活剂。
本申请还涉及所述化合物在制备用于促进哺乳动物组织或复杂结构或器官再生修复能力的药物或试剂中的应用。
本申请还涉及所述化合物在制备用于治疗与哺乳动物组织或复杂结构或器官的再生修复相关的疾病的药物或试剂中的应用。
视黄酸受体RARs是视黄酸的核受体，可以被视黄酸RA激活。视黄酸是动物体内维生素A的代谢中间产物，主要影响骨的生长和促进上皮细胞增生、分化、角质溶解等代谢作用。RA的核受体主要有视黄酸受体RARs和视黄类X受体RXRs。RARs和RXRs在核内作为配体依赖的转录调节因子来调控特异性目的基因的表达。RARs有三个亚型RARα、RARβ、RARγ，均可被同等激活。通过对各靶点因子进行干扰，其结果显示，激活RARα对再生修复无显著促进作用，激活RARβ会抑制基底水平愈合，而激活RARγ能够促进再生修复。
在本申请的具体实施方式中，所述促进组织器官再生修复能力是通过对视黄酸或视黄酸受体γ的激活来实现的。具体的，本申请中所述视黄酸受体激活剂包括对视黄酸受体RARs各通路可产生同等激活作用的化合物或几种化合物的组合，以及可对视黄酸受体RARs中的RARγ通路产生选择性激活的化合物或几种化合物的组合。
在一些具体的实施方式中，所述视黄酸受体激活剂为全反式视黄酸，其可对视黄酸受体RARs各通路可产生同等激活作用。全反式视黄酸(ATRA)为视黄酸的两种活性形式之一，其既是维甲酸受体(RAR)，也是视黄醇X受体(RXR)。RAR和RXR作为转录因子，共同调节正常细胞和肿瘤细胞的生长和分化。
在另一些具体的实施方式中，所述视黄酸受体激活剂为选择性视黄酸受体γ激动剂，优选的，所述选择性视黄酸受体γ激动剂选自CD437和/或BMS961
在本申请的实施方式中，所述再生修复是指哺乳动物生物体的整体、器官或局部组织发生创伤而部分丢失，在剩余部分的基础上又生长出与丢失部分在形态与功能上相同的结构的修复过程。
在一个具体的实施方式中，所述再生修复为促进哺乳动物组织或器官切除或损伤后的组织或复杂结构或器官的再生。
在一些具体的实施方式中，所述组织为表皮、真皮、肌肉、骨骼、脂肪、毛囊、血管或神经。
在一些具体的实施方式中，所述复杂结构为至少包括皮肤、毛囊、腺体、软骨、肌肉、脂肪、血管、神经或与肢体中的两种以上的机体结构。
在一些具体的实施方式中，所述器官为肺、皮肤、心脏、肝脏、肾、胃、肠等。
本申请所述的复杂结构，是由不同组织构成的机体结构部件或能够完成特定生理功能或功能活动的机体功能部件，如耳朵、各脏器、肢体、眼睛、鼻等。
在一个优选的实施方式中，所述复杂结构为耳洞。
在一个具体的实施方式中，所述再生修复为促进耳朵被部分切除后的再生。
在一个具体的实施方式中，所述再生修复为促进皮肤损伤后的再生修复，脱发后的毛发再生，软骨肌肉损伤再生修复，肺、肝、皮肤、心、肾、肌肉纤维化的再生以及血管、神经与肢体损伤后的再生。
在一个具体的实施方式中，所述再生修复为促进烫伤皮肤的再生修复。
在本申请的实施方式中，所述与组织器官的再生修复相关的疾病包括但不限于皮肤烫伤、器官的纤维化、肌肉/软骨损伤或神经***疾病等，优选为皮肤烧/烫/创伤、脱发、软骨肌肉损伤、肺纤维化、肝纤维化、肾纤维化、心肌纤维化、肢体创伤或多种神经***疾病等。
在一个具体的实施方式中，包含所述视黄酸受体激活剂的组合物中还可以包括其他具有再生修复能力的化合物。
在一些具体的实施方式中，所述组合物中还包括蛋白质合成抑制剂。
在一些具体的实施方式中，所述蛋白质合成抑制剂选自环己酰亚胺(Cycloheximide(CHX))、茴香霉素(Anisomycin(Ani))、膜海鞘素B(Didemnin B(DIDB))、波凡霉素(Bouvardin(BVD))、石蒜科植物提取物或石蒜科生物碱。
在一些具体的实施方式中，所述石蒜科植物提取物或石蒜科生物碱包括水仙环素(Narciclasine)或水鬼蕉碱(Pancratistatin)。
在一个具体的实施方式中，所述组合物包括蛋白质合成抑制剂、视黄酸受体激活剂和BMP激活剂。
在一个优选的实施方式中，所述组合物包括环己酰亚胺(Cycloheximide(CHX))，全反式视黄酸和和BMP signaling agonist sb4。进一步的，以所述组合物中的环己酰亚胺为1重量份计，全反式视黄酸为0.25～8重量份，BMP signaling agonist sb4为0.25～4重量份。
在另一个优选的实施方式中，所述组合物包括水仙环素(Narciclasine)，全反式视黄酸和BMP signaling agonist sb4。进一步的，以所述组合物中的水仙环素(Narciclasine)为1重量份计，全反式视黄酸为0.25～10重量份，BMP signaling agonist sb4为2～10重量份。
在本申请的实施方式中，所述药物或试剂中还可以添加药物学上可接受的载体或辅料。
具体的，所述药物或试剂可以以如下形式制备：将所述蛋白质合成抑制剂或包含其的组合物与药学上可接受的载体混合，例如得到口服制剂，诸如片剂(包括糖衣片剂、薄膜包衣片剂、舌下片剂、口腔崩解片剂)、胶囊剂(包括软胶囊剂、微囊剂)、颗粒剂、粉末剂、锭剂、糖浆剂、乳剂、混悬剂、薄膜(例如、口服崩解性的薄膜)等、肠胃外制剂如注射剂(例如皮下注射剂、静脉内注射剂、肌内注射剂、腹腔注射剂、滴注剂)、外用制剂(例如皮肤制剂、软膏剂)、栓剂(例如直肠栓剂、***栓剂)、丸剂、滴鼻剂、呼吸制剂(吸入剂)、眼药水等。除此之外，这些制剂可作为控释制剂(例如持续释放微囊剂)、诸如立即释放制剂、持续释放制剂等。这样的制剂可通过本技术领域中常规使用的制备方法获得。
具体的，上述药学上可接受的载体的例子包括赋形剂(例如，淀粉，乳糖，蔗糖，碳酸钙，磷酸钙等)，粘合剂(例如，淀粉，***胶，羧甲纤维素，羟丙基纤维素，结晶纤维素，海藻酸，凝胶，聚乙烯吡咯烷酮等)，润滑剂(例如，硬脂酸镁，硬脂酸钙，滑石粉等)，崩解剂(例如，羧甲纤维素钙，滑石粉等)，稀释剂(例如，注射用水，盐水等)，添加剂(例如，稳定剂，防腐剂，着色剂，调味剂，溶解助剂，乳化剂，缓冲剂，等渗剂等)，等等。
在本申请的实施方式中，所述组合物、药物或试剂的给药方式可以为腹腔注射、静脉注射、灌胃、口服或皮肤涂抹。具体的，向受试者的给药量根据给药途径、症状、患者年龄等等而不同，临床医生可以实际确定。
实施例
近年来研究显示，如MRL小鼠1及P21-/-突变鼠2可以闭合2mm(毫米)耳洞，而野生型实验小鼠均不能闭合，因此耳洞闭合可作为评估再生能力良好的模型来筛选促进提高再生能力的细胞、信号通路、基因。主要筛选靶点为调控个体发育过程中形态发生、器官发育、免疫与应激发应(代谢、翻译等)等重要信号通路；同时也包括参与肿瘤发生及低等生物再生的基因及信号通路。
耳洞创伤小鼠模型的建立：选用7周龄C57BL/6小鼠，使用5％水合氯醛对小鼠实施麻醉，腹腔注射10mL/kg，按体重进行麻醉。将麻醉好的小鼠进行绑定，使用75％的乙醇对小鼠耳朵和器械进行消毒。以小鼠的耳廓中心，使用直径为2mm的耳洞打孔器对小鼠左右耳廓分别打孔，对创伤后小鼠通过腹腔、静脉注射或灌胃的方式给药(靶向筛选靶点的小分子或生长因子溶于生理盐水或DMSO)，以不含药物的溶剂作为对照。DMSO溶解的给药体系为：2-5％DMSO+30-40％PEG400+2-5％Tween 80按终浓度(体积比)依次加入。每2天给药一次，每7天进行观察，使用游标卡尺对小鼠耳洞的近-远轴(proximal–distal，DPD)和前后轴(anterior–posterior，DAP)进行测量，并计算小鼠被割除耳洞的面积，面积的计算公式为S＝πDPD×DAP/4，对小鼠的耳洞闭合情况进行记录。
如无特别说明，本申请以下实施例中均采用上述耳洞创伤小鼠模型进行耳洞再生实验，其中7周龄C57BL/6小鼠购自北京维通利华实验动物技术有限公司；Nsun2敲除小鼠由申请人实验室制备，利用CRISPR/Cas9介导基因敲除技术，通过将注射Cas9mRNA与Nsun2的sgRNA注射到小鼠受精卵获得基因敲除胚胎，进一步繁殖获得稳定敲除的小鼠；CHX:(即4-((R)-2-((1S,3S,5S)-3,5-dimethyl-2-oxocyclohexyl)-2-hydroxyethyl)piperidine-2,6-dione)购自MedCamExpress。其他材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例一：翻译抑制(环己酰亚胺(Cycloheximide，后缩写为CHX或C)；茴香霉素(Anisomycin，缩写为Ani))促进小鼠直径2mm耳洞割处再生。
实验方法：对耳洞创伤小鼠以不同剂量CHX(溶于生理盐水或DMSO)分别采取腹腔注射给药(图1I实验中增加灌胃给药)，生理盐水或DMSO作为对照组(标记为Vehicle)。对药物处理过的小鼠创伤耳廓进行鉴定。具体实验结果如图1所示。
图1A转录组分析再生的非洲刺鼠与非再生的小鼠(C57BL/6小鼠)之间差异基因，并通过功能富集分析，结果显示再生非洲刺鼠相比于非再生小鼠相比显著下调是核糖体亚基及翻译相关。
图1B显示，基于图1A分析结果，对非再生小鼠利用翻译的小分子抑制剂Ani,CHX分别进行药物处理21天后，观察其对2mm耳洞愈合效果，结果显示相比较于对照组(Vehicle)，翻译抑制剂Ani(10mg/kg),CHX(20mg/kg)均能显著促进耳洞愈合，CHX愈合效果更佳，由此发现翻译抑制能显著促进非再生物种的再生。后续实验基于CHX开展。
图1C的示意图显示了经不同浓度的Vehicle/CHX进行处理后，小鼠耳洞的闭合情况：各浓度的CHX对于直径2mm的耳洞闭合都有促进作用，其中浓度大于8mg/kg以上，药物处理三周后，小鼠耳洞创口完全闭合。n≥8。
图1D为经Vehicle/CHX(20mg/kg)处理耳洞创伤小鼠2mm耳洞30天后的愈合情况照片，其中以CHX处理的耳洞创伤已经完全闭合。
图1E的小鼠耳廓组织HE染色图显示，经CHX(20mg/kg)处理后，耳洞创伤小鼠耳洞部分的皮肤，***等多种组织结构生长连接在一起，创伤已经完全愈合。
图1F的小鼠耳廓组织HE染色图显示，耳洞创伤小鼠经DMSO/CHX(20mg/kg)处理后，创伤后第1天(D1组)两组织均局部可见灶性坏死，坏死灶内炎性细胞弥漫性浸润，如箭头①所示；组织内真皮层内可见炎性细胞弥散性浸润，如箭头②所示；其中CHX组中部分细胞内可见含铁血黄素沉积，如箭头③所示。第15天DMSO组的组织中部分细胞内可见含铁血黄素沉积，如箭头③所示；可见组织水肿，真皮下***间隙增大，组织结构疏松，如箭头④所示。第15天CHX组的组织损伤后愈合形成的肉芽组织，肉芽组织内成纤维细胞和血管大量增生，如箭头⑤所示；并可见炎性细胞弥散性分布，如箭头②所示。
图1G的小鼠耳廓组织的KI67免疫组化染色图显示，CHX(20mg/kg)处理7天后，小鼠基底层细胞大量表达细胞增殖的标志性蛋白KI67，如箭头显示，对照组表达相对较少。
图1H的小鼠耳廓组织HE染色图显示，经CHX(20mg/kg)处理的小鼠耳洞闭合180天后，伤口部分有毛囊，腺体，软骨，肌肉与血管等组织和组织衍生物的再生。
图1I的示意图显示，采用灌胃和腹腔注射等不同给药方式对耳洞创伤小鼠进行处理，3周后均产生愈合效果(损伤后21天)。其中n≥6。***p<0.001，ns：无显著性差异，t检验。
此外，Nsun2敲除被报道可以通过调控tRNA稳定性来抑制翻译，因此用Nsun2敲除小鼠作为遗传学模型来验证翻译抑制对再生作用。具体方法为：分别以野生型小鼠(WT)与Nsun2敲除小鼠(KO)建立耳洞创伤小鼠模型，制造直径2mm耳洞(方法同上述实施例)，3周后分别观察并测量耳洞愈合情况。结果显示Nsun2敲除的小鼠相比野生型小鼠，耳洞面积明显减小。这也表明翻译抑制对再生具有作用(图1J)。
以上实验结果显示：不同剂量的蛋白质合成抑制剂CHX都能显著促进2mm耳洞愈合，且这种促进效果具有剂量依赖性，大于8mg/kg剂量可以促进闭合。闭合耳洞可以再生处毛囊，腺体，软骨和肌肉等组织和组织衍生物。同时也证实不同给药方式均具有促进再生的效果。
实施例二：环己酰亚胺CHX促进小鼠直径4mm耳洞割处再生修复。
研究发现极少数的哺乳动物如非洲刺鼠具有较强再生能力，能够再生4mm耳洞，同时还发现4mm耳洞闭合模型可以区分再生能力强弱，目前没有任何人工实现4mm耳洞闭合再生，即使是被报道“超级再生”的MRL小鼠也不能完全闭合。因此4mm耳洞可以作为评价哺乳动物割处再生的良好模型，其模型建立采取实施例1的方法，其中以直径为4mm的耳洞打孔器对小鼠左右耳廓分别打孔。
实验方法：对4mm耳洞创伤小鼠以不同剂量的CHX(20,125,175mg/kg,溶于DMSO或生理盐水)采取腹腔注射给药，DMSO作为对照组。对药物处理过的小鼠创伤耳廓进行鉴定。具体实验结果如图2所示。
图2A的示意图显示，不同剂量的CHX对于直径4mm的耳洞闭合效果，发现20mg/kg与超级愈合小鼠MRL/lpr效果类似，有促进耳洞伤口变小作用，但不能完全闭合，当剂量大于20mg/kg(125,175mg/kg)能实现耳洞的闭合。
图2B为DMSO/CHX药物处理90天后的耳洞愈合照片，其显示经CHX(125mg/kg)处理的小鼠4mm耳洞，创伤的面积显著减小。
实施例三：环己酰亚胺CHX促进耳洞再生不依赖于铁死亡与自噬的抑制。
CHX作为一种抗真菌的抗生素，除了抑制真核生物蛋白质合成和RNA合成外，还可抑制铁死亡和细胞自噬。为了验证其在再生过程中通过哪个靶点发挥作用，分别针对铁死亡和自噬进行小分子验证。
实验方法：采用上述相同的耳洞创伤小鼠模型进行实验，分别用铁死亡和自噬的抑制剂代替CHX观察其促进耳洞愈合的效果。DMSO替代小分子的对照组。Auto:Autophinib(MCE，HY-101920)，抑制自噬；3BDO：3BDO(MCE，HY-U00434)，抑制自噬；UAMC：UAMC-3203(MCE，MCE，HY-112909A)，抑制铁死亡；EBSE：Ebselen(MCE，HY-13750)，抑制铁死亡。使用浓度为10-20mg/kg，给药方式同CHX，n≥8，**p<0.01，***p<0.001，ns：无显著性差异，t检验。鉴定不同抑制剂对2mm耳洞愈合效果(损伤后21天测定)。结果如图3所示：分别用铁死亡和自噬的抑制剂均不能像CHX一样促进耳洞愈合，这说明CHX促进耳洞再生不依赖于其对铁死亡或自噬的抑制活性。
实施例四：抑制RARs抑制CHX再生作用。
为了进一步验证CHX促进耳洞再生中发挥作用的信号通路，通过在添加CHX同时干扰发育及低等生物再生相关通路进行筛选。
实验方法：对2mm耳洞创伤小鼠分别以CHX(20mg/kg)、CHX+AGN、CHX+RA、CHX+RA+AGN进行腹腔注射给药处理。其中，AGN为视黄酸受体RARs特异性抑制剂AGN193109(AGN，MCE，HY-U00449，0.75mg/kg)，拮抗RARs，RARs；RA为RARs激活剂全反式视黄酸(RA，MCE，HY-14649，5mg/kg)，激活RARs。以DMSO作为对照组。n≥6。***p<0.001，t检验。损伤后21天对各组药物处理过的小鼠创伤耳洞面积进行测定。具体实验结果如图4所示。
结果显示RARs通路抑制剂与激活剂分别对CHX产生抑制和协同作用。其中，视黄酸受体RARs特异性抑制剂AGN193109可以完全抑制CHX对耳洞的愈合。而联合使用CHX与RARs激活剂全反式视黄酸可以产生协同促进耳洞闭合的作用，且这种协调作用仍然可以被RARs抑制剂AGN所抑制。这显示RARs可能在耳洞再生模型中发挥重要作用。
实施例五：激活RARγ促进耳洞再生。
进一步验证直接激活RARs是否可以发挥类似于CHX一样促进耳洞再生的作用，以DMSO/RA(5mg/kg，20mg/kg)分别进行耳洞创伤实验。图5A的结果(耳洞损伤后21天)显示，单独使用RA也可以促进耳洞闭合，且具有剂量依赖性。
由于RA能够同等激活RAR受体的三个亚型：RARα、RARβ、RARγ，另外还能够作用并激活PPARβ/δ6，以及抑制转录因子Nrf27，为了进一步确认其发挥作用促进耳洞再生的具体靶点，对各潜在靶点进行干扰以进行验证。实验方法为：分别以如下试剂给药于耳洞创伤小鼠模型，检测创伤后21天的耳洞愈合情况。
Tazarotene(MCE，HY-15388，20mg/kg)与Bexarotene(MCE，HY-14171，20mg/kg)，视黄酸受体激动剂；VA(VitaminA，Selleck，S5592，20mg/kg)，维生素A，体内代谢产生RA；Talarozole(MCE，HY-14531，10mg/kg)，抑制CYP26A1、CYP26B1，提高内源性全反式维甲酸；AGN 195183(MCE，HY-16684，10mg/kg)，选择性RARα激动剂；CD437(MCE，HY-100532，10mg/kg)与BMS 961(Glpbio，GC17382，10mg/kg)，选择性RARγ激动剂；GW0742(MCE，HY-13928，20mg/mg)，激活PPARβ/δ；ML385(MCE，HY-100523，10mg/kg)，Nrf2特异抑制剂。n≥5，**p<0.01，***p<0.001，ns：无显著性差异，t检验。实验结果如图5B所示。
结果显示，激活RARγ(包括RARs激活剂及RARγ选择激活剂)能够促进耳洞再生，而激活RARα无显著促进效果，激活PPARβ/δ及抑制Nrf2抑制基底水平的愈合。另外，通过补充维生素A或使用全反式维甲酸代谢阻断剂Talarozole，提高内源性RA水平也可以促进耳洞再生，这丰富了激活RARs的调控靶点。
实施例六：组合CRB(C:蛋白质合成抑制剂CHX；R:RARs激活剂全反式视黄酸(All-trans retinoic acid)；B:BMP激活剂BMP(signaling agonist sb4))促进小鼠4mm耳洞闭合及割处再生。
以7周龄小鼠为例，使用5％水合氯醛对小鼠实施麻醉，腹腔注射10mL/kg，按体重进行麻醉。将麻醉好的小鼠进行绑定，使用75％的乙醇对小鼠耳朵和器械进行消毒。以小鼠的耳廓中心，使用直径为4mm的耳洞打孔器对小鼠左右耳廓分别打孔。对于创伤后的小鼠，采用腹腔注射DMSO/CRB(CRB给药量为CHX 20mg/kg、全ATRA 20mg/kg，BMP signaling agonist sb4 10-20mg/kg)的方式每2天给药一次，并且每7天对小鼠进行麻醉，使用游标卡尺对小鼠耳洞的近-远轴(proximal–distal，DPD)和前后轴(anterior–posterior，DAP)进行测量，并计算小鼠被割除耳洞的面积，面积的计算公式为S＝Πx DPD x DAP/4。对药物处理过的小鼠创伤耳廓进行进一步鉴定。
图6A显示分别经DMSO/CRB处理后，小鼠耳洞的闭合情况。结果显示CRB对于直径4mm的耳洞闭合都有促进作用，在给药处理30天后，小鼠耳洞完创口全闭合。
图6B显示小鼠4mm耳洞经药物处理30天后的闭合情况。可以看出，经药物处理后的小鼠，耳洞创伤已经闭合。
图6C显示经药物处理7天后，小鼠耳廓组织HE染色结果和表皮厚度统计。比例尺为200um。经药物处理后的小鼠耳廓有芽基形成，表皮厚度明显降低。n≥3，t检验。
图6D显示经药物处理7天后，小鼠耳廓组织α-SMA免疫荧光染色结果。比例尺为100um。创伤7天后，对照组与药物处理组均出现大量的α-SMA表达，与对照组相比，药物处理组的α-SMA表达呈线性有序排列，而对照组为无序堆积，表达模式与疤痕形成方式类似。
图6E显示经药物(CRB)诱导大于90天后各个组织再生情况，显示表皮、真皮、腺体、毛囊、肌肉、软骨、脂肪、肌肉的再生。“e”指示表皮再生；“d”指示再生的真皮；“g”指示再生的腺体；“ad”指示再生的脂肪组织；“hf”指示再生的毛囊；“c”指示再生的软骨；“m”指示再生的肌肉。
图6F免疫荧光染色血管标记物CD31进一步鉴定血管再生，三角形指示再生的血管。
实施例七：RA降低小鼠烫伤后疤痕形成，促进皮肤再生。
实验方法：使用7周龄小鼠，(1)腹腔注射0.5％戊巴比妥钠(100μl/10g b.w.)麻醉小鼠；(2)背部剃毛，75％酒精皮肤消毒。(3)开水烫10-15s，烫伤面积为直径1.5cm的圆。(4)烫伤后平均分成两组，分别以Vehicle组(DMSO)和RA组(20mg/kg，溶解于DMSO)进行给药处理。(5)检测伤口修复情况。实验结果如图7所示。
结果显示：给药40天后，剃毛检测疤痕，显示RA经处理可显著降低疤痕面积。
实施例八：环己酰亚胺CHX(C)和全反式视黄酸RA(R)优化组合促进小鼠肢端***结构(Outgrowth)再生修复
基于耳洞的再生效果，接下来进行了更复杂的损伤表型的再生诱导实验，选用肢端切除作为损伤模型。对ICR小鼠进行肢端造模，并联合使用适当剂量的CHX(100mg/kg)与RARs激活剂全反式视黄酸(RA，20mg/kg)，腹腔连续隔天给药8周，观察再生表型。
[根据细则91更正 10.07.2023]
实验方法：以8周龄ICR小鼠为模型，使用5％水合氯醛对小鼠实施麻醉，腹腔注射10mL/kg，按体重进行麻醉。将麻醉好的小鼠进行绑定，使用75％的乙醇对小鼠左上肢端和手术器械进行消毒。这里为了准确定义再生的发生，测量肘关节至桡尺骨的距离，保留10mm，其余至手掌、手指等节段进行切除造模(尺骨和桡骨属于前臂的两根骨头，最简单的区别的方法为拇指一侧为桡骨、小指一侧为尺骨)。腹腔连续隔天给药8周，CHX(100mg/kg,溶于DMSO或生理盐水)、RA(20mg/kg)采取腹腔注射给药，DMSO作为对照组，持续观察表型。不同天数后，对药物处理过的小鼠肢端创伤进行鉴定。具体实验结果如图8A-8G所示。图8A为肢端造模的模式图。图8B显示经药物处理20、40、120天后的肢端诱导再生现象。可以看出，经药物处理后的小鼠截肢部位出现outgrowth的生长。比例尺为2mm。图8C为肢端outgrowth长度的统计结果，可以看到CR给药组很好诱导肢端再生，长度与对照组存在极显著差异。n≥3，***p<0.001，t检验。图8D显示经药物CR处理20天和40天后，利用电子计算机断层扫描(CT)获取骨组织重建的过程高清图，CR处理组出现很好的肢端骨组织诱导生长过程，CR处理20天后即可观察到骨组织的伸长重建碎片(右图，三角箭头指示)，而对照组呈现出钝化状态，显示为骨组织的增生积累，未呈现出向前的生长趋势(左图，三角箭头指示)，是一种类似瘢痕修复的发生。图8E为骨再生的更直接关键证据，药物CR处理30后，取出完整桡尺骨进行观察，与CT扫描结果观察一致，可以明显看到骨组织的再生趋势。此外，HE切片染色发现该组合可以促进截肢肢端多组织类型的再生，如皮肤、毛囊、毛细血管、新生骨等组织(图8F，“Ep”指示表皮再生；“CT”指示***再生；“CV”指示毛细血管再生；“HF”指示毛囊再生；“OT”指示骨组织再生。)。图8G显示经药物处理21、50、120天后，小鼠肢端再生组织Masson染色结果，对照组可以观察到骨组织的钝化形态，与CT结果相符，药物处理组的骨组织呈现生长趋势，重要的是实验组在120天染色观察到肌肉组织的再生，而对照组为无序堆积，表达模式与疤痕形成方式类似。比例尺为2mm，n≥3，t检验。
以上结果显示，环己酰亚胺CHX和全反式视黄酸RA的组合物能促进肢端切除后***结构的再生修复，包括骨、肌肉、皮肤、毛囊、毛细血管、结蹄组织、新生骨组织等再生。
实施例九：组合NRB(N:水仙环素Narciclasine；R:RARs激活剂全反式视黄酸(All-trans retinoic acid)；B:BMP激活剂BMP(signaling agonist sb4))促进小鼠4mm耳洞割处再生。
实验方法：小鼠模型构建方法同实施例二，对4mm耳洞创伤后的小鼠，采用腹腔注射DMSO/NRB(NRB：Narciclasine 3mg/kg、ATRA 20mg/kg，BMP signaling agonist sb4 10-20mg/kg)的方式每2天给药一次，并且每7天对小鼠进行麻醉，使用游标卡尺对小鼠耳洞的近-远轴(proximal–distal，DPD)和前后轴(anterior–posterior，DAP)进行测量，并计算小鼠被割除耳洞的面积，面积的计算公式为S＝π×DPD×DAP/4。对药物处理过的小鼠创伤耳廓进行进一步鉴定。实验结果如图9A～9C所示。
其中，图9A显示了小鼠4mm耳洞经NRB处理30天后的愈合效果，并显示具有愈合促进作用，在给药处理30天后，小鼠耳洞完创口全闭合，且鉴定为再生事件。图9B，9C显示，HE切片染色数据很好地指征了软骨(黑色长箭头)、毛囊(星号)、腺体/皮脂腺(三角形箭头)、肌肉(虚线框选区域)等结构的再生结构。
以上实验结果显示，NRB小分子组合物也可以促进4mm耳洞闭合并促进再生。
前面仅仅示出了本发明的原理，应理解，本发明的范围不预期限制在本文所述的示例性方面，而应包括所有当前已知的和未来开发的等同物。另外，应当指出，在不脱离本发明技术原理的前提下，还可以作出若干改进和修改，这些改进和修改也应被视为本发明的范围。

Claims (17)

  1. 视黄酸受体激活剂或包含视黄酸受体激活剂的组合物在促进哺乳动物组织或复杂结构或器官再生修复能力中的应用。
  2. 视黄酸受体激活剂或包含视黄酸受体激活剂的组合物在制备用于促进哺乳动物组织或复杂结构或器官再生修复能力的药物或试剂中的应用。
  3. 视黄酸受体激活剂或包含视黄酸受体激活剂的组合物在制备用于治疗与哺乳动物组织或复杂结构或器官的再生修复相关的疾病的药物或试剂中的应用。
  4. 根据权利要求1-3中任一项所述的应用，其特征在于，所述视黄酸受体激活剂包括全反式视黄酸、选择性视黄酸受体γ激动剂的一种或两种以上，优选的，所述选择性视黄酸受体γ激动剂为CD437和/或BMS961。
  5. 根据权利要求1-4中任一项所述的应用，其特征在于，所述再生修复为促进组织或器官切除或损伤后的组织或复杂结构或器官的再生。
  6. 根据权利要求1-5中任一项所述的应用，其特征在于，
    所述组织为皮肤、脂肪、肌肉、骨骼、毛囊、血管或神经，
    所述复杂结构为至少包括皮肤、毛囊、腺体、软骨、肌肉、脂肪、血管、神经或与肢体中的两种以上的机体结构，
    所述器官为肺、肝、心、胰岛或肾。
  7. 根据权利要求1～6中任一项所述的应用，其特征在于，所述复杂结构为耳朵、肢体、手指、眼或鼻。
  8. 根据权利要求1-6中任一项所述的应用，其特征在于，所述再生修复为促进皮肤损伤后的再生修复，脱发后的毛发再生，软骨肌肉损伤再生修复，肺、肝、皮肤、心、肾、肌肉纤维化的再生以及血管、神经与肢体损伤后的再生。
  9. 根据权利要求3～8中任一项所述的应用，其特征在于，所述疾病为皮肤烫伤、皮肤创伤、皮肤烧伤、脱发、软骨肌肉损伤、肝纤维化、肺纤维化或肢体损伤。
  10. 根据权利要求1-9中任一项所述的应用，其特征在于，所述促进哺乳动物组织或复杂结构或器官再生修复能力是通过对视黄酸的激活实现的， 优选的，通过对视黄酸受体γ的激活实现的。
  11. 一种组合物，其特征在于，包括蛋白质合成抑制剂、全反式视黄酸和BMP激活剂，优选的，所述蛋白质合成抑制剂为环己酰亚胺(Cycloheximide(CHX))或水仙环素(Narciclasine)，所述BMP激活剂为BMP signaling agonist sb4。
  12. 根据权利要求11所述的组合物，其特征在于，以所述组合物中的环己酰亚胺为1重量份计，全反式视黄酸为0.25～8重量份，BMP signaling agonist sb4为0.25～4重量份。
  13. 根据权利要求11所述的组合物，其特征在于，以所述组合物中的水仙环素(Narciclasine)为1重量份计，全反式视黄酸为0.25～10重量份，BMP signaling agonist sb4为2～10重量份。
  14. 根据权利要求11～13中任一项所述的组合物，其特征在于，所述组合物的给药方式为腹腔注射、静脉注射、灌胃、口服或皮肤涂抹。
  15. 根据权利要求1-10中任一项所述的应用，其特征在于，所述组合物为权利要求11-14中任一项所述的组合物。
  16. 一种促进哺乳动物组织或复杂结构或器官再生修复的方法，其包括向有需要的受试者施用视黄酸受体激活剂或包含视黄酸受体激活剂的组合物。
  17. 根据权利要求16所述的方法，其中，视黄酸受体激活剂或包含视黄酸受体激活剂的组合物为权利要求1～10中所涉及的视黄酸受体激活剂或包含视黄酸受体激活剂的组合物或权利要求11-14中任一项所述的组合物。
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