虎杖苷改善氧化应激介导的肺泡上皮细胞线粒体损伤的机制  

Mechanism of polydatin in promoting oxidative stress-mediated mitochondrial damage in alveolar epithelial cells

作　　者：曹媛媛[1] 李桂成[1] 邓加雄 李云峰[1] 段智[1] CAO Yuan-Yuan;LI Gui-Cheng;DENG Jia-Xiong;LI Yun-Feng;DUAN Zhi(Department of Critical Care Medicine,First People′s Hospital of Chenzhou in Hunan Province,Chenzhou 423000,China)

机构地区：[1]湖南省郴州市第一人民医院重症医学科

出　　处：《中华老年多器官疾病杂志》2020年第2期141-145,共5页Chinese Journal of Multiple Organ Diseases in the Elderly

基　　金：湖南省自然科学基金(2018JJ6004,2018JJ3015,2019JJ40010);郴州市科技计划(CZ2014018)~~

摘　　要：目的探讨虎杖苷对氧化应激介导的肺泡上皮细胞线粒体损伤的影响及其可能机制。方法A549细胞分为4组:对照组接受0.1%浓度的DMSO处理60 min;模型组给予同等浓度DMSO预处理30 min后以H 2O 2250μmol/L刺激30 min;治疗组接受虎杖苷50μmol/L预处理30 min后予以H 2O 2250μmol/L刺激30 min;抑制剂组接受线粒体自噬抑制剂mdivi-110μmol/L及虎杖苷50μmol/L预处理30 min后以H 2O 2250μmol/L刺激30 min。Western blotting法检测线粒体膜蛋白[线粒体外膜转位酶(TOM20)和线粒体内膜转位酶(TIM23)],及线粒体生成调节因子[线粒体转录因子(mTFA)和过氧化物酶体增殖物激活受体γ共激活因子1α(PGC-1α)],同时Keima法检测细胞红色/绿色荧光面积以反映线粒体自噬水平。JC-1探针检测线粒体膜电位(MMP)。DCFH-DA荧光探针检测细胞活性氧(ROS)水平。荧光素-荧光素酶法检测细胞ATP水平。细胞计数试剂盒(CCK-8)检测细胞活性。应用SPSS 20.0软件进行统计分析。结果(1)线粒体自噬水平。与对照组比较,模型组细胞TOM20及TIM23表达显著下降;与模型组比较,治疗组TOM20及TIM23表达显著下降;与治疗组比较,抑制剂组TOM20及TIM23显著增加,差异均有统计学意义(P<0.01)。但4组细胞mTFA及PGC-1α水平比较差异均无统计学意义(P>0.05)。Keima法显示,4组细胞红色/绿色荧光面积比值变化趋势与TOM20及TIM23水平变化相反。(2)MMP水平。对照组、模型组、治疗组与抑制剂组MMP分别为(100.0±5.9)%、(54.2±4.8)%、(70.8±3.6)%和(56.0±6.1)%。与对照组比较,模型组细胞MMP显著下降,治疗组MMP较模型组显著增加,而抑制剂组MMP较治疗组显著下降,差异有统计学意义(P<0.01)。(3)ROS水平、细胞活性及ATP水平。与对照组比较,模型组细胞ROS水平显著上升,细胞活性、ATP水平显著下降;与模型组比较,治疗组ROS水平显著下降,细胞活性、ATP水平显著增加;与治疗组比较,抑制剂组细胞ROS水平显Objective To investigate the effect of polydatin(PD)on oxidative stress-induced mitochondrial damage in alveolar epithelium and its possible mechanism.Methods A549 cells were assigned to four groups:control group in which cells were treated with 0.1%DMSO for 60 min;model group in which cells received the pretreatment of 0.1%DMSO for 30 min and exposure to H 2O 2(250μmol/L)for 30 min;treatment group in which cells received the pretreatment of PD(50μmol/L)for 30 min and exposure to H 2O 2(250μmol/L)for 30 min;and inhibitor group in which cells received the pretreatment of PD(50μmol/L)and mitochondrial division inhibitor 1(mdivi-1,10μmol/L)for 30 min,and exposure to H 2O 2(250μmol/L)for 30 min.Western blotting was performed for expression of mitochondrial membrane protein[translocase of outer mitochondrial membrane 20(TOM20),translocase of inner mitochondrial membrane 23(TIM23)]and mitogen regulatory factor[mitochondrial transcription factor A(mTFA),and peroxisome proliferator-activated receptor-gamma coactivator-1α(PGC-1α)].At the same time,Keima method was used to detect the red/green fluorescent area of cells as a reflection of mitochondrial autophagy.ATP was measured by fluorescein-luciferase kits,mitochondrial membrane potential(MMP)was determined using the fluorescent dye JC-1,reactive oxygen species(ROS)level was evaluated by DCFH-DA,and cell viability was determined by cell count kit-8(CCK-8).SPSS statistics 20.0 was used for analysis.Results(1)Mitochondrial autophagy level.The expression of TOM20 and TIM23 was significantly lower in the model group than in the control group,in the treatment group than in model group,but significantly higher in the inhibitor group than in the treatment group(<0.01).However,there was no significant differences in mTFA and PGC-1αbetween the four groups(P>0.05).Keima method showed that the change of red/green fluorescent area ratio in the four groups trended conversely with the changes of expression level of TOM20 and TIM23.(2)MMP.MMP was(100.0±5.9)%in the control gro

关 键 词：线粒体 自噬 虎杖苷 氧化应激 肺泡上皮细胞 

分 类 号：R563[医药卫生—呼吸系统]