Activated PPARa Regulate OA Cartilage Matrix Metabolism By Increasing The Level Of Autophagy

Objective:Autophagy plays an important role in the pathological process of osteoarthritis(OA).The aim of this study is to investigate the effects of PPARa on cartilage matrix metabolism and autophagy in mice and human OA chondrocytes,as well as OA animal models,and to clarify the molecular mechanism of PPARa in protecting articular cartilage,so as to provide new ideas and theoretical basis for the prevention and treatment of osteoarthritis.Methods:1.In Vitro Cell ExperimentsC57BL/6 mice(48-72 hours of birth)and human OA chondrocytes were isolated and cultured in vitro.Mouse chondrocytes were treated with LPS to simulate the state of OA or IL-lbeta to maintain the environment of human OA cells.Firstly,the expression levels of Aggrencan,Collagen ?,ADAMTS5 and MMP13 in cartilage matrix were detected by Western Blotting method,the concentration of GAGs in supernatant of culture medium was detected by DMMB staining,and the morphology and quantity of autophagy of cartilage cells treated with WY14643 were observed by transmission electron microscopy.Secondly,the expression levels of Aggrencan,Collagen II,ADAMTS5 and MMP13 were detected by Western Blotting,and the levels of autophagy markers LC3B ?/?,P62 and the changes of some signal molecules regulating autophagy were also detected.The concentration of GAGs in culture supernatant was detected by DMMB staining.The morphology of autophagy of chondrocytes treated with WY14643 was observed by transmission electron microscopy.And quantity.Subsequently,the changes of p-Akt and p-ERK in chondrocytes treated with WY14643 and the changes of matrix metabolism,autophagy and related signal molecules in chondrocytes treated with Akt inhibitors and ERK inhibitors were further detected.In addition,the effects of WY14643-activated PARalpha on chondrocyte-related lipid metabolism genes Abcal,Pdk4,Cptla and Lpl were detected by RT-PCR.2.1n Vivo Animal Experiments(1)Establish the OA model of C57BL/6 mice:?8-10 weeks old C57BL/6 mice were induced by medial meniscus ligament amputation(DMM);? WY14643 and DMSO were injected into the joint cavity after successful modeling(solvent control),twice a week(see the experimental method specifically),and the mice were killed four weeks later.(2)Establishing the OA model of PPAR alpha KO mice:?Inducing the OA model by medial meniscus ligament amputation(DMM);?Executing the mice four weeks after the successful establishment of the model.(3)All animals were killed and knee joint samples were collected.Pathological sections were routinely made and stained with Safranine O fixing green staining.Pathological morphology of articular cartilage was observed.Matrix metabolic related proteins(Aggrecan,ADAMTS5,Collagen II),autophagy markers(LC3,P62)and signal molecules(p-Akt,p-ERK)were detected by immunohistochemistry.Results:1.In C57BL/6 mouse chondrocyte and human OA chondrocyte experiments,we obtained the following results:? WY14643,an activator of PPARalpha,can inhibit the degradation of ECM in chondrocyte under OA environment;?WY 14643 activates PPARalpha to inhibit the degradation of ECM in chondrocyte by increasing the level of cell autophagy;?WY14643 activates PPARalpha to regulate autophagy and inhibit the degradation of ECM.These include:activated PARalpha initiates Akt to inhibit ULK1(PPARalpha/Akt-ULK 1),ERK to inhibit ULK1(PPARalpha/ERK-ULK1),and activated PARalpha to promote the dissociation of Beclin1 and Bcl2 complexes.2.In C57BL/6 mice OA model,intra-articular injection of WY14643 could delay the injury of OA cartilage,promote cartilage autophagy,and increase the phosphorylation of Akt and ERK.3.In the OA model of PPARa knockout mice,the knee joint of DMM mice showed obvious degradation of articular cartilage matrix,accompanied by the decrease of autophagy level and phosphorylation of Akt and ERK.Conclusion:The results of in vitro and in vivo experiments were summarized as follows:(1)Activated PARa promotes matrix synthesis of chondrocyte by increasing autophagy;(2)Activated PARa can enhance phosphorylation of Akt and ERK in chondrocyte,thereby inhibiting the activity of ULK1(Ser757)to enhance autophagy;(3)Activated PARa promotes the dissociation of Beclinl and Bcl 2 in chondrocyte,thereby improving autophagy.