Effect of Yishen Jiangu Pill on AMPK/Cyclin Y/CDK 16 pathway activity and cellular autophagy in KOA chondrocytes
Objective To explore the effect of Yishen Jiangu Pill on AMPK/CyclinY/CDK16 pathway activity and autophagy of chondrocytes in knee osteoarthritis(KOA).Methods Chondrocytes were extracted from the cartilage of KOA patients undergoing total knee arthroplasty.After screening the optimal concentration and time of administration,the chondrocytes were divided into model group(Modle),control group(Control),AMPK activator group(AICAR),AMPK inhibitor group(CompoundC),Yish-en Jiangu pill group(YSJGP)and AMPK inhibitor+Yishen Jiangu pill group(Compound C+YSJGP).Except the model group and control group,the other groups were treated with corresponding drugs.The effect of Yishen Jiangu Pill on the activity of chon-drocytes was detected by CCK8 method.The expression of AMPK,CyclinY,CDK16mRNA and protein,the expression of ULK-1,Beclin1,p62 mRNA and protein and the ratio of LC3 Ⅱ/Ⅰ in chondrocytes were detected by qRT-PCR and Westernblot.Au-tophagosomes were observed by transmission electron microscope,and the relative expression of LC3 Ⅱ protein was observed by la-ser confocal.Results YSJGP could significantly increase the activity of AMPK/Cyclin Y/CDK16 pathway in chondrocytes induced by LPS,increase the activities of ULK-1 and Beclin 1 and the ratio of LC3 Ⅱ/Ⅰ,decrease the expression of p62,and increase the number of autophagosomes and fluorescence intensity of LC3 Ⅱ in chondrocytes.Its effect on chondrocytes was consistent with the trend of AICAR,while the intervention of Compound C was on the contrary.In addition,Compound C significantly inhibited the regulation of AMPK/Cyclin Y/CDK16 pathway activity and ULK-1,Beclin-1,p62,LC3 Ⅱ/Ⅰ by YSJGP,and reversed the regulation of YSJGP on autophagy activity of chondrocytes.Conclusion Yishen Jiangu Pill can enhance the autophagy activity of KOA chondrocytes,and its mechanism is closely related to the up-regulation of AMPK/CyclinY/CDK16 pathway activity.
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