The Effect Of Autophagy On Advanced Glycation End Product-induced Apoptosis And Expression Of MMP-3 And MMP-13 In Rat Chondrocytes

Background and Objective:Aging is one of the most prominent risk factors for the pathological progression of osteoarthritis(OA).One feature of age related changes in OA is advanced glycation end products(AGEs)accumulation in articular cartilage.Apoptosis and catabolism of chondrocytes in the extracellular matrix(ECM)both play pivotal roles in the progress of OA.Matrix metalloproteinases(MMPs)are important regulators of the degradation of ECM during OA development.Autophagy plays a cellular housekeeping role by removing dysfunctional cellular organelles and proteins.Thus,the aim of this study is to determine whether autophagy participates in the pathology of AGEs-treated chondrocytes and to investigate the exact role of autophagy in AGEs-induced cell apoptosis and expression of MMP-3 and MMP-13.Methods:(1)Rat chondrocytes were isolated and cultured in vitro.The second generation of cultured rat chondrocytes were identified by toluidineblue staining,Alcian blue staining and immunocytochemical staining for type II collagen;(2)After chondrocytes were treated with AGEs at different concentrations for 24 h,cell viability and the expression of MMP-3 and MMP-13 were detected by CCK-8 and Western blot;(3)After chondrocytes were treated with AGEs at different concentrations for 24 h,the apoptotic incidence was detected using an Annexin V/FITC apoptosis detection kit,and the expression of MMP-3 and MMP-13 were determined by Western blot;(4)After chondrocytes were treated with AGEs for various times,the expression of LC3 B and Beclin-1 were determined by Western blot,and chondrocytes were transfected with mCherry-GFP-LC3B-adenovirus to monitor autophagic flux;(5)Chondrocytes were pretreated with 3-MA or RA 1 h before the addition of AGEs,then the apoptotic incidence was detected using an Annexin V/FITC apoptosis detection kit,and the expression of MMP-3 and MMP-13 were determined by Western blot.Results:(1)Chondrocytes grew with adherence and the shape of cells was triangle or polygonal mostly.Toluidine blue stain shows the cytoplasm of chondrocytes is blue and the nuclei is purplish blue.The cytoplasm of chondrocytes change to nattier blue after alcian blue staining.After immunocytochemical staining for type II collagen,the cytoplasm of chondrocytes turn to yellow,and the brown yellow particles are found around the nucleus;(2)Cell viability decreased after chondrocytes were treated with AGEs at different concentrations(0,10,25,50,100 and 200 ?g/m L)for 24 h( < 0.01).After treatments with AGEs at different concentrations(25,50 and 100 ?g/mL)for 24 h,Western blot analysis showed that AGEs significantly increased protein levels of MMP-3 and MMP-13 in a dose-dependent manner( < 0.01);(3)After chondrocytes were treated with 100?g/mL AGEs at various time points(0,3,6,12,24h),Western blot analysis showed that the expression of Beclin1 and the ratio of LC3-II to LC3-I were significantly increased in chondrocytes treated with AGEs,peaking at 6 h and then decreasing gradually( < 0.01).Chondrocytes were transfected with adenovirus expressing m Cherry-GFP-LC3 B and the treated with 100?g/m L AGEs.Fluorescent microscopy showed that the number of red puncta increased after treated with 6h( < 0.01);(4)After chondrocytes were treated with 100?g/mL AGEs at various time points(0,3,6,12,24h),the number of apoptotic cells significantly increased after treated with 6h( < 0.01).Western blot analysis showed that the expression of MMP-3 and MMP-13 were significantly increased after treated with 3h( < 0.05).(5)The chondrocytes were pretreated with 3-MA(5m M)or RA(5 M)for 1 h before the treatment of AGEs for 6h.Compared to cells treated with AGEs alone,3-MA+AGEs significantly increased the number of apoptotic cells and levels of MMP-3 and MMP-13( < 0.01).Whereas RA+AGEs remarkably attenuated AGE-induced apoptosis in chondrocytes,and prevented the increase of MMP-3 and MMP-13 expression induced by AGEs( < 0.01).Conclusion:(1)AGEs can induce apoptosis and increase the level of MMP-3 and MMP-13 in chondrocytes;(2)Autophagy is linked with AGE-related pathological changes of OA,and it plays a protective role in AGE-induced apoptosis and in the upregulation of MMP-3 and MMP-13 in rat chondrocytes.