电针调控lncRNA GAS5抑制SNP诱导软骨细胞线粒体凋亡的机制研究OA

Objective:To investigate the molecular mechanism by which electroacupuncture(EA)inhibits sodium nitroprusside(SNP)-induced mitochondrial apoptosis in chondrocytes by regulating long non-coding RNA GAS5(lncRNA GAS5),thereby providing experimental evidence for elucidating the therapeutic targets of EA in osteoarthritis(OA).Methods:Twenty SPF-grade male SD rats were randomly allocated into a normal control group and an EA group(n=10).Rats in the EA group were treated with EA at bilateral Neixiyan(EX-LE4)and Waixiyan(ST-35)acupoints(2 Hz,30 min per session,once daily)for one week to prepare medicated serum.Primary rat chondrocytes were isolated and randomly divided into six groups:blank+sh-NC,blank+sh-GAS5,SNP+sh-NC,SNP+sh-GAS5,SNP+EA,and SNP+sh-GAS5+EA.Chondrocyte injury was induced by 1 mmol/L SNP(except for the blank groups).Lentiviral transfection was used to silence lncRNA GAS5.The EA and SNP+sh-GAS5+EA groups were treated with 10%EA-containing serum.Fluorescence in situhybridization(FISH)was employed to detect the subcellular localization and fluorescence intensity of lncRNA GAS5.Real-time PCR was used to measure lncRNA GAS5 mRNA levels.Western blot analysis was performed to detect the protein expression of mitochondrial apoptosis-related markers(Caspase-3,Bax,Cytochrome C[Cyt-C])and the matrix-degrading enzyme MMP-13.Results:FISH indicated that lncRNA GAS5 was primarily located in the nucleus of chondrocytes.Compared with the blank+sh-NC group,the fluorescence intensity in the SNP+sh-NC group increased significantly(P<0.01),whereas it decreased markedly in the SNP+EA group compared to the SNP+sh-NC group(P<0.05).Real-time PCR showed that lncRNA GAS5 mRNA expression was upregulated in the SNP+sh-NC group relative to the blank+sh-NC group(P<0.01),but downregulated in both the SNP+EA and SNP+sh-GAS5 groups(P<0.05 or P<0.01).Western blot analysis revealed that protein levels of Caspase-3,Bax,MMP-13,and Cyt-C were significantly elevated in the SNP+sh-NC group compared to the blank+sh-NC group(P<0.05).These protein levels were significantly reduced in the SNP+EA group versus the SNP+sh-NC group(P<0.05).Furthermore,silencing lncRNA GAS5 followed by EA intervention(SNP+sh-GAS5+EA group)resulted in even lower expression of these proteins compared to the SNP+sh-GAS5 group(P<0.05).Conclusion:Electroacupuncture exerts a protective effect against OA chondrocyte injury by downregulating lncRNA GAS5 expression,thereby suppressing the mitochondrial apoptosis pathway and matrix degradation.lncRNA GAS5 represents a potential key molecular target for the treatment of OA with EA.