Use of Nell-1 and its derivative products in the preparation of a medicament for preventing and / or treating mechanical cartilage degeneration

Abstract

The application provides application of Nell-1 and a derivative product thereof in preparation of a medicine for preventing and / or treating mechanical cartilage degeneration, and belongs to the technical field of biological medicines. The application explores response, function and molecular mechanism of Nell-1 in mechanical cartilage degeneration, and innovatively reveals that pathological mechanical stress inhibits expression of Nell-1 of chondrocytes. 2+ Ca internal flow and abnormal activation of YAP, breaks the vicious cycle of "mechanical overload-mechanical hypersensitivity" from the source. Nell-1 has multiple effects of regulating the mechanical adaptability of cartilage, inhibiting the terminal differentiation of chondrocytes, reducing the release of inflammatory factors and cell apoptosis, and has significant and comprehensive protective effect on cartilage damage caused by mechanical overload. This shows that Nell-1 or the derivative product of Nell-1 provides a new means for clinical treatment of mechanical cartilage degeneration.

Description

Technical Field

[0001] This application belongs to the field of biopharmaceutical technology, specifically relating to the use of Nell-1 and its derivatives in the preparation of drugs for the prevention and / or treatment of mechanical cartilage degeneration. Background Technology

[0002] Mechanical degeneration of cartilage refers to the degenerative pathological changes in articular cartilage under long-term, repeated, and abnormal mechanical stress, characterized by extracellular matrix degradation, decreased chondrocyte function, and progressive destruction of cartilage structure. Mechanical degeneration of cartilage can subsequently develop into mechanical temporomandibular joint osteoarthritis (TMJOA), significantly impacting patients' quality of life. Mechanical stimulation exceeding physiological limits is considered one of the main causes of TMJOA. However, the precise mechanism by which excessive mechanical force leads to cartilage degeneration remains unclear, thus insufficient understanding of this pathological process hinders the development of effective clinical treatments for TMJOA.

[0003] Nell-1 is a novel chondrogenic molecule that promotes chondrocyte proliferation, cartilage differentiation, and collagen synthesis, and has been shown to effectively repair cartilage defects. For example, intra-articular injection of Nell-1 can alleviate degenerative changes in knee cartilage caused by IL-1β accumulation. However, it should be noted that the main cartilage component of the condyle is fibrocartilage, which differs from the dominant hyaline cartilage in the knee joint. Furthermore, temporomandibular joint arthritis is not essentially an inflammatory disease, but rather a degenerative process induced by mechanical overload. Currently, there are no reports on the role of Nell-1 in the pathological mechanism of mechanical overload-induced fibrocartilage degeneration. Summary of the Invention

[0004] The purpose of this invention is to provide a new use for Nell-1, namely, the use of Nell-1 in the preparation of medicaments for the prevention and / or treatment of mechanical degenerative changes in cartilage.

[0005] This invention provides the use of Nell-1 or Nell-1 derivatives in the preparation of medicaments for the prevention and / or treatment of mechanical cartilage degeneration.

[0006] Preferably, the Nell-1 includes the Nell-1 protein and a nucleic acid molecule encoding the Nell-1 protein.

[0007] Preferably, the Nell-1 derivative includes at least one of the following: A1) an expression cassette containing a nucleic acid molecule encoding the Nell-1 protein; A2) A recombinant vector containing a nucleic acid molecule encoding the Nell-1 protein; A3) A recombinant vector containing the expression cassette described in A1); A4) Recombinant microorganisms or transgenic cells containing nucleic acid molecules encoding the Nell-1 protein; A5) Recombinant microorganisms or transgenic cells containing the expression cassette described in A1); A6) Recombinant microorganisms or transgenic cells containing the recombinant vector described in A2); A7) Recombinant microorganisms or transgenic cells containing the recombinant vector described in A3).

[0008] Preferably, the recombinant microorganism includes a recombinant virus; The transgenic cells include transgenic stem cells and / or transgenic human fibrochondrocytes.

[0009] Preferably, the use of Nell-1 or a derivative thereof in combination with a Piezo1 inhibitor in the preparation of a medicament for the prevention and / or treatment of mechanical temporomandibular joint arthritis.

[0010] Preferably, the Piezo1 inhibitor includes GsMTx4.

[0011] This invention provides a medicament for the prevention and treatment of mechanical temporomandibular osteoarthritis, the active ingredients of which include Nell-1 or a derivative of Nell-1 and / or a Piezo1 inhibitor.

[0012] This invention provides the application of a reagent for detecting the expression level of the Nell-1 gene or Nell-1 protein in the preparation of a kit for diagnosing mechanical cartilage degeneration.

[0013] Preferably, the mechanical cartilage degeneration includes mechanical temporomandibular osteoarthritis.

[0014] This invention provides the application of Nell-1 or Nell-1 derivatives in the preparation of drugs for the prevention and / or treatment of mechanotrophic cartilage degeneration. This invention investigates the response, function, and molecular mechanism of Nell-1 in mechanotrophic cartilage degeneration, and innovatively reveals that pathological mechanical stress inhibits Nell-1 expression in chondrocytes. Furthermore, exogenous addition of Nell-1 blocks Ca2+ expression. 2+ Influx and abnormal activation of YAP break the vicious cycle of "mechanical overload-mechanical hypersensitivity" at its source. Nell-1 has multiple functions, including regulating cartilage's mechanical adaptability, inhibiting chondrocyte terminal differentiation, reducing the release of inflammatory factors, and inhibiting apoptosis, demonstrating a significant and comprehensive protective effect against cartilage damage caused by mechanical overload. This indicates that Nell-1 or its derivatives provide a new approach for the clinical treatment of mechanical cartilage degeneration.

[0015] This invention further provides the application of Nell-1 or Nell-1 derivatives in combination with a Piezo1 inhibitor in the preparation of a medicament for the prevention and / or treatment of mechanical temporomandibular joint arthritis (TMJOA). Experiments of this invention demonstrate that Nell-1 can directly bind to and inhibit the function of the mechanosensitive ion channel Piezo1, innovatively revealing a "Nell-1 / Piezo1-YAP" feedforward regulatory mechanism, providing a novel targeted therapeutic modality for mechanical TMJOA. Attached Figure Description

[0016] Figure 1 The results are for Nell-1 intrachondral conditional knockout mice (cKO). A shows the temporomandibular joint arthritis (TMJOA) phenotype in cKO mice and wild-type mice (WT). B shows the inhibition of Piezo1 channel current by recombinant NELL-1 protein (rmNELL-1). C shows the simulated binding mode of NELL-1 and PIEZO1. Detailed Implementation

[0017] This invention provides the use of Nell-1 or Nell-1 derivatives in the preparation of medicaments for the prevention and / or treatment of mechanical cartilage degeneration.

[0018] In this invention, the Nell-1 preferably comprises the Nell-1 protein and a nucleic acid molecule encoding the Nell-1 protein. The Nell-1 protein and nucleic acid molecule encoding the Nell-1 protein are selected according to the species adaptability of the mechano-chondrogenic degeneration. For example, when treating human mechano-chondrogenic degeneration, a human-derived Nell-1 protein and a nucleic acid molecule encoding the Nell-1 protein are selected; when treating mouse mechano-chondrogenic degeneration, a mouse-derived Nell-1 protein and a nucleic acid molecule encoding the Nell-1 protein are selected.

[0019] In this invention, the Nell-1 derivative preferably includes at least one of the following: A1) an expression cassette containing the nucleic acid molecule encoding the Nell-1 protein; A2) A recombinant vector containing a nucleic acid molecule encoding the Nell-1 protein; A3) A recombinant vector containing the expression cassette described in A1); A4) Recombinant microorganisms or transgenic cells containing nucleic acid molecules encoding the Nell-1 protein; A5) Recombinant microorganisms or transgenic cells containing the expression cassette described in A1); A6) Recombinant microorganisms or transgenic cells containing the recombinant vector described in A2); A7) Recombinant microorganisms or transgenic cells containing the recombinant vector described in A3).

[0020] In this invention, the expression level preferably includes promoters, enhancers, and terminators. This invention does not impose special restrictions on the selection of promoters and enhancers; any species-homologous promoters and / or enhancers known in the art can be used. The recombinant vector preferably includes a recombinant viral expression vector, such as a recombinant lentiviral expression vector or a recombinant adeno-associated virus expression vector. The recombinant microorganism preferably includes a recombinant virus. The virus used in the recombinant virus is preferably a lentivirus or adeno-associated virus. The transgenic cells preferably include transgenic stem cells and / or transgenic human fibrochondrocytes. The host of the transgenic stem cells is preferably mesenchymal stem cells. This invention does not impose special restrictions on the preparation methods of the recombinant vector, recombinant microorganism, and transgenic cells; any preparation methods known to those skilled in the art can be used.

[0021] In this invention, the mechanical cartilage degeneration preferably includes mechanical temporomandibular joint arthritis. The mechanical temporomandibular joint arthritis is studied by inducing a unilateral anterior tooth shift model to investigate the therapeutic effect on this disease. In embodiments of this invention, Nell-1 conditional knockout (cKO) mice or shNell-1 transfected cells, compared to wild-type (WT) or empty vector transfected cells, exhibited more severe cartilage homeostasis imbalance and mechanosensitive responses in all experiments (histological, molecular biological, and mechanical tests), directly demonstrating the necessity of Nell-1 function. Furthermore, treatment of chondrocytes with recombinant mouse NELL-1 protein (rmNELL-1) can alleviate chondrocyte damage caused by mechanical overload and reverse the harmful phenotype induced by shNell-1, demonstrating the therapeutic potential of Nell-1.

[0022] In this invention, to further explore the molecular mechanism by which Nell-1 regulates the mechanical degeneration of cartilage, RNA-seq was performed on ATDC5 cells transfected with shNell-1 to screen for differentially expressed genes and enriched pathways. The shNell-1 group... Piezo1 Expression was significantly upregulated, and differentially expressed genes were enriched in the Hippo pathway. Yap1 Gene expression was upregulated. Simultaneously, YAP nuclear translocation increased and the p-YAP / YAP ratio decreased in cKO mice and shNell-1 transfected cells, confirming that Nell-1 deficiency promotes YAP activation. Under mechanical overload, YAP nuclear translocation was more pronounced in the shNell-1 group, indicating that Nell-1 affects cartilage metabolism by regulating the YAP pathway.

[0023] In this invention, the Nell-1 or its derivatives are preferably used in combination with a Piezo1 inhibitor in the preparation of a medicament for the prevention and / or treatment of mechanical temporomandibular joint arthritis. The Piezo1 inhibitor preferably includes GsMTx4. In one embodiment of this invention, a unilateral anterior tooth deviation model was constructed using Nell-1 knockout mice. Compared to the WT+UAC group, the cKO+UAC group showed more severe cartilage degeneration, with further reductions in cartilage thickness and cell number, increased apoptosis rate, lower COLII expression, higher Mmp13 expression, and a significantly increased Mankin score. This confirms that Nell-1 deficiency disrupts cartilage homeostasis and reduces mechanical adaptation. Simultaneously, in cKO mouse cartilage and shNell-1 transfected cells, both Piezo1 mRNA and protein expression were significantly increased, while rmNELL-1 treatment significantly reduced Piezo1 channel currents. Co-IP confirmed that Nell-1 directly binds to Piezo1. By injecting GsMTx4 (3 μM) into the joint cavity of the model and pretreating shNell-1 transfected cells with GsMTx4 (500 nM) for 24 h before applying mechanical force, the results showed that after GsMTx4 intervention, cKO+UAC mice had increased cartilage thickness, increased proteoglycan content, upregulated COLII expression, downregulated MMP13 expression, decreased apoptosis rate, and decreased Mankin score. In vitro, compared with the shNell-1 group, the shNell-1+GsMTx4 group had upregulated Col2a1 and Acan expression, downregulated Mmp13 expression, and reduced apoptosis and mortality, confirming that inhibiting Piezo1 can alleviate cartilage degeneration caused by Nell-1 deficiency.

[0024] In one embodiment of the present invention, treatment of cKO mice or shNell-1 transfected cells with the Piezo1-specific inhibitor GsMTx4 significantly alleviated cartilage degeneration exacerbated by Nell-1 deficiency, indicating that Piezo1 is a key downstream effector molecule of Nell-1. Inhibition of Piezo1 can partially compensate for the effects of Nell-1 deficiency. Therefore, Piezo1 inhibitors can be combined with Nell-1 or its derivatives to treat mechanical cartilage degeneration, especially mechanical temporomandibular joint arthritis.

[0025] This invention provides a medicament for the prevention and treatment of mechanical temporomandibular osteoarthritis, the active ingredients of which include Nell-1 or a derivative of Nell-1 and / or a Piezo1 inhibitor.

[0026] This invention provides the application of a reagent for detecting the expression level of the Nell-1 gene or Nell-1 protein in the preparation of a kit for diagnosing mechanical cartilage degeneration. The mechanical cartilage degeneration preferably includes mechanical temporomandibular joint arthritis.

[0027] In another embodiment of the present invention, the expression level and cellular localization of the Nell-1 gene were detected by single-cell RNA sequencing (scRNA-seq) and a unilateral anterior tooth offset model (UAC). The results showed that the Nell-1 gene was mainly located in chondrocytes of human TMJOA tissue. The Nell-1 positive area in the condylar cartilage of the UAC group was significantly less than that of the SHAM group, indicating that Nell-1 expression was downregulated under TMJOA conditions. Comparison of the results from Nell-1 conditional knockout (cKO) mice or cells transfected with shNell-1 with wild-type (WT) or empty vector transfected cells showed that Nell-1 deficiency disrupted cartilage homeostasis and reduced mechanical adaptability. Treatment of chondrocytes with recombinant mouse NELL-1 protein (rmNELL-1) alleviated chondrocyte damage caused by mechanical overload and reversed the harmful phenotype induced by shNell-1, demonstrating the correlation between Nell-1 expression and cartilage degeneration and the alleviation of cartilage damage. Therefore, Nell-1 expression levels can be used to diagnose mechanical cartilage degeneration, and compared with healthy samples, the significantly reduced expression level of Nell-1 indicates that the tested samples have mechanical cartilage degeneration.

[0028] This invention investigates the expression and function of Nell-1 in condylar fibrocartilage, revealing the response mechanism of Nell-1 as a mechanosensitive protein to the abnormal mechanical environment of TMJOA. It elucidates the regulatory role of Nell-1 in the adaptive capacity and mechanical degeneration of condylar fibrocartilage and its downstream mechanisms, and confirms Piezo1 as a key signal transduction molecule in this process. This invention deepens our understanding of the pathological progression of mechanoarthritis of the temporomandibular joint and lays a theoretical foundation for using Nell-1 as a novel therapeutic target for mechanoarthritis of the temporomandibular joint.

[0029] The following examples illustrate the application of Nell-1 and its derivatives provided by the present invention in the preparation of medicaments for the prevention and / or treatment of mechanical cartilage degeneration, but these should not be construed as limiting the scope of protection of the present invention.

[0030] Example 1 A Preliminary Study on the Expression Characteristics and Mechanical Response Properties of Nell-1 in TMJOA I. Experimental Methods 1. Single-cell RNA sequencing (scRNA-seq): Human TMJOA tissue samples (provided by the Stomatological Hospital of Jilin University) were collected, digested, and single-cell sequencing was performed to analyze cell clustering and cellular localization of the Nell-1 gene; at the same time, scRNA-seq data (GSE169454) of knee arthritis and normal cartilage samples were analyzed to clarify the expression pattern of Nell-1 in fibrochondrocyte subsets.

[0031] 2. Animal model experiments 2.1 Construction of animal model: 6-week-old C57BL / 6 mice were randomly divided into UAC group (unilateral anterior teeth bonded with a 135˚ curved metal tube) and SHAM group (surgery without bonding of metal tube). Condylar cartilage tissue was collected 6 weeks after modeling.

[0032] 2.2 Histological examination: Cartilage tissue was stained with HE, Safranin O-Fixed Green, and Toluidine Blue to assess cartilage thickness, cell number, and proteoglycan content. Immunohistochemistry was used to detect COLII and MMP13 expression, and TUNEL staining was used to detect cell apoptosis. In addition, immunohistochemistry and immunofluorescence techniques were used to detect Nell-1 expression.

[0033] 3. In vitro mechanical loading: Mouse-derived chondrogenic cell line ATDC5 cells were cultured in vitro and subjected to mechanical forces of 1000 μstrain (mild), 3000 μstrain (moderate), and 5000 μstrain (severe), respectively. RT-qPCR and Western blot were used to detect Nell-1 mRNA and protein expression. The primers for RT-qPCR were: Forward: 5´-GCGTTGGTGCGCCCTGCT-3´ (SEQ ID NO:1); Reverse: 5´-CTGTCCTGGCGGTGCACACA-3´ (SEQ ID NO:2). The reaction system consisted of 2 µl cDNA, 0.4 µl each of forward and reverse primers, 10 µl SYBR buffer, and 7.2 µl DEPC water. After mixing, RT-qPCR amplification was performed. The reaction program is shown in the table, using 2... -ΔΔCt The method is to conduct data analysis.

[0034] Table 1 RT-PCR reaction procedure

[0035] II. Test Results 1. scRNA-seq results showed that the Nell-1 gene was mainly located in chondrocytes of human TMJOA tissue, and was highly expressed in the fibrochondrocyte subset of knee arthritis cartilage. The expression of Nell-1 in this subset showed a down-regulation trend in the arthritis group. The fibrochondrocyte subset in TMJOA cartilage was the dominant subset and was an active hub for signal transduction.

[0036] 2. The UAC model successfully induced mechanical TMJOA. Compared with the SHAM group, the UAC group showed reduced cartilage thickness, decreased cell number, reduced proteoglycan content, downregulated COLII expression, upregulated MMP13 expression, increased apoptosis rate, and significantly lower Mankin score. The Nell-1 positive area in the condylar cartilage of the UAC group was significantly less than that in the SHAM group, confirming the downregulation of Nell-1 expression under TMJOA conditions.

[0037] 3. In vitro mechanical loading showed that mild and moderate mechanical forces promoted Nell-1 expression, while severe mechanical forces (5000 μstrain) significantly inhibited Nell-1 mRNA and protein expression, clarifying its mechanical response characteristics.

[0038] Example 2 The impact of Nell-1 deficiency on cartilage homeostasis and mechanical damage I. Experimental Methods 1. Conditional knockout mouse construction: Nell-1 flox / flox European mouse mutant archive mice were crossed with Acan-cre ERT2 mice (purchased from Jicui Yaokang). Six-week-old offspring were intraperitoneally injected with tamoxifen (100 mg / kg) for 5 consecutive days to induce cartilage-specific Nell-1 knockout (cKO group). Littermates with Nell-1... flox / flox Mice were placed in the WT group, and cartilage tissue was collected after 4 weeks.

[0039] 2. Cartilage homeostasis assessment: HE, Safranin O-Fix Green, and Toluidine Blue staining were used to assess cartilage morphology and metabolism; immunohistochemistry was used to detect COLII and COLX expression; and atomic force microscopy was used to detect the elastic modulus of chondrocytes. 3. ATDC5 cells were transfected with shRNA (see Table 2) to inhibit Nell-1, and RT-qPCR was used for detection. Nell-1 , Col2a1, Acan, Mmp13 Gene expression was recorded, and the specific sequences are shown in Table 3. The detection method is the same as above.

[0040] Table 2. DNA sequence of shRNA targeting Nell-1 (shNell-1)

[0041] Table 3 Primer Sequences

[0042] 4. Mechanical damage assessment: UAC model was constructed in both cKO and WT mice according to the method in Example 1. Cartilage degeneration indicators were detected after 6 weeks (using the same histological detection method as in Example 1). 5. In vitro mechanical loading cell experiments: ATDC5 cells were transfected with shNell-1 and pretreated with recombinant Nell-1 protein (rmNELL-1). The shNell-1 transfection method involved seeding healthy target cells into 12-well plates. After cell adhesion, Nell-1 shRNA lentiviral plasmid was added for transfection. Control wells used culture medium instead of lentivirus. Sixteen hours after lentivirus transfection, fresh culture medium was added for further culture. After 48 hours of culture, the medium was replaced with 30 µg / ml puromycin to select stable cell lines. The medium was changed every 2-3 days until all untransfected cells died, thus obtaining stable Nell-1 knockdown cell lines. The rmNELL-1 pretreatment method involved pretreating ATDC5 cells with recombinant mouse Nell-1 protein (2 ng / ml, 72 h). After pretreatment, ATDC5 cells in different treatment groups were subjected to a mechanical force of 5000 μstrain. RT-qPCR detection was performed. Col2a1, Mmp13, Acan The expression of cartilage characteristic factors and inflammatory factors was measured, and apoptosis and mortality were detected by TUNEL staining and live / dead staining, respectively.

[0043] II. Test Results 1. Results of cartilage homeostasis and cell detection in conditional knockout mice See results Figure 1 In A. cKO mice, condylar cartilage thickness was reduced, cell number decreased, proteoglycan content decreased, COLII expression was downregulated, and COLX expression was upregulated. The elastic modulus of chondrocytes decreased from 6.65 MPa in the WT group to 5.00 MPa. shNell-1 transfection downregulated Col2a1 and Acan expression and upregulated Mmp13, Adamts5, and Col10a1 expression in ATDC5 cells, confirming that Nell-1 deficiency disrupts cartilage homeostasis and reduces mechanical adaptability.

[0044] 2. Results of Mechanical Damage Assessment Compared with the WT+UAC group, the cKO+UAC group showed more severe cartilage degeneration, with further reductions in cartilage thickness and cell number, increased apoptosis rate, lower COLII expression, higher Mmp13 expression, and significantly increased Mankin score.

[0045] 3. Results of in vitro mechanical loading cell detection Under external mechanical overload, shNell-1 group Col2a1, Acan The expression is downgraded. Mmp13, Adamts5, Col10a1, IL-1β, Nlrp3 Upregulation of expression increased apoptosis and death rates; however, pretreatment with rmNELL-1 reversed these changes, confirming that Nell-1 has a protective effect against mechanical cartilage damage.

[0046] Example 3 Molecular mechanisms by which Nell-1 regulates cartilage mechanical degeneration I. Experimental Methods 1. Downstream target screening: RNA-seq was performed on ATDC5 cells transfected with shNell-1 in Example 2 to screen for differentially expressed genes and enriched pathways.

[0047] 2. Piezo1-related detection: Immunohistochemistry and immunofluorescence were used to detect Piezo1 expression in the cartilage of cKO mice in Example 2; RT-qPCR (Piezo1: F5´-CTTACACGGTTGCTGGTTGG-3´, SEQ ID NO:13); R: 5´-CACTTGATGAGGGCGGAAT-3´, SEQ ID NO:14), Western blot, and immunofluorescence were used to detect the mRNA and protein expression of Piezo1 in shNell-1 transfected cells; calcium ion staining was used to detect intracellular calcium. 2+ Concentration; whole-cell patch-clamp assay to detect the effect of rmNELL-1 on Piezo1 channel current in ATDC5 cells; co-immunoprecipitation (Co-IP), immunofluorescence co-localization and protein docking analysis to analyze the interaction between Nell-1 and Piezo1.

[0048] 3. Piezo1 inhibitor intervention: After constructing a UAC model in cKO mice, GsMTx4 (3μM) was injected into the joint cavity once every other day for 3 consecutive weeks; after pretreating shNell-1 transfected cells with GsMTx4 (500nM) for 24h, mechanical force was applied, and cartilage degeneration-related indicators were detected.

[0049] 4. YAP pathway detection: Immunofluorescence was used to detect the expression and localization of YAP in cKO mice and shNell-1 transfected cells in Example 2; Western blot was used to detect the levels of YAP and phosphorylated YAP (p-YAP) to assess the activation status of YAP.

[0050] II. Test Results 1. RNA-seq showed that Piezo1 expression was significantly upregulated in the shNell-1 group, and differentially expressed genes were enriched in the Hippo pathway. Yap1 Gene expression is upregulated.

[0051] 2. In cKO mouse cartilage and shNell-1 transfected cells, the mRNA and protein expression of Piezo1 were significantly increased; intracellular Ca2+ in shNell-1 group cells was also significantly increased. 2+ Increased concentration, while rmNELL-1 treatment significantly reduced the Piezo1 channel current ( Figure 1(See Figure B); Co-IP confirmed that Nell-1 and Piezo1 bind directly, and immunofluorescence co-localization showed that the two significantly overlapped in the cytoplasm (Pearson's Rr = 0.901). Protein docking showed that the binding site was located at the top of the "ion channel" outside the Piezo1 membrane (see Figure B). Figure 1 (C)

[0052] 3. After GsMTx4 intervention, cKO+UAC mice showed increased cartilage thickness, elevated proteoglycan content, upregulated COLII expression, downregulated MMP13 expression, decreased apoptosis rate, and reduced Mankin score. Compared with the shNell-1 group, the shNell-1+GsMTx4 group showed upregulated Col2a1 and Acan expression, downregulated Mmp13 expression, and reduced apoptosis and mortality, confirming that inhibiting Piezo1 can alleviate cartilage degeneration caused by Nell-1 deficiency.

[0053] 4. In cKO mice and shNell-1 transfected cells, YAP nuclear translocation was increased and the p-YAP / YAP ratio was decreased, confirming that Nell-1 deficiency promotes YAP activation; 5. Under mechanical overload, YAP nuclear translocation was more pronounced in the shNell-1 group, indicating that Nell-1 affects cartilage metabolism by regulating the YAP pathway.

[0054] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Use of Nell-1 or its derivatives in the preparation of medicaments for the prevention and / or treatment of mechanical cartilage degeneration.

2. The application according to claim 1, characterized in that, The Nell-1 includes the Nell-1 protein and the nucleic acid molecule encoding the Nell-1 protein.

3. The application according to claim 1, characterized in that, The Nell-1 derivatives include at least one of the following: A1) an expression cassette containing a nucleic acid molecule encoding the Nell-1 protein; A2) A recombinant vector containing a nucleic acid molecule encoding the Nell-1 protein; A3) A recombinant vector containing the expression cassette described in A1); A4) Recombinant microorganisms or transgenic cells containing nucleic acid molecules encoding the Nell-1 protein; A5) Recombinant microorganisms or transgenic cells containing the expression cassette described in A1); A6) Recombinant microorganisms or transgenic cells containing the recombinant vector described in A2); A7) Recombinant microorganisms or transgenic cells containing the recombinant vector described in A3).

4. The application according to claim 3, characterized in that, The recombinant microorganisms include recombinant viruses; The transgenic cells include transgenic stem cells and / or transgenic human fibrochondrocytes.

5. The application according to any one of claims 1 to 4, characterized in that, The use of Nell-1 or a Nell-1 derivative in combination with a Piezo1 inhibitor in the preparation of a medicament for the prevention and / or treatment of mechanical temporomandibular joint arthritis.

6. The application according to claim 5, characterized in that, The Piezo1 inhibitors include GsMTx4.

7. A drug for preventing and treating mechanical temporomandibular joint arthritis, characterized in that, The active ingredients include Nell-1 or Nell-1 derivatives and / or Piezo1 inhibitors.

8. The application according to claim 7, characterized in that, The Piezo1 inhibitors include GsMTx4.

9. The application of a reagent for detecting the expression level of the Nell-1 gene or Nell-1 protein in the preparation of a kit for diagnosing mechanical cartilage degeneration.

10. The application according to claim 1 or 9, characterized in that, The mechanical cartilage degeneration includes mechanical temporomandibular joint arthritis.

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