Advanced Glycation End Products (AGEs) Induce Osteoarthritis Through Inhibition Of AMPK-PGC-1α-SIRT3 Pathway

Objective: The etiology and pathogenesis of osteoarthritis(OA)are unclear,with age being one of the most important causes,and the most significant change in the body with aging is the massive accumulation of advanced glycation end products(AGEs).The mechanism of osteoarthritic lesions is not clear.The present study aimed to investigate the role of AGEs on mitochondrial function of chondrocytes and related mechanisms through animal and in vitro cultured chondrocytes,and to provide new intervention targets for the prevention and treatment of OA lesions.Methods: In this study,OA lesions were induced by direct injection of exogenous AGEs into the knee joint cavity of mice,and OA lesions were assessed by staining with saffron Osolid green.Phosphorylation-Adenosine monophosphate activated protein kinase(p-AMPK),Peroxisome proliferator-activated receptor-gamma coactivator-1α(ΔΨm),and peroxisome proliferator-activated receptor-gamma coactivator-1α(ΔΨm)were examined by western blot.receptor-gamma coactivator-1alpha(PGC-1alpha)and silencing information regulator 2related enzyme 3(SIRT3)to explore the mechanisms involved in the regulation of mitochondrial function;The expression of matrix metalloproteinase-3(MMP-3)and matrix metalloproteinase-13(MMP-13)was detected by western blot to assess the changes of chondrocyte catabolism.Results:(1)In comparison with normal mice in the knee joint injection AGEs model group,the articular cartilage layer became thinner,the chondrocyte nuclear morphology was fixed,the cell distribution was uneven,more vacuolated cells(without nucleus)appeared in the lower cartilage,the staining was light,and the subchondral bone with solid green staining was blue-green.Compared with the model group of OA lesion induced by ACL resection,the changes were more consistent,and the results suggested that joint cavity injection of AGEs could induce OA lesion.(2)Purified chondrocytes in the AGEs model group showed significantly lower ΔΨm and significantly higher expression of MMP-3 and MMP-13 compared with the control group.(3)In chondrocytes cultured in vitro,different concentrations of AGEs could significantly inhibit the expression of p-AMPK compared with normal controls,while the difference in the effect on t-AMPK was not statistically significant;the expression results of SIRT3 and PGC-1α were consistent with the changes in p-AMPK,and AGEs could concentration-dependently inhibit the protein expression of SIRT3 and PGC-1α,with 160 μg/m L concentration group had the highest effect,and the difference was statistically significant.(4)In chondrocytes cultured in vitro,AGEs could significantly induce a decrease in chondrocyte ΔΨm compared with the control group,while pretreatment with the AMPK-selective agonist AICAR significantly inhibited the impairing effect of AGEs on mitochondrial function.Further,the protective effect on mitochondrial damage induced by AGEs in chondrocytes was significantly reduced after conditional knockdown of SIRT3 and PGC-1α in chondrocytes using si RNA SIRT3 or si RNA PGC-1α,followed by administration of AICAR.(5)The addition of AICAR significantly increased the protein expression of AMPK,PGC-1α and SIRT3 inhibited by AGEs,while the administration of nicotinamide ribosyl chloride,an agonist of SIRT3,also significantly increased the protein expression of PGC-1α inhibited by AGEs.(6)Compared with the control group,AGEs significantly induced MMP-3 and MMP-13 protein expression in chondrocytes,whereas administration of the AMPK-selective agonist AICAR pretreatment significantly inhibited AGEs-induced MMP-3 and MMP-13 expression;in si RNA SIRT3 or si RNA PGC-1α conditionally knocked down the chondrocyte SIRT3,PGC-1α,followed by the administration of AMPK selective agonist AICAR,its inhibitory effect on AGEs-induced MMP-3 and MMP-13 expression in chondrocytes was significantly reduced,and the difference was statistically significant.Conclusion: AGEs induce mitochondrial functional impairment in chondrocytes through down-regulation of AMPK-PGC-1α-SIRT3 pathway,which in turn promotes OA lesions,and agonizing this pathway significantly alleviates AGEs-induced OA lesions.