CN117244070A

CN117244070A - Use of Nr4a1 agonist/overexpression formulations in osteogenesis

Info

Publication number: CN117244070A
Application number: CN202311335064.2A
Authority: CN
Inventors: 徐佳佳; 张忠民; 高阳帅
Original assignee: Southern Hospital Southern Medical University
Current assignee: Southern Hospital Southern Medical University
Priority date: 2023-10-16
Filing date: 2023-10-16
Publication date: 2023-12-19

Abstract

The invention provides application of an Nr4a1 agonist/overexpression preparation in preparation of a medicine/preparation for relieving or reversing immune rejection caused by mesenchymal stem cell BMSCs and biomaterial treatment.

Description

Use of Nr4a1 agonist/overexpression formulations in osteogenesis

Technical Field

The present invention relates to the field of biology and pharmacy, in particular to the use of Nr4a1 agonist/overexpression formulations in osteogenesis.

Background

Musculoskeletal disease is a major cause of global disability, severely limiting mobility and flexibility. In the united states alone, more than 200 tens of thousands of procedures are used annually to repair damaged bones. The number of patients with limb dysfunction caused by bone defects in China per year is up to 1,000 ten thousand. Bone injury repair is a multi-tissue-involved, multicellular, inter-regulated process.

As far as the existing studies are concerned, mesenchymal Stem Cells (MSCs) can differentiate into osteoblastic progenitor cells, which serve as a main source of osteoblasts and play an important role in bone regeneration. Repair of large area bone defects remains a challenge for orthopedics because these defects are not self-repairing. Tissue repair based on stem cells and biological materials is a very promising therapeutic approach. However, when biomaterial-loaded bone marrow mesenchymal stem cells (BMSCs) are implanted into the body for bone repair, the implant stimulates the host immune response in the body and causes an acute inflammatory response as a long-term inflammatory stimulus, prolonging the inflammatory phase of tissue repair. Immune cells are recruited to the surface of biological material, interact with implants and release inflammatory factors, inhibiting osteogenic differentiation of BMSCs and leading to tissue fibrosis.

It was found that in C57BL/6J mice, the cytokine with highest local expression of the implant is transforming growth factor- β1 (TGF- β1), which is expressed at a much higher level than other inflammatory factors. TGF-. Beta.1 is also an immunomodulator that regulates immune cell function and affects the local immune microenvironment. TGF- β1 can promote bone remodeling at the appropriate time and at the appropriate location; however, in pathological conditions, high doses of TGF- β1 can inhibit the expression of bone morphogenic protein 2 (Bmp 2) and impair BMSCs-mediated bone formation. Similar phenomena were also observed by other researchers. TGF-beta 1 levels in aged mouse and human bone samples are increased, TGF-beta 1 inhibits differentiation of mesenchymal progenitor cells into osteoblasts by degrading TNF receptor-related factor 3, and aging cells are found to release a large amount of TGF-beta 1 by age-related osteoporosis Lianping Xing and the like, so that functions of the mesenchymal progenitor cells are hindered. Thus, reducing the overactive TGF- β1 signal may promote bone formation in vivo. However, TGF- β1 signaling pathways play a critical role in tissue development and maintenance of body function, and systemic inhibition of TGF- β1 function can produce strong adverse and side effects.

In summary, since the bone marrow mesenchymal stem cells have a potential and therapeutic effect for bone formation differentiation damaged by strong inflammatory reaction caused by immune rejection and infection, how to solve the problem of immune rejection caused by stem cell therapy, and how to find an effective method for improving BMSCs differentiation and bone repair have yet to be elucidated, which is also a key problem to be rapidly solved for promoting clinical application of stem cells.

Disclosure of Invention

The present invention aims to overcome the above drawbacks, and through further research, a reliable and safe molecular target and therapy are determined to antagonize TGF- β1 overactivation and promote bone regeneration in vivo. The method comprises the following steps:

the invention provides application of an Nr4a1 agonist/over-expression preparation in preparing a medicine/preparation for relieving or reversing immune rejection caused by mesenchymal stem cell BMSCs and biological material treatment.

The invention provides application of an Nr4a1 agonist/overexpression preparation in preparation of a medicament/preparation for promoting bone formation differentiation of mesenchymal stem cells (BMSCs).

The invention provides the use of an Nr4a1 agonist/overexpression formulation for the manufacture of a medicament/formulation for antagonising osteogenesis inhibition caused by excessive TGF- β1 activation.

The invention provides application of an Nr4a1 agonist/overexpression preparation in preparation of a medicament/preparation for treating/relieving inflammatory bone diseases.

The invention provides the use of an Nr4a1 agonist/overexpression formulation for the preparation of a medicament/formulation for slowing or reversing matrix mineralization.

The invention provides application of an Nr4a1 agonist/over-expression preparation in preparing a medicine/preparation for promoting ectopic osteogenesis in vivo.

The invention provides application of an Nr4a1 agonist/overexpression preparation in preparing a medicament/preparation for accelerating/promoting skull defect repair.

The invention provides application of an Nr4a1 agonist/over-expression preparation in preparation of a medicament/preparation for promoting fracture healing.

In addition, the invention also indicates that the dosage of the Nr4a1 agonist/over-expression preparation is not higher than 10mg/kg.

In addition, the invention also teaches that the Nr4a1 agonist/overexpression formulation can be replaced by an agonist or expression formulation targeting Wnt4 and Wnt pathway.

The invention has the following functions and effects:

in the study of the invention, the over-expression of Nr4a1 can not only reverse the osteogenic differentiation inhibited by high-dose TGF-beta 1, but also directly promote BMSC osteogenesis. Nr4a1 promotes transcription of Wnt proteins, thereby activating Wnt/beta-catenin signaling pathway and enhancing osteogenic differentiation of BMSCs.

In addition, nr4a1 was overexpressed in BMSCs using gene therapy or with agonists thereof, such as: cytosporane B (Csn-B) and other treatments promote bone regeneration and bone healing.

Therefore, the research result of the invention shows that Nr4a1 can block excessive TGF-beta 1 signal transduction and accelerate bone repair, and the invention reveals that Nr4a1 is used as a treatment target to improve the effectiveness of inflammation and inhibiting bone regeneration, thereby providing a new method for treating clinical bone defects and inflammatory bone diseases.

Drawings

FIG. 1, TGF-. Beta.1 inhibits Nr4a1 expression;

among them, (A, B) qPCR (A) and Western Blot (B) showed that TGF-. Beta.1 (50 ng/mL) inhibited the expression of Nr4a1 in BMSCs. (C) Immunohistochemistry showed TGF- β1 and Nr4a1 expression levels in C57BL/6J and nude mice ectopic osteogenic implants. Scale bar: 50 μm. Each point represents a separate sample and the data are expressed as mean ± standard deviation. * P <0.05.

FIG. 2, nr4a1 enhancing the osteogenic capacity of BMSCs and rescuing osteogenic differentiation from high dose TGF- β1 inhibition

(A) Overexpression of Nr4a1 promotes bone formation in BMSCs and rescue of matrix mineralization by TGF- β1 inhibition. (B, C) qPCR detects the expression of osteoblast marker genes in BMSCs, including (B) Runx2 and (C) Bglap. (D) Alizarin red staining shows that knock-down of Nr4a1 enhances TGF- β1-induced osteogenesis inhibition. (E, F) qPCR detects the expression of bone formation related genes. NC: negative control. Each point represents a separate sample and the data are expressed as mean ± standard deviation. * P <0.05; * P <0.01; * P <0.001

FIG. 3, nr4a1 regulating Wnt4 transcription

(A-E) overexpression of Nr4a1 in BMSCs qPCR examined the expression of Wnt4, cnnb 1, ccnd1, lef1 and Dkk 1. (F) Western Blot detects protein levels of Nr4a1 and beta-catenin. (G) The Luciferase assay detects the promoting effect of Nr4a1 on Wnt4 transcription. OE: and (5) over-expression. Each point represents a separate sample and the data are expressed as mean ± standard deviation. * P <0.05; * P <0.001

FIG. 4, nr4a1 promoting ectopic bone formation in vivo

(A) Microscopic CT reconstruction showed new bone formation in mice. (B) quantitative analysis of bone volume fraction (BV/TV). (C) H & E staining showed localized osteogenesis of the implant. Scale bar: 100 μm. B: bone; CT: connective tissue. (D) quantitative analysis of percent bone formation by H & E images. OE: and (5) over-expression. Each dot represents an individual mouse and data are expressed as mean ± standard deviation. * P <0.01; * P <0.001

FIG. 5, nr4a1, promoting repair of bone defects

(A) The reconstruction of the micro CT shows the repair condition of the skull defect of the mice. (B, C) quantitative analysis of bone volume fraction (BV/TV) and Bone Formation Area (BFA). (D) H & E staining showed new bone formation at the defect site. Scale bar: 100 μm. OE: and (5) over-expression. Each dot represents an individual mouse and data are expressed as mean ± standard deviation. * P <0.01

FIG. 6, csn-B treatment to improve osteogenic differentiation and promote bone regeneration of TGF- β1 inhibition

(A) Alizarin red staining detects the regulation of osteogenic differentiation by Csn-B stimulation. (B) quantitatively analyzing the extracellular matrix deposition. (C) microscopic CT reconstruction showed partial osteogenesis of the implant. Mice were intraperitoneally injected twice a week with 5mg/kg Csn-B. (D) quantitative analysis of bone volume fraction (BV/TV). (E) H & E staining and quantitative analysis showed new bone formation in the implant. Scale bar: 100 μm. B: bone; CT: connective tissue; OM: osteogenesis inducing solution. Each point represents a separate sample and the data are expressed as mean ± standard deviation. * P <0.05; * P <0.001

FIG. 7, csn-B treatment to promote repair of bone defects

(A) The microscopic CT reconstruction shows the repair of 1.8mm circular skull defects. (B, C) quantitative analysis of bone volume fraction and area of new bone formation. (D) H & E staining showed bone repair. (E) Microscopic CT reconstruction showed 1.0mm round tibial defect repair. (F, G) quantitative analysis of bone volume fraction and bone trabecular number (Tb. N). (H) H & E staining showed new strand formation at tibial cortical bone defects. Mice were intraperitoneally injected twice a week with 5mg/kg Csn-B. Scale bar: 100 μm. B: bone; BM: bone marrow. Each point represents a separate sample and the data are expressed as mean ± standard deviation. * P <0.05

FIG. 8 Csn-B treatment enhances fracture healing

(A) The micro CT reconstruction shows the tibial fracture repair condition. (B, C) quantitative analysis of bone volume fraction and bone trabecular number. (D) H & E staining showed new bone formation in the callus. Mice were intraperitoneally injected twice a week with 5mg/kg Csn-B. Scale bar: 50 μm. Each point represents a separate sample and the data are expressed as mean ± standard deviation. * P <0.05.

FIG. 9 high dose Csn-B has no promoting effect on bone formation

(A, B) microscopic CT quantitative analysis showed bone volume fraction and bone trabecular number. Mice were intraperitoneally injected twice a week with 10mg/kg Csn-B. Each point represents a separate sample and the data are expressed as mean ± standard deviation.

Detailed Description

The invention is capable of many modifications and various embodiments and its several specific embodiments are illustrated in the drawings and described herein. It is not intended to limit the invention to the particular embodiments but is to be understood to include all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.

1. Materials and methods

1. Animals

Male C57BL/6J mice and nude mice

2. Cells

Mouse bone marrow mesenchymal stem cells (BMSCs)

3. Materials and reagents

Beta tricalcium phosphate particles, hystem-HP Hydrogel, TGF-beta 1 cytokine, alpha-MEM, FBS

Green streptomycin, dexamethasone, beta phosphoglycerate, ascorbic acid, sodium hydroxide, trizol, reverse transcriptase, hiScript IIIall-in-one RT SuperMix Perfect for qPCR, lipofectamine 2000 transfection reagent, PVDF membrane, methanol, running buffer, transfer buffer, filter paper, pcDNA3.1 (+) vector, pGL3 vector, pRL-SV40 vector, nr4a1 lentivirus, puromycin, csn-B, OCT,4% paraformaldehyde, H & E dye solution

4. Experimental method

4.1BMSCs sorting

After 8-week-old C57BL/6J mice were sacrificed, they were soaked in 75% alcohol and sterilized for 10 minutes. The femur and tibia of the hind limb of the mouse are removed in an ultra clean bench, the femur and tibia epiphysis are removed by surgical scissors after muscle and soft tissue are removed, and the bone marrow is flushed out of the bone marrow cavity by sucking the culture medium through a 2ml syringe and repeated for several times until the bone becomes white. The cells were blown up into a single cell suspension by a syringe, and after passing through a 70 μm sieve, the cell pellet was collected by centrifugation at 300g for 10 minutes, and 10ml of a culture solution (a-MEM containing 10% FBS,1% diabody) was added and seeded in a 10cm dish. After culturing in a 5% CO2 incubator at 37℃for 3 days, the cells which were not attached were removed by pipetting, and pipetting was performed every 3 days thereafter. When the cells had grown to 90% confluence, the medium was removed, washed once with PBS, digested again with 0.25% Trypsin-EDTA for 1 min, and the reaction was stopped with complete medium. Cells were blown down, centrifuged at 300g for 5 min to harvest cells, and passaged at a ratio of 1:2 to become first generation cells. After 4 passages the experiment was started.

4.2 osteogenesis Induction

The osteogenesis inducing liquid formula comprises: alpha-MEM+10% FBS+1% Green streptomycin+100 nM dexamethasone+50. Mu.M ascorbic acid+10 mM beta-phosphoglycerate. After BMSCs are attached, the BMSCs are replaced by osteoinductive liquid, cultured for 10-14 days, and added with TGF-beta 1 and Csn-B for stimulation, and the liquid is replaced every 3 days. Cells were fixed with 4% pfa, stained with alizarin red dye, and the red pellet was dissolved with 0.1N sodium hydroxide for quantitative analysis.

4.3 immunohistochemistry

The implant was decalcified in 12.5% edta decalcification solution after 24 hours fixation with 4% paraformaldehyde. Serial sections were performed after paraffin embedding, immunohistochemical staining was performed according to the instructions, and antibodies were used with TGF- β1 and Nr4a1.

4.4 Dual luciferase reporter assay

A total of 1313bp upstream of 2,000bp to 688bp upstream of the transcription initiation site of the Nr4a1 gene was synthesized as a promoter sequence of Nr4a1 and constructed on pGL3 vector. pcDNA3.1 vector, pGL3 vector and pRL-SV40 reporter vector were co-transfected in 293T cells. After 72 hours the fluorescence activity was measured with the Luciferase assay kit.

4.5 construction of Nr4a1 stably transformed BMSCs

Nr4a1 lentiviruses were transfected into BMSCs and screened 72h later with puromycin (3 ug/mL) to kill uninfected cells. The overexpression efficiency was verified by qPCR detection.

4.6 animal model

Ectopic osteogenic model: BMSCs (3×10) ⁶ ) The mixture was compounded with beta-TCP (45 mg), then implanted under the back skin of C57BL/6J mice, and after suturing the skin, the mixture was fed for 2 months.

Skull defect model: after anesthesia of the C57BL/6J mice, the top skin was dissected away to expose the skull, and circular defects were made using circular cutting drills on the frontal (1.8 mm diameter) or parietal (4.0 mm diameter) bones of the skull. BMSCs were wrapped with Hystem-HP Hydrogel for 4.0mm defects (2X 10) ⁶ ) And (5) planting the bone to the defect part of the top bone for repairing.

Tibia defect model: after C57BL/6J mice were anesthetized, the leg skin was dissected away to expose the tibia, and a round single cortical defect was made at the upper end of the tibia using a 1.0mm drill.

Fracture model: the tibia was fixed after C57BL/6J mice were anesthetized, and closed fractures were made in the tibia using a three-point fracture method.

Csn-B treatment: after Csn-B was dissolved with corn oil, the mixture was injected into the abdominal cavity of mice at a dose of 5mg/kg or 10mg/kg, twice weekly until the end of the experiment.

4.7 micro CT (micro-CT)

Implants or skull samples were fixed overnight with 4% paraformaldehyde and scanned for analysis with a Scanco Medical CT-80 instrument after running water rinse.

4.8H & E staining

The implant or skull was decalcified in 12.5% edta decalcification solution after fixation with 4% paraformaldehyde for 24 hours. OCT embedding was followed by serial sectioning and H & E staining was performed using white shark organism reagent according to the instructions.

Discussion of results

1. TGF-beta 1 inhibits expression of Nr4a1

As shown in FIG. 1, nr4a1 is an inhibitory molecule for TGF- β1, and high concentrations of TGF- β1 can reduce the expression of Nr4a1 (FIGS. 1A, B), which in turn enhances the osteogenesis inhibitory effect of TGF- β1. Ectopic bone formation experiments also demonstrated high expression of TGF- β1 in C57BL/6J mouse in vivo implants. In contrast, the level of Nr4a1 was significantly suppressed (fig. 1C). Thus, high concentrations of TGF-. Beta.1 may inhibit expression of Nr4a1.

2.Nr4a1 promotes osteogenic differentiation of BMSCs and reverses osteogenic differentiation inhibited by high doses of TGF-beta 1

To further investigate the effect of Nr4a1 on BMSCs osteogenesis and reversing TGF- β1 inhibition, this example, over-expressing Nr4a1 in BMSCs, as shown in fig. 2, alizarin red staining showed that Nr4a1 over-expression enhanced osteogenic potential while effectively reversing TGF- β1 inhibited osteogenic differentiation (fig. 2A). Quantitative analysis of osteogenesis-specific gene expression (qPCR) also demonstrated positive regulation of osteogenesis by Nr4a1 (fig. 2b, c), including run-related transcription factor 2 (Runx 2) and osteocalcin (bglpap). Conversely, down-regulation of Nr4a1 prevented osteogenic differentiation of BMSCs and exacerbated the inhibition of TGF- β1 (fig. 2D-F). Thus, nr4a1 is not only an endogenous inhibitor of TGF- β1, which alleviates the osteogenesis inhibitory effect of TGF- β1, but also itself promotes osteogenic differentiation of BMSCs.

3.Nr4a1 promotes Wnt4 transcription to enhance osteogenic differentiation of BMSCs

To elucidate the molecular mechanism by which Nr4a1 promotes osteogenic differentiation, it was found by transcriptome sequencing that the expression of Wnt4, a key molecule, was up-regulated after overexpression of Nr4a1, in BMSCs. As shown in fig. 3, qPCR validated increased transcription of Wnt4 (fig. 3A) while expression of Wnt pathway downstream molecules Ctnnb1, ccnd1, and Lef1 were elevated (fig. 3B-D). In contrast, wnt pathway-inhibiting molecule Dkk1 expression decreased (fig. 3E). Western Blot experiments also demonstrated activation of the Wnt pathway by over-expression Nr4a1 (fig. 3F). Finally, we constructed a Luciferase plasmid containing Wnt4 promoter, and cotransfection of Nr4a1 and pGL3-Wnt4 promoter plasmids increased Luciferase fluorescence, indicating regulation of Wnt4 transcription by Nr4a1 (fig. 3G).

4.Nr4a1 overexpression promotes ectopic osteogenesis in vivo

To examine the effect of Nr4a1 on bone formation in vivo, BMSCs overexpressing Nr4a1 were first constructed and, after complexing with β -TCP, implanted subcutaneously in C57BL/6J mice. As shown in fig. 4, overexpression of Nr4A1 significantly promoted bone formation in vivo (fig. 4A) and increased new bone mass (fig. 4B). H & E staining showed that the control group was mostly connective tissue, while the Nr4a1 over-expressed group had more new bone formation (fig. 4c, d). Thus, nr4a1 may promote BMSCs-mediated bone regeneration.

5. Overexpression of Nr4a1 accelerates repair of in vivo skull defects

We also examined the therapeutic effect of Nr4a1 in the mouse skull defect model. First, a 4mm circular critical bone defect was constructed in the top bone of C57BL/6J mice using a cranial drill, and the BMSCs infected with control plasmid or overexpressing Nr4a1 were implanted for repair, respectively. As shown in fig. 5, microscopic computer tomography (micro-CT) analysis showed that the Nr4a1 over-expressed group significantly promoted defect healing at 8 weeks post-surgery (fig. 5A), increased bone volume fraction (BV/TV, fig. 5B) and bone formation area (BFA, fig. 5C) compared to the control group. H & E staining showed a significant shortening of the skull defect spacing after implantation of Nr4a1 overexpressing BMSCs (fig. 5D). Taken together, these data indicate that targeting Nr4a1 can improve BMSCs-mediated repair of bone defects.

6. Treatment with the Nr4a1 small molecule agonist Csn-B reverses TGF-beta 1 inhibited osteogenic differentiation and promotes bone regeneration in vivo

To facilitate clinical transformation applications, BMSCs were stimulated with the Nr4a1 agonist Csn-B. As shown in FIG. 6, csn-B reversed the osteogenesis inhibitory effect of high concentrations of TGF- β1 (FIGS. 6A, B). Furthermore, we used Csn-B for in vivo treatment, and in vivo ex vivo osteogenesis experiments demonstrated that Csn-B treatment improved bone regeneration with inflammation inhibition and promoted BMSCs-mediated bone formation (fig. 6C-E).

7. Csn-B treatment to promote repair of bone defects

As shown in FIG. 7, the therapeutic effect of Csn-B on the skull defects of mice was verified. A1.8 mm circular defect was constructed in the frontal bone of C57BL/6J mice, and control vector and Csn-B were injected for treatment, respectively. Micro-CT analysis showed that Csn-B treatment group significantly promoted defect healing at 4 weeks post-surgery (fig. 7A), increased bone volume fraction (BV/TV, fig. 7B) and bone formation area (BFA, fig. 7C). H & E staining showed significant shortening of the skull defect spacing after Csn-B treatment (fig. 7D). Likewise, csn-B treatment in the mouse tibial defect model improved bone regeneration and increased bone mass (fig. 7E-H). Thus, the Nr4a1 small molecule agonist Csn-B can be used as a therapeutic agent to improve bone defect repair.

8. Csn-B treatment promotes fracture healing

As shown in FIG. 8, the promotion of bone regeneration by Csn-B was again confirmed in the mouse tibial fracture model. Micro-CT showed that Csn-B treatment increased bone mass and fracture healing rate (FIGS. 8A-C), and H & E staining also demonstrated more new bone formation in the Csn-B treated group (FIG. 8D). In conclusion, csn-B has remarkable promotion effect on both intramembranous osteogenesis and endochondral osteogenesis, and shows that the Csn-B has feasibility for treating bone diseases.

9. High doses of Csn-B lead to ineffective treatment

In vivo treatment with Csn-B, it was found that different doses of Csn-B did not have the same therapeutic effect and not as much as better. As shown in FIG. 9, the use of 5mg/kg Csn-B promoted bone regeneration, but the use of 10mg/kg Csn-B had no therapeutic effect, showing new bone formation comparable to the control group (FIG. 9). Thus, appropriate dosages should be used in performing in vivo treatment.

In the study of this example, it was also found that Csn-B treatment also inhibited inflammatory response and tissue fibrosis, improving the bone repair environment. Meanwhile, medicines or agonists targeting Nr4a1, wnt4 and Wnt channels can replace Csn-B to treat.

Although the embodiments have been described above mainly, this is merely illustrative, and not restrictive of the invention, and it will be apparent to those skilled in the art that various modifications and applications not illustrated above can be made without departing from the essential characteristics of the present embodiments. For example, each component specifically shown in the embodiments can be implemented by being modified. Moreover, various points related to such modifications and applications should be construed as including the scope of the present invention as defined in the appended claims.

Claims

Use of an nr4a1 agonist/overexpression formulation for the preparation of a medicament/formulation for alleviating or reversing immune rejection reactions caused by mesenchymal stem cell BMSCs and biomaterial therapies.
Use of an nr4a1 agonist/overexpression formulation for the preparation of a medicament/formulation for promoting osteogenic differentiation of mesenchymal stem cells BMSCs.
Use of an nr4a1 agonist/overexpression formulation for the manufacture of a medicament/formulation for antagonizing osteogenesis inhibition caused by overactivation of TGF- β1.
Use of an nr4a1 agonist/overexpression formulation for the preparation of a medicament/formulation for the treatment/alleviation of inflammatory bone diseases.
Use of an nr4a1 agonist/overexpression formulation in the manufacture of a medicament/formulation to slow or reverse matrix mineralization.
Use of an nr4a1 agonist/overexpression formulation for the preparation of a medicament/formulation for promoting ectopic osteogenesis in vivo.
Use of an nr4a1 agonist/overexpression formulation for the preparation of a medicament/formulation for accelerating/promoting repair of a skull defect.
Use of an nr4a1 agonist/overexpression formulation in the manufacture of a medicament/formulation for promoting fracture healing.
9. Use according to any one of claims 1-8, characterized in that:

the Nr4a1 agonist/overexpression formulation is used in an amount of no more than 10mg/kg.
10. The use according to any one of claims 1-9, wherein:

the Nr4a1 agonist/overexpression formulation is replaced by an agonist or expression formulation targeting Wnt4 and Wnt pathway.