Application of Chchd6 protein as target to preparation of medicine for treating and preventing osteoarthritis

Abstract

The invention discloses an application of Chchd6 protein as a target spot in preparation of a medicine for treating and preventing osteoarthritis. Research finds that pathological mineralization can be inhibited by adjusting the mitochondrial inner membrane protein Chchd6, and a new target is provided for intervening pathological calcification from the source. On the basis, the hydrogel microspheres for treating osteoarthritis by delivering Chchd6 protein through active oxygen response hydrogel of targeted mitochondria are designed, the microspheres can respond to the rising active oxygen level in an osteoarthritis articular cavity, Chchd6 is released to chondrocyte mitochondria, and by inhibiting abnormal aggregation of mitochondrial DNA and formation of a mineral precursor which occurs accordingly, the osteoarthritis can be treated. The starting of pathological cartilage calcification is blocked from the source, so that the progress of osteoarthritis is effectively delayed.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials and osteoarthritis treatment technology, specifically relating to a smart hydrogel microsphere that targets mitochondria and responds to reactive oxygen species, its preparation method, and its application in the early treatment of osteoarthritis. Background Technology

[0002] Osteoarthritis is a common degenerative joint disease characterized by articular cartilage degradation, synovial inflammation, and pathological calcification. Pathological cartilage calcification plays a crucial role in the progression of osteoarthritis, as its resulting mineral deposits disrupt joint homeostasis and exacerbate matrix degradation.

[0003] Current research indicates that chondrocyte-derived matrix vesicles provide the minerals that induce pathological chondrocyte calcification. However, the origin of these minerals within osteoarthritis chondrocytes remains unclear, severely hindering the development of targeted therapies.

[0004] Traditional treatments, such as nonsteroidal anti-inflammatory drugs and intra-articular injections of hyaluronic acid, mainly focus on relieving symptoms and cannot fundamentally block the initial pathological process of the disease, especially the occurrence of early pathological calcification. Summary of the Invention

[0005] This invention has discovered that regulating the mitochondrial inner membrane protein Chchd6 can inhibit pathological mineralization, providing a new target for intervening in pathological calcification at its source. Based on this, the invention further provides the application of the aforementioned hydrogel microspheres or pharmaceutical composition in the preparation of medicaments for the treatment or prevention of osteoarthritis, particularly achieving therapeutic effects by inhibiting pathological calcification of chondrocytes.

[0006] Based on the inventors' research findings, this application provides the application of using the Chchd6 protein and its encoding gene as targets to prepare drugs for the treatment and prevention of osteoarthritis. Furthermore, the drug is a mitochondrial-targeting drug. The drug comprises the Chchd6 protein and a mitochondrial target. The treatment or prevention is achieved by inhibiting pathological calcification of chondrocytes. The inhibition of pathological calcification of chondrocytes is achieved by preventing the formation of mineral precursors.

[0007] The present invention also provides a medicament for the treatment of osteoarthritis, the medicament comprising Chchd6 protein and mitochondrial targets.

[0008] An alternative approach is that the drug comprises liposomes loaded with Chchd6 protein and a mitochondrial target. The drug formulation is a hydrogel microsphere, on which liposomes loaded with Chchd6 protein and the mitochondrial target are coated. The mitochondrial target is SS31. The hydrogel matrix of the hydrogel microsphere comprises methacrylamide hyaluronic acid and phenylboronic acid-modified hyaluronic acid. The mass ratio of methacrylamide hyaluronic acid to phenylboronic acid-modified hyaluronic acid is 1:1.

[0009] An alternative approach is that the preparation method of the above-mentioned drug includes:

[0010] (1) Prepare liposomes loaded with Chchd6, and then link SS31 peptide to the liposomes to obtain mitochondrial-targeting liposomes; (2) The liposomes obtained in step (1) are dispersed in a physiological saline solution containing methacrylamide hyaluronic acid and phenylboronic acid modified hyaluronic acid to form a mixed reaction solution; (3) The mixed reaction solution obtained in step (2) was prepared into hydrogel microspheres using microfluidic technology.

[0011] Further optionally, in step (2), the concentration of the liposomes in the mixed reaction solution is 1-10 mg / mL.

[0012] This invention is the first to intervene in mitochondria as the source of pathological calcification mineral precursors in osteoarthritis, targeting the Chchd6 protein to fundamentally prevent calcification initiation. Specifically, leveraging the elevated levels of reactive oxygen species (ROS) within the joint cavity of osteoarthritis patients, a ROS-responsive hydrogel shell is designed to achieve lesion-specific release of therapeutic drugs, improving targeting and efficacy while reducing systemic side effects. Furthermore, the core-shell structure design combines the organelle targeting capabilities of liposomes with the sustained-release and drug-protective properties of hydrogel microspheres, constructing a highly efficient cascade delivery system. Attached Figure Description

[0013] Figure 1 This invention presents a heatmap of differentially expressed proteins obtained through mitochondrial proteomics analysis.

[0014] Figure 2 shows the Alizarin Red staining (A) and quantification (B) of the key proteins used in this invention to screen for inhibiting pathological calcification in osteoarthritis.

[0015] Figure 3 shows the tissue observation (A) and quantification (B) of the effect of in vivo upregulation of Chchd6 in chondrocytes on the progression of osteoarthritis.

[0016] Figure 4 This is a schematic diagram illustrating the preparation principle of mitochondrial-targeted liposomes in an embodiment of the present invention.

[0017] Figure 5 shows a scanning electron microscope (A) and a confocal fluorescence microscope (B) image of the reactive oxygen species responsive hydrogel microspheres prepared in the embodiment of the present invention, showing that the microspheres have a regular morphology and that liposomes (green) are successfully encapsulated inside the microspheres.

[0018] Figure 6 shows the immunofluorescence staining (A) and quantitative analysis (B) of Chchd6 protein in the cells after co-culturing hydrogel microspheres with chondrocytes in this embodiment of the invention. The results show that the fluorescence intensity of Chchd6 in the Lipo-RRM group is significantly higher than that in the Control-RRM group.

[0019] Figure 7 shows the immunofluorescence staining (A) and quantitative analysis (B) of Chchd6 protein and mitochondrial marker Tomm20 in articular chondrocytes after treatment of a rat model of osteoarthritis with hydrogel microspheres in this embodiment of the invention. The results show that Chchd6 in the Lipo-RRM group is distributed in the mitochondria, indicating that Chchd6 protein was successfully delivered to the mitochondria.

[0020] Figure 8 shows the HE staining and Safranin O staining images of articular cartilage (A), as well as the quantitative analysis of cartilage tissue and OARSI score (B) after hydrogel microsphere treatment of a rat model of osteoarthritis in this embodiment of the invention. The results show that the loss of cartilage proteoglycans in the Lipo-RRM group was reduced, and the degree of osteoarthritis was alleviated.

[0021] Figure 9 The images shown are scanning electron microscope (SEM) images and elemental mapping images of the articular cartilage calcification areas in the hydrogel microsphere treatment group and the control group in this embodiment of the invention. The images show that the Lipo-RRM group has a significant reduction in pathological calcification deposition. Detailed Implementation

[0022] Unless otherwise specified, the scientific and technical terms used in this article are intended for understanding by those skilled in the art.

[0023] This invention has discovered that regulating the mitochondrial inner membrane protein Chchd6 can inhibit pathological mineralization, providing a new target for intervening in pathological calcification at its source. Based on this, related drugs for the treatment and prevention of osteoarthritis can be developed. This article further explains the invention by designing a reactive oxygen species-responsive hydrogel targeting mitochondria to deliver Chchd6 protein for the treatment of osteoarthritis.

[0024] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. The test specimens and research materials used in the following embodiments are all commercially available products.

[0025] Example 1: Screening of key regulatory proteins in osteoarthritis (1) Differential protein analysis of mitochondrial proteomics Eight-week-old SPF-grade female C57BL / 6 mice (weighing 17–19 g) were purchased from the Experimental Animal Center of Air Force Medical University. All animal experimental protocols were approved by the university's Animal Ethics Committee (approval number: IACUC-20241371), and the experiments were conducted in accordance with the ARRIVE guidelines.

[0026] Modeling: Mice were randomly divided into two groups for temporomandibular joint surgery: a TMJ-OA group induced by a unilateral UAC appliance (n = 21) and a sham control group (n = 21). The specific procedures were as follows: In the OA group, a 1.5 mm long metal tube was fixed to the left maxillary incisor using zinc phosphate cement, while a 5 mm long metal tube bent at a 135° angle was adhered to the left mandibular incisor. Anterior crossbite contact (overbite of -1.2 mm) was confirmed using a stereomicroscope. Mice in the control group received the same anesthesia procedure but did not have the appliance fitted.

[0027] One week after modeling, mitochondrial samples were isolated from the articular cartilage of the TMJ-OA group and the Control group of mice, respectively. To meet the minimum protein content requirements for sequencing, samples from every 7 mice were pooled to form one sequencing sample. Total protein was extracted using the iST8x Proteomics Sample Preparation Kit (Suzhou Panomics Biotechnology Co., Ltd.), followed by nano-level liquid chromatography-tandem mass spectrometry analysis.

[0028] The entire proteomics workflow (including sample preparation, mass spectrometry detection, and data analysis) was completed by Suzhou Panomics Biotechnology Co., Ltd. Raw mass spectrometry data were retrieved from the database using the Proteome Discoverer software platform (v2.4.1.15, Thermo Fisher Scientific) for probabilistic model-based peptide identification and quantification. The data quality assessment system included three dimensions: peptide matching accuracy (mass error <10 ppm), protein identification reliability (FDR <1%), and precursor ion mass tolerance control (± 0.02 Da).

[0029] After rigorous quality control, a quantitative proteomics dataset based on unique peptides was established. MitoCARTA 3.0 (Sneha et al., 2020) was used for mitochondrial protein classification, and multilevel functional annotation analysis was performed: 1) Gene Ontology enrichment analysis was used to identify significantly enriched biological processes; 2) KEGG pathway mapping was used to resolve metabolic network associations of differentially expressed proteins; 3) the STRING database was used to construct a protein-protein interaction network. Data standardization was performed using Lowess regression to eliminate systematic errors. Proteins meeting the quantitative criteria (≥2 unique peptides) underwent: 1) hierarchical clustering analysis based on Pearson correlation coefficients; 2) principal component analysis (PCA) was used to assess heterogeneity between groups; 3) two-tailed t-tests (n = 3 biological replicates) were used to screen for differentially expressed proteins (threshold: log2|fold change| ≥ 1.3 and P < 0.05). Data visualization was performed using the Python 3.8 ecosystem (matplotlib / seaborn library) to generate heatmaps. Results are shown below. Figure 1 As shown.

[0030] (2) Upregulation of chondrocyte proteins by overexpressing lentivirus: Based on the aforementioned mitochondrial proteomics and literature review, proteins associated with pathological calcification in osteoarthritis were screened. Two proteins, Chchd6 (sequence number Q91VN4) and Phb2 (sequence number O35129), which are regulated in osteoarthritis, were upregulated using lentiviral transfection overexpression. The specific method was as follows: Chondrocytes were harvested from the TMJ cartilage of 5-day-old C57BL / 6J mice. The specific steps were as follows: the cartilage was first digested with 0.25% trypsin for 20 minutes, followed by digestion with 0.2% type II collagenase for 2–3 hours. The isolated chondrocytes were resuspended and cultured in a humid environment at 37°C and 5% CO2. The culture medium consisted of DMEM containing 10% fetal bovine serum and 1% penicillin / streptomycin. The third-generation chondrocytes were plated in 12-well plates and transfected with Chchd6 (200 pfu / cell), Phb2 (200 pfu / cell) overexpressing lentiviruses, and the corresponding pLent-EF1aFH-CMV-copGFP-P2A-Puro control virus. The lentiviruses were provided by Shandong Weizhen Biotechnology Co., Ltd., and the transfection process was completed within 24 hours (n = 3). (3) Downregulation of chondrocyte proteins via shRNA plasmid transfection: For the screened osteoarthritis-related proteins, two proteins upregulated in osteoarthritis, Mtfp1 and Dnajc11 (Mtfp1 sequence number Q9CRB8 and Dnajc11 sequence number Q5U458), were downregulated by transfecting plasmids containing shRNA. The specific method was as follows: Chondrocytes were plated in 12-well plates and transfected with plasmids containing Mtfp1 shRNA and Dnajc11 shRNA, as well as corresponding control plasmids. Transfection was performed using Lipofectamine™ 3000 transfection reagent. All plasmids were provided by Hunan Fenghui Biotechnology Co., Ltd. The transfection process was completed within 8 hours (n = 3).

[0031] The sequences of the Mtfp1 shRNAs are as follows: Mtfp1-shRNA-A is 5'-CTTTGTATGGCAGGCTCTAGCCTCTGTGG- 3', Mtfp1-shRNA-B is 5'-CCATTGACAGGTCGGTAGACTTCCTCCTG- 3', Mtfp1-shRNA-C is 5'-TCCAGCTCCTATGTCTTGGCCGATGCCAT- 3', and Mtfp1-shRNA-D is 5'-AGAAGGCAGGAGAGGTGCAAGCCCTGAA- 3'. Among the four different Mtfp1-shRNA plasmids, Mtfp1-shRNA-D showed the most significant knockdown effect on Mtfp1 expression. Therefore, Mtfp1-shRNA-D was used in subsequent experiments. As a negative control, the sense strand sequence of the missense shRNA is 5'-GCACTACCAGAGCTAACTCAGATAGTACT-3', and the antisense strand sequence is 5'-AGTACTATCTGAGTTAGCTCTGGTAGTGC-3'.

[0032] The sequences of Dnajc11 shRNAs are as follows: Dnajc11-shRNA-A is 5'-GCAGACATAATGCAAGCCAAA- 3', Dnajc11-shRNA-B is 5'-CGTTATGATGAGGAATATGAA- 3', and Dnajc11-shRNA-C is 5'-CCCGCAGATTGAGATTAATAA- 3'. Among the three different Dnajc11-shRNA plasmids, Dnajc11-shRNAB showed the most significant knockdown effect on Dnajc11 expression. Therefore, Dnajc11-shRNA-B was used in subsequent experiments. As a negative control, the sense strand sequence of the missense shRNA was 5'-GCACTACCAGAGCTAACTCAGATAGTACT- 3', and the antisense strand sequence was 5'-AGTACTATCTGAGTTAGCTCTGGTAGTGC- 3'.

[0033] (4) Transfected cell culture: Cells in each group were cultured simultaneously in DMEM medium supplemented with 10 nM dexamethasone, 100 μM L-ascorbic acid and 10 ng / mL interleukin-1β.

[0034] (5) Alizarin Red S staining analysis After two weeks of culture, samples were fixed in 10% formaldehyde and stained with Alizarin Red S (40 mmol / L, pH 4.2). Thirty minutes after staining, the samples were washed with PBS to remove the dye. The stained samples were then imaged using a Zeiss microscope. Quantitative analysis of the stained areas was performed using ImageJ software to determine the calcification levels in each group (n=5).

[0035] The results are shown in Figure 2. The figure shows that the staining area in the Chchd6 upregulation intervention group was significantly reduced compared with the control group, indicating that Chchd6 upregulation intervention can significantly inhibit pathological calcification of cells.

[0036] Example 2: Chchd6 overexpression in chondrocytes inhibits the progression of osteoarthritis. (1) Construction of osteoarthritis model One week after unilateral anterior crossbite induction or medial meniscus instability surgery, each C57BL / 6 mouse (from the Animal Experiment Center of Air Force Medical University) was anesthetized with a mixture of 2% isoflurane and oxygen, followed by deep anesthesia via pentobarbital. Recombinant adeno-associated virus type 2 (AAV-Chchd6OE, 1.14 × 10⁻⁶) overexpressing Chchd6 and targeting chondrocytes was used in the experiment. 13 vg / mL, Shandong Weizhen Biotechnology Co., Ltd., Jinan, Shandong, China) and the corresponding control virus pAV-COL2A1-P2A-GFP (AAV-Control, 1.27×10 vg / mL, Shandong Weizhen Biotechnology Co., Ltd., Jinan, Shandong, China) and corresponding control virus pAV-COL2A1-P2A-GFP (AAV-Control, 1.27×10 vg / mL, Shandong Weizhen Biotechnology Co., Ltd., 13 (vg / mL, Shandong Weizhen Biotechnology Co., Ltd.) Both viruses were injected into the temporomandibular joint cavity at a dose of 300 nL, and into the knee joint cavity at a dose of 500 nL (n=5).

[0037] (2) Staining analysis Four weeks after unilateral anterior crossbite induction and six weeks after medial meniscus instability surgery, histological examinations were performed on the temporomandibular joint condyles and femoral condyles (n = 5). After fixation and complete decalcification with EDTA, the samples were embedded in paraffin. Paraffin-impregnated samples were selected, and central sagittal sections of the condyles and knee joints were cut into 5 μm thick sections and stained with Safranin O-Fix Green (n = 5). The sections were observed under a stereomicroscope, and the severity of osteoarthritis was assessed by quantifying the percentage of proteoglycan area and using the OARSI histological scoring system.

[0038] As shown in Figure 3, upregulation of Chchd6 in chondrocytes significantly inhibited the progression of osteoarthritis.

[0039] Example 3: Preparation of mitochondrial-targeting liposomes (Lipo-Chchd6) Step 1: Prepare liposomes loaded with Chchd6 protein using a thin-film hydration method: Phospholipids DSPC, Cholesterol, and DSPE-PEG2000 were dissolved in a chloroform / methanol mixture (volume ratio 2:1) at a molar ratio of 5:4:1. The solution was vortexed to completely dissolve the lipids, forming a clear and transparent solution. The flask was then connected to a rotary evaporator and evaporated under reduced pressure at 120 rpm for 30-45 minutes in a 40°C water bath until the solvent was completely evaporated, forming a uniform lipid film on the flask wall. To further remove residual solvent, the flask containing the lipid film was placed in a vacuum drying oven overnight.

[0040] Add 2 mL of PBS buffer (pH 7.4) containing Chchd6 protein (concentration 1 mg / mL, obtained by expression and purification from Chchd6) to the flask containing the dried lipid film; seal the flask and gently shake it in a 50°C water bath for 60 minutes to allow the lipid film to completely detach from the flask wall and form a multilayered liposome suspension. To ensure uniform liposome size and that the liposomes were monocompartments, the pre-hydrated liposome suspension was extruded sequentially through polycarbonate membranes (purchased from Waterman, UK): first, five extrusions were performed using a 400 nm pore size membrane (using the material from the previous extrusion as feed for the next), then ten more extrusions were performed using a 100 nm pore size membrane. The entire extrusion process was carried out at 65°C to maintain the lipids in a liquid crystal state and ensure smooth extrusion; the final product was a Chchd6-loaded liposome suspension with a size of approximately 100-200 nm.

[0041] The extruded liposome suspension was purified by ultracentrifugation (100,000 g, 4°C, 60 min) or by centrifugation using ultrafiltration centrifuge tubes (molecular weight cutoff 100 kDa) (in this example, an Optima XPN series ultracentrifuge was used for centrifugation purification). The purified liposomes were collected and resuspended in fresh PBS buffer (pH 7.4) to obtain the purified liposome suspension.

[0042] Step 2: To endow liposomes with mitochondrial targeting capability, the SS31 peptide was covalently linked to the PEG chain terminus on the liposome surface. First, the carboxyl terminus of the SS31 peptide was activated: the SS31 peptide was dissolved in MES buffer (pH 6.0), EDC·HCl and NHS were added, and the activation reaction was carried out at room temperature for 15 minutes. Then, the activated SS31 peptide solution was mixed with the purified liposome suspension obtained in Step 1, and the reaction was carried out with gentle stirring at room temperature for 4 hours. After the reaction, unreacted peptides and byproducts were removed again by ultracentrifugation or dialysis to obtain the mitochondrial-targeting liposome Lipo-Chchd6 (see schematic diagram). Figure 4 ).

[0043] Example 4: Preparation of reactive oxygen species responsive hydrogel microspheres (Lipo-RRM) Step 1, Synthesis of PBA-HA: Weigh 4.5 g of hyaluronic acid (HA) into a 1000 mL clean beaker, add 400 mL of deionized water, and stir magnetically at 500 rpm for 4 hours at room temperature until HA is completely dissolved, resulting in a colorless, transparent, viscous solution. Then, add 3.94 g of EDC·HCl to the HA solution and stir until completely dissolved to activate the carboxyl groups on the HA chain. Next, add 1.96 g of 3-APBA and continue stirring at room temperature in the dark for 24 hours. After the reaction is complete, transfer the reaction solution to a dialysis bag with a molecular weight cutoff of 3500 Da and dialyze with deionized water for 7 days, changing the water 3 times a day to thoroughly remove unreacted 3-APBA, the byproduct urea, and salt ions. Filter the dialyzed solution through a 0.22 μm microporous membrane, then pre-freeze it in an ultra-low temperature freezer at -80°C, and finally freeze-dry it in a freeze dryer for 48 hours to obtain a white, flocculent PBA-HA powder.

[0044] Step 2, Preparation of microsphere precursor solution: Accurately weigh HAMA powder and PBA-HA powder, mix them in a 1:1 mass ratio (2.5g each) in a 20 mL glass bottle, add 10 mL of physiological saline, and magnetically stir at room temperature in the dark for 6-8 hours until the powder is completely dissolved, forming a homogeneous and transparent mixed hydrogel prepolymer solution; then, add the Lipo-Chchd6 liposomes prepared in Example 3 to this mixed solution, so that the final concentration of Lipo-Chchd6 liposomes is 5 mg / mL (based on lipids). To promote photocrosslinking, LAP photoinitiator is added to the mixed solution at the same time, so that the final concentration is 0.25% (w / v); then, the mixed solution is gently stirred for 2 hours in the dark to ensure that the components are fully mixed and the liposomes are uniformly dispersed, to obtain the final microsphere precursor solution (Lipo-RRM precursor solution).

[0045] Step 3, Microfluidic Preparation of Microspheres: Using the microsphere precursor solution as raw material, Lipo-RRM microspheres were prepared using a flow-focusing microfluidic chip device (see Figure 2 for a schematic diagram). This example was commissioned to a professional company (Shanghai Pengzan Biotechnology Co., Ltd.). https: / / www.fluidiclab.com / product / droplet-generator / The operation was performed using FluidicLab's automated microsphere fabrication system, which uses pressure-driven mobile and dispersed phases that enter the microsphere generation chip at a constant flow rate via a flow sensor. By adjusting the flow rate and chip parameters, the microsphere diameter was controlled to be between 8 and 12 μm.

[0046] Scanning electron microscopy (SEM) revealed the morphology of the microspheres, as shown in Figure 5. The microspheres were regular spherical with smooth surfaces. Confocal microscopy, also shown in Figure 5, revealed that the liposomes (labeled with green fluorescence) were uniformly distributed within the microspheres.

[0047] Example 5: In vitro performance testing of Lipo-RRM microspheres Chondrocytes were harvested from the TMJ cartilage of 5-day-old C57BL / 6J mice. The cells were first digested with 0.25% trypsin for 20 minutes, followed by digestion with 0.2% type II collagenase for 2–3 hours. The isolated chondrocytes were resuspended and cultured in a humid environment at 37°C and 5% CO2 in DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin. Third-generation primary chondrocytes from mice in good growth condition were divided into two groups for intervention: (1) Lipo-RRM group: Lipo-RRM microspheres (final concentration 5 mg / mL) were added to OA simulated medium and the cells were cultured. (2) Control-RRM group: The cells were cultured in OA simulated medium with blank hydrogel microspheres (final concentration 5 mg / mL, prepared by the method in Example 4 above without Lipo-Chchd6 liposomes) without liposomes.

[0048] Three days later, the cells were fixed and subjected to Chchd6 immunofluorescence staining. Confocal microscopy observation showed, as shown in Figure 6, that the Chchd6 fluorescence signal in the cells of the Lipo-RRM group was significantly higher than that in the Control-RRM group, proving that the microspheres can effectively deliver Chchd6 to chondrocytes.

[0049] Example 6: Evaluation of the therapeutic effect of Lipo-RRM microspheres in an animal model of osteoarthritis Animal model establishment: Eight-week-old female C57BL / 6J mice were selected to induce a temporomandibular joint osteoarthritis model through unilateral anterior tooth reverse dentition.

[0050] Treatment intervention: One week after modeling, the mice were randomly divided into two groups: (1) Lipo-RRM treatment group: 100 μL of PBS suspension of 1.5 mg / mL Lipo-RRM microspheres was injected into the joint cavity; (2) Control-RRM control group: 100 μL of blank microsphere suspension without liposomes was injected.

[0051] Efficacy evaluation: Two weeks after modeling, the mice were sacrificed and the condylar joints were collected for immunofluorescence staining of Chchd6 and the mitochondrial marker TOMM20. As shown in Figure 7, the mitochondrial marker TOMM20 in the Lipo-RRM group was colocalized with Chchd6 and was significantly higher than that in the Control-RRM control group, indicating that the in vivo administration method can deliver Chchd6 into the mitochondria of chondrocytes.

[0052] Histological analysis: Mice were sacrificed six weeks after modeling. Condylar joint sections were stained with hematoxylin and eosin (HE) and Safranin O / Fixed Green to assess chondroitin proteoglycan content and joint structure, and OARSI scores were calculated. The results are shown in Figure 8. The Lipo-RRM group showed significantly less cartilage damage and the lowest OARSI score compared to the Control-RRM group.

[0053] Calcification analysis: Mice were sacrificed six weeks after modeling, and the condylar articular cartilage was examined by scanning electron microscopy and elemental mapping analysis. Results are as follows: Figure 9 As shown, the calcium and phosphorus signal intensity and calcification deposits on the cartilage surface of the Lipo-RRM group were significantly less than those of the other two groups, indicating that pathological calcification was effectively inhibited.

[0054] The results of the above examples show that the reactive oxygen species-responsive mitochondrial-targeting hydrogel microspheres prepared based on the present invention can effectively deliver Chchd6 to the mitochondria of osteoarthritis chondrocytes, inhibit the formation of pathological mineral precursors and cartilage calcification, and delay the progression of osteoarthritis, showing good application prospects.

[0055] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Application of Chchd6 protein and its encoding gene as targets in the preparation of drugs for the treatment and prevention of osteoarthritis.

2. The application according to claim 1, characterized in that, The drug is a mitochondrial-targeting drug.

3. The application according to claim 1, characterized in that, The drug includes the Chchd6 protein and a mitochondrial target.

4. The application according to claim 1, characterized in that, The treatment or prevention is achieved by inhibiting pathological calcification of chondrocytes.

5. The application according to claim 4, characterized in that, The inhibition of pathological calcification of chondrocytes is achieved by preventing the formation of mineral precursors.

6. A medicament for the treatment of osteoarthritis, characterized in that, The drug includes the Chchd6 protein and a mitochondrial target.

7. The medicament for treating osteoarthritis according to claim 6, characterized in that, The drug comprises liposomes loaded with the Chchd6 protein and mitochondrial targets.

8. The medicament for treating osteoarthritis according to claim 6, characterized in that, The pharmaceutical formulation is a hydrogel microsphere, which is coated with liposomes loaded with Chchd6 protein and mitochondrial targets.

9. The medicament for treating osteoarthritis according to claim 6, 7, or 8, characterized in that, The mitochondrial target is SS31.

10. The medicament according to claim 8, characterized in that, The hydrogel matrix of the hydrogel microspheres includes methacrylamide hyaluronic acid and phenylboronic acid modified hyaluronic acid.

11. The medicament according to claim 10, characterized in that, The mass ratio of the methacrylamide hyaluronic acid to the phenylboronic acid-modified hyaluronic acid is 1:

1.

12. The medicament according to claim 8, characterized in that, The method for preparing the drug includes: (1) Prepare liposomes loaded with Chchd6, and then link SS31 peptide to the liposomes to obtain mitochondrial-targeting liposomes; (2) The liposomes obtained in step (1) are dispersed in a physiological saline solution containing methacrylamide hyaluronic acid and phenylboronic acid modified hyaluronic acid to form a mixed reaction solution; (3) The mixed reaction solution obtained in step (2) was prepared into hydrogel microspheres using microfluidic technology.

13. The medicament according to claim 5, characterized in that, In step (2), the concentration of the liposomes in the mixed reaction solution is 1-10 mg / mL.

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