摘要
目的通过体外实验明确AMPK敲除对IL-1β诱导的大鼠软骨细胞的影响,并基于PINK 1/Parkin介导的线粒体自噬探讨其机制。方法将细胞分为空白组(Control)、模型组(Model)、转染组(siRNA)、转染加模型组(siRNA+M),通过siRNA技术敲除软骨细胞AMPK的表达,流式细胞技术检测线粒体膜电位、ROS表达水平,ELISA检测ATP水平,RT-PCR技术检测AMPK、MMP-13、LC 3B、TOMM 20、VDAC 1、PINK 1、Parkin基因表达水平,Western blot技术与免疫荧光染色技术检测AMPK、MMP-13、LC 3B、TOMM 20、VDAC 1、PINK 1、Parkin蛋白表达水平。结果经siRNA技术敲除AMPK后,siRNA组、siRNA+M组AMPK表达下降,Model组在IL-1β诱导下AMPK表达下降,siRNA组、siRNA+M组、Model组中线粒体荧光染色强度、线粒体膜电位下降、ATP生成减少,ROS增多,线粒体自噬通路蛋白PINK 1、Parkin表达下降,自噬蛋白LC 3B表达下降,线粒体自噬标志物TOMM 20、VDAC 1表达上升,软骨分解代谢标志物MMP-13表达上升。结论IL-1β预处理后大鼠软骨细胞中线粒体膜电位下降、ROS增多、ATP生成减少,软骨分解代谢标志物表达增加,敲除AMPK能够抑制PINK 1/Parkin介导的线粒体自噬,增加软骨分解代谢标志物表达。
Objective To explore the effect of AMPK knockout in chondrocytes induced by interleukin-1βbased on cell experiments and the mechanism of PINK 1/Parkin-mediated mitophagy.Methods The chondrocytes were divided into blank group(Control),model group(Model),transfection group(siRNA)and transfection plus model group(siRNA+M).The siRNA technology was applied to knock out the expression of AMPK in primary rat chondrocytes.Flow cytometry was applied to detect the mitochondrial membrane potential and the ROS expression level.ELISA was applied to detect the ATP levels.Real-time fluorescence quantitative PCR(qRT-PCR)was applied to detect the mRNA expression levels of AMPK,MMP-13,LC 3B,TOMM 20,VDAC 1,PINK 1 and Parkin.Western blot technology and immunofluorescence staining technology were applied to detect the proteins expression levels AMPK,MMP-13,LC 3B,TOMM 20,VDAC 1,PINK 1and Parkin protein.Results After knocking out AMPK using siRNA technology,the expression of AMPK decreased in the siRNA group and the siRNA+M group,while the model group showed a decrease in IL-1 expressionβunder induction,the expression of AMPK decreased,and in the siRNA group,siRNA+M group,and model group,the intensity of mitochondrial fluorescence staining,mitochondrial membrane potential decreased,ATP generation decreased,ROS increased.The expression of mitophagy pathway proteins PINK 1 and Parkin decreased,while the expression of autophagy protein LC 3B decreased.The expression of mitophagy markers TOMM 20 and VDAC 1 increased,and the expression of cartilage breakdown metabolism marker MMP-13 increased.Conclusion The mitochondrial membrane potential decreased,ROS increased,ATP production decreased,and the expression of cartilage catabolism markers increased in the chondrocytes induced by IL-1βpretreatment.The AMPK knockout of could inhibit PINK 1/Parkin mediated mitophagy and increase the expression of cartilage catabolism markers.
作者
王晓萍
周明旺
杨波
吉星
李盛华
WANG Xiaoping;ZHOU Mingwang;YANG Bo;JI Xing;LI Shenghua(Gansu Traditional Chinese Medicine,Lanzhou 730050,China;Gansu Provincial Academy of Chinese Medicine,Lanzhou 730050,China;Gansu University of Chinese Medicine,Lanzhou 730050,China)
出处
《中国骨质疏松杂志》
北大核心
2025年第8期1114-1120,共7页
Chinese Journal of Osteoporosis
基金
甘肃省骨关节退行性疾病临床医学研究中心项目(18JR2FA009,2020-0411-SFC-0002)
甘肃省重点研发计划-社会发展领域项目(22YF7FA103)
全国名老中医药专家传承工作室[(2022)75号]。
