Method for preparing marcks agonist to promote cartilage regeneration and application thereof

Abstract

The application discloses a preparation method and application of a MARCKS agonist for promoting cartilage regeneration, determines MARCKS protein as a key target point for cartilage regeneration through cross-species and cross-development stage conservation screening, improves the phosphorylation level of the MARCKS protein, and prepares a MARCKS phosphorylation activator; the MARCKS phosphorylation activator is a PKC epsilon selective agonistic peptide, contains an active effect sequence and a membrane penetrating sequence, and the two are connected through an amide bond or a disulfide bond; the application of the MARCKS protein screened as the target point in screening or preparing a product for promoting cartilage repair, and the application of the MARCKS phosphorylation activator in preparing a drug for treating osteoarthritis or promoting cartilage injury repair prove that the MARCKS protein and the MARCKS phosphorylation activator can effectively promote stem cell chondrogenic differentiation, reduce osteoarthritis cartilage injury and improve joint function, have a clear molecular target point, and have a good clinical transformation prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a screening method for MARCKS, a protein related to promoting cartilage regeneration, a preparation method for MARCKS agonists, and their applications. Background Technology

[0002] Osteoarthritis, accompanied by cartilage degeneration, is an irreversible, age-related degenerative pathological process. Due to its extremely high prevalence and disability risk, it has become a heavy health burden worldwide. Currently, treatment strategies for osteoarthritis mainly focus on symptomatic drug therapy and end-stage joint replacement surgery. While these methods can slow disease progression and improve patients' quality of life to some extent, they cannot achieve true regeneration of cartilage structure or fundamental restoration of function.

[0003] Proteomics can efficiently obtain protein expression information from biological samples and is currently widely used in the discovery and development of biopharmaceuticals. However, there is still a lack of mature technical solutions for systematically integrating proteomics with transcriptomics and single-cell omics, and for screening cross-species conserved cartilage regeneration regulatory proteins from natural regeneration models.

[0004] Deer antlers are the only organ in mammals capable of periodic, complete cartilage regeneration, with their cartilage undergoing complete structural reconstruction. This characteristic provides a valuable natural model for research into cartilage regeneration therapies. However, previous studies have primarily relied on conventional experimental animal models, making it difficult to elucidate the core molecular processes underlying complete cartilage regeneration in mammals. Current technologies lack endogenous regulatory molecules and targeted intervention strategies that can effectively drive functional regeneration of adult hyaline cartilage, hindering structural regeneration and functional recovery in degenerative cartilage diseases such as osteoarthritis. Summary of the Invention

[0005] The purpose of this invention is to provide a method for screening MARCKS, a protein associated with promoting cartilage regeneration.

[0006] Another objective of this invention is to provide a method for increasing the phosphorylation level of protein MARCKS.

[0007] The present invention also aims to provide the application of MARCKS protein and MARCKS phosphorylation activator, which solves the problem in the prior art of lacking endogenous regulatory molecules and targeted intervention strategies that can effectively drive the functional regeneration of hyaline cartilage, and thus preventing structural regeneration and functional recovery in degenerative cartilage diseases such as osteoarthritis.

[0008] The first technical solution adopted in this invention is a screening method for MARCKS, a protein related to promoting cartilage regeneration, as follows: Step 1: Collect cartilage tissue samples at different time points during antler cartilage regeneration; Step 2: Perform TMT-labeled quantitative proteomics detection on cartilage tissue samples to obtain time-series protein expression data; Step 3: Perform time-series cluster analysis on the protein expression data to screen for protein modules that show sustained increases during the rapid growth phase of cartilage; Step 4: Perform correlation analysis between the screened protein modules and the antler transcriptome data to obtain candidate genes with consistent transcription-protein levels; Step 5: Through conserved screening across species and developmental stages, the MARCKS protein was identified as a key target for cartilage regeneration.

[0009] The invention is further characterized in that, The different time points mentioned in step 1 include day 0, day 2, day 5, day 10, and day 60 after the antlers naturally fall off.

[0010] In step 4, candidate genes with a correlation > 0.8 are selected.

[0011] The second technical solution adopted in this invention is: a method for increasing the phosphorylation level of protein MARCKS, wherein the screened MARCKS protein is phosphorylated in any of the following ways: a) Utilizing protein MARCKS phosphorylation activator; b) Overexpressing or enhancing MARCKS protein expression; c) Activate PKCε protein kinase activity; d) Inhibit MARCKS dephosphorylation.

[0012] Activating the MARCKS-related pathway by increasing MARCKS phosphorylation levels upregulates the expression of SP1, SIRT1, SOX9, COL2A1, and ACAN proteins. SOX9 is a key transcription factor controlling chondrocyte differentiation and phenotype maintenance; COL2A1 and ACAN are the main structural protein (type II collagen) and major aggregate protein (ACAN) of the extracellular matrix of chondrocytes, respectively, and upregulation of all three directly promotes cartilage matrix synthesis and deposition. SIRT1 is known to inhibit chondrocyte senescence and inflammatory responses through deacetylation. SP1, as discovered in this invention, regulates multiple cartilage-related genes by binding to MARCKS.

[0013] The MARCKS phosphorylation activator (ψεRACK) is a PKCε selective agonist peptide containing an active effect sequence and a membrane-penetrating sequence. The active effect sequence contains HDAPIGYD, and the membrane-penetrating sequence contains YGRKKRRQRRR, which are linked by an amide bond or a disulfide bond. It is synthesized using the Fmoc solid-phase synthesis strategy and purified by HPLC.

[0014] The amino acid sequence contained in the MARCKS phosphorylation activator is SEQ ID No. 1.

[0015] The third technical solution adopted in this invention is the application of MARCKS protein, which is used as a target in screening or preparing products that promote cartilage repair.

[0016] The fourth technical solution adopted in this invention is the application of MARCKS phosphorylation activator in the preparation of drugs for treating osteoarthritis or promoting cartilage damage repair.

[0017] The concentration of MARCKS phosphorylation activator (ψεRACK) used is 1 μM to 50 μM, more preferably 10 μM; the intra-articular injection volume is 5 μL to 15 μL, more preferably 5 μL; the dosing frequency is once every 1 to 4 weeks, more preferably once every 2 weeks.

[0018] The beneficial effects of this invention are: (1) A systematic and efficient method for screening cartilage regeneration-related proteins is provided, which can discover conserved cartilage regeneration regulators in mammals from natural regeneration models; (2) MARCKS was identified for the first time as a key protein for cartilage regeneration, and a new mechanism by which it promotes chondrogenic differentiation through phosphorylated nuclear translocation and the SP1-SIRT1 axis was elucidated. (3) It provides a method to activate the MARCKS pathway to promote cartilage repair, which can promote the differentiation of stem cells into cartilage in vitro and effectively treat osteoarthritis in vivo; (4) Through in vitro and in vivo experiments, MARCKS phosphorylation activator can effectively reduce cartilage damage in osteoarthritis, protect subchondral bone, and improve joint function. It has a clear molecular target and good prospects for clinical translation. Attached Figure Description

[0019] Figure 1 It is a proteomics sampling design diagram; Figure 2 It is a multi-omics joint analysis diagram; Figure 3 This is a diagram showing the cross-species conservation of the MARCKS protein; Figure 4 This is a diagram showing the reduced chondrogenic ability of synovial mesenchymal stem cells after MARCKS knockout. Figure 5 This is a translocation map of ψεRACK-activated MARCKS protein from the cytoplasm to the nucleus; Figure 6 This is a WB image showing the activation of MARCKS-related pathways to promote cartilage regeneration; Figure 7This is a micro-CT scan of a mouse OA model after treatment, along with bone density and joint space statistics. Figure 8 These are HE staining and safranin-fast green staining images of a mouse OA model after treatment. Figure 9 This is an immunofluorescence staining image of COL2A1 and MMP13 in a mouse OA model after treatment. Figure 10 This is a graph showing the changes in molecular transcriptome levels after treatment in a mouse OA model. Figure 11 This is a gait analysis graph showing gait frequency and step length in a mouse OA model after treatment. Detailed Implementation

[0020] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0021] Example 1 Screening of cartilage regeneration-related proteins MARCKS 1.1 Sample Collection On days 0, 2, 5, 10, and 60 after the antlers naturally shed, cartilage tissue from the distal end of the antler was aseptically harvested under anesthesia. Bone and connective tissue were removed, and the tissue was flash-frozen in liquid nitrogen and stored at -80°C. Three animals were used at each time point as biological replicates.

[0022] 1.2 TMT-labeled quantitative proteomics detection The cartilage tissue described above was collected and lysed with 6M urea, 2M thiourea, and 100mM TEAB buffer using pressure cycling. Proteins were reduced and alkylated, then sequentially digested with Lys-C (1:40) and trypsin (1:50). Desalted peptides were labeled with TMTpro 16-plex reagent. The labeled peptides were fractionated into 30 fractions by high-pH reversed-phase chromatography (5%-35% acetonitrile / 10mM ammonium hydroxide), and data-dependent acquisition was performed using an EASY-nLC 1200 tandem Q Exactive HF-X mass spectrometer. MS1 scan resolution was 60000 (m / z 350-1800), and the first 18 precursor ions were selected for MS / MS (resolution 45000). Raw data were retrieved from the sika deer protein database using Proteome Discoverer v2.4.

[0023] 1.3 Temporal Clustering and Multi-omics Integration Analysis Protein quantification values ​​at each time point were subjected to Mfuzz fuzzy clustering to divide expression modules. The C5 module, which showed sustained increases and enrichment in the chondrogenesis pathway during the rapid chondrogenesis phase (days 10-60), was selected. Spearman correlation analysis was performed on C5 module proteins and antler transcriptome data (bulk RNA-seq and single-cell RNA-seq) to select candidate genes with p > 0.8. Single-cell pseudo-time series analysis (Monocle2) was used to determine the expression peak of candidate genes in the early differentiation of antler stem cells into chondrocytes. The above analysis showed that MARCKS and GALNT1 exhibited high consistency between transcriptional and protein levels, and showed expression peaks in the early pseudo-time series differentiation. Western blot validation confirmed that the expression level of MARCKS protein remained continuously elevated during antler cartilage regeneration.

[0024] 1.4 Cross-species conservation screening Hyaline cartilage from the knee joints of sika deer embryos (approximately 190 days gestation), 2-year-old deer, and 12-year-old deer was collected and subjected to proteomics analysis as described in step 1.2 to obtain a developmental-senescence proteomic atlas. The intersection of highly expressed proteins from antler regeneration and highly expressed proteins from embryonic knee joints (fold difference >2, p < 0.05) yielded 46 co-upregulated proteins. These intersection proteins were mapped to a mouse cartilage developmental time-series proteome (3 weeks, 2 months, 5 months, 12 months, 18 months), and proteins highly expressed in mice at 3 weeks of age (the repair capacity window) were screened. Cross-species sequence alignment identified MARCKS as a protein with a highly conserved effector domain.

[0025] Example 2: Preparation of MARCKS phosphorylation activator This embodiment uses the PKCε selective agonist peptide as a representative of the MARCKS phosphorylation activator for verification. It contains the active effect sequence HDAPIGYD and the transmembrane sequence YGRKKRRQRRR, which are linked by a disulfide bond. The amino acid sequence is SEQ ID No. 1: His-Asp-Ala-Pro-Ile-Gly-Tyr-Asp-Cys-Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Cys.

[0026] A solid-phase synthesis strategy using Fmoc was employed, with Wang resin (0.3 mmol / g substitution level) as the support. Fmoc-protected amino acids were sequentially coupled from the C-terminus to the N-terminus. For peptide 1 (HDAPIGYD-C), the first amino acid at the C-terminus was Fmoc-Cys(Trt)-OH; for peptide 2 (YGRKKRRQRRR-C), the first amino acid at the C-terminus was also Fmoc-Cys(Trt)-OH. The Arg side chain was protected with Pbf, the Lys side chain with Boc, the Tyr side chain with tBu, the Asp side chain with OtBu, the His side chain with Trt, and the Gln side chain with Trt. Each coupling step involved activation in DMF using 3 equivalents of Fmoc-AA-OH, 2.85 equivalents of HBTU, 3 equivalents of HOBt, and 6 equivalents of DIEA, followed by a 1-hour reaction. Fmoc was removed using a 20% piperidine / DMF solution, and coupling efficiency was monitored using the ninhydrin method. After the full-length sequence was assembled, it was cleaved with TFA / cleavage agent for 3 hours and precipitated with diethyl ether to obtain two linear crude peptides with free thiol groups at the C-terminus.

[0027] Two crude peptides were dissolved separately in 50 mM Tris-HCl buffer (pH 7.5) containing 6 M guanidine hydrochloride, and mixed in an equimolar ratio to achieve a total peptide concentration of approximately 0.5 mg / mL. The mixture was stirred at room temperature for 24 hours to allow intermolecular disulfide bonds to form between the C-terminal Cys groups of the two peptides. After the reaction, the product was purified by preparative reversed-phase high-performance liquid chromatography (C18 column, mobile phase A: 0.1% TFA / water, mobile phase B: 0.1% TFA / acetonitrile, gradient elution). The target heterodimer fraction was collected and freeze-dried. The final product was analyzed by RP-HPLC (C18 column, 214 nm detection, area normalization method) to determine a purity greater than 95%.

[0028] Example 3: MARCKS phosphorylation activator promotes chondrogenic differentiation in vitro 3.1 Isolation and Culture of Synovial Mesenchymal Stem Cells (SMSCs) Synovial tissue from the knee joints of 6-8 week old C57BL / 6 mice was collected, minced, and digested with 0.2% type II collagenase at 37°C for 12 hours. The tissue was filtered through a 200-mesh sieve, resuspended in DMEM / F12 complete medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin), and seeded in T25 culture flasks. The culture was incubated at 37°C with 5% CO2. The medium was changed every 2 days, and non-adherent cells were removed after 4 days. Cells were passaged when they reached 80% confluence. Cells from passages 3-5 were used for experiments. Flow cytometry was used to identify cell surface markers (CD105). + CD90 + CD73 + CD34 - CD45 - This confirms that the SMSCs were successfully separated.

[0029] 3.2 Treatment with MARCKS phosphorylation activator promotes chondrogenic differentiation SMSCs were 2 × 10 5 Cells were seeded in 6-well plates and cultured in chondrogenic induction medium (containing 10 ng / mL insulin and 100 ng / mL TGF-β1), with MARCKS phosphorylation activator added to a final concentration of 10 μM. The medium was changed every 2 days. An equal volume of physiological saline was added to the control group (Veh was the solvent control group, used to eliminate interference from the solvent itself; si-MARCKS is a small interfering RNA targeting MARCKS, used to knock down MARCKS expression). Alsin blue staining was performed on days 7 and 14 of induction to assess matrix deposition and chondrogenic phenotype. Figure 4 The results showed that the MARCKS phosphorylation activator treatment group had significantly stronger Alsin blue staining on days 7 and 14 than the control group, indicating that it significantly enhanced the chondrogenic differentiation capacity of SMSCs.

[0030] 3.3 Marks Knockdown Verification To further verify that the above-mentioned effect depends on MARCKS, SMSCs were seeded in 6-well plates. When the confluence reached 70%, si-MARCKS (final concentration 50 nM) or the negative control si-NC were transfected using Lipofectamine 3000. The culture medium was changed 6 hours after transfection, and the cells were cultured for another 48 hours. Western blot was used to verify the knockdown efficiency. After knockdown, chondrogenic induction and treatment with MARCKS phosphorylation activator were performed according to step 3.2. The results showed that after MARCKS knockdown, the chondrogenic differentiation effect induced by MARCKS phosphorylation activator was significantly weakened (Alsin blue staining was weakened), proving that the effect of MARCKS phosphorylation activator depends on MARCKS.

[0031] 3.4 MARCKS nuclear translocation detection SMSCs were seeded in confocal culture dishes and treated with a MARCKS phosphorylation activator for 24 hours, followed by immunofluorescence staining. In the control group, MARCKS were mainly located in the cytoplasm. After treatment with the MARCKS phosphorylation activator, MARCKS underwent a significant redistribution from the cytoplasm to the nucleus. Western blot analysis after nucleocytoplasmic separation further confirmed that treatment with the MARCKS phosphorylation activator significantly increased the number of intranuclear MARCKS and phosphorylated MARCKS (p-MARCKS), while the PKC inhibitor Gö6983 produced the opposite effect.

[0032] 3.5 Mechanism Verification: Activation of SP1 / SIRT1 Axis ATAC-seq and bulk RNA-seq analyses were performed on SMSCs treated with MARCKS phosphorylation activator. ATAC-seq identified 6303 regions with increased chromatin accessibility, while RNA-seq identified 2857 upregulated genes. Motif enrichment analysis showed significant enrichment at binding sites for bZIP and C2H2 zinc finger transcription factors. Footprint analysis identified SP1 as a key downstream transcription factor.

[0033] Co-IP experiments confirmed a physical interaction between p-MARCKS and SP1, while unphosphorylated MARCKS did not significantly bind to SP1. SP1 knockdown significantly suppressed the protein expression of cartilage marker genes SOX9, COL2A1, and ACAN, while SP1 overexpression reversed this inhibitory effect. Network analysis further revealed that SIRT1 is located at a downstream regulatory node of SP1. Interference with either SP1 or SIRT1 significantly reduced the expression of cartilage marker genes.

[0034] Example 4: In vivo treatment of osteoarthritis in mice with MARCKS phosphorylation activator 4.1 Establishment of osteoarthritis (OA) model and drug administration Eight-week-old male C57BL / 6 mice were anesthetized with sodium pentobarbital. A longitudinal incision was made on the medial side of the right knee, and the knee joint was exposed via a medial approach through the patellar ligament. The anterior cruciate ligament was then cut (ACLT method) to establish an osteoarthritis (OA) model. In the sham surgery group, only the joint capsule was cut, without cutting the ligaments.

[0035] After ACLT surgery, animals were randomly assigned to a model group (ACLT + saline) and a treatment group (ACLT + MARCKS phosphorylation activator), with 5 animals in each group. The treatment group received an intra-articular injection of 5 μL (10 μM) of MARCKS phosphorylation activator immediately after surgery and every two weeks thereafter. The model group received an equal volume of sterile saline.

[0036] 4.2 Gait Analysis Gait analysis was performed at week 8 post-surgery (16 weeks of age for mice). Stride length and walking speed were recorded using the CatWalk system. Results showed that the model control group had significantly shorter stride length and significantly slower walking speed, while the MARCKS phosphorylation activator treatment group showed significant recovery in stride length and walking speed, approaching the levels of the sham-operated group.

[0037] 4.3 Micro-CT Detection Mice were sacrificed after gait analysis, and the knee joints were harvested for Micro-CT scans to measure bone volume fraction (BV / TV) and trabecular bone separation (Tb.Sp). Results showed that the model control group had significantly reduced bone volume fraction and significantly increased trabecular bone separation. The MARCKS phosphorylation activator treatment group showed significant improvement in these indicators, approaching the levels of the sham-operated group.

[0038] 4.4 Histological examination Knee joints were decalcified, paraffin-embedded, and sectioned (5 μm) for Safranin O-Fix Green (SafO-FG) and H&E staining. Cartilage damage was scored according to the OARSI criteria. Results showed that the model control group exhibited severe cartilage matrix loss and significant surface wear, with a significantly elevated OARSI score. The MARCKS phosphorylation activator treatment group showed intact cartilage matrix and a significantly reduced OARSI score.

[0039] Immunofluorescence staining was used to detect the proportion of COL2A1 and MMP13 positive cells. The results showed that the proportion of COL2A1 positive cells in the MARCKS phosphorylation activator treatment group was significantly higher than that in the model control group, while the proportion of MMP13 positive cells was significantly lower than that in the model control group.

[0040] 4.5 Transcriptome Analysis Total RNA was extracted from knee cartilage of mice in each group, constructed into libraries, and sequenced on the Illumina NovaSeq platform (150bp paired ends). Differentially expressed genes were selected based on |log2FC|>1, p<0.05. Results showed that the MARCKS phosphorylation activator reversed the widespread transcriptional dysregulation induced by ACLT. Venn analysis identified 721 downregulated genes in osteoarthritis (OA) that were restored by the MARCKS phosphorylation activator, and 782 upregulated genes in OA that were suppressed. GO enrichment analysis showed that the restored downregulated genes were enriched in anabolic pathways such as chondrogenic differentiation, SMAD signaling, and glycosaminoglycan metabolism; the suppressed upregulated genes were enriched in degenerative pathways such as bone mineralization, osteogenic differentiation, and abnormal collagen remodeling. Key gene heatmaps showed that the MARCKS phosphorylation activator significantly upregulated Sp1, Sirt1, Col2a1, and Acan, and inhibited inflammatory and catabolic factors such as Il6 and Mmp13.

[0041] Example 5: Validation of MARCKS as a universal target To further verify the feasibility of MARCKS as a universal target for cartilage repair, the expression level of MARCKS in donor-derived cells of different ages was examined, as well as the correlation between MARCKS phosphorylation level and chondrogenic differentiation capacity. The results showed that MARCKS expression and phosphorylation levels were negatively correlated with donor age and positively correlated with chondrogenic differentiation capacity. These results indicate that MARCKS is a key molecular node regulating cartilage regeneration capacity.

[0042] The application principle of MARCKS phosphorylation activator: This invention reveals that the core mechanism of MARCKS's role in cartilage regeneration lies in the following: after phosphorylation mediated by PKCε, MARCKS translocates from the cytoplasm to the nucleus, directly interacting with the transcription factor SP1, thereby activating the downstream SIRT1 signaling pathway and initiating the cartilage differentiation program. The advantages of using the PKCε-selective agonist peptide ψεRACK (composed of the active sequence HDAPIGYD linked with the membrane-penetrating peptide TAT) are: it specifically enhances MARCKS phosphorylation and nuclear translocation, avoiding the off-target effects of non-selective PKC agonists; the membrane-penetrating peptide TAT ensures effective entry of the peptide into cells, making it suitable for in vivo intra-articular injection.

[0043] In the treatment of osteoarthritis, the principle of intra-articular injection of ψεRACK lies in its direct action on synovial mesenchymal stem cells and residual chondrocytes within the joint cavity, activating the endogenous MARCKS / SP1 / SIRT1 pathway. This inhibits cartilage matrix degradation (downregulating MMP13), promotes matrix synthesis (upregulating COL2A1 and ACAN), and improves subchondral bone microstructure. Its advantages include: it does not rely on exogenous cells or gene transfection, is simple to administer, and has good clinical translational feasibility.

[0044] Example 6 The screening method described in this invention and the MARCKS phosphorylation activator ψεRACK were tested, and the results are as follows: like Figure 1 As shown, the proteomics sampling design for antler cartilage regeneration at five time points (0, 2, 5, 10, and 60 dac) covered the entire process from injury response to rapid cartilage growth.

[0045] like Figure 2 As shown, multi-omics joint analysis screened out 23 candidate genes that were highly consistent at the transcriptional and protein levels, among which MARCKS showed the most significant expression peak in the early stage of the pseudo-time-sequence differentiation trajectory.

[0046] like Figure 3 As shown, the effector domains of the MARCKS protein are highly conserved in mammals (deer, mice, and humans), suggesting that its cartilage regeneration regulation function has cross-species universality.

[0047] like Figure 4 As shown, knocking down MARCKS in synovial mesenchymal stem cells significantly reduced the chondrogenic differentiation capacity induced by ψεRACK (decreased Alsin blue staining), demonstrating that the effect of ψεRACK depends on MARCKS.

[0048] like Figure 5As shown, after ψεRACK treatment, MARCKS translocated from the cytoplasm to the nucleus. Nucleocytoplasmic separation experiments further confirmed that phosphorylated MARCKS were enriched in the nucleus, while the PKC inhibitor Gö6983 inhibited this translocation.

[0049] like Figure 6 As shown, Western blot results indicate that activation of the MARCKS-related pathway upregulates the expression of SP1, SIRT1, SOX9, COL2A1, and ACAN proteins, while knocking down SP1 or SIRT1 eliminates this effect.

[0050] like Figure 7 As shown, Micro-CT scans of the mouse osteoarthritis (OA) model induced by anterior cruciate ligament transection after treatment showed that the bone volume fraction in the ψεRACK treatment group was significantly higher than that in the model control group, the trabecular separation was significantly reduced, and the joint structure was improved. Among them, Sham represents the sham surgery group, and ctrl is the control group. In the sham surgery group, Sham only cuts the skin to expose the joint without damaging the cartilage, while in the control group, ctrl is the model group that causes cartilage damage but does not treat it.

[0051] like Figure 8 As shown, hematoxylin-eosin staining and safranin-fast green staining revealed that the cartilage matrix in the ψεRACK treatment group remained intact.

[0052] like Figure 9 As shown, immunofluorescence staining revealed that the proportion of COL2A1 positive cells in the ψεRACK treatment group was significantly higher than that in the model control group, while the proportion of MMP13 positive cells was significantly lower.

[0053] Figure 10 It is the OARSI score after staining. The higher the score, the more severe the cartilage damage. 0 points means intact, and 6 points means that the cartilage has completely eroded into the bone.

[0054] like Figure 11 As shown in the figure, the footprint analysis results show that the stride length and walking speed of the ψεRACK treatment group were significantly higher than those of the control group.

[0055] It should be noted that the present invention is not limited to the specific embodiments described above. For those skilled in the art, several improvements or substitutions can be made based on the concept of the present invention, and all such improvements or substitutions should be considered to fall within the scope of the present invention.

[0056] sequence list <110> Northwestern Polytechnical University <120> Preparation method and application of MARCKS agonists that promote cartilage regeneration <130> none <160> 1 <210> 1 <212> SEQ ID No. 1 <213> Artificial sequence <400> HDAPIGYDCYGRKKRRQRRRC

Claims

1. A method for screening MARCKS, a protein associated with promoting cartilage regeneration, characterized in that, Specifically as follows: Step 1: Collect cartilage tissue samples at different time points during antler cartilage regeneration; Step 2: Perform TMT-labeled quantitative proteomics detection on cartilage tissue samples to obtain time-series protein expression data; Step 3: Perform time-series cluster analysis on the protein expression data to screen for protein modules that show sustained increases during the rapid growth phase of cartilage; Step 4: Perform correlation analysis between the screened protein modules and the antler transcriptome data to obtain candidate genes with consistent transcription-protein levels; Step 5: Through conserved screening across species and developmental stages, the MARCKS protein was identified as a key target for cartilage regeneration.

2. The screening method for MARCKS, a protein related to promoting cartilage regeneration, according to claim 1, is characterized in that, The different time points mentioned in step 1 include day 0, day 2, day 5, day 10, and day 60 after the antlers naturally fall off.

3. The screening method for MARCKS, a protein related to promoting cartilage regeneration, according to claim 1, is characterized in that... In step 4, candidate genes with a correlation > 0.8 are selected.

4. A method for increasing the phosphorylation level of protein MARCKS, characterized in that, Use any of the following methods: a) MARCKS phosphorylation activator obtained using protein MARCKS; b) Overexpressing or enhancing MARCKS protein expression; c) Activate PKCε protein kinase activity; d) Inhibit MARCKS dephosphorylation.

5. The method for increasing the phosphorylation level of protein MARCKS according to claim 4, characterized in that, The MARCKS phosphorylation activator is a PKCε selective agonist peptide containing an active effect sequence and a membrane-penetrating sequence; the active effect sequence contains HDAPIGYD, and the membrane-penetrating sequence contains YGRKKRRQRRR, which are linked by an amide bond or a disulfide bond; it is synthesized using the Fmoc solid-phase synthesis strategy and obtained by HPLC purification.

6. The method for phosphorylation treatment of chondrocyte regeneration protein MARCKS according to claim 4, characterized in that, The amino acid sequence contained in the MARCKS phosphorylation activator is SEQ ID No.

1.

7. Applications of the MARCKS protein, characterized in that, Application of MARCKS protein as a target in screening or preparing products that promote cartilage repair.

8. The application of MARCKS phosphorylation activator, characterized in that, Applications in the preparation of drugs for treating osteoarthritis or promoting the repair of cartilage damage.

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