WO2023241716A1 - 一种诱导哺乳动物原位再生的方法及其应用 - Google Patents

一种诱导哺乳动物原位再生的方法及其应用 Download PDF

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WO2023241716A1
WO2023241716A1 PCT/CN2023/100867 CN2023100867W WO2023241716A1 WO 2023241716 A1 WO2023241716 A1 WO 2023241716A1 CN 2023100867 W CN2023100867 W CN 2023100867W WO 2023241716 A1 WO2023241716 A1 WO 2023241716A1
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regeneration
protein synthesis
synthesis inhibitor
chx
repair
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PCT/CN2023/100867
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English (en)
French (fr)
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李伟
周琪
何正泉
袁雪薇
卢宗宝
王馨
王帅
李宇飞
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 WO2023241716A1 publication Critical patent/WO2023241716A1/zh

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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
    • A61K31/185—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
    • A61K31/203—Retinoic acids ; Salts thereof
    • 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
    • A61K31/33—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
    • 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
    • A61K31/33—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
    • 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
    • A61K31/33—Heterocyclic compounds
    • 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
    • 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
    • A61K31/33—Heterocyclic compounds
    • 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
    • A61K31/44—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
    • 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
    • A61K31/33—Heterocyclic compounds
    • 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
    • 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
    • A61K31/66—Phosphorus compounds
    • A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/05—Dipeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the invention relates to the field of biotechnology, and specifically relates to a method for inducing in situ regeneration of mammals with small molecule compounds 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.
  • Pulmonary fibrosis is a pathological change characterized by the proliferation of fibroblasts and the accumulation of large amounts of extracellular matrix, accompanied by inflammatory damage and tissue structure destruction. That is, the normal alveolar tissue is damaged and undergoes abnormal repair resulting in structural abnormalities (scar formation). Pulmonary fibrosis will seriously affect the human respiratory function, manifesting as various dyspnea, which will continue to worsen with the aggravation of the condition and lung damage and the patient's respiratory function. It is reported that the morbidity and mortality of idiopathic pulmonary fibrosis are increasing year by year worldwide, and the average survival time after diagnosis is less than 3 years, which is higher than most tumors, so it is also called a "tumor-like disease" . Therefore, promoting the regeneration of damaged tissue is the most fundamental means to treat and prevent pulmonary fibrosis-related diseases and has important application value.
  • 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 mammalian tissues or complex structures or organs.
  • the compound is a protein synthesis inhibitor.
  • the present application provides the use of the protein synthesis inhibitor or a composition comprising the protein synthesis inhibitor in promoting the regeneration and repair ability of mammalian tissues or complex structures or organs.
  • the present application provides the use of the protein synthesis inhibitor or a composition comprising the protein synthesis inhibitor in the preparation of a medicament or agent for promoting the regeneration and repair ability of mammalian tissues or complex structures or organs.
  • the present application provides the use of the protein synthesis inhibitor or a composition comprising the protein synthesis inhibitor in the preparation of a medicament or agent for the treatment of diseases associated with the regeneration and repair of mammalian tissues or complex structures or organs.
  • 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 complex structures described in this application are ears, limbs, fingers, eyes or noses.
  • the organ described in this application is lung, liver, heart, pancreatic islet or kidney.
  • 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 fibrosis of lungs, liver, skin, heart, kidney, muscles and other tissues and organs, and blood vessels. , Regeneration after nerve and limb injury.
  • the regeneration and repair described in this application is to promote the regeneration and repair of scalded skin.
  • the diseases related to the regeneration and repair of tissues and organs described in this application are skin scalds, skin wounds, skin burns, aging or unexplained hair loss, cartilage and muscle damage, liver fibrosis or pulmonary fibrosis.
  • the protein synthesis inhibitor provided in this application is used to promote tissue and organ regeneration and repair capabilities by inducing life quiescence.
  • the protein synthesis inhibitor provided in this application is used to promote the regeneration and repair ability of mammalian tissues and complex structures and organs by activating the STING-TBK1-IRF3 signal.
  • the protein synthesis inhibitor provided by this application can be cycloheximide (CHX), anisomycin (Ani), Didemnin B (DIDB), Bofanomycin Bouvardin (BVD), Amaryllidaceae plant extract or Amaryllidaceae alkaloids.
  • the Amaryllidaceae plant extract or Amaryllidaceae alkaloid is Narciclasine or Pancratisatin.
  • the composition comprising the protein synthesis inhibitor provided by the present application includes a protein synthesis inhibitor, all-trans retinoic acid and a BMP activator.
  • the protein synthesis inhibitor in the composition is cycloheximide (CHX), and the BMP activator is BMP signaling agonist sb4.
  • the total The amount of trans-retinoic acid is 0.25 to 8 parts by weight, and the amount of BMP signaling agonist sb4 is 0.25 to 4 parts by weight.
  • the protein synthesis inhibitor in the composition is Narciclasine
  • the BMP activator is BMP signaling agonist sb4.
  • composition based on 1 part by weight of Narciclasine, 0.25-10 parts by weight of all-trans retinoic acid, and 2-10 parts by weight of BMP signaling agonist sb4.
  • protein synthesis inhibitor or its composition may be administered by intraperitoneal injection, intravenous injection, intragastric administration, oral administration, or skin application.
  • the present application also provides a method of promoting regeneration and repair of mammalian tissue or complex structures or organs, which includes administering a protein synthesis inhibitor or a composition comprising a protein synthesis inhibitor to a subject in need thereof.
  • the protein synthesis inhibitor or the composition containing the protein synthesis inhibitor in the method is the protein synthesis inhibitor or the composition containing the protein synthesis inhibitor provided in this application.
  • the protein synthesis inhibitor or the composition containing the protein synthesis inhibitor can be administered to the subject in need by administration methods such as intraperitoneal injection, intravenous injection, intragastric administration, oral administration, or skin application.
  • Figure 1A shows the differential gene analysis and functional enrichment results between the transcriptomes of regenerated African agouti and non-regenerated mice.
  • 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 1E shows the results of HE staining of mouse auricle tissue after treatment with CHX drugs.
  • the scale bar is 200um.
  • Figure 1F shows the results of HE staining of mouse auricle tissue after treatment with CHX drugs.
  • the scale bar is 1 mm.
  • Figure 1G shows KI67 immunohistochemical staining of mouse auricle tissue after treatment with CHX drugs for 7 days. Schematic diagram of the results, scale bar is 100um.
  • 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 2A is a schematic diagram of the closure of a 4 mm diameter mouse ear hole after treatment with Vehicle/CHX.
  • Figure 2B is a schematic diagram of the closure of mouse ear holes 90 days after CHX treatment after trauma from a 4 mm diameter ear hole punch.
  • the scale bar is 1 mm.
  • Figure 3 shows the healing effect of mouse ear piercings treated with inhibitors of ferroptosis and autophagy (21 days after injury)
  • Figure 4A is a schematic diagram of the healing of mouse ear holes after Vehicle/CRB treatment.
  • Figure 4B shows the healing effect of a 4mm ear hole in mice treated with Vehicle/CRB for 30 days.
  • Figure 4C is a HE staining picture of mouse auricle tissue after Vehicle/CRB treatment.
  • Figure 4D is a picture of ⁇ -SMA immunofluorescence staining of mouse auricle tissue after being treated with Vehicle/CRB for 7 days.
  • Figure 4E is a schematic diagram of the HE staining results of mouse auricle tissue after the 4mm ear piercing of mice was treated with Vehicle/CRB for more than 180 days.
  • the scale bar is 100 ⁇ m.
  • FIG. 4F Immunofluorescence staining results of vascular marker CD31, scale bar is 20 ⁇ m.
  • Figure 5A is a flow chart of CHX administration experiments on pulmonary fibrosis injury model mice.
  • Figure 5B is a schematic diagram of the survival rate of mice in the lung fibrosis injury model, Vehicle (V)/CHX (C).
  • Figure 5C is a schematic diagram of body weight changes in pulmonary fibrosis injury model mice.
  • Figures 5D and 5E are schematic diagrams of changes in respiratory frequency and primary ventilation volume in pulmonary fibrosis injury model mice.
  • Figure 5F is a schematic diagram of the results of HE staining and Sirius red staining in pulmonary fibrosis injury model mice.
  • Figure 5G is a schematic diagram of the sequencing results of fibrosis-related genes in mice with pulmonary fibrosis injury model.
  • Figure 5H is a schematic diagram of the sequencing results of genes related to respiratory system development in mice with pulmonary fibrosis injury model.
  • Figures 5I and 5J are schematic diagrams of HE staining results of pulmonary fibrosis injury model mice after administration of Vehicle/CHX for 9 days after injury.
  • Figure 6A is a schematic diagram of changes in scar area in burn model mice.
  • Figure 6B is a schematic diagram of the Sirius red staining results in scald model mice.
  • Figure 6C is a schematic diagram of the histochemical staining results in scald model mice.
  • Figure 7A shows the incorporation experiment of Puromycin (PURO) to verify that Narciclasine (Nar) has translation inhibitory effect. Create a schematic diagram of the experimental results.
  • PURO Puromycin
  • Nar Narciclasine
  • Figure 7B is a schematic diagram of the closure of mouse ear holes traumatized by a 2 mm diameter ear hole punch and treated with Nar drug for 30 days.
  • the scale bar is 1 mm.
  • Figure 7C is a schematic diagram of the closure of mouse ear holes with a diameter of 2 mm after treatment with Vehicle and different doses of Nar drugs.
  • Figure 7D is a schematic diagram of the immunohistochemistry results after treatment with Nar drug.
  • Figure 7E is a schematic diagram of the staining results of Masson sections after treatment with Nar drug.
  • Figure 7F is a schematic diagram of the healing status of a 4mm ear hole in mice after treatment with Vehicle/NRB.
  • Figure 7G is a schematic diagram of the immunohistochemistry results after NRB treatment.
  • Figure 7H is a schematic diagram of the staining results of HE sections after NRB treatment.
  • Figure 8A is a schematic diagram of the experimental results of the puromycin incorporation experiment to verify that Pancratistatin has a translation inhibitory effect.
  • Figure 8B is a schematic diagram of the closure of mouse ear holes traumatized by a 2 mm diameter ear hole punch after being treated with Pan drug for 21 days.
  • the scale bar is 1 mm.
  • Figure 8C is a statistical diagram of the effect of Pan drug treatment on mice with 2mm diameter ear hole punch trauma for 21 days, with DMSO replacing Pan treatment as the control.
  • Figure 8D is a schematic diagram of the HE section results after treatment with Pan drug.
  • Figure 8E is a schematic diagram of the HE section results after treatment with Pan drug.
  • Figures 9A and 9B are schematic diagrams of water ghost banana bulbs to be treated.
  • FIGS 9C and 9D are schematic diagrams of the mother liquor obtained after homogenizing bulbs and tissues.
  • Figure 9E is a schematic diagram of the statistical results of ear piercing area after 9 weeks of administration.
  • Figure 9F is a statistical diagram of the ear piercing area after treatment with 70% ethanol extraction method of homogenized wall-broken extract for 3 weeks, 6 weeks, and 9 weeks respectively.
  • Figure 10A is a schematic diagram of analysis of genes that are co-upregulated at the transcription and translation levels due to CHX.
  • Figure 10B is a schematic diagram of interferon-responsive genes and their enrichment.
  • Figures 10C and 10D are schematic diagrams of experimental results of quantitative qPCR detection of CHX-induced ISG gene expression.
  • Figure 10E is a schematic diagram of the results of immunofluorescence staining to identify CHX activation of the STING/TBK1/IRF3 signaling pathway.
  • Figure 10F is a schematic diagram of the results of quantitative qPCR detection of GSK86126 and Des inhibiting STING/TBK1/IRF3 pathway activity and CHX-induced ISG gene expression.
  • Figure 10G is a schematic diagram of the regeneration results of GSK86126 and Des inhibiting STING/TBK1/IRF3 pathway activity and inhibiting CHX-induced regeneration.
  • Figure 10H is a schematic diagram of the results of protein Westernblot experiment revealing that Des inhibits the STING/TBK1/IRF3 pathway activity but does not inhibit the CHX protein synthesis inhibitory function.
  • Figure 11A is a schematic diagram of extremity modeling.
  • Figure 11B shows the induced regeneration phenomenon of the limbs after 20, 40, and 120 days of drug treatment. Scale bar is 2mm.
  • Figure 11C shows the statistical results of limb outgrowth length. n ⁇ 3, ***p ⁇ 0.001, t test.
  • Figure 11D shows high-definition images of the process of bone tissue reconstruction using computed tomography (CT) after 20 and 40 days of drug CR treatment.
  • CT computed tomography
  • Figure 11E shows the regeneration results of bone tissue after 30 days of drug CR treatment.
  • Figure 11F 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.
  • the present application mainly relates to the use of protein synthesis inhibitors or compositions containing said protein synthesis inhibitors in promoting the regeneration and repair ability of mammalian tissues or complex structures or organs.
  • Protein synthesis inhibitors are a class of substances that affect protein biosynthesis. They can act on DNA replication and RNA transcription, and have an indirect effect on protein biosynthesis. They can act on all aspects of protein synthesis, including inhibiting initiation factors and elongation factors. and the role of ribonucleosomes, etc. Among them, common protein synthesis inhibitors are mainly blockers that can inhibit the protein biosynthesis and translation process.
  • the protein synthesis inhibitor can be cycloheximide (CHX), Didemnin B (DIDB), and Bouvardin (BVD) .
  • the protein synthesis inhibitor is cycloheximide, abbreviated as CHX, and its chemical formula is: 4-((R)-2-((1S,3S,5S)-3 ,5-Dimethyl-2-oxycyclohexyl)-2-hydroxyethyl)piperidine-2,6-dione (4-((R)-2-((1S,3S,5S)-3 ,5-dimethyl-2-oxocyclohexyl)-2-hydroxyethyl)piperidine- 2,6-dione)
  • CHX is a commonly used protein synthesis inhibitor that can inhibit protein synthesis in eukaryotes. It mainly acts on the translation process in protein synthesis.
  • the protein synthesis inhibitor is an Amaryllidaceae herb alkaloid.
  • the alkaloid of the Amaryllidaceae herbaceous plant is Narciclasine; in another preferred embodiment, the alkaloid of the Amaryllidaceae herbaceous plant is Pancratisatin. ).
  • Nar Nar
  • Pancratistatin (abbreviated as Pan) is an analog of narcissine.
  • the protein synthesis inhibitor is an Amaryllidaceae plant extract.
  • the Amaryllidaceae plant is Hymenocallis littoralis (Jacq.) Scalisb. Banana bulbs are rich in pancratistatine and narciclasine.
  • the Amaryllidaceae plant extract is an alcohol extract of the Amaryllidaceae plant.
  • the alcohol extract is obtained by homogenization and wall-breaking extraction using an ethanol extraction method.
  • the present application also provides a composition comprising a protein synthesis inhibitor, all-trans retinoic acid and a BMP activator.
  • the protein synthesis inhibitor is cycloheximide (CHX), and the BMP activator is BMP signaling agonist sb4.
  • the composition includes cycloheximide, BMP signaling agonist sb4 and all-trans retinoic acid.
  • Retinoic acid is a metabolic intermediate product of vitamin A in animals. It mainly affects bone growth and promotes epithelial cell proliferation, differentiation, keratinolysis and other metabolic effects.
  • All-trans retinoic acid (ATRA) is one of the two active forms of retinoic acid, which is both a retinoic acid receptor (RAR) and a retinoid X receptor (RXR). RAR and RXR, as transcription factors, jointly regulate the growth and differentiation of normal cells and tumor cells.
  • BMP signaling agonist sb4 is a benzoxazole bone morphogenetic protein 4 (BMP4) signaling agonist that activates BMP signaling by stabilizing intracellular p-SMAD-1/5/9. It also activates BMP4 target genes (DNA binding inhibitors, Id1 and Id3) in the canonical BMP signaling pathway.
  • BMP4 target genes DNA binding inhibitors, Id1 and Id3
  • the BMP signaling pathway can regulate cell proliferation and differentiation and plays an important regulatory role in embryonic development.
  • the composition includes cycloheximide 5-40 mg/kg, all-trans retinoic acid 10-40 mg/kg and BMP signaling agonist sb4 10-20 mg/kg, that is, including
  • the dosage of cycloheximide is 5-40 mg/kg, for example, it can be 5, 10, 15, 20, 25, 30, 35, 40 mg/kg; the dosage is 10-40 mg/kg of all-trans Xanthate, for example, can be 10, 15, 20, 25, 30, 35, 40mg/kg; BMP signaling agonist sb4 with a dosage of 10-20mg/kg, for example, can be 10, 12, 15, 18, 20mg/ kg.
  • the composition includes Narciclasine 0.5-3 mg/kg, all-trans retinoic acid 10-50 mg/kg and BMP signaling agonist sb4 20-50 mg/kg, namely Including cycloheximide with a dosage of 5-40 mg/kg, for example, 0.5, 1, 1.5, 2, 2.5, 3 mg/kg; all-trans retinoic acid with a dosage of 10-50 mg/kg , for example, it can be 10, 15, 20, 25, 30, 35, 40, 45, 50mg/kg; the dosage of BMP signaling agonist sb4 is 20-50mg/kg, for example, it can be 20, 25, 30, 35, 40, 45, 50mg/kg.
  • the composition also contains pharmaceutically acceptable excipients.
  • the protein synthesis inhibitor or a composition containing the protein synthesis inhibitor can be used in the preparation of drugs or agents for promoting the regeneration and repair ability of mammalian tissues or complex structures or organs.
  • the protein synthesis inhibitor or a composition comprising the protein synthesis inhibitor can be used in the preparation of a medicament or agent for the treatment of diseases related to the regeneration and repair of mammalian tissues or complex structures or organs.
  • the regenerative repair refers to the partial loss of the whole body, organ or local tissue due to trauma, and the growth of a structure that is the same in form and function as the lost part based on the remaining part. Repair process.
  • the ability to promote the regeneration and repair of mammalian tissues or complex structures or organs is achieved by inducing life quiescence.
  • the regenerative repair is to promote the regeneration of tissue or complex structures or organs after tissue or organ removal or damage.
  • the tissue in the regeneration repair is epidermis, dermis, muscle, bone, fat, hair follicle, blood vessel or nerve
  • the complex structure at least includes skin, hair follicle, gland, cartilage, Muscles, fats, blood vessels, nerves, or two or more body structures in limbs.
  • the organs are lungs, skin, heart, liver, kidneys, stomach, intestines, 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 regenerative repair 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, muscles, and blood vessels and nerves. and regeneration after limb injury.
  • 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 scalds, skin wounds, skin burns, hair loss, organ fibrosis, muscle/cartilage damage or neurological diseases, etc., preferably For skin burns/scalds, pulmonary fibrosis, liver fibrosis, kidney fibrosis, myocardial fibrosis, limb trauma or various neurological diseases, etc.
  • pharmaceutically acceptable carriers or excipients may also be added to the medicine or reagent.
  • 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.,
  • examples of the above-mentioned pharmaceutically acceptable carriers include excipients (for example, starch, lactose, sucrose, calcium carbonate, calcium phosphate, etc.), binders (for example, starch, gum arabic, carboxymethylcellulose Vitamins, hydroxypropyl cellulose, crystalline cellulose, alginic acid, gel, polyvinylpyrrolidone, etc.), lubricants (e.g., magnesium stearate, calcium stearate, talc, etc.), disintegrants (e.g.
  • carboxymethylcellulose calcium, talc, etc. carboxymethylcellulose calcium, talc, etc.
  • diluents e.g., water for injection, saline, etc.
  • additives e.g., stabilizers, preservatives, colorants, flavorings, dissolution aids, emulsifiers, buffers, Isotonic agents, etc.
  • the administration method of the composition, drug or agent may be intraperitoneal injection, intravenous injection, gavage, oral administration or skin application.
  • the amount of administration to a subject varies according to the route of administration, symptoms, age of the patient, etc., and can be determined practically by the clinician.
  • MRL mice 1 and P21-/- mutant mice 2 can close 2 mm (mm) ear holes, but wild-type experimental mice cannot close them. Therefore, ear hole closure can be used as a good model to evaluate regeneration ability.
  • the main screening targets are important signaling pathways that regulate morphogenesis, organ development, immunity and stress responses (metabolism, translation, etc.) during individual development; they also include genes and signaling pathways involved in tumorigenesis and regeneration of lower organisms.
  • mice 7-week-old C57BL/6 mice were selected, anesthetized with 0.5% sodium pentobarbital (100 ⁇ l/10g b.w.), intraperitoneally injected 10mL/kg, and anesthetized according to body weight. The anesthetized mice were bound, and 75% ethanol was used to sterilize the mouse ears and instruments.
  • auricle At the center of the mouse's auricle, use an ear hole punch with a diameter of 2 mm to punch holes in the left and right auricles of the mouse respectively, and administer the drug to the post-traumatic mice through intraperitoneal, intravenous injection or intragastric administration (targeted screening target Dotted small molecules or growth factors were dissolved in physiological saline or DMSO), and drug-free solvents were used as controls.
  • the DMSO-dissolved drug delivery system is: 2-5% DMSO + 30-40% PEG400 + 2-5% Tween 80, added in sequence according to the final concentration (volume ratio). Administration was given every 2 days, and observations were made every 7 days.
  • mice were prepared by the applicant's laboratory, using CRISPR/Cas9-mediated gene knockout technology, by injecting Cas9mRNA and Nsun2 sgRNA into mouse fertilized eggs. Gene knockout embryos were obtained and further bred to obtain stable knockout mice; CHX: (i.e.
  • Example 1 Translation inhibition (Cycloheximide (hereinafter abbreviated as CHX or C); Anisomycin (abbreviated as Ani)) promotes the regeneration of mouse ear piercings with a diameter of 2mm.
  • CHX Cycloheximide
  • Ani Anisomycin
  • FIG. 1A Transcriptome analysis of differential genes between regenerated African agouti and non-regenerated mice (C57BL/6 mice), and through functional enrichment analysis, the results show that regenerated African agouti compared with non-regenerated mice Significantly down-regulated were ribosomal subunits and translation-related.
  • Figure 1B shows that based on the analysis results in Figure 1A, non-regenerative mice were treated with the translated small molecule inhibitors Ani and CHX for 21 days respectively, and the healing effect on the 2mm ear hole was observed.
  • the results showed that compared with the control group (Vehicle ), translation inhibitor Ani (10 mg/kg), and CHX (20 mg/kg) can significantly promote ear piercing healing, and CHX has a better healing effect. It was found that translation inhibition can significantly promote the regeneration of non-regenerative species. Subsequent experiments were carried out based on CHX.
  • the schematic diagram in Figure 1C shows the closure of mouse ear holes after treatment with different concentrations of Vehicle/CHX: CHX at each concentration has a promoting effect on the closure of ear holes with a diameter of 2 mm, and the concentration is greater than 8 mg/kg. After three weeks of treatment, the mouse ear piercing wounds were completely closed. n ⁇ 8.
  • Figure 1D is a photo of the healing status of the 2 mm ear piercing in mice treated with Vehicle/CHX (20 mg/kg) for 30 days. The ear piercing wound treated with CHX has been completely closed.
  • the HE staining picture of mouse auricle tissue in Figure 1E shows that after treatment with CHX (20 mg/kg), the skin, connective tissue and other tissue structures of the ear pierced part of the mouse ear piercing trauma have grown and connected together, and the wound has completely healed. .
  • 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; inflammatory cells can be seen in the dermis of the tissue. There was diffuse infiltration of cells, as shown by arrow 2; hemosiderin deposition could be seen in some cells in the CHX group, as shown by arrow 3.
  • the KI67 immunohistochemical staining of mouse auricle tissue in Figure 1G shows that after 7 days of CHX (20 mg/kg) treatment, the mouse basal layer cells express a large number of KI67, a marker protein for cell proliferation. As shown by the arrow, the expression in the control group is relatively less.
  • 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.
  • FIG. 1I The schematic diagram in Figure 1I shows that mice with ear piercing trauma were treated with different administration methods such as intragastric administration and intraperitoneal injection, and the healing effect was produced after 3 weeks (21 days after injury).
  • n ⁇ 6 mice with ear piercing trauma were treated with different administration methods such as intragastric administration and intraperitoneal injection, and the healing effect was produced after 3 weeks (21 days after injury).
  • ns no significant difference, t test.
  • 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 2 Cycloheximide CHX promotes regeneration and repair of pierced ears with a diameter of 4 mm in mice.
  • FIG. 2A shows the effect of different doses of CHX on the closure of ear holes with a diameter of 4 mm. It was found that 20 mg/kg has a similar effect to MRL/lpr in super-healing mice. It can promote the ear hole wound to become smaller, but cannot completely close it. When the dose Greater than 20mg/kg (125,175mg/kg) can achieve closure of the ear hole.
  • Figure 2B is a photo of ear piercing healing after 90 days of DMSO/CHX drug treatment. It shows that the area of the 4mm ear piercing of mice treated with CHX (125 mg/kg) was significantly reduced.
  • Example 3 Cycloheximide CHX promotes ear piercing regeneration independent of the inhibition of ferroptosis and autophagy.
  • CHX can inhibit ferroptosis and autophagy in addition to inhibiting eukaryotic protein synthesis and RNA synthesis.
  • small molecules were verified for ferroptosis and autophagy respectively.
  • 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.
  • the effects of different inhibitors on the healing of 2mm ear holes were identified. The results are shown in Figure 3: Ferroptosis and autophagy inhibitors were not able to promote the healing of ear holes like CHX, which shows that CHX promotes ear hole healing. Hole regeneration is independent of its inhibitory activity on ferroptosis or autophagy.
  • Example 4 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 hole closure and cut area regeneration.
  • 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 hole closure and cut area regeneration.
  • mice 7-week-old mice as an example, 0.5% sodium pentobarbital (100 ⁇ l/10 gb.w.) was used to test the mice. Apply anesthesia, intraperitoneally inject 10mL/kg, and anesthetize 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 4A shows the closure of mouse ear holes after DMSO/CRB treatment. 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 4B 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 4C 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 4D 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.
  • FIG. 4E tissue staining shows the regeneration of various tissues after drug (CRB) induction for more than 180 days, showing complete regeneration, mainly including the regeneration of epidermis, dermis, glands, hair follicles, muscles, cartilage, fat, muscles, etc.
  • CRB drug
  • FIG. 4F Immunofluorescence staining of the vascular marker CD31 further identifies vascular regeneration, and triangles indicate regenerated blood vessels.
  • CRB combined treatment can induce the regeneration of epidermis, dermis, glands, hair follicles, muscles, cartilage, fat, muscles and blood vessels.
  • Example 5 Protein synthesis inhibitor CHX inhibits lung injury fibrosis and promotes lung regeneration.
  • a chronic injury model of pulmonary fibrosis was successfully constructed by instilling bleomycin (BLM) into the trachea of C57B1/6 mice, and treated with intraperitoneal administration of CHX, verifying that CHX inhibits pulmonary fibrosis and promotes lung regeneration and microbial growth. The effect of mouse survival was determined, and the clinical anti-idiopathic pulmonary fibrosis drug nintedanib was selected as a positive control.
  • BBM bleomycin
  • Bleomycin dilution dilute 50 times of mother solution 50 mg/ml to a final concentration of 1 mg/ml;
  • mice Anesthetize mice by intraperitoneal injection of 0.5% sodium pentobarbital (100 ⁇ l/10g b.w.);
  • mice were intraperitoneally administered CHX (C, 20 mg/kg, once every two days) 2 days after surgery (Figure 5A). Record the survival status of mice. The results are shown in Figure 5B.
  • the control group (V) all died on the 18th day of treatment, while the survival rate of the CHX treatment group on the 18th day of treatment was 75%.
  • Body weight tracking test found that after mycin-induced lung injury, the weight of the control group gradually decreased, by 30% after 15 days, while CHX significantly inhibited weight loss (***P ⁇ 0.001), and was basically maintained at 85% after 15 days (as shown in Figure 5C Show).
  • RNA-Seq sequencing analysis showed that compared with the wild-type (WT) control group after injury, fibrosis-related genes such as extracellular matrix and collagen were significantly up-regulated, while the CHX-treated group could significantly down-regulate these genes, which are related to the development of blood vessels and circulatory systems.
  • Example 6 CHX reduces scar formation and promotes skin regeneration in mice after burns.
  • mice Anesthetize mice by intraperitoneal injection of 0.5% sodium pentobarbital (100 ⁇ l/10 gb.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 being scalded, the subjects were divided into two groups and treated with Vehicle (normal saline) and CHX (20 mg/kg, dissolved in normal saline) respectively. (5) Detect wound repair status.
  • Vehicle normal saline
  • CHX 20 mg/kg, dissolved in normal saline
  • CHX can significantly reduce skin regeneration after burns, mainly including the regeneration of epidermis, hair follicles, glands, dermis, and blood vessels.
  • Example 7 Narciclasine and combination with NRB promote the regeneration of complex structures in mice.
  • Puromycin (abbreviated as Puro) was involved in the experiment.
  • the Puro labeling method refers to the literature "Kearse, et al. Ribosome queuing enables non-AUG translation to be resistant to multiple protein synthesis inhibitors, 2019, Genes&Development”.
  • Figure 7B shows the healing status of mouse ear holes with 2 mm diameter ear punch wounds treated with Narciclasine for 30 days.
  • Figure 7C shows the regeneration effects of different doses of Narciclasine on mouse ear piercings.
  • Figures 7D and 7E show histochemistry and Masson section staining data respectively.
  • the regeneration structure of cartilage (long black arrow), hair follicle (asterisk), gland/sebaceous gland (triangular arrow) and other structures is characterized. From the figure, multiple centers of cartilage can be seen. It is speculated that such multiple starting points for regeneration will greatly speed up regeneration. speed.
  • 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-(N-(D)) 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 7F to 7H.
  • Figure 7F 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 7G and 7H 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 box area) and other structures. .
  • Example 8 Pancrastatin promotes regeneration of pierced ears in mice.
  • mice with 2mm ear hole trauma with 2 mg/kg Pancrastatin (dissolved in DMSO, administration system 2-5% DMSO+30-40% PEG400+2-5% Tween80+physiological saline).
  • Adopt intraperitoneal injection of DMSO without drug as the control group (labeled Vehicle). Characterization of injured auricles in drug-treated mice. The specific experimental results are shown in the figure Shown in 8B ⁇ 8E.
  • Figure 8B shows the effect of pancratistatin administration on promoting the healing of the ear piercing in the treatment group 21 days after the ear piercing injury, showing that the ear piercing is completely closed.
  • Figure 8C shows the closure of the ear holes after Pancratistatin treatment for 21-28 days.
  • the results of Figures 8D (HE staining) and 8E (Masson staining) show that the mouse ear piercing wound is completely closed and identified as a regeneration event.
  • the staining data better shows multiple cartilage development centers (long black arrows), hair follicles ( Regenerative structures of structures such as asterisks), glands/sebaceous glands (triangular arrows), and muscles (areas selected by dotted lines).
  • Example 9 The primary extract of the Amaryllidaceae plant of the genus Amaryllis promotes the regeneration of the 2mm ear piercing.
  • Hymenocallis littoralis (Jacq.) Scalisb.) is a plant of the genus Hymenocallis in the Amaryllidaceae family. Banana bulbs are rich in pancratistatine and narciclasine. Therefore, it is further verified whether the extract of Amaranthus japonica can promote the in-situ regeneration of ear piercings.
  • Figure 9E shows the statistical results of the ear hole area after 9 weeks of administration, showing the regeneration promotion of the two extracts. There is a very significant difference in the effect.
  • the extract obtained by homogenizing and breaking the wall using 70% ethanol extraction method can significantly promote the healing of ear piercings.
  • Figure 9F shows the statistical graph of the ear piercing area after treatment with 70% ethanol extraction method of homogenized wall-broken extract for 3 weeks, 6 weeks, and 9 weeks respectively. A gradual healing trend can be seen. And the thickness of the epidermis of the mouse auricle tissue is close to that of the wild type, n ⁇ 3, t test.
  • Example 10 Cycloheximide CHX activates the STING-TBK1-IRF3-interferon-stimulated genes (ISGs) pathway, and ISG gene expression is necessary for CHX-induced regeneration.
  • CHX CHX was used to treat mouse primary fibroblasts and macrophages respectively, and large-scale RNA-seq (transcription level) and Ribo-seq (ribosome imprint sequencing, translatomics) were performed. ).
  • RNA-seq transcription level
  • Ribo-seq ribosome imprint sequencing, translatomics
  • Example 11 Optimized combination of cycloheximide CHX (C) and all-trans retinoic acid RA (R) promotes regeneration and repair of the outgrowth structure in mice
  • mice 8-week-old ICR mice as a model, the 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.
  • 75% ethanol was used to sterilize the mouse's left upper limb and surgical instruments.
  • 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. The simplest way to distinguish them is The radius is on the thumb side and the ulna is on the little finger side).
  • FIG. 11A is a schematic diagram of extremity modeling.
  • Figure 11B shows the induced regeneration phenomenon of the limbs 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. Scale bar is 2mm.
  • Figure 11C shows the statistical results of the extremity outgrowth length.
  • FIG. 11D 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.
  • CT computerized tomography
  • FIG. 11E is more direct key evidence of bone regeneration. After 30 days of drug CR treatment, the complete radius and ulna were removed for observation. The observation is consistent with the CT scan results, and the regeneration trend of bone tissue can be clearly seen.
  • Figure 11F shows the Masson staining results of the mouse acral regenerated tissue after drug treatment for 21, 50, and 120 days.
  • 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.
  • the regeneration of muscle tissue was observed in the 120-day staining, while the control group showed disordered accumulation, and the expression pattern was similar to scar formation.
  • Scale bar is 2mm, n ⁇ 3, t test.

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Abstract

蛋白质合成抑制剂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，蛋白质合成抑制剂为环己酰亚胺或石蒜科提取物中任一种可促进哺乳动物组织器官再生的小分子化合物。

Description

一种诱导哺乳动物原位再生的方法及其应用 技术领域
本发明涉及生物技术领域，具体涉及一种小分子化合物诱导哺乳动物原位再生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、退行性神经系统疾病、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，其特点是用大量结缔组织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哺乳动物组织或复杂结构或器官再生修复能力的化合物，具体的，该化合物为蛋白质合成抑制剂。
本申请提供了所述蛋白质合成抑制剂或包含所述蛋白质合成抑制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。
本申请提供了所述蛋白质合成抑制剂或包含所述蛋白质合成抑制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。
本申请提供了所述蛋白质合成抑制剂或包含所述蛋白质合成抑制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，本申请所述复杂结构为耳朵、肢体、手指、眼或鼻。
优选地，本申请所述器官为肺、肝、心、胰岛或肾。
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、肌肉等组织器官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、衰老或不明原因脱发、软骨肌肉损伤、肝纤维化或肺纤维化。
本申请所提供的所述蛋白质合成抑制剂，在促进组织器官再生修复能力的应用中，是通过诱导生命静息实现的。
本申请所提供的所述蛋白质合成抑制剂，在促进哺乳动物组织及复杂结构及器官再生修复能力的应用中，是通过激活STING-TBK1-IRF3信号实现的。
本申请所提供的所述蛋白质合成抑制剂可以为环己酰亚胺(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。
优选地，所述石蒜科植物提取物或石蒜科生物碱为水仙环素(Narciclasine)或水鬼蕉碱(Pancratistatin)。
优选地，本申请提供的包含所述蛋白质合成抑制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蛋白质合成抑制剂或其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蛋白质合成抑制剂或包含蛋白质合成抑制剂的组合物。
进一步的，所述方法中的蛋白质合成抑制剂或包含蛋白质合成抑制剂的组合物即为本申请中所提供的蛋白质合成抑制剂或包含蛋白质合成抑制剂的组合物。
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蛋白质合成抑制剂或包含蛋白质合成抑制剂的组合物。
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Vehicle/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，90天后的闭合情况示意图，比例尺为1mm。
图3为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4A为经Vehicle/CRB处理后，小鼠耳洞的愈合情况示意图。
图4B为小鼠4mm耳洞经Vehicle/CRB处理30天后的愈合效果图。
图4C为经Vehicle/CRB处理后小鼠耳廓组织HE染色图。
图4D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4E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4F免疫荧光染色血管标记物CD31染色结果，比例尺为20μm。
图5A为对肺纤维损伤模型小鼠进行CHX给药实验流程图。
图5B为对肺纤维损伤模型小鼠存活率示意图，Vehicle(V)/CHX(C)。
图5C为肺纤维损伤模型小鼠体重变化示意图。
图5D、5E为肺纤维损伤模型小鼠呼吸频率和一次换气量变化示意图。
图5F为肺纤维损伤模型小鼠HE染色和天狼星红染色结果示意图。
图5G为肺纤维损伤模型小鼠纤维化相关基因测序结果示意图。
图5H为肺纤维损伤模型小鼠呼吸系统发育相关基因测序结果示意图。
图5I、5J为肺纤维损伤模型小鼠损伤9天给药Vehicle/CHX后的HE染色结果示意图。
图6A为烫伤模型小鼠疤痕面积变化示意图。
图6B为烫伤模型小鼠天狼星红染色结果示意图。
图6C为烫伤模型小鼠组化染色结果示意图。
图7A为Puromycin(PURO)参入实验验证Narciclasine(Nar)具有翻译抑 制作用实验结果示意图。
图7B为直径2mm耳洞打孔器创伤的小鼠耳洞经Nar药物处理30天的闭合情况示意图，比例尺为1mm。
图7C为经Vehicle与不同剂量的Nar药物处理后，直径2mm的小鼠耳洞闭合情况示意图。
图7D为经Nar药物处理后的免疫组化结果示意图。
图7E为经Nar药物处理后的Masson切片染色结果示意图。
图7F为经Vehicle/NRB处理后，小鼠4mm耳洞的愈合情况示意图。
图7G为经NRB处理后的免疫组化结果示意图。
图7H为经NRB处理后的，HE切片染色结果示意图。
图8A为Puromycin参入实验验证Pancratistatin具有翻译抑制作用实验结果示意图。
图8B为直径2mm耳洞打孔器创伤的小鼠耳洞经Pan药物处理21天的闭合情况示意图，比例尺为1mm。
图8C为直径2mm耳洞打孔器创伤的小鼠耳洞经Pan药物处理21天效果统计图，DMSO替代Pan处理为对照。
图8D为经Pan药物处理后的HE切片结果示意图。
图8E为经Pan药物处理后的HE切片结果示意图。
图9A、9B为待处理水鬼蕉鳞茎示意图。
图9C、9D为鳞茎及组织匀浆后获得的母液示意图。
图9E为给药9周后的耳洞面积统计结果示意图。
图9F为利用70％乙醇萃取法匀浆破壁提取物分别处理3周、6周、9周后耳洞面积统计图。
图10A为CHX导致转录和翻译水平共同上调基因分析示意图。
图10B为干扰素响应基因及富集情况示意图。
图10C、10D为定量qPCR检测CHX诱导ISG基因表达实验结果示意图。
图10E为免疫荧光染色鉴定CHX激活STING/TBK1/IRF3信号通路结果示意图。
图10F为定量qPCR检测GSK86126与Des抑制STING/TBK1/IRF3通路活性抑制CHX诱导ISG基因表达结果示意图。
图10G为GSK86126与Des抑制STING/TBK1/IRF3通路活性抑制CHX诱导的再生结果示意图。
图10H为蛋白Westernblot实验揭示Des抑制STING/TBK1/IRF3通路活性并不抑制CHX蛋白合成抑制功能结果示意图。
图11A为肢端造模的模式图。
图11B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11C为肢端outgrowth长度的统计结果。n≥3，***p<0.001，t检验。
图11D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11E为药物CR处理30后骨组织的再生结果。
图11F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。
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。
本申请主要涉蛋白质合成抑制剂或包含所述蛋白质合成抑制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。
蛋白质合成抑制剂是一类影响蛋白质生物合成的物质，它们可以作用于DNA复制和RNA转录，对蛋白质的生物合成起间接作用，可作用于蛋白质合成的各个环节，包括抑制起始因子，延长因子及核糖核蛋白体的作用等等。其中，常见的蛋白质合成抑制剂主要为可以抑制蛋白质生物合成翻译过程的阻断剂等。
在一些具体的实施方式中，所述蛋白质合成抑制剂可以为环己酰亚胺(Cycloheximide(CHX))、膜海鞘素B(Didemnin B(DIDB))和波凡霉素(Bouvardin(BVD))。
在本申请一个具体的实施方式中，所述蛋白质合成抑制剂为环己酰亚胺，缩写为CHX，其化学式为：4-((R)-2-((1S,3S,5S)-3,5-二甲基-2-氧基环己基)-2-羟基乙基)哌啶-2,6-二酮(4-((R)-2-((1S,3S,5S)-3,5-dimethyl-2-oxocyclohexyl)-2-hydroxyethyl)piperidine- 2,6-dione)
CHX是一种常用的蛋白合成抑制剂，可抑制真核生物的蛋白合成，其主要作用于蛋白质合成中的翻译过程。
在另一个具体的实施方式中，所述蛋白质合成抑制剂为石蒜科草本植物生物碱。
在一个优选的实施方式中，所述石蒜科草本植物生物碱为水仙环素(Narciclasine)；在另一个优选的实施方式中，所述石蒜科草本植物生物碱为水鬼焦碱(Pancratistatin)。
水仙环素(Narciclasine，缩写为Nar)是石蒜科中所富含的一种生物碱，被报道也具有抑制蛋白质翻译过程的作用，且翻译延伸因子eEF1A被发现作为Narciclasine新的直接靶点。水鬼蕉碱(Pancratistatin，缩写为Pan)是一种水仙环素类似物。
在另一个具体的实施方式中，所述蛋白质合成抑制剂为石蒜科植物提取物。在一个优选的实施方式中，所述石蒜科植物为水鬼蕉(学名：Hymenocallis littoralis(Jacq.)Scalisb.)。水鬼蕉鳞茎富含水鬼蕉碱(pancratistatine)与水仙环素(narciclasine)等。
在一个优选的实施方式中，所述石蒜科植物提取物为石蒜科植物的醇提物。
在一个优选的实施方式中，所述醇提物为采用乙醇萃取法匀浆破壁提取得到的。
本申请还提供一种组合物，其包括蛋白质合成抑制剂、全反式视黄酸和BMP激活剂。
在一个优选的实施方式中，所述蛋白质合成抑制剂为环己酰亚胺(Cycloheximide(CHX))，所述BMP激活剂为BMP signaling agonist sb4。
在一个优选的实施方式中，所述组合物包括环己酰亚胺、BMP signaling agonist sb4和全反式视黄酸。
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。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。
BMP signaling agonist sb4是一种苯并恶唑骨形态发生蛋白4(BMP4)信号激动剂，通过稳定细胞内p-SMAD-1/5/9激活BMP信号。其还能激活典型BMP信号通路中的BMP4靶基因(DNA结合抑制剂，Id1和Id3)。BMP信号通路可以调节细胞的增殖和分化,在胚胎发育过程中具有重要的调控作用。
在一个具体的实施方式中，所述组合物中包括环己酰亚胺5-40mg/kg、全反式视黄酸10-40mg/kg和BMP signaling agonist sb4 10-20mg/kg，即包括给药量为5-40mg/kg的环己酰亚胺，例如可以为5、10、15、20、25、30、35、40mg/kg；给药量为10-40mg/kg的全反式视黄酸，例如可以为10、15、20、25、30、35、40mg/kg；给药量为10-20mg/kg的BMP signaling agonist sb4，例如可以为10、12、15、18、20mg/kg。
在另一个具体的实施方式中，所述组合物中包括水仙环素(Narciclasine)0.5-3mg/kg、全反式视黄酸10-50mg/kg和BMP signaling agonist sb4 20-50mg/kg，即包括给药量为5-40mg/kg的环己酰亚胺，例如可以为0.5、1、1.5、2、2.5、3mg/kg；给药量为10-50mg/kg的全反式视黄酸，例如可以为10、15、20、25、30、35、40、45、50mg/kg；给药量为20-50mg/kg的BMP signaling agonist sb4，例如可以为20、25、30、35、40、45、50mg/kg。
在一个具体的实施方式中，所述组合物中还包含药学上可用的赋形剂。
在本申请中，所述蛋白质合成抑制剂或包含所述蛋白质合成抑制剂的组合物可用于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。
在本申请中，所述蛋白质合成抑制剂或包含所述蛋白质合成抑制剂的组合物可用于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。
在本申请的实施方式中，所述再生修复是指生物体的整体、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。
在本申请的实施方式中，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诱导生命静息实现的。
在一个具体的实施方式中，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。
在一个具体的实施方式中，所述再生修复中的所述组织为表皮、真皮、肌肉、骨骼、脂肪、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、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、皮肤创伤、皮肤烧伤、脱发、器官的纤维化、肌肉/软骨损伤或神经系统疾病等，优选为皮肤烧/烫伤、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系统疾病等。
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、阴道栓剂)、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，阿拉伯胶，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0.5％戊巴比妥钠(100μl/10g b.w.)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，结缔组织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，真皮下结缔组织间隙增大，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(Vehicle，V)。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，还可以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。其中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：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0.5％戊巴比妥钠(100μl/10gb.w.)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4A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4B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4C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4D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4E组织染色显示经药物(CRB)诱导大于180天后各个组织再生情况，显示完整再生，主要包括表皮、真皮、腺体、毛囊、肌肉、软骨、脂肪、肌肉等的再生。“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4F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。
上述结果显示CRB联合处理可诱导包括表皮、真皮、腺体、毛囊、肌肉、软骨、脂肪、肌肉与血管等的再生。
实施例五：蛋白质合成抑制剂CHX抑制肺损伤纤维化促进肺再生。
采用博来霉素(bleomycin，BLM)肺气管滴注C57B1/6小鼠成功构建肺纤维化的慢性损伤模型，并通过腹腔给药CHX进行治疗，验证CHX抑制肺纤维化及促进肺再生和小鼠存活的作用，并选取临床上抗特发性肺纤维化药物尼达尼布作为阳性对照。
肺纤维化慢性损伤模型构建：
(1)博来霉素稀释：母液50mg/ml稀释50倍终浓度为1mg/ml；
(2)腹腔注射0.5％戊巴比妥钠(100μl/10g b.w.)麻醉小鼠；
(3)75％酒精颈部皮肤消毒，切开颈部皮肤后钝性分离气管上粘膜与肌肉，暴露气管，注意勿损伤甲状腺；
(4)用胰岛素注射器按2.5mg/kg剂量于气管软骨间隙注入50μl(20g体重)博来霉素，拔出针头后，立即将操作台直立，左右旋转1min，缝合皮肤，小鼠自然苏醒后自由饮水进食。
实验方法：术后2天小鼠腹腔给药CHX(C,20mg/kg，每两天一次)(图5A)。记录小鼠的生存状态。结果如图5B所示，对照组(V)在治疗18天已全部死亡，而CHX治疗组在治疗第18天的存活率在75％。统计分析CHX显著提高博来霉素诱导肺损伤的存活率(P＝0.002)，每组至少8只小鼠。体重跟踪检测发现霉素诱导肺损伤后，对照组体重逐渐下降，15天后下降30％,而CHX显著抑制体重下降(***P＜0.001)，15天后基本维持在85％(如图5C所示)。进一步检测肺功能，发现CHX显著维持了正常的呼吸频率和一次换气量(Tidalvolume)(如图5D,5E所示)。HE染色和天狼星红染色结果显示，处理14天后，对照组肺组织发生大量损伤和纤维化，而CHX组显著抑制纤维化(如图5F所示)。进一步通过RNA-Seq测序分析显示损伤后对照组相比于野生型(WT)胞外基质、胶原等纤维化相关基因显著上调，而CHX处理组能显著下调这些基因，血管，循环系统等发育相关基因上调(如图5G所示)。进一步分析发现CHX促进呼吸系统发育相关基因上调(如图5H所示)。以上实验结果说明CHX抑制了纤维化，促进了肺损伤后再生。进一步，在博来霉素损伤9天后(更严重的肺纤维化模型)进行CHX给药治疗，发现类似的CHX处理显著回复组织完整性，降低纤维化(图5I,5J)。
上述结果显示CHX可以显著降低肺损伤后纤维化，并促进肺再生。
实施例六：CHX降低小鼠烫伤后疤痕形成，促进皮肤再生。
实验方法：(1)腹腔注射0.5％戊巴比妥钠(100μl/10gb.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(生理盐水)和CHX(20mg/kg，溶解生理盐水)给药处理。(5)检测伤口修复情况。
结果：给药40天后，剃毛检测疤痕，显示CHX处理后显著降低疤痕面积(如图6A所示)，天狼星红染色结果显示CHX降低了胶原的堆积(如图6B所示)，进一步组化染色显示CHX显著促进表皮、毛囊、腺体、真皮、血管的再生(如图6C所示)。
上述结果显示CHX可以显著降低烫伤后皮肤再生，主要包括表皮、毛囊、腺体、真皮、血管的再生。
实施例七：水仙环素(Narciclasine)及组合NRB促进小鼠的复杂结构再生。
(1)水仙环素(Narciclasine)的翻译抑制功能验证。
实验方法：嘌呤霉素(Puromycin，缩写为Puro)参入实验，Puro标记方法参考文献《Kearse,et al.Ribosome queuing enables non-AUG translationto be resistantto multiple protein synthesis inhibitors,2019,Genes&Development》。
结果如图7A所示。其中，Cycloheximide(CHX)为对照，“+”代表添加，“-”代表不添加。实验结果表明水仙环素(Narciclasine)可显著抑制蛋白翻译过程。
(2)水仙环素促进小鼠2mm耳洞割处再生。
实验方法：对2mm耳洞创伤小鼠以不同剂量(1-3mg/kg)Narciclasine(溶于DMSO，给药体系2-5％DMSO+30-40％PEG400+2-5％Tween80+生理盐水)分别采取腹腔注射给药，不含药物的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7B～7E所示。
其中，图7B的示意图显示了直径2mm耳洞打孔器创伤的小鼠耳洞经Narciclasine药物处理30天的愈合情况。图7C显示了不同剂量的Narciclasine对于小鼠耳洞的再生效果。图7D、7E分别为组化与Masson切片染色数据 表征了软骨(黑色长箭头)、毛囊(星号)、腺体/皮脂腺(三角形箭头)等结构的再生结构，从图中可看到软骨的多个发生中心，推测这样多起点再生大大加快再生速度。
以上实验结果显示，不同剂量的Narciclasine均可显著促进2mm耳洞愈合，闭合耳洞可以再生出毛囊，腺体，软骨和肌肉等组织和组织衍生物，证实了Narciclasine的再生促进作用。
(3)组合NRB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7F～7H所示。
其中，图7F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7G，7H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。
实施例八：水鬼蕉碱(Pancratistatin)促进小鼠耳洞割处再生。
(1)水鬼蕉碱(Pancratistatin)的翻译抑制功能验证。
实验方法：嘌呤霉素参入实验，同实施例七。实验结果如图8A所示，表明水鬼蕉碱(Pancratistatin)可显著抑制蛋白翻译过程。
(2)水鬼蕉碱(Pancratistatin)促进小鼠2mm耳洞割处再生。
实验方法：对2mm耳洞创伤小鼠以2mg/kg水鬼蕉碱(Pancratistatin)(溶于DMSO，给药体系2-5％DMSO+30-40％PEG400+2-5％Tween80+生理盐水)分别采取腹腔注射给药，不含药物的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 8B～8E所示。
其中，图8B为耳洞损伤后21天时处理组Pancratistatin给药对促进耳洞愈合的效果，显示耳洞完全闭合。图8C为Pancratistatin处理21-28天，观察耳洞闭合情况。图8D(HE染色)、8E(Masson染色)的结果显示，小鼠耳洞创口完全闭合，且鉴定为再生事件，染色数据较好地显示了软骨多个发生中心(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。
以上结果显示，Pancratistatin小分子可以促进耳洞闭合，并促进再生。
实施例九：石蒜科水鬼蕉属植物初提物促进2mm耳洞割处再生。
水鬼蕉(学名：Hymenocallis littoralis(Jacq.)Scalisb.)，石蒜科水鬼蕉属植物。水鬼蕉鳞茎富含水鬼蕉碱(pancratistatine)与水仙环素(narciclasine)等。因此，进一步验证认为水鬼蕉的提取物是否可以起到促进耳洞原位再生的效果。
实验方法：
(1)将野生型石蒜科水鬼蕉属植物水鬼蕉的鳞茎除根除叶，清洗干净获得80g干净的鳞茎，将干净鳞茎切成1cm×1cm×1cm的小方块，方便破壁匀浆，将鳞茎分成相等的两部分，分别利用70％乙醇、10％DMSO萃取法匀浆破壁提取。具体方法为将鳞茎分别放入小型组织匀浆机，加入等体积(1:1)的70％乙醇或10％DMSO，接通电源，将组织打成匀浆，室温静置48h，用干净滤布过去除去残渣，过滤后的滤液为可使用的提取母液，4℃存储，备用。图9A、9B为待处理水鬼蕉鳞茎，图9C、9D为鳞茎组织匀浆后获得的母液。
(2)以8周龄小鼠为模型，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。对于创伤后的小鼠，采用喂水方式分别以步骤(1)中得到的两种提取母液给药，给药浓度选用提取的母液:水＝1:5的稀释，并且每7天对小鼠进行麻醉，测量并计算小鼠耳洞的面积，对药物处理过的小鼠创伤耳廓进行进一步鉴定。
图9E为给药9周后的耳洞面积统计结果，显示两种提取物的促进再生 效果存在极显著差异，利用70％乙醇萃取法匀浆破壁得到的提取物显著促进耳洞愈合。图9F显示利用70％乙醇萃取法匀浆破壁提取物分别处理3周、6周、9周后耳洞面积统计图，可以看到逐渐愈合趋势。并且小鼠耳廓组织表皮厚度接近野生型，n≥3，t检验。
以上结果显示，石蒜科水鬼蕉属植物鳞茎的乙醇抽提物可以促进再生。
实施例十：环己酰亚胺CHX激活STING-TBK1-IRF3-干扰素刺激基因(Interferon-stimulated genes,ISGs)通路，且ISG基因表达是CHX诱导再生所必需的。
为了进一步验证CHX诱导再生的机制，利用CHX分别处理小鼠原代成纤维细胞和巨噬细胞，并进行大样本量RNA-seq(转录水平)和Ribo-seq(核糖体印记测序，翻译组学)。通过生信息学分析发现CHX导致两类细胞转录水平和翻译水平共同上调基因32个(G1，图10A)，功能分析发现这些基因为干扰素响应基因，功能主要富集到响应干扰素和病毒(图10B)。进一步通过qPCR实验验证CHX确实能够上调经典ISG表达，如Mx2,Ifit1,Cxcl10,Ifih1等(图10C显示成纤维细胞ISG基因表达，图10D显示巨噬细胞ISG基因表达)。为了验证ISG基因表达是否是CHX诱导再生所必需的，我们首先在体外发现CHX通过激活STING-TBK1-IRF3信号通路激活ISG，主要体现STING蛋白核周富集，IRF3蛋白入核(图10E)，且利用小分子抑制剂GSK8612(5μM)或Dexamethasone(Dex,10μM)抑制TBK1能显著抑制能显著CHX(1μg/ml)诱导的ISG基因的表达(图10F)，在体内抑制TBK1(GSK8612(25mg/kg)，Dexamethasone(Dex,10mg/kg))能显著抑制CHX(20mg/kg)诱导的耳洞再生(图10G)。此外通过Puromycin(PURO)参入实验(见参考文献《Kearse,et al.Ribosome queuing enables non-AUG translation to be resistant to multiple protein synthesis inhibitors,2019,Genes&Development》)发现Dexamethasone并不影响CHX对整体翻译的抑制，预示CHX介导的翻译抑制位于诱导ISG的上游(图10H)。以上说明ISG基因表达是CHX诱导再生所必需的。
实施例十一：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隆起结构(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。
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11所示。图11A为肢端造模的模式图。图11B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11C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11D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11E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。图11F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，且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的组合物促进肢端切除后隆起结构的再生修复，包括骨、肌肉组织等再生。
前面仅仅示出了本申请的原理，应理解，本申请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，在不脱离本申请技术原理的前提下，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申请的范围。

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  1. 蛋白质合成抑制剂或包含蛋白质合成抑制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. 蛋白质合成抑制剂或包含蛋白质合成抑制剂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. 蛋白质合成抑制剂或包含蛋白质合成抑制剂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. 如权利要求1-3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。
  5. 根据权利要求1-3中任一项所述的应用，其特征在于，
    所述组织为皮肤、肌肉、软骨、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。
  6. 根据权利要求5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。
  7. 根据权利要求1-3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、肌肉的再生以及血管、神经与肢体损伤后的再生。
  8. 根据权利要求3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。
  9. 如权利要求1-8中任一项所述的应用，其特征在于，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。
  10. 根据权利要求9所述的应用，其特征在于，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)。
  11. 根据权利要求1-10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激活STING-TBK1-IRF3信号实现的。
  12. 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。
  13. 根据权利要求12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。
  14. 根据权利要求12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。
  15. 根据权利要求12～14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。
  16. 如权利要求1-11中任一项所述的应用，其特征在于，所述组合物为权利要求12-15中任一项所述的组合物。
  17. 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蛋白质合成抑制剂或包含蛋白质合成抑制剂的组合物。
  18. 根据权利要求17所述的方法，其中，蛋白质合成抑制剂或包含蛋白质合成抑制剂的组合物为权利要求1～11中所涉及的蛋白质合成抑制剂或包含蛋白质合成抑制剂的组合物或权利要求12-15中任一项所述的组合物。
PCT/CN2023/100867 2022-06-16 2023-06-16 一种诱导哺乳动物原位再生的方法及其应用 WO2023241716A1 (zh)

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