荣筋拈痛方治疗膝骨关节炎作用机制OA

Objective To study the mechanism of Rongjin Niantong Formula(RJNTF)in treating knee osteoarthritis(KOA)by regulating the glucose metabolism of rat chondrocytes through the miR-140-Sirt1/AMPK signaling axis.Methods Experiment 1:Chondrocytes were divided into control group(blank serum),interleukin-1 β(IL-1β)group(IL-1β+blank serum),and RJNTF group(IL-1β+RJNTF containing serum).IL-1β intervention lasted for 24 h,while blank serum and RJNTF containing serum intervened for 48 h.Using mitochondrial membrane potential probe(JC-1)to detect changes in mitochondrial membrane potential.Enzyme linked immunosorbent assay(ELISA)was used to detect the content of adenosine triphosphate(ATP)in cells.Real-time fluorescent quantitative nucleic acid amplification detection system(RT-qPCR)were used to detect miR-140-3p,Sirt1,gene expressions of peroxisome proliferator activated receptor gamma co-activator factor-1 alpha(PGC-1 alpha),hexokinase 2(HK2),and lactate dehydrogenase A(LDHA).Western Blot was used to detect the expressions of Sirt1,phosphorylated adenosine monophosphate activated protein kinase(p-AMPK),AMPK,PGC-1α,HK2,and LDHA proteins.Experiment 2:Chondrocytes were divided into Group A(blank serum+negative control virus),Group B(blank serum+IL-1β+negative control virus),Group C(RJNTF+IL-1β+negative control virus),Group D(blank serum+miR-140-3p knockdown lentivirus),Group E(blank serum+IL-1β+miR-140-3p knockdown lentivirus),and Group F(RJNTF drug containing serum+IL-1β+miR-140-3p knockdown lentivirus),with the same intervention time for IL-1β and serum.Using ELISA method to detect ATP content in cells.RT-qPCR was used to detect the expressions of miR-140-3p,Sirt1,PGC-1α,HK2,and LDHA genes in cells.Western Blot was used to detect the expressions of Sirt1,p-AMPK,AMPK,PGC-1α,HK2,and LDHA proteins in cells.Results Experiment 1:Compared with the control group,the IL-1β group showed a decrease in mitochondrial membrane potential and ATP content(P＜0.05),miR-140-3p gene expression and p-AMPK protein expression(P＜0.05),Sirt1 and PGC-1 α gene and protein expressions(P＜0.05),and an increase in HK2 and LDHA gene and protein expressions(P＜0.05).Compared with the IL-1β group,the RJNTF group showed an increase in mitochondrial membrane potential and ATP content(P＜0.05),miR-140-3p gene expression and p-AMPK protein expression(P＜0.05),Sirt1 and PGC-1 α gene and protein expressions(P＜0.05),and a decrease in HK2 and LDHA gene and protein expressions(P＜0.05).Experiment 2:Compared with Group A,Group B showed a decrease in ATP content(P＜0.05),a decrease in p-AMPK protein expression(P＜0.05),a decrease in Sirt1 and PGC-1 α gene and protein expressions(P＜0.05),and an increase in HK2 and LDHA gene and protein expressions(P＜0.05).Compared with group B,group C showed an increase in ATP content(P＜0.05),an increase in p-AMPK protein expression(P＜0.05),an increase in Sirt1 and PGC-1α gene and protein expressions(P＜0.05),and a decrease in HK2 and LDHA gene and protein expressions(P＜0.05).Compared with group C,group F showed a decrease in ATP content and a decrease in p-AMPK protein expression(P＜0.05),a decrease in Sirt1 and PGC-1 α gene and protein expressions(P＜0.05),and an increase in HK2 and LDHA gene and protein expressions(P＜0.05).Compared with Group E,the expression trends of various indicators in Group D were consistent with those in Group A and Group B.Compared with Group F,the expression trends of various indicators in Group E were consistent with those in Group B and Group C.Conclusion RJNTF treated KOA possibly by up-regulating miR-140-3p,Sirt1,p-AMPK,PGC-1 α expressions,and down-regulating HK2 and LDHA expressions,and regulating chondrocyte glucose metabolism.