Application of ROS (reactive oxygen species) response hydrogel coated tiopronin to preparation of medicine for treating osteoarthritis

Abstract

The invention discloses an application of tiopronin wrapped by ROS (reactive oxygen species) response hydrogel in preparation of a medicine for treating osteoarthritis. The ROS response hydrogel is prepared from the following components: tiopronin; the compound group of lecithin and ketal thiol (TK) is TK-NH22, 2 '-(propane-2, 2-diyl bis (sulfanilamide diyl)) diethylamine (sulfanediyl), and the compound group of lecithin and ketal thiol (TK) is TK-NH22, 2'-(propane-2, 2-diyl bis (sulfanilamide diyl)); 12 wt% of methyl cellulose, Na2HPO4. 12H2O and xylitol; according to the hydrogel, controlled release of tiopronin is achieved through an ROS response group, mitochondrial autophagy is enhanced by up-regulating a Bnip3-Pink1-Parkin signal channel, oxidative stress is inhibited, and the progress of osteoarthritis is delayed; compared with the prior art, the invention has the advantages that: the invention discloses an osteoarthritis treatment medicine with the effects of resisting inflammation, relieving pain and inhibiting cartilage degeneration for the first time, and discloses a knee joint intracavity administration route of tiopronin for the first time. Particularly, the tiopronin targeted activation Bnip3-Pink1-Parkin signal channel provided by the invention has a remarkable effect in the aspect of inhibiting the progress of OA, which proves that the medicine has a strong effect on the treatment of osteoarthritis.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to an application of a ROS-responsive hydrogel encapsulating tiopronin in preparing a medicine for treating osteoarthritis. Background Art

[0002] Abnormal oxidative stress is an important pathological event that disrupts the homeostasis of the cartilage microenvironment. Abnormal oxidative stress leads to increased levels of reactive oxygen species (ROS), accumulation of matrix metalloproteinases (MMPs), and impaired cell function. At the cellular level, excessive oxidative stress can cause severe damage to mitochondrial DNA (mtDNA) and nuclear DNA, affecting cell signaling pathways and protein transcription. During the onset of osteoarthritis, elevated oxidative stress levels increase ROS levels and inhibit mitochondrial autophagy, disrupting cell function. Mitochondrial autophagy reduces ROS levels and restores the homeostasis of the cartilage microenvironment by eliminating damaged mitochondria. Enhancing mitochondrial autophagy to reduce oxidative stress has been identified as a key strategy for treating osteoarthritis (OA) and preventing its progression. The extensive clinical application of tiopronin has shown that after the use of this drug, patients have very few adverse reactions and the price is affordable. Therefore, it is relatively safe and has unique advantages in cost-effectiveness, and has great potential in the treatment of osteoarthritis. The intra-articular injection of tiopronin for the treatment of OA is a more targeted route of administration that can maximize the advantages of local administration.

[0003] Our study showed that Tiopronin has a significant effect on upregulating mitophagy and preventing cartilage degeneration in osteoarthritis (OA). We first determined that Bnip3 gene expression was significantly different after Tiopronin treatment. Bnip3 expression increased both in vitro and in vivo after Tiopronin treatment. As a hypoxia-induced mitochondrial adaptor protein, Bnip3 is upregulated and anchored to the mitochondrial outer membrane (MOM) under hypoxic stimulation, and acts on the MOM together with Pink1, interacting to promote the accumulation of Pink1 on the MOM, thereby promoting Parkin recruitment and Pink1 / Parkin-mediated mitophagy. Further studies showed that Tiopronin promoted mitophagy levels mainly by significantly activating Bnip3 expression, thereby increasing the expression of Pink1 and Parkin, that is, Tiopronin delayed the progression of osteoarthritis (OA) by enhancing mitophagy levels through the Bnip3-Pink1-Parkin signaling pathway. Compared with current clinical drugs, tiopronin has advantages and advancements in terms of economic security, in addition to its analgesic and anti-inflammatory effects, especially in inhibiting cartilage degradation. Furthermore, we have achieved the effect of sustained drug release by constructing a ROS-responsive tiopronin drug controlled release system, which is highly superior in reducing the frequency of drug administration to reduce the damage caused by injection and prolonging the duration of drug action.

[0004] At present, research on various age-related diseases, including degenerative joint diseases, is still in its infancy, and only emphasizes the importance of maintaining mitochondrial stability and function. In summary, as the relationship between mitochondrial function and osteoarthritis becomes clearer, there are few studies on drugs targeting mitochondrial oxidative stress. It is urgent to screen and design more efficient, low-toxic and well-targeted oxidative stress inhibitory drugs and use them in combination with effective administration methods for the treatment of osteoarthritis, as well as for the study of the mechanism and drug development of diseases related to cellular oxidative stress. Summary of the invention

[0005] 1. Technical issues

[0006] The present invention aims to provide the use of tiopronin and ROS-responsive hydrogel in the preparation of drugs for preventing, alleviating and treating osteoarthritis; provide a mechanism of action of tiopronin, which enhances mitochondrial function and inhibits the generation of ROS by increasing the level of mitochondrial autophagy; provide the use of tiopronin as a drug in drugs for treating osteoarthritis through intra-articular administration of the knee joint; and verify the use of a ROS-responsive tiopronin drug controlled release system for the treatment of osteoarthritis.

[0007] (II) Technical content

[0008] In order to solve the above technical problems, the technical solution of the present invention is: use of a ROS-responsive hydrogel encapsulating tiopronin in the preparation of a drug for treating osteoarthritis, wherein the ROS-responsive hydrogel is prepared from the following components:

[0009] (a) Tiopronin;

[0010] (b) lecithin and thioketal (TK) complex group TK-NH22,2\'-(propane-2,2-diylbis(sulfonamide))diethylamine (sulfanediyl);

[0011] (c) 12 wt% methyl cellulose, Na 2 HPO 4 12H 2 O and xylitol;

[0012] The hydrogel achieves controlled release of tiopronin through ROS-responsive groups, and enhances mitochondrial autophagy by upregulating the Bnip3-Pink1-Parkin signaling pathway, inhibits oxidative stress, and delays the progression of osteoarthritis;

[0013] The drug is administered through the knee joint cavity administration route, the administration dosage is 10-15 μL, and the effective dosage of tiopronin is 60-90 mg / kg.

[0014] Furthermore, the ROS-responsive hydrogel is a Tio-LIP-TK@MC-Gel system, which is prepared by the following steps:

[0015] (1) preparing liposomes (Tio-LIP) by combining lecithin and tiopronin;

[0016] (2) dissolving the thioketal (TK) complex group TK-NH22,2\'-(propane-2,2-diylbis(sulfonamide))diethylamine (sulfanediyl) in a solution, adding the liposomes of step (1), and mixing to form Tio-LIP-TK microparticles;

[0017] (3) The microparticles are mixed with methylcellulose (MC-Gel) to form Tio-LIP-TK@MC-Gel hydrogel.

[0018] Furthermore, the osteoarthritis includes one or more of knee osteoarthritis, hip osteoarthritis, shoulder osteoarthritis and hand (wrist and finger) osteoarthritis.

[0019] Furthermore, the drug controlled release system of the ROS-responsive hydrogel achieves sustained release by:

[0020] (a) ROS-responsive groups respond to elevated ROS levels in the osteoarthritis microenvironment and trigger hydrogel degradation;

[0021] (b) Sustained release of tiopronin from the hydrogel inhibits cartilage degradation and promotes mitophagy.

[0022] Furthermore, the specific injection site for the intra-articular administration of the drug is the inner depression below the patella.

[0023] Furthermore, a pharmaceutical composition for treating osteoarthritis comprises tiopronin encapsulated in the ROS-responsive hydrogel according to claim 1, and pharmaceutically acceptable excipients; the composition is used to treat osteoarthritis by upregulating mitochondrial autophagy and inhibiting oxidative stress.

[0024] Furthermore, a method for preparing a drug for treating osteoarthritis and cartilage degeneration mediated by oxidative stress is provided, wherein the drug enhances mitochondrial autophagy by activating the Bnip3-Pink1-Parkin signaling pathway and combines with a ROS-responsive hydrogel to achieve targeted controlled release.

[0025] (III) Technical Effect

[0026] Compared with the prior art, the present invention has the following advantages:

[0027] The present invention discloses for the first time a drug for treating osteoarthritis that has both anti-inflammatory and analgesic effects and the effect of inhibiting cartilage degradation, and discloses for the first time a knee joint intra-articular administration route of tiopronin. In particular, the targeted activation of the Bnip3-Pink1-Parkin signaling pathway by tiopronin provided by the present invention is effective in inhibiting the progression of OA, proving that the drug has a potent effect on the treatment of osteoarthritis. In addition, knee joint intra-articular administration, as a new, safe and targeted lesion administration route, is expected to provide a new treatment for such intractable diseases in clinical practice. At the same time, the present invention is a new use for an old drug, the pharmacokinetic data of the drug molecule is relatively detailed, it is safe and reliable, and the side effects are very mild. The development of a new administration method can soon enter clinical evaluation, shortening the research and development cycle and saving development costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a schematic diagram of the chemical structural formula of the tiopronin molecule in the present invention. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below in conjunction with the embodiments.

[0030] The invention discloses an application of a ROS-responsive hydrogel encapsulating tiopronin in the preparation of a drug for treating osteoarthritis. The ROS-responsive hydrogel is prepared from the following components: (a) tiopronin; (b) lecithin and thioketal (TK) composite group TK-NH22,2\'-(propane-2,2-diylbis(sulfonamide))diethylamine (sulfanediyl); (c) 12wt% of methylcellulose, Na 2 HPO 4 12H 2 O and xylitol; the hydrogel realizes the controlled release of tiopronin through ROS responsive groups, enhances mitochondrial autophagy by upregulating the Bnip3-Pink1-Parkin signaling pathway, inhibits oxidative stress, and delays the progression of osteoarthritis; the drug is administered through the knee joint cavity, the dosage is 10-15 μL, and the effective dose of tiopronin is 60-90 mg / kg.

[0031] The ROS responsive hydrogel is a Tio-LIP-TK@MC-Gel system, which is prepared by the following steps: (1) preparing liposomes (Tio-LIP) with lecithin and thiopronil; (2) dissolving the thioketal (TK) complex group TK-NH22,2\'-(propane-2,2-diylbis(sulfonamide))diethylamine (sulfanediyl) in a solution, adding the liposomes of step (1), and mixing to form Tio-LIP-TK microparticles; (3) mixing the microparticles with methylcellulose (MC-Gel) to form a Tio-LIP-TK@MC-Gel hydrogel.

[0032] The osteoarthritis includes one or more of knee osteoarthritis, hip osteoarthritis, shoulder osteoarthritis and hand (wrist and finger) osteoarthritis.

[0033] The drug controlled release system of the ROS-responsive hydrogel achieves sustained release in the following ways: (a) the ROS-responsive group responds to the elevated ROS level in the osteoarthritis microenvironment, triggering the degradation of the hydrogel; and (b) tiopronin is continuously released from the hydrogel, inhibiting cartilage degradation and promoting mitochondrial autophagy.

[0034] The preparation of the drug also includes a pharmaceutically acceptable carrier or excipient, such as physiological saline.

[0035] The specific injection site for the knee joint intracavitary administration is the inner depression below the patella.

[0036] A pharmaceutical composition for treating osteoarthritis, comprising tiopronin encapsulated in the ROS-responsive hydrogel according to claim 1, and pharmaceutically acceptable excipients; the composition is used for treating osteoarthritis by upregulating mitochondrial autophagy and inhibiting oxidative stress.

[0037] A use of tiopronin in the preparation of a drug for treating osteoarthritis and cartilage degeneration mediated by oxidative stress, wherein the tiopronin enhances mitochondrial autophagy by activating the Bnip3-Pink1-Parkin signaling pathway and combines with a ROS-responsive hydrogel to achieve targeted controlled release.

[0038] The raw materials and equipment used in the specific embodiments of the present invention are all known products, which are purchased from the market. The experimental method is a conventional method.

[0039] 1. Experimental Materials

[0040] 1.1 Instruments and Equipment

[0041] Synergy TM2 multi-function microplate reader (BioTek, USA); equipment required for Western Blot including Mini-Protean Tetra System, Trans-Blot Turbo System and Chemidoc+XRS (Bio-Rad, USA); Milli-Q Gradient A10 ultrapure water device (Millipore, USA); Forma-86C ultra-low temperature refrigerator, HERAcell150i CO2 incubator and MSC 1.2 biological safety cabinet (Thermo Fisher Scientific, USA); cell counter Cellometer Mini (Nexcelom, USA); Soniprep150 ultrasonic disruptor (SANYO, Japan); 5810R high-speed centrifuge (Eppendorf, Germany); Shimadzu AW120 electronic analytical balance (Shimadzu, Japan); Micro-CT (Bruker, Beijing); slicer (Thermo, Germany); laser confocal microscope (Zeiss, Germany); and inverted fluorescence microscope (Nikon, Japan).

[0042] 1.2 Reagents

[0043] Collagenase 2 (Gibco, USA), Tiopronin (#HY-B0373, MCE), Mdivi-1 (#HY-15886, MCE), siRNAs (Hippobio, Nan Jing, China), Tert-butyl hydroperoxide (Sigma, USA), Safranin-O / FastGreen (#G1371, Solarbio, Beijing, China), Hematoxylin-Eosin (H&E) (#C0105S, Beyotime), BCA protein quantification kit (Beyotime Biotechnology Co., Ltd.), Cell Counting Kit-8 (Dojindo Co, Japan), CF488-Tunel Detection Kit (#G1504-50T, Servicebio), JC-1 staining kit (Beyotime), DCFH-DA (Beyotime), MitoSOX Red (Beyotime), other experimental-related solvents, chemical reagents, etc. (Beyotime Biotechnology Co., Ltd.).

[0044] 1.3 Experimental animals

[0045] 25 g 8-week-old SPF-grade C57 / BL6J male mice were purchased from the Experimental Animal Center of Wenzhou Medical University.

[0046] 2. Experimental Methods

[0047] 2.1 Solution and drug preparation

[0048] Tiopronin was diluted with saline to a working concentration for intra-articular administration in mice. Opti-Medium and Lipofectamine were used to dilute si-RNA into a working solution for molecular level administration, and PBS and DMSO were used to dilute Tiopronin and Mdivi-1 into working concentrations for cellular level administration.

[0049] PBS buffer formula: 0.20g KCl, 8.00g NaCl, 0.20g KH2PO4, 2.080g Na2HPO4·12H2O, accurately weighed and placed in a beaker, dilute to 1L with deionized water, place on a magnetic stirrer and stir thoroughly, adjust the pH to 7.5, and place at room temperature for use.

[0050] 2.2 Establishment of mouse osteoarthritis model (DMM)

[0051] Surgical osteoarthritis was induced by the previously described medial meniscus destabilization (DMM) method. After anesthesia with sodium pentobarbital, the right knee joint of mice underwent DMM surgery, which included cutting the medial meniscus-medial tibial ligament and fixing the medial meniscus to the tibial plateau. Control mice underwent sham surgery, with the same surgical procedure but without cutting the ligament. Mice were euthanized 8 weeks after surgery.

[0052] 2.3 Isolation and culture of primary mouse chondrocytes

[0053] Primary mouse chondrocytes were harvested and cultured as previously described. Briefly, rib cartilage was removed from 3-day-old C57BL / 6 mice and digested using 0.2% collagenase 2 (Gibco, USA) at 37°C for 4 h. After removal of soft tissue, chondrocytes were cultured in Dulbecco's Modified Eagle's Medium with 1 g / L glucose (Gibco, Carlsbad, CA) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Gibco) at 37°C and 5% CO2. The cell culture medium was changed every 72 h.

[0054] 2.4 Construction of a ROS-responsive tiopronin controlled release system for the treatment of osteoarthritis

[0055] 1) Preparation and detection of ROS-responsive thiopronin drug controlled release system materials: Phosphatidylcholine and thiopronin were used to prepare liposomes (Tio-LIP). The complex group TK-NH22,2'-(propane-2,2-diylbis(sulfonamide))diethylamine (sulfanediyl) of ketalthiool (TK) was used to synthesize a solution with ROS-responsive groups. The lipid droplets of liposomes containing (Tio-LIP) were added to the solution containing ROS-responsive groups, mixed thoroughly, and left at room temperature overnight to obtain microparticles (Tio-LIP-TK-particel) containing thiopronin drugs in the inner layer and grafted with ROS-responsive groups. Na2HPO4·12H2O and xylitol were further added to 12wt% methyl cellulose as additives to prepare MC-Gel with thermosensitive effect, and the above-mentioned Tio-LIP-TK particles were further added to MC-Gel to obtain Tio-LIP-TK@MC-Gel system, and the response control group was LIP-TK@MC-Gel. The obtained delivery system was subjected to corresponding physical and chemical tests, including particle size, zeta potential of particles, microstructure, mass spectrometry analysis of each component, hydrogel gelation temperature detection, storage modulus (G') and loss modulus (G") detection, and drug release curve of the sustained release system.

[0056] 2) Biosafety test of LIP-TK@MC-Gel system materials: Osteoclasts and osteoblasts were incubated in the LIP-TK@MC-Gel sustained-release system. After 2 days of culture, cell live and dead staining was performed to test the cytotoxic effect of LIP-TK@MC-Gel and evaluate its biosafety.

[0057] 3) Evaluation of the therapeutic effect of Tio-LIP-TK@MC-Gel's ROS-responsive siopronin drug delivery system on particle-induced osteolysis: C57BL / 6 male mice were selected to construct a polyethylene particle (diameter 0.1-0.8μm)-induced osteolysis model. After modeling, Tio-LIP-TK@MC-Gel and LIP-TK@MC-Gel were locally injected, and another group of animals were treated with siopronin alone. The specific groups were: sham operation group, particle induction + LIP-TK@MC-Gel treatment group, particle induction Tio-LIP-TK@MC-Gel treatment group, particle induction + siopronin treatment group. Skull tissue was obtained 2 weeks after modeling, and Micro-CT scanning was performed to evaluate bone mass. The level of osteoblasts and osteoclasts was evaluated by relevant histological staining, including hematoxylin and eosin staining (H&E), Masson staining, Trap staining, etc. Immunohistochemical staining was used to evaluate osteoclast-related indicators (Ctsk, Nfatc1, c-Fos), osteogenesis-related indicators (Alp, Ocn, Runx2, Sp7, Bmp2), oxidative stress indicators (Ros, Sod1, Ho1), and mitochondrial autophagy-related indicators (Pink1, Parkin, Tomm20).

[0058] 2.5 Western Blot to detect indicators related to mitochondrial autophagy and cartilage degeneration:

[0059] 1) Prepare SDS-PAGE electrophoresis gel according to the kit instructions.

[0060] 2) Prepare electrophoresis buffer: weigh 18.9 g of glycine, 3.02 g of Tris base and 1 g of SDS powder and add them into 1000 mL of pure water and fully dissolve.

[0061] 3) Electrophoresis: Add 20 μL of protein sample into the gel loading wells in the order of samples, add appropriate amount of electrophoresis buffer into the electrophoresis tank, and perform electrophoresis at a constant voltage of 120 V for 60 min-90 min.

[0062] 4) Prepare electrotransfer buffer: weigh 14.42 g of glycine and 3.02 g of Tris base and add them to 800 mL of pure water to fully dissolve. Use methanol to make up to 1,000 mL and pre-cool at low temperature for later use.

[0063] 5) Electrotransfer: Cut the PVDF membrane to the appropriate size and pre-activate it in methanol. In a low temperature environment, install the black side of the transfer chuck, sponge and filter paper, electrophoresis gel, PVDF membrane, filter paper and sponge, and transparent side of the transfer chuck in the order. Electrotransfer at a constant current of 300mA for 90 minutes in the electrotransfer tank. The electrotransfer process should be carried out on ice.

[0064] 6) Blocking: Place the PVDF membrane onto which the protein has been transferred in a 5% skim milk solution for 90 minutes to block non-specific macromolecular proteins.

[0065] 7) Primary antibody incubation: After blocking, wash the PVDF membrane three times with TBST buffer. Prepare the primary antibody solution according to the antibody instructions, and incubate the PVDF membrane in the primary antibody working solution at 4°C overnight.

[0066] 8) Secondary antibody incubation: After the primary antibody incubation, wash the PVDF membrane three times with TBST buffer. Prepare the secondary antibody solution according to the antibody instructions, transfer the PVDF membrane to the secondary antibody working solution, and incubate on a shaker at room temperature for 1 hour.

[0067] 9) Exposure: After the secondary antibody incubation, the PVDF membrane was washed three times with TBST buffer. ECL ultrasensitive colorimetric solution A and solution B were prepared in equal proportions and then dropped onto the PVDF membrane for exposure imaging in Chemidoc+XRS (Bio-Rad, USA).

[0068] 10) Image analysis: Use Image J software to analyze and process the experimental results.

[0069] 2.6 Protein concentration determination

[0070] The protein concentration was determined by the BCA method: 0.5 mg / mL standard protein gradient was added to the well plate and made up to 20 μL with PBS; an appropriate volume (3 μL) of protein sample was added to the well plate and made up to 20 μL with PBS; 200 μL of BCA working solution (prepared before use, ready for use) was added to each well and incubated at 37°C for 30 min; the absorbance at a wavelength of 562 nm was measured and the protein concentration was calculated using the standard curve and sample volume.

[0071] 2.7 Cell Counting Kit-8 assay

[0072] To evaluate the cytotoxic effect of tert-butyl hydroperoxide (tbhp) on chondrocytes treated with or without tiopronin, and on chondrocytes treated with or without the mitochondrial autophagy inhibitor Mdivi-1, we performed the Cell Counting Kit-8 (CCK-8) kit (Dojindo Co, Kumamoto, Japan) experiment according to the manufacturer's instructions. Equal amounts of chondrocytes were seeded into 96-well plates and treated with tbhp and / or tiopronin and / or Mdivi-1 for 24 hours. After washing with PBS, the cells in each well were incubated with DMEM / F12 medium containing 10% (v / v) CCK-8 solution at 37°C for 2 hours. The absorbance was measured at a wavelength of 450 nm using a microplate reader (Thermo Scientific, Logan, UT, USA).

[0073] 2.8 Micro-computed tomography (Mirco-CT) analysis

[0074] After soft tissue dissection, the collected knee joints were fixed in 4% formaldehyde solution overnight. Subsequently, the specimens were scanned and reconstructed using a high-resolution microcomputed tomography (CT) device (SkyScan 1172) and CT reconstruction software (NRecon v1.6). For three-dimensional model visualization and further data analysis, CTAn v1.9 and μCTVol v2.0 software tools were used. The scanning parameters were set at 100 kVp voltage, 200 μA current, and a pixel resolution of 9.066683 μm. The entire subchondral bone of the specimen was defined as the region of interest, and the bone volume fraction (BV / TV) was quantified.

[0075] 2.9 Immunofluorescence and immunohistochemistry analysis

[0076] Animal and human osteoarthritis (OA) cartilage sections were incubated with primary antibodies overnight at 4°C for the relevant primary antibodies. For immunofluorescence staining, FITC- or tetramethylrhodamine isothiocyanate (TRITC)-labeled secondary antibodies were applied to the sections and left in the dark for 1 hour. Alternatively, for immunohistochemical staining, sections were exposed to HRP-labeled secondary antibodies. The stained sections were imaged using a fluorescence microscope (Zeiss, Heidelberg, Germany).

[0077] 2.10 Culture and processing of human OA cartilage grafts

[0078] Articular cartilage from patients with osteoarthritis (OA) undergoing total knee replacement was cultured. Briefly, full-thickness cartilage samples were obtained and rinsed with sterile saline solution. The cartilage was then cut into small pieces of 0.5 cm per side and cultured overnight in low-glucose Dulbecco's Modified Eagle's Medium (DMEM) at 37°C. Subsequently, the cartilage blocks were randomly assigned to three groups: (1) control group; (2) 50 μM tert-butyl hydroperoxide (tbhp); and (3) 50 μM tbhp + 5 mM tiopronin. The culture medium containing the corresponding drugs was replaced every 72 hours. After 7 days of treatment, the cartilage blocks were fixed with 4% formaldehyde, embedded in paraffin, and cut into 5 μm thick sections for histological analysis.

[0079] The present invention and its embodiments are described above, and such description is not restrictive. The drawings show only one embodiment of the present invention, and the actual structure is not limited thereto. In short, if ordinary technicians in the field are inspired by it, without departing from the purpose of the invention, they can design a structure and embodiment similar to the technical solution without creativity, which should belong to the protection scope of the present invention.

Claims

1. Use of a ROS-responsive hydrogel encapsulating tiopronin in the preparation of a drug for treating osteoarthritis, characterized in that: The ROS-responsive hydrogel is prepared from the following components: (a) Tiopronin; (b) lecithin and thioketal (TK) complex group TK-NH22,2\'-(propane-2,2-diylbis(sulfonamide))diethylamine (sulfanediyl); (c) 12 wt % methylcellulose, Na2HPO4·12H2O and xylitol; The hydrogel achieves controlled release of tiopronin through ROS-responsive groups, and enhances mitochondrial autophagy by upregulating the Bnip3-Pink1-Parkin signaling pathway, inhibits oxidative stress, and delays the progression of osteoarthritis; The drug is administered through the knee joint cavity administration route, the administration dosage is 10-15 μL, and the effective dosage of tiopronin is 60-90 mg / kg.

2. The use of a ROS-responsive hydrogel encapsulated with tiopronin according to claim 1 in the preparation of a drug for treating osteoarthritis, characterized in that: The ROS-responsive hydrogel is a Tio-LIP-TK@MC-Gel system, which is prepared by the following steps: (1) preparing liposomes (Tio-LIP) by combining lecithin and tiopronin; (2) dissolving the thioketal (TK) complex group TK-NH22,2\'-(propane-2,2-diylbis(sulfonamide))diethylamine (sulfanediyl) in a solution, adding the liposomes of step (1), and mixing to form Tio-LIP-TK microparticles; (3) The microparticles are mixed with methylcellulose (MC-Gel) to form Tio-LIP-TK@MC-Gel hydrogel.

3. Use of a ROS-responsive hydrogel encapsulated with tiopronin according to claim 1 or 2 in the preparation of a drug for treating osteoarthritis, characterized in that: The osteoarthritis includes one or more of knee osteoarthritis, hip osteoarthritis, shoulder osteoarthritis and hand (wrist and finger) osteoarthritis.

4. The use of a ROS-responsive hydrogel encapsulated with tiopronin according to claim 1 in the preparation of a drug for treating osteoarthritis, characterized in that: The drug controlled release system of the ROS-responsive hydrogel achieves sustained release in the following ways: (a) ROS-responsive groups respond to elevated ROS levels in the osteoarthritis microenvironment and trigger hydrogel degradation; (b) Sustained release of tiopronin from the hydrogel inhibits cartilage degradation and promotes mitophagy.

5. The use of a ROS-responsive hydrogel encapsulated with tiopronin according to claim 1 in the preparation of a drug for treating osteoarthritis, characterized in that: The specific injection site for the knee joint intracavitary administration is the inner depression below the patella.

6. A pharmaceutical composition for treating osteoarthritis, characterized in that: The composition comprises tiopronin encapsulated by the ROS-responsive hydrogel according to claim 1, and pharmaceutically acceptable excipients; the composition is used for treating osteoarthritis by upregulating mitochondrial autophagy and inhibiting oxidative stress.

7. A use of tiopronin in the preparation of a drug for treating osteoarthritis and cartilage degradation mediated by oxidative stress, characterized in that: The tiopronin enhances mitochondrial autophagy by activating the Bnip3-Pink1-Parkin signaling pathway, and combines with ROS-responsive hydrogel to achieve targeted controlled release.

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