机械敏感性通道蛋白对膝骨关节炎滑膜巨噬细胞焦亡的调控效应分析  

Study of the Regulatory Efficacy of Mechanical-Sensitive ChannelProtein(Piezo1)on Cell Pyrolysis in Knee Osteoarthritis

作　　者：廖太阳 王培民[1,2] 徐波[1,2] 张力[1,2] 李晓辰[1,2] 吴鹏 邢润麟 黄正泉[1,2] 张农山[1,2] LIAO Taiyang;WANG Peimin;XU Bo;ZHANG Li;LI Xiaochen;WU Peng;XIN Runlin;HUANG Zhengquan;ZHANG Nongshan(Affiliated Hospital of Nanjing University of Chinese Medicine,Nanjing 210029,China;Jiangsu Province Hospital of Chinese Medicine,Nanjing 210029,China)

机构地区：[1]南京中医药大学附属医院,南京210029 [2]江苏省中医院

出　　处：《中国中医骨伤科杂志》2021年第8期12-17,共6页Chinese Journal of Traditional Medical Traumatology & Orthopedics

基　　金：江苏省自然科学基金项目(BK20171513);江苏省第十六批“六大人才高峰”高层次人才项目(WSW-014)。

摘　　要：目的:探讨机械敏感性通道蛋白Piezo1对膝骨关节炎细胞焦亡的调控效应。方法:选取5只SPF级雄性SD大鼠,提取腹腔巨噬细胞,分为空白对照组、应力拉伸组及应力拉伸+Piezo1抑制剂组。使用FX-5000T细胞牵张拉伸应力加载系统以6个循环/min的周期性频率对BioFlex板内巨噬细胞进行表面干预。Western Blot检测Caspase-1,Caspase-1 p10,GSDMD蛋白表达;酶联免疫吸附测定(ELISA)检测细胞上清IL-1β及IL-18含量;Hoechst33342/PI细胞双染免疫荧光检测巨噬细胞膜的完整性;乳酸脱氢酶(LDH)试剂盒检测巨噬细胞培养上清乳酸脱氢酶含量;荧光倒置显微镜观察线粒体活性氧(ROS)荧光强度。结果:应力拉伸组Caspase-1,Caspase-1 p10及GSDMD蛋白表达水平较空白对照组增加(P<0.05),而应力拉伸+Piezo1抑制剂组较应力拉伸组有所降低,差异有统计学意义(P<0.05);此外,应力拉伸组较空白对照组培养上清中IL-1β和IL-18含量显著上升,差异有统计学意义(P<0.05),应力拉伸+Piezo1抑制剂组较应力拉伸组含量显著下降,差异有统计学意义(P<0.05);Ho/PI双染发现应力拉伸组细胞膜遭到破坏,碘化丙啶(PI)染色呈现红色荧光,应力拉伸+Piezo1抑制剂组细胞膜较为完整,PI不能染色或者染色较少;同时,应力拉伸组上清液中LDH含量显著上升,差异有统计学意义(P<0.05);应力拉伸+抑制剂LDH含量较应力拉伸组则显著下降,差异有统计学意义(P<0.05);最后ROS荧光检测发现应力拉伸组产生大量红色荧光,而应力拉伸+抑制剂组荧光强度较低。结论:异常机械应力可通过Piezo1离子通道,加剧滑膜巨噬细胞焦亡及推动膝骨关节炎的进程。Objective:To explore the regulatory efficacy of mechanical-sensitive channel protein(Piezo1)on cell pyrolysis in knee osteoarthritis(KOA).Methods:5 SPF male SD rats were selected,and macrophages were extracted and divided into blank control group,stress stretching group and stress stretching + inhibitor group.FX-5000 T-intermittent cyclic mechanical tension(FX-5000 T-ICMT)was used to perform surface elongation of synovial macrophages in the BioFlex plate at a periodic frequency of 6 cycles/min.Protein expression of Caspase-1,Caspase-1 p10 and GSDMD were detected by Western Blot.Enzyme-linked immunosorbent assay(ELISA)was used to detect the content of IL-1βand IL-18 in cell supernatant.The integrity of macrophage membrane was detected by Hoechst33342/PI cell double-staining immunofluorescence.The content of lactate dehydrogenase(LDH)in the supernatant of macrophage culture was detected by LDH kit.Fluorescence inverted microscope was used to observe the fluorescence intensity of reactive oxygen species(ROS).Results:The protein expression levels of Caspase-1,Caspase-1 p10 and GSDMD in the stress stretching group were increased compared with the blank control group(P<0.05),while that in the stress stretching group + inhibitor group were decreased compared with the stress stretching group(P<0.05).In addition,the content of IL-1βand IL-18 in the supernatant of the stress stretching group was significantly increased compared to the blank control group(P<0.05),while the content of IL-1βand IL-18 in the stress stretching+inhibitor group was significantly decreased compared to the stress stretching group(P<0.05).Ho/PI double staining revealed that the cell membrane of the stress stretching group was damaged,and PI staining showed red fluorescence,while the cell membrane of stress stretching+inhibitor group was more complete,and PI could not be stained or stained less.At the same time,the LDH content in the supernatant of the stress stretching group was significantly increased(P<0.05).LDH content of stress stretching +in

关 键 词：异常机械应力 机械敏感性通道蛋白 膝骨关节炎 滑膜巨噬细胞焦亡 

分 类 号：R-33[医药卫生]