青藤碱调节AMPK/mTOR/ULK1信号通路对IL-1β诱导的关节软骨细胞自噬和凋亡的影响  

Impacts of sinomenine on autophagy and apoptosis in articular chondrocytes induced by IL-1βby regulating AMPK/mTOR/ULK1 signaling pathway

作　　者：胡宏志[1] 汪能[1] 李娟[1] 李冰[1] 姚金龙 Hu Hongzhi;Wang Neng;Li Juan;Li Bing;Yao Jinlong(Department of Orthopedics and Trauma,Second Affiliated Hospital of Hunan University of Traditional Chinese Medicine,Hunan Province,Changsha 410005,China)

机构地区：[1]湖南中医药大学第二附属医院骨伤四科,长沙410005

出　　处：《疑难病杂志》2024年第11期1379-1384,1398,共7页Chinese Journal of Difficult and Complicated Cases

基　　金：湖南省自然科学基金省市联合项目(2022JJ50049)。

摘　　要：目的探讨青藤碱(SN)调节单磷酸腺苷活化蛋白激酶(AMPK)/雷帕霉素靶蛋白(mTOR)/UNC-51样激酶1(ULK1)信号通路对白介素-1β(IL-1β)诱导的关节软骨细胞自噬和凋亡的影响。方法将关节软骨细胞分为Control组(正常培养)、IL-1β组(10μg/L的IL-1β诱导12 h)、L-SN、M-SN、H-SN组(在IL-1β诱导的基础上添加25、50、100μmol/L的SN)、SN+Compound C组(在H-SN组的基础上添加10μmol/L AMPK抑制剂Compound C)。MTT法、透射电子显微镜(TEM)、流式细胞仪分别检测SN对各组关节软骨细胞增殖、自噬、凋亡的影响;ELISA试剂盒检测各组细胞中COX-2、TNF-α、MMP-3、MMP-13的表达;蛋白印迹实验(WB)检测各组细胞中p-AMPK、AMPK、p-mTOR、mTOR、p-ULK1、ULK1蛋白水平。结果与Control组比较,IL-1β组关节软骨细胞的A 490值、p-AMPK/AMPK、p-ULK1/ULK1蛋白水平降低,自噬空泡数、凋亡率、COX-2、TNF-α、MMP-3、MMP-13、p-mTOR/mTOR蛋白水平升高(P<0.05);与IL-1β组比较,L-SN组、M-SN组、H-SN组A 490值、自噬空泡数、p-AMPK/AMPK、p-ULK1/ULK1蛋白水平升高,凋亡率、COX-2、TNF-α、MMP-3、MMP-13、p-mTOR/mTOR蛋白水平降低(P<0.05);与H-SN组比较,SN+Compound C组A 490值、自噬空泡数、p-AMPK/AMPK、p-ULK1/ULK1蛋白水平降低,凋亡率、COX-2、TNF-α、MMP-3、MMP-13、p-mTOR/mTOR蛋白水平升高(P<0.05)。结论SN可以通过促进IL-1β诱导的关节软骨细胞自噬,抑制细胞凋亡,其机制可能是通过激活AMPK/mTOR/ULK1信号通路实现的。Objective To investigate the impacts of sinomenine(SN)on interleukin-1β(IL-1β)induced autophagy and apoptosis in articular chondrocytes by regulating the adenosine monophosphate activated protein kinase(AMPK)/mammalian target of rapamycin(mTOR)/UNC-51-like kinase 1(ULK1)signaling pathway.Methods Joint chondrocytes were grouped into control group(normal culture),IL-1βgroup(induced with 10 ng/mL IL-1βfor 12 hours),L-SN,M-SN,H-SN groups(adding SNs of 25,50,and 100μM on the basis of IL-1βinduction),and SN+Compound C group(adding 10μmol/L AMPK inhibitor Compound C pn the basis of the H-SN group).MTT assay,transmission electron microscopy(TEM),and flow cytometry were used to detect the effects of SN on proliferation,autophagy,and apoptosis of articular chondrocytes in each group.ELISA kit was applied to detect the expression of COX-2,TNF-α,MMP-3,and MMP-13 of cells in various groups.Western blotting(WB)was applied to detect the protein expression of p-AMPK,AMPK,p-mTOR,mTOR,p-ULK1,and ULK1 in the cells of each group.Results Compared with the Control group,the A490 values of articular chondrocytes,the expression of p-AMPK/AMPK and p-ULK1/ULK1 proteins in the IL-1βgroup were decreased,the autophagy vacuole count,apoptosis rate,the expression of COX-2,TNF-α,MMP-3,MMP-13,and p-mTOR/mTOR proteins were increased(P<0.05).Compared with IL-1βgroup,the A490 value,number of autophagic vacuoles,the expression of p-AMPK/AMPK and p-ULK1/ULK1 proteins in the L-SN group,M-SN group,and H-SN group were increased,the apoptosis rate,the expression of COX-2,TNF-α,MMP-3,MMP-13,and p-mTOR/mTOR proteins were decreased(P<0.05).Compared with H-SN group,the A490 value,number of autophagic vacuoles,the expression of p-AMPK/AMPK and p-ULK1/ULK1 proteins in the SN+Compound C group were decreased,the apoptosis rate,the expression of COX-2,TNF-α,MMP-3,MMP-13,and p-mTOR/mTOR proteins were increased(P<0.05).Conclusion SN can promote IL-1βinduced autophagy in articular chondrocytes and inhibit cell apoptosis,which may be achieved by activat

关 键 词：骨关节炎 青藤碱 单磷酸腺苷活化蛋白激酶 雷帕霉素靶蛋白 UNC-51样激酶1 白介素-1Β 关节软骨细胞 自噬 凋亡 

分 类 号：R684.3[医药卫生—骨科学]