The Effect And Corresponding Molecular Mechanisms Of Celastrol In Osteoarthritis

Background:Osteoarthritis(OA)is one of the most prevalent joint disorder that profoundly affects millions of individuals around the globe,particularly the elderly.It is reported that the number of Americans with osteoarthritis will reach 50 million by 2020,accounting for 25%of the adult population.According to the "guidelines for diagnosis and treatment of osteoarthritis" published by the Chinese journal of orthopedics in 2018,more than 50%of people over the age of 65 are patients with osteoarthritis,involving hip,knee,ankle,hand,cervical and lumbar joints.The prevalence rate of symptomatic knee osteoarthritis in China is 8.1%,higher in women than men and higher in rural areas than in urban areas.In addition,with the aging of China's population,the prevalence rate of osteoarthritis has a gradually increasing trend,which seriously affects the health and quality of life of middle-aged and elderly people and brings a huge burden to the national economy and social development.Osteoarthritis pathological changes mainly in the mechanical and biological factors together under the action of damaged cartilage cells,extracellular matrix and the subchondral bone synthesis and degradation of smooth,cause articular cartilage degeneration,fibrosis,and sclerosis of subchondral bone,bone hyperplasia,joint gap Narrows and around synovitis,etc.And the most important link is degeneration of articular cartilage.Articular cartilage consists of less chondrocytes and more extracellular matrix(ECM).Many studies have confirmed that IL-1? can block the synthesis of chondrocytes in ECM components and interfere with the synthesis of key structural proteins,such as collagen II and aggrecan.Cartilage cells by IL-1?stimulation,often rapidly aging and inducing cell apoptosis,can observe IL-1? have various effects on cartilage,including the possibility of its repair,directly by enzyme accelerates cartilage degeneration and side effects on the cartilage cells,therefore IL-1? is widely used as in vitro model of cartilage cells induced inflammation.Matrix metalloproteinases(MMPs)are a super family of neutral proteases whose enzyme activity depends on zinc ions.Among them,MMP-3,MMP-9 and MMP-13 plays an important role in articular cartilage damage,they mainly consists of cartilage,synovial cells,inflammatory cells and macrophages to secrete,these three enzymes are involved in the destruction of the cartilage matrix,stellate exposed to damage the activity of collagen enzyme and activate enzymes,destroying the reticular structure of collagen,finally make it broken.Collagen II and aggrecan are the main components in cartilage matrix,and their expression and apoptosis can be used as indicators to reflect the degree of cartilage degeneration.The reduction of their activity will lead to ECM regeneration disorder and eventually complete cartilage degradation.SDF-1 is produced in the synovium,while CXCR4 is predominantly expressed by the articular chondrocytes.Together they have been shown to be key players in the migration of hematopoietic progenitors from the bone marrow to the periphery.Recent findings strongly suggest that SDF-1/CXCR4 signaling also plays crucial roles in the pathology of OA.It was demonstrated by in vitro studies that the interaction between SDF1 and CXCR4 stimulated the catabolic activities of human chondrocytes through upregulation of MMPs release.Following the same line of investigations,Kanbe and colleagues found that SDF-1 levels in synovial fluid was elevated in OA patients,but could be reduced by synovectomy,providing further evidence for the involvement of SDF-1/CXCR4 in the progression of OA.Some scholars through genetic manipulation or pharmacological blocking cut SDF-1/CXCR4 expression,can strong people cartilage cells in vitro ECM degradation,and can improve the animal model of OA,the results show that the SDF-1 is through stimulation of MMPs expression to influence chondrocytes and matrix changes,thus inhibition of SDF-1/CXCR4 signaling pathways is a potential drug targets in the treatment of OA.Clinically,the early treatment of OA is mainly based on basic treatment and drug treatment.Although it can reduce pain and relieve some symptoms,it cannot delay the progress of the disease.In addition,oral drugs still have non-negligible side effects,such as potential damage to the digestive system and cardiovascular system risk.Although artificial joint replacement is a measure that can truly block the progress of OA and achieve clinical cure,it is mainly applicable to patients with advanced OA with severe symptoms due to the existence of complications such as wear and tear of artificial joint materials,high price and surgical risk.For patients with early and middle OA,there is still a lack of effective ways to intervene the disease progression.Therefore,the in-depth study of the pathogenesis and treatment of OA is of great practical significance and value.Plant-derived herbal products possess tremendous potential as therapeutic tools against arthritis,since they are more natural and generally less tolerated by patients,among which celastrol,a quinone methide triterpenoid extracted from the root of Trypterigium wilfordii(Thunder of God Vine),has attracted wide interest for its anti-cancer and anti-inflammatory effects.Previous reports clearly revealed the ability of celastrol to modulate various transcriptional factors,signaling pathways,adhesion molecules,cytokines and chemokines,including CXCR4.Consistent with the beneficial activities of celastrol in cancer and inflammation,it has been observed both in the lab and in the clinic that celastrol exert potent curative effects in different forms of arthritis,namely rheumatoid arthritis(RA)and adjuvant-induced arthritis(AIA)as well.In the previous report,Yadav et al found in the experiment that celastrol has the potential to inhibit the invasion and metastasis of cancer cells by down-regulating the expression of CXCR4.Similarly,celastrol improves RA by inhibiting CXCR4 migration and invasion of fibroblast-like synovial cells(FLSs)under hypoxia.Other reports have also revealed the inhibitory effect of celastrol on MMPs and inflammatory cytokines,which may be involved in the pathological process of arthritis.In conclusion,these results provide a mechanistic explanation for the efficacy of celastrol in treating RA patients.However,data on the precise function of celastrol in OA is limited.Considering the key role of SDF-1/CXCR4 in the development of OA,it is reasonable to speculate that celastrol,as a Chinese herbal product that affects SDF-1/CXCR4,has a significant application potential in the treatment of OA.Part I Celastrol play anti-inflammatory and anti-catabolic in rat chondrocytesObjective:To observe the toxicity of celastrol on cultured rat articular chondrocytes and to observe the antagonistic effect of celastrol on inflammatory degradation of chondrocytes stimulated by IL-1?.Methods:Chondrocytes from the femoral head of 4-week-old SD rats were isolated and cultured in vitro to the third generation,and the chondrocytes were identified by saffron O staining and collagen ? fluorescence immunofluorescence.After the action of tripterine on rat chondrocytes,the differentiation activity of chondrocytes was detected by cell count activity method(CCK-8).Different concentrations of celastrol were added to interfere with chondrocytes stimulated by IL-1?(10ng/ml),and the expression changes of mmp-13 and adams-5 genes in the cells were detected by real-time quantitative PCR.Cellular proteins were extracted and the expressions of mmp-13 and adamts-5 in chondrocytes were detected by Western blot,which was verified from the protein levels.The changes of cox-2 and iNOS in the supernatant were detected by ELISA.Rat chondrocytes were treated with celastrol and the expression of collagen ? and aggrecan gene was detected by real-time quantitative PCR.Results:Rat chondrocytes were isolated and cultured successfully,and the extracted cells were identified as chondrocytes by saffron O staining and collagen ?immunofluorescence.CCK8 showed that celastrol had no obvious toxic effect on chondrocytes in the concentration range of 0.2?M-1.2?M.It was found that celastrol with 0.8?M concentration had the best activity on chondrocytes.Celastrol can inhibit the expression of MMP-13 and ADAMTS-5,also inhibit the expression of inflammatory cytokines COX-2 and iNOS.Conclusions:Celastrol has no obvious toxic effect on chondrocytes in the concentration range of 0.2?M-1.2?M.Celastrol has anti-inflammatory and anti-degradation effects on OA chondrocytes.Celastrol can promote the repair of cartilage extracellular matrix to some extent.Part ? Celastrol can delay the progression of osteoarthritis in ratsObjective:To investigate the changes of joint pain and cartilage injury in rats treated with celastrol before and after OA treatment.To observe the effects of celastrol on MMPs,COMP fragment,collagen II,Aggrecan and Aggrecan neo-epitope in rat OA.Methods:Twenty-four SD rats weighing about 200-220g were randomly divided into four groups,Sham group,OA group,low-dose celastrol group and high-dose celastrol group,with 6 rats in each group.MIA-induced OA rat model was used.Different doses of celastrol were injected into the articular cavity on the second day after modeling,and the changes of walking distance,standing times and weight distribution of hind PAWS on days 0,7 and 14 were used to evaluate the changes of osteoarthritis pain in rats before and after modeling and treatment.Real-time quantitative PCR was used to detect the expression changes of pain genes CCR-2 and MCP-1.On the 14th day after treatment,femoral condyle and knee joint specimens were collected,fixed with formalin,decalcified,dehydrated and made of wax blocks.After successful preparation,the changes of articular cartilage were observed by means of saffrine O staining and OOCHAS scoring system.Elisa was performed on COMP fragments in rat serum,and immunohistochemical staining of COMP fragments was performed on tissue sections to observe whether celastrol could inhibit COMP degradation.Immunohistochemistry was used to observe the changes of MMP-13.WesternBlot method was adopted to detect the expression of MMP-3,MMP-9,MMP-13,collagen II and aggrecan in the MIA induced OA rat model.Results:After intraarticular injection of MIA in rats,the walking distance and standing times of OA group were significantly reduced compared with Sham group,and the weight distribution of hind PAWS was significantly reduced(P<0.01).Compared with the OA group,the walking distance and standing times of the celastrol group were significantly increased on day 7 and 14,and the weight distribution of the hind PAWS was significantly increased(P<0.01).The expression of pain genes CCR-2 and MCP-1 in the treatment group was lower than that in the OA group(P<0.01),which was dose-dependent.Safranin-O staining of cartilage tissue:there was no significant decrease in safranin-O staining of articular cartilage in the normal group,and the tidal line was intact The content of proteoglycan(PG)was normal.In the OA group,the number of chondrocytes was significantly reduced,local cells were disordered and irregular,and the content of proteoglycan(PG)was significantly reduced.The local chondrocytes were clustered in the celastrol injection group,the fading degree was lighter than that in the control group,and the content of proteoglycan(PG)was significantly increased.OOCHAS score in OA group was significantly higher than that in normal group,while OOCHAS score in celastrol group was significantly lower than that in OA group,and the difference was statistically significant(P<0.01).The serum COMP fragment concentration in the rat arthritis model was detected,which showed that the serum concentration of celastrol treatment group was lower than that of OA group.Immunohistochemical staining of tissue sections showed that COMP fragment and MMP-13 in the celastrol treatment group were all less than those in OA group.Celastrol could significantly inhibit the expression of MMP-3,MMP-9 and MMP-13,and the inhibition effect of celastrol in the high-dose group(2mg/kg)was more obvious than that in the low-dose group(lmg/kg),with statistically significant differences(Cell:P<0.01,Cel2:P<0.05,compared with that in the OA group).Celastrol could up-regulate protein expression of collagen ? and aggrecan,showing statistically significant differences(Cell:P<0.01,Cel2:P<0.05,compared with OA group),and there was a dose-dependent nature.Conclusions:Celastrol can reduce OA pain symptoms and cartilage injury in rats,inhibit the expression of MMP-3,MMP-9 and MMP-13,up-regulate collagen II and aggrecan protein expression,and reduce the degradation of cartilage extracellular matrix,thereby delaying the progress of OA in rats.Part ? Molecular mechanism of anti-inflammatory and anti-catabolic of celastrol in rat chondrocytes and OA animal models.Objective:To investigate the molecular mechanism of anti-inflammatory and anti-catabolic of celastrol in rat chondrocytes and OA animal models.Methods:In our in vivo experiment,MIA induced rat OA was used as the research object,and the expression levels of SDF-1 and CXCR4 in the normal group,OA group and different doses of celastrol group were detected by western blot.In vitro experiments,articular chondrocytes of the femoral head of SD rats aged 6-8 weeks were isolated and cultured in vitro and transferred to the third generation.CXCR4 expression levels in chondrocytes were detected by PCR after different doses of celastrol were added for 12 h,and IL-1?(10ng/ml)stimulation was added for 18 h.the messenger levels of the ECM degradation enzyme MMP-13 and the proteoglycan enzyme adamt-5 were detected by PCR.Results:Celastrol inhibited the expression of SDF-1 and CXCR4 in the cartilage tissue of OA rat model.In addition,the inhibitory effect of celastrol in the high-dose group was obvious in the low-dose group.Celastrol can inhibit CXCR4 expression in il-1-stimulated chondrocytes,and the higher the concentration,the more obvious the inhibitory effect of tripterine on CXCR4.Chondrocytes overexpressed CXCR4 gene by lentivirus transfection.On the premise of the normal expression of CXCR4,the expression of MMP-13 and ADAMTS-5 in chondrocytes after the intervention of celastrol was significantly lower than that in the non-intervention group.Under the premise of the same dose of celastrol intervention,the ability of celastrol to inhibit MMP-13 and ADAMTS-5 genes in CXCR4 overexpressed genome was significantly reduced compared with that in CXCR4 normal expression group.Without the intervention of celastrol,the expression of MMP-13 and ADAMTS-5 in the CXCR4 overexpressed genome was significantly increased compared with that in the CXCR4 normal expression group.Conclusions:Celastrol inhibited the expression of SDF-1 and CXCR4 in rat OA model.Celastrol inhibits the expression of CXCR4 in IL-1?-mediated chondrocytes in dose-dependently.Celastrol reduces the expression levels of MMP-13 and ADAMTS-5 by inhibiting SDF-1/CXCR4 signaling pathway.