阅读说明：本技术 成纤维细胞激活蛋白作为药物靶点在治疗骨关节炎中的用途 (Application of fibroblast activation protein as drug target in treating osteoarthritis ) 是由 岳锐 尹峰 范骜元 于 2020-12-18 设计创作，主要内容包括：本发明提供了成纤维细胞激活蛋白(Fap)作为靶点在筛选用于治疗骨关节炎或抑制软骨损伤或骨赘生成或滑膜炎症药物中的应用。还提供了Fap基因作为靶点在筛选用于治疗骨关节炎或抑制软骨损伤或骨赘生成或滑膜炎症药物中的应用。还提供了靶向Fap抑制剂在制备用于治疗骨关节炎或抑制软骨损伤或骨赘生成或滑膜炎症药物中的应用。还提供了骨凝集素(Oln)在制备用于治疗骨关节炎或抑制软骨损伤或骨赘生成或滑膜炎症药物中的应用。本发明发现Fap高表达于骨关节炎滑膜,遗传学及药理学抑制Fap能缓解小鼠骨关节炎的软骨损伤、骨赘生成、滑膜炎症及软骨聚集蛋白多糖丢失,Fap小分子抑制剂和内源性蛋白抑制剂Oln均可治疗骨关节炎。(The invention provides application of fibroblast activation protein (Fap) as a target spot in screening medicaments for treating osteoarthritis or inhibiting cartilage damage or osteophyte generation or synovium inflammation. Also provides the application of the Fap gene as a target point in screening medicaments for treating osteoarthritis or inhibiting cartilage damage or osteophyte generation or synovium inflammation. Also provides the application of the targeted Fap inhibitor in preparing a medicament for treating osteoarthritis or inhibiting cartilage damage or osteophyte generation or synovium inflammation. Also provided is the use of a bone lectin (Oln) in the manufacture of a medicament for the treatment of osteoarthritis or for inhibiting cartilage damage or osteophyte production or synovial inflammation. The invention discovers that Fap is highly expressed in osteoarthritis synovium, genetic and pharmacological inhibition of Fap can relieve cartilage damage, osteophyte generation, synovium inflammation and cartilage aggrecan loss of osteoarthritis of mice, and Fap small molecule inhibitor and endogenous protein inhibitor Oln can treat osteoarthritis.)

1. The use of fibroblast activation protein as a target in the screening of medicaments for treating osteoarthritis or inhibiting cartilage damage or osteophyte generation or synovial inflammation.

2. The use according to claim 1, wherein the medicament is a medicament that inhibits the expression level of fibroblast activation protein.

3. The use according to claim 1, wherein the medicament is a medicament that inhibits the activity of fibroblast activation protein.

4. The use of fibroblast activation protein gene as a drug target in the screening of drugs for treating osteoarthritis or inhibiting cartilage damage or osteophyte production or synovial inflammation.

5. Use of an inhibitor targeting fibroblast activation protein for the manufacture of a medicament for the treatment of osteoarthritis or for inhibiting cartilage damage or osteophyte production or synovial inflammation.

6. Use of fibroblast activation protein in the manufacture of a biomarker for diagnosis or prognosis of patients with osteoarthritis or inhibited cartilage damage or osteophyte production or synovial inflammation.

7. Use of an osteolectin in the manufacture of a medicament for the treatment of osteoarthritis or for inhibiting cartilage damage or osteophyte production or synovial inflammation.

Technical Field

The invention belongs to the technical field of biology, and relates to a fibroblast activation protein, in particular to an application of the fibroblast activation protein as a drug target in treating osteoarthritis.

Background

Osteoarthritis (OA) is one of the most common orthopaedic diseases, with a prevalence of OA of up to 26% in people over the age of 45. The most prominent pathological changes in OA are articular cartilage degeneration, combined with osteophyte production, lower levels of inflammation, and subchondral bone remodeling. The mechanical abrasion of articular cartilage at the beginning of OA causes the secretion of a variety of proinflammatory factors and matrix degrading enzymes from tissues such as cartilage and synovium, further aggravating the degradation of cartilage matrix. Early OA can be treated by microfracture, osteochondral mosaicism, autologous cartilage transplantation, etc., but the efficacy is still controversial. End-stage OA patients only improve quality of life through joint replacement surgery. In the aspect of non-operative treatment, only non-steroidal anti-inflammatory drugs for relieving symptoms and glucocorticoid injected into articular cavities and other drugs including chondroitin and hyaluronic acid have been controversial in the curative effect commonly recognized by domestic and foreign guidelines, so that more effective therapeutic drugs are urgently needed for OA treatment.

Aggrecan and Type II collagen (Type II collagen, Col II) are the major components of the cartilage matrix. Aggrecan degradation in the cartilage matrix is mainly accomplished by ADAMTS family proteases, occurring early in the lesion and reversible; whereas degradation of Col II is mainly accomplished by Matrix Metalloproteinases (MMPs), and this process is irreversible. MMPs should therefore be good targets for OA treatment, however existing non-selective MMP inhibitors produce dose-dependent side effects (joint stiffness, inflammation and pain) in clinical trials. However, the selective MMP inhibitor developed at present has the defects of low solubility and low permeability, and the clinical application value of the MMP as a therapeutic target is hindered. In addition to MMPs, researchers have demonstrated the role of serine proteases, including thrombin, proteolytic enzyme (Matriptase) and furin, in the pathogenesis of OA, but there are currently no FDA-approved serine proteases as targets for drugs for treating OA.

Fibroblast activation protein (Fap) is a membrane-bound serine protease with dipeptidyl peptidase and endopeptidase activities. Currently known Fap substrates include type I collagen, α 2 antiplasmin, and fibroblast growth factor 21. Fap is highly expressed in activated fibroblasts and is involved in the process of tumor microenvironment formation, inflammation and injury repair. In a musculoskeletal system, Fap is highly expressed in bone marrow mesenchymal cells and osteoblasts and has the function of inhibiting osteogenesis; the genetic and pharmacological inhibition of Fap can promote osteogenesis and inhibit bone resorption, and is a potential target for treating osteoporosis. Fap is also highly expressed in synovial membranes and synovial fluid of patients with rheumatoid arthritis, and the expression level is positively correlated with the severity of rheumatoid arthritis. In the rheumatoid arthritis model, the Fap knockout can relieve the articular cartilage damage of mice. Expression of Fap is also present in synovial tissue of patients with OA, but is significantly lower than that of rheumatoid arthritis, but it is unclear whether Fap plays an important role in the pathogenesis of OA.

Bone agglutinin (ostolectin, Oln) is a growth factor that promotes hematopoietic colony formation in vitro, also known as stem cell growth factor or C-type lectin protein 11 a. Our previous studies showed Oln high expression and osteochondral lineage cells are able to promote osteogenesis through the Wnt pathway. Our recent studies showed Oln to inhibit the enzymatic activity of Fap, thereby regulating osteogenesis and osteogenic differentiation. As an in vivo inhibitor of Fap, Oln has not been studied as to whether it is involved in OA disease progression by inhibiting Fap enzyme activity.

As described above, the current therapeutic effects of OA are limited, and there is a high demand for drugs that can effectively alleviate cartilage damage. The medicine capable of effectively relieving cartilage injury can improve the life quality of patients, reduce the economic burden of replacement surgery on the patients and the society, and has great clinical significance.

Disclosure of Invention

Aiming at the technical problems in the prior art, the invention provides the application of the fibroblast activation protein as a drug target in treating osteoarthritis, and the application of the fibroblast activation protein as the drug target in treating osteoarthritis aims to solve the technical problem that the effect of the drug in the prior art on treating osteoarthritis or inhibiting cartilage damage or osteophyte generation or synovium inflammation is poor.

The invention provides application of fibroblast activation protein as a target in screening medicaments for treating osteoarthritis or inhibiting cartilage damage or osteophyte generation or synovium inflammation.

Further, the drug is a drug that inhibits the expression level of fibroblast activation protein.

Further, the drug is a drug that inhibits the activity of fibroblast activation protein.

The invention also provides application of the gene of the fibroblast activation protein as a drug target in screening drugs for treating osteoarthritis or inhibiting cartilage damage or osteophyte generation or synovium inflammation.

The invention also provides the use of an inhibitor targeting fibroblast activation protein in the manufacture of a medicament for the treatment of osteoarthritis or for inhibiting cartilage damage or osteophyte production or synovial inflammation.

The invention also provides application of the fibroblast activation protein in preparing a biomarker for diagnosing or prognosing osteoarthritis patients.

The invention also provides the use of an osteolectin in the manufacture of a medicament for the treatment of osteoarthritis or for inhibiting cartilage damage or osteophyte production or synovial inflammation.

In some embodiments of the invention, the agent is an agent for inhibiting the activity of a Fap enzyme, including but not limited to Fap pharmacological inhibitors, Fap blocking antibodies, sirnas of Fap.

In some embodiments of the invention, the medicament is a medicament for inhibiting Fap enzyme activity in synovial cells and/or chondrocytes and/or synovial fluid.

In some embodiments of the invention, the OA is primary OA and/or secondary OA.

In some embodiments of the invention, the drug is a single pharmacodynamic ingredient drug targeting Fap.

In another aspect, the invention provides a pharmaceutical composition, which is characterized by comprising a pharmaceutical effective component with Fap as a target point.

Through a large amount of research and research, the inventor of the invention finds that Fap is taken as a medicine target for treating OA, inhibits the enzyme digestion activity of Fap, can relieve OA cartilage injury, osteophyte generation and synovium inflammation, and can also inhibit the degradation of Fap to a cartilage matrix Col II, thereby achieving the purpose of relieving OA, and further having good industrialization prospect.

In a first aspect, the invention provides the use of Fap as a therapeutic target for OA in a medicament for: 1) treatment of OA; and/or, 2) inhibiting cartilage damage; and/or, 3) inhibiting osteophyte production; and/or, 4) inhibiting synovial inflammation.

The Fap inhibitor is any substance capable of reducing the enzyme activity of the Fap, reducing the stability of the Fap, inhibiting the expression of the Fap, reducing the effective action time of the Fap or inhibiting the transcriptional activation processing of the Fap, and includes but is not limited to a Fap-specific inhibitor. In some embodiments of the disclosure, the Fap inhibitor is, for example, Ac-Gly-BoroPro.

The Fap inhibitor provided by the invention is dissolved in Phosphate Buffered Saline (PBS), and the use mode is intra-articular injection, and the injection amount is 40 mug/kg of mouse body weight.

In a second aspect the invention provides a pharmaceutical composition comprising a component of a Fap inhibitor, for use in: 1) treatment of OA; and/or, 2) inhibiting cartilage damage; and/or, 3) inhibiting osteophyte production; and/or, 4) inhibiting synovial inflammation.

The pharmaceutical composition may also include a pharmaceutically acceptable carrier. The carrier may include various excipients and diluents, which are not essential active ingredients per se and which are not unduly toxic after administration. On the other hand, the dosage to be considered in administering the pharmaceutical composition should vary depending on the frequency and mode of administration, the age, sex, weight and general condition of the subject to be treated, the condition and severity of the treatment, and the route of administration, any concomitant diseases to be treated, and other factors apparent to those skilled in the art. Also, depending on the condition of the subject and other pathological conditions, a pharmaceutical composition comprising the present invention may be administered or applied in combination with one or more other therapeutically active compounds or substances.

The inventor finds that Fap is highly expressed in OA synovium, the genetic and pharmacological inhibition of Fap activity can relieve cartilage injury, osteophyte formation, synovium inflammation and reduced expression of cartilage tissue aggrecan in OA model mice, Fap inhibitor can be used for treating OA in mice, and furthermore, Fap can degrade Col II in cartilage matrix, and the effect can be inhibited by Fap inhibitor and another Fap in vivo inhibition protein Oln. Further, Oln knock-out mice constructed OA models with significantly increased cartilage damage, osteophyte formation, synovial inflammation and significantly decreased expression of aggrecan in cartilage tissue, recombinant Oln can be used to treat OA in mice.

Experiments to determine the expression of Fap in synovial tissue were also performed, and in one example, high expression of Fap in OA synovial membrane was determined by immunofluorescence, qRT-PCR and Western blot experiments, thereby confirming that Fap may be a target for OA therapeutic pharmaceutical composition.

The invention also carries out a detection experiment of the OA severity degree of a Medial meniscal Destabilization (DMM) mouse, and in one embodiment, the invention can relieve the cartilage injury, osteophyte formation, synovitis and the reduction of the expression of cartilage tissue aggrecan polysaccharide of an OA model mouse by inhibiting the Fap activity genetically and pharmacologically; in another example, we started injecting the Fap inhibitor in the joint cavity every week after 4 weeks after DMM surgery, i.e. after the mice had developed a degree of osteoarthritis, and found that cartilage damage, osteophyte formation, synovial inflammation were also significantly alleviated and cartilage tissue aggrecan expression was also significantly increased after 8 weeks of injection.

The invention also carries out the detection experiment of the co-incubation product of the recombinant Fap and the main component Col II of the cartilage matrix, in one embodiment, the recombinant Fap can degrade the denatured Col II, and the effect has time and dosage dependence. Both small molecule Fap inhibitors and in vivo Fap inhibitor Oln can inhibit the degradation of denatured Col II by recombinant Fap. Recombinant Fap can also further degrade native Col II after digestion by MMP 13.

The invention also carries out an in vivo FAP inhibitor Oln knock-out mouse OA severity detection experiment, and in one embodiment, through the knock-out Oln, the OA model mouse can be promoted to have cartilage injury, osteophyte formation, synovial inflammation and cartilage tissue aggrecan expression reduction.

In one embodiment, after 4 weeks after DMM operation, namely after the mice have had a certain degree of osteoarthritis, the mice begin to carry out intra-articular injection of recombinant Oln every week, after 8 weeks of injection, cartilage damage, osteophyte formation and synovium inflammation are also remarkably relieved, and the expression of aggrecan in cartilage tissues is also remarkably increased.

Drawings

FIG. 1 is a graph showing the results of the up-regulation of Fap expression in OA synovium; wherein, A is the expression of synovial membrane Fap of an immunofluorescence detection control and an OA patient; b is qRT-PCR detection control and expression of OA patient synovial membrane Fap; c is expression of synovial membrane Fap of a western blot detection control and an OA patient; d is the expression of synovial membrane Fap of an immunofluorescence detection control and an OA model mouse.

FIG. 2 is a graph showing the effect of genetic and pharmacological inhibition of Fap remission in OA progression in mice; wherein, A-C is safranin fast green staining of knee joint 8 weeks after Fap inhibitor/vector injection for control and Fap knockout mice constructing OA model, showing cartilage damage (A), osteophyte generation (B) and synovium inflammation (C), respectively; d is OARSI score to assess the extent of cartilage damage (a) in each group; e assessing the extent of osteophyte generation (B) for each group for osteophyte score; f, assessment of the degree of synovial inflammation (C) for each group for synovial score; g is the expression of each group of aggrecan polysaccharide detected by immunofluorescence; h is quantitative analysis of the fluorescence area of the aggrecan in the G picture.

FIG. 3 is a graph showing the effect of intra-articular injection of a Fap inhibitor on the treatment of OA in mice; wherein, A-C is safranin fast green staining of knee joint after 8 weeks of mouse OA model injection with Fap inhibitor/vector treatment, which respectively shows cartilage damage (A), osteophyte generation (B) and synovium inflammation (C); d is OARSI score to assess the extent of cartilage damage (a) in each group; e assessing the extent of osteophyte generation (B) for each group for osteophyte score; f, assessment of the degree of synovial inflammation (C) for each group for synovial score; g is the expression of each group of aggrecan polysaccharide detected by immunofluorescence; h is quantitative analysis of the fluorescence area of the aggrecan in the G picture.

FIG. 4 shows a schematic representation of the effect of Col II after Fap degradation denaturation or matrix metalloproteinase digestion. Wherein, A is a product obtained by performing co-incubation on denatured Col II and different doses of recombinant Fap through colloidal blue staining analysis and gray scale quantitative analysis of the product; b, analyzing co-incubation products of the denatured Col II and the recombinant Fap for different incubation times by using colloidal blue staining and quantitatively analyzing gray scales of the co-incubation products; c, analyzing co-incubation products of the Fap inhibitor, the recombinant Fap and the denatured Col II with different doses and gray scale quantitative analysis by using colloidal blue staining; d is a Fap sample of the over-expression of the co-immunoprecipitate 293T cell by Western blot analysis; e is a product incubated by the denatured Col II and the co-immunoprecipitation Fap after the colloidal blue staining analysis and the gray scale quantitative analysis thereof; f is a Col II, EDTA and recombinant Fap co-incubation product after digestion of the recombinant matrix metalloproteinase 13 by colloidal blue staining analysis and gray scale quantitative analysis thereof; g is the grayscale quantification of the 55kDa, 40kDa and 30kDa bands of the F panel.

FIG. 5 is a schematic illustration of the effect of knock-out Oln in promoting OA progression. Wherein, A-C is wild type and safranin fast green staining of knee joint 8 weeks after OA model of Oln knockout mouse, showing cartilage injury (A), osteophyte generation (B) and synovitis (C), respectively; d is OARSI score to assess the extent of cartilage damage (a) in each group; e assessing the extent of osteophyte generation (B) for each group for osteophyte score; f, assessment of the degree of synovial inflammation (C) for each group for synovial score; g is the expression of each group of aggrecan polysaccharide detected by immunofluorescence; h is quantitative analysis of the fluorescence area of the aggrecan in the G picture.

FIG. 6 is a graph showing the therapeutic effect of recombinant Oln injected intra-articular into mice with OA. Wherein, A-C is safranine fast green staining of knee joint after 8 weeks of starting injection of recombinant Oln/vector treatment 4 weeks after mouse OA model modeling, and cartilage injury (A), osteophyte generation (B) and synovium inflammation (C) are respectively shown; d is OARSI score to assess the extent of cartilage damage (a) in each group; e assessing the extent of osteophyte generation (B) for each group for osteophyte score; f, assessment of the degree of synovial inflammation (C) for each group for synovial score; g is the expression of each group of aggrecan polysaccharide detected by immunofluorescence; h is quantitative analysis of the fluorescence area of the aggrecan in the G picture.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

Example 1 expression of Fap in OA synovium

In this example, Fap expression in synovial tissue was determined by immunofluorescence, qRT-PCR and Western blotting.

The synovium of the control group (non-OA acute injury) and the synovium of the OA patient are collected, frozen sections are taken after fixed dehydration, and the expression of the Fap in the synovium tissue is detected through an immunofluorescence experiment, so that the expression of the Fap in the synovium tissue of the control group is low, and the expression of the Fap in the synovium tissue of the OA patient is high (FIG. 1A).

Synovium of the control group and the OA patients is collected, expression of Fap in the synovium is detected through qRT-PCR and Western blot experiments, and the mRNA and protein level of Fap in the synovium tissue of the OA patients is found to be remarkably higher than that of the synovium tissue of the control group in accordance with the result of immunofluorescence (FIGS. 1B and 1C).

Collecting knee joints of wild mice in a sham operation group and a DMM group, fixing, decalcifying and dehydrating, carrying out frozen section, detecting the expression condition of Fap in joints of the mice through an immunofluorescence experiment, and finding that the Fap is mainly expressed in synovial tissues and is remarkably increased in OA mouse synovial membranes simulated by the DMM operation (figure 1D), wherein the immunofluorescence result is consistent with that of human synovial tissues.

Example 2 genetic and pharmacological inhibition of Fap Mitigation of mouse OA progression

To investigate whether Fap modulates the progression of OA, we constructed DMM models simulating mouse OA for wild-type and Fap knockout mice and performed intra-articular injection of Fap-specific small molecule inhibitors or vehicle controls (PBS) weekly starting on day 3 post-surgery. Wild-type mice modeled exhibited significant cartilage erosion, osteophyte production, and synovial inflammation, while on one hand cartilage erosion and synovial inflammation were significantly reduced and osteophyte production tended to be reduced in Fap knockout mice, and on the other hand wild-type mice injected intra-articular with Fap inhibitors also exhibited significantly reduced cartilage erosion, osteophyte production, and synovial inflammation (fig. 2A-F). Intra-articular injection of Fap inhibitors (Ac-Gly-BoroPro) had no therapeutic effect on Fap knockout mice, suggesting that the therapeutic effect of Fap inhibitors is Fap dependent (fig. 2A-F). Immunofluorescence experiments with aggrecan demonstrated that genetic and pharmacological inhibition of Fap activity can alleviate loss of aggrecan in articular cartilage (fig. 2G, H).

Example 3 Intra-articular injection of Fap inhibitors has therapeutic effects on OA in mice

To examine whether the Fap inhibitor (Ac-Gly-BoroPro) could alleviate joint symptoms of OA after the onset of OA, we injected the Fap inhibitor (Ac-Gly-BoroPro) or vehicle control (PBS) into the knee joint cavity weekly in mice 4 weeks after DMM molding. The vehicle control group had significant cartilage erosion, osteophyte formation, and synovial inflammation after 8 weeks of injection compared to the sham operated group, while OA was significantly reduced in the Fap inhibitor group (fig. 3A-F). Immunofluorescence experiments with aggrecan demonstrated that Fap inhibitors (Ac-Gly-BoroPro) can alleviate aggrecan loss in articular cartilage (FIG. 3G, H).

Example 4 Fap degradation of Col II after denaturation or matrix Metalloproteinase digestion

Fap, a serine protease, degrades denatured type I collagen. To investigate whether Fap could degrade Col II, which is a major component of cartilage matrix, recombinant Fap (R & D Systems, commercial number: 8647-SE-010) (recombinant Fap is purified Fap purchased from R & D Systems, and is identical in sequence to Fap) was incubated with heat-denatured Col II (10 minutes at 95 ℃) and was found to have a dose-dependent and time-dependent degradation effect on heat-denatured Col II (FIG. 4A, B). In contrast, when the recombinant Fap was incubated with Fap inhibitor (Ac-Gly-BoroPro) for half an hour in advance and then heat-denatured Col II was added for incubation, it was found that the Fap inhibitor (Ac-Gly-BoroPro) could inhibit the degradation of denatured Col II by the recombinant Fap (FIG. 4C). After overexpression of Fap by 293T cells, co-immunoprecipitating Fap, and then adding heat-denatured Col II for co-incubation, the co-immunoprecipitated Fap can degrade denatured Col II; while degradation of denatured Col II by co-immunoprecipitated Fap after co-overexpression of Fap with Oln was inhibited (fig. 4D, E). We further tested whether Fap could further degrade native Col II, which was initially degraded by MMP 13. We incubated MMP13 with native Col II for 12 hours, then EDTA was added to stop MMP13 enzyme activity and recombinant Fap was added to continue incubation for 12 hours, and Fap was found to be able to further degrade the native Col II fragment cleaved by MMP13 (fig. 4F-G).

Example 5 knock-out Oln promotes progression of OA

In example 4 we found that Fap in vivo inhibitor Oln can inhibit Fap degradation of denatured Col II, and to further investigate whether Oln could modulate OA progression, we constructed DMM models for Oln knockout mice versus their difference in OA progression from wild type mice. Histomorphometric analysis revealed no significant difference between wild-type and Oln knockout mice in the contralateral knee joint without DMM, suggesting that Oln did not affect joint development or function at steady state (fig. 5A-F). Whereas Oln knockout mice, which constructed the DMM model, had more severe cartilage damage, osteophyte formation and synovial inflammation than wild-type mice (fig. 5A-F). Immunofluorescence experiments with aggrecan also demonstrated a significant reduction in aggrecan signal following Oln knockout (FIG. 5G, H).

EXAMPLE 6 Intra-articular injection Oln has therapeutic effect on OA in mice

To test Oln for the ability to alleviate joint symptoms of OA after its onset, we performed a weekly intra-articular injection Oln (available from R & D Systems under the trade designation: 3729-SC-025) into wild-type mice 4 weeks after construction of the DMM model, and we found that Oln injection significantly alleviated cartilage wear, osteophyte production, and synovial inflammation by histomorphometric analysis 8 weeks after injection (fig. 6A-F). Immunofluorescence experiments with aggrecan also demonstrated a significant increase in aggrecan signal following Oln injection (FIG. 6G, H).