线粒体辅酶Q通过PI3K/Akt途径减轻脂多糖诱导的Ⅱ型肺泡上皮细胞线粒体依赖性凋亡  

Mitochondrial coenzyme Q attenuates lipopolysaccharide-induced mitochondria-dependent apoptosis in typeⅡalveolar epithelial cells via phosphatidylinositol 3-kinase/Akt pathway

作　　者：周家奇 高静 方强[1] Zhou Jiaqi;Gao Jing;Fang Qiang(Department of Critical Care Medicine,the First Affiliated Hospital of Zhejiang University School of Medicine,Hangzhou 310003,Zhejiang,China;Department of Anesthesiology,the Children's Hospital of Zhejiang University School of Medicine,Hangzhou 310052,Zhejiang,China)

机构地区：[1]浙江大学医学院附属第一医院重症医学科,杭州310003 [2]浙江大学医学院附属儿童医院麻醉科,杭州310052

出　　处：《中华危重病急救医学》2022年第4期378-382,共5页Chinese Critical Care Medicine

基　　金：国家自然科学基金(81901937)。

摘　　要：目的观察线粒体辅酶Q(MitoQ)在脂多糖(LPS)诱导的Ⅱ型肺泡上皮细胞线粒体依赖性凋亡中的保护作用及分子机制。方法体外培养Ⅱ型肺泡上皮细胞株A549,采用不同浓度LPS处理细胞,构建急性肺损伤(ALI)模型,根据半数抑制浓度(IC_(50))得出LPS最佳刺激浓度;采用不同浓度MitoQ对细胞进行预处理,确定MitoQ最佳干预浓度。将细胞分为4组:空白对照组使用正常培养基;LPS组在正常培养基中加入10 mg/L的LPS刺激细胞24 h;MitoQ+LPS组用1μmol/L的MitoQ预处理细胞60 min,然后用含10 mg/L LPS的培养基处理细胞24 h;MitoQ+磷脂酰肌醇3激酶(PI3K)选择性抑制剂LY294002+LPS组用1μmol/L的MitoQ和20μmol/L的LY294002预处理细胞60 min,然后用含10 mg/L LPS的培养基处理细胞24 h。采用细胞增殖与毒性检测试剂盒(CCK-8)检测细胞活性;采用流式细胞仪和原位末端缺刻标记法(TUNEL)检测细胞凋亡率;采用蛋白质免疫印迹试验(Western blotting)检测凋亡蛋白Bax、抗凋亡蛋白Bcl-2及PI3K/丝氨酸-苏氨酸蛋白激酶(Akt)通路蛋白PI3K的表达和Akt磷酸化水平。结果根据抑制率曲线计算得出LPS对A549细胞的IC_(50)为11.06 mg/L,故选择10 mg/L作为LPS的刺激浓度。随着MitoQ预处理浓度增加,经10 mg/L的LPS刺激后,细胞活性呈先上升后下降趋势。根据细胞活性曲线计算得出MitoQ对细胞的最适浓度为1μmol/L。MitoQ预处理可减弱LPS诱导的线粒体依赖性凋亡,表现为细胞凋亡率较LPS组明显降低〔流式细胞仪:(8.73±0.25)%比(18.10±0.70)%,TUNEL:(12.30±0.82)%比(21.43±0.86)%,均P<0.05〕,Bax表达显著下降(Bax/β-actin:0.58±0.03比1.06±0.10,P<0.05),Bcl-2表达显著上升(Bcl-2/β-actin:1.03±0.06比0.53±0.07,P<0.05),且PI3K表达及Akt磷酸化水平显著升高〔PI3K蛋白(PI3K/β-actin):1.20±0.02比0.96±0.04,磷酸化Akt(p-Akt)蛋白(p-Akt/t-Akt):1.22±0.08比0.92±0.04,均P<0.05〕。LY294002预处理可抑制MitoQ对细胞的抗凋亡作用,表现为凋亡�Objective To investigate the protective effect and potential mechanism of mitochondrial coenzyme Q(MitoQ)on mitochondria-dependent apoptosis in typeⅡalveolar epithelial cells induced by lipopolysaccharide(LPS).Methods The typeⅡlung epithelial cell line(A549)were cultured with different concentrations of LPS in vitro,a cell model of acute lung injury(ALI)was reproduced,the optimal concentration of LPS was obtained according to the half maximal inhibitory concentration(IC_(50)).The cells were pretreated with different concentrations of MitoQ to determine the best intervention concentration of MitoQ.The cells were divided into four groups:the cells in blank control group were cultured in DMEM;the cells in LPS group were stimulated with 10 mg/L of LPS for 24 hours;the cells in MitoQ+LPS group were pretreated with 1μmol/L MitoQ for 60 minutes,and then were co-cultured with 10 mg/L of LPS for 24 hours;and the cells in MitoQ+phosphatidylinositol 3-kinase(PI3K)selective inhibitor LY294002+LPS group were pretreated with 1μmol/L MitoQ and 20μmol/L LY294002 for 60 minutes,and then were co-cultured with 10 mg/L of LPS for 24 hours.Cell viability was measured using cell counting kit-8(CCK-8).The cell apoptosis rate was determined by flow cytometry and TdT-mediated dUTP-nick end labeling(TUNEL)method.The protein expression levels of apoptosis protein Bax,anti-apoptotic protein Bcl-2 and PI3K-serine/threonine kinase(Akt)protein PI3K expression and Akt phosphorylation level were detected by Western blotting.Results According to the inhibition rate curve,the IC_(50)of LPS on A549 cells was 11.06 mg/L.Therefore,10 mg/L was selected as the stimulating concentration of LPS.After stimulation with 10 mg/L LPS,the cell viability first increased and then decreased with the increase in MitoQ pretreatment concentration.According to the cell viability curve,1μmol/L was selected as the optimum concentration of MitoQ.Compared with LPS group,after pretreated with 1μmol/L MitoQ,cell mitochondrial dependent apoptosis was significantly

关 键 词：脂多糖 线粒体辅酶Q 急性肺损伤 凋亡 

分 类 号：R563[医药卫生—呼吸系统]