冬虫夏草调控PERK-eIF2α信号通路抑制香烟烟雾提取物刺激下的人支气管上皮细胞凋亡研究  

Cordyceps Sinensis Regulates the PERK-eIF2αSignaling Pathway to Inhibit the Apoptosis of Human Bronchial Epithelial Cells Stimulated by Cigarette Smoke Extract

作　　者：刘玉[1] 谢纬[1] 祝庆华[1] 李敏芳[1] 唐明文[1] 陈生[1] Liu Yu;Xie wei;Zhu Qinghua;Li Minfang;Tang Mingwen;Chen Sheng(Shenzhen Traditional Chinese Medicine Hospital,The Fourth Clinical Medical College of Guanghzhou University of Traditional Chinese Medicine,Guangdong,Shenzhen 518003,China)

机构地区：[1]广东省深圳市中医院,广州中医药大学第四临床医学院,广东深圳518003

出　　处：《中国中医急症》2021年第10期1696-1700,1728,共6页Journal of Emergency in Traditional Chinese Medicine

基　　金：国家自然科学基金项目(81904103);广东省中医药管理局项目(20192083);深圳市科技计划项目(JCYJ20190812155412785);深圳市“医疗卫生三名工程”(SZSM201812063)。

摘　　要：目的观察冬虫夏草对慢性阻塞性肺疾病细胞模型的内质网应激(ERS)信号通路蛋白激酶R样内质网激酶(PERK)-真核起始因子2α(eIF2α)和细胞凋亡的影响。方法用香烟烟雾提取物(CSE)诱导的人肺泡上皮细胞(A549)制作COPD细胞模型,MTT法测定CSE最佳干预浓度和时间,孵育不同质量浓度的虫草菌液(10、50、100、200 mg/L),MTT法检测不同质量浓度冬虫夏草和CSE干预细胞24 h后的存活率,根据实验结果确定冬虫夏草最佳干预质量浓度。随后将A549细胞分为空白对照组、CSE组、冬虫夏草组。空白组加入培养基,CSE组加入2%CSE,冬虫夏草组加入虫草菌液(100 mg/L)和2%CSE。共作用24 h后采用Hoechst 33342染色和Annexin V-FITC/PI流式检测各组细胞凋亡情况,蛋白免疫印迹法(Western blotting)检测PERK、pPERK、eIF2α、p-eIF2α、凋亡相关蛋白Bcl-2和BAX水平。结果细胞存活率与CSE呈浓度、时间依赖性负相关;除200 mg/L的虫草菌液外,余不同质量浓度的虫草菌液对A549细胞无明显毒性;与CSE组比较,虫草菌液100 mg/L组显著提高细胞存活率;与空白组比较,CSE组的细胞凋亡率升高(P<0.01),且染色质凝集,核萎缩,凋亡小体增多,与CSE组比较,冬虫夏草组细胞凋亡率降低(P<0.01),且染色质凝集,核萎缩和凋亡小体较少;与空白组比较,CSE组p-PERK、p-eIF2α、Bax和Bcl-2的表达增多(P<0.01),Bax表达降低;与CSE组比较,冬虫夏草组p-PERK、p-eIF2α、Bcl-2的表达减少,Bax表达升高(P<0.01)。结论冬虫夏草通过抑制PERKeIF2α信号通路过度激活减轻内质网应激反应从而减少细胞凋亡,最终发挥对细胞的保护作用。Objective:To investigate the effects of Cordyceps sinensis on endoplasmic reticulum stress(ERS)signaling pathway PERK-eIF2αand apoptosis in chronic obstructive pulmonary disease(COPD)cell model.Methods:Cigarette smoke extract(CSE)induced A549 was used to make COPD cell model,and MTT method was used to determine the optimal intervention concentration and time of CSE.Different concentrations of Cordyceps sinensis solution(10,50,100,200 mg/L)were incubated,and MTT assay was used to detect the survival rate of cells treated with Cordyceps sinensis and CSE at different concentrations for 24 h;the optimal intervention concentration of Cordyceps sinensis was determined according to the experimental results.Then,A549 cells were divided into blank control group,CSE group and Cordyceps sinensis group.The blank group was added with medium,the CSE group was added with 2%CSE,and the Cordyceps sinensis group was added with Cordyceps sinensis solution(100 mg/L)and 2%CSE.After co-treatment for 24 h,apoptosis was detected by Hoechst 33342 staining and Annexin V-FITC/PI flow cytometry.The levels of PERK,p-PERK,eIF2α,p-eIF2α,and apoptosis-related proteins Bcl-2 and Bax were detected by Western blotting.Results:The cell survival rate was negatively correlated with CSE in concentration and time dependence;except for 200 mg/L Cordyceps sinensis solution,the other different concentrations of Cordyceps sinensis solution had no obvious toxicity to A549 cells;compared with CSE group,100 mg/L Cordyceps sinensis solution significantly increased cell survival rate;compared with the blank group,the apoptosis rate of CSE group was increased(P<0.01),chromatin agglutination,nuclear atrophy and apoptotic bodies were increased;compared with the CSE group,cell apoptosis rate in Cordyceps sinensis group was decreased(P<0.01),the chromatin agglutination,nuclear atrophy and apoptotic bodies were less;compared with the blank group,the expressions of p-PERK,p-eIF2α,Bax and Bcl-2 in the CSE group were increased(P<0.01),while the expression of Bax was

关 键 词：慢性阻塞性肺疾病 冬虫夏草 A549细胞 炎症 细胞凋亡 

分 类 号：R285.5[医药卫生—中药学]