参附注射液通过调控PI3K-AKT通路对LPS诱导的小鼠肺上皮细胞自噬与凋亡的影响  

Effects of Shenfu Injection on LPS-Induced Autophagy and Apoptosis of Mice Lung Epithelial Cells by Regulating PI3K-AKT Pathway

作　　者：陈娟[1,2,3] 姬晓航 王蒙蒙 冯靖 孙兆瑞 聂时南 CHEN Juan;JI Xiao-hang;WANG Meng-meng;FENG Jing;SUN Zhao-rui;NIE Shi-nan(Nanjing University of Chinese Medicine,Nanjing 210023,China;Jinling Hospital,Nanjing University School of Medicine/Department of Emergency Medicine,General Hospital of the Eastern Theater Command of the Chinese People′s Liberation Army,Nanjing 210002,China;Department of Emergency Medicine,Xuzhou First People′s Hospital,Xuzhou 221006,China)

机构地区：[1]南京中医药大学,江苏南京210023 [2]南京大学医学院附属金陵医院/中国人民解放军东部战区总医院急诊医学科,江苏南京210002 [3]徐州市第一人民医院急诊医学科,江苏徐州221006

出　　处：《中药材》2023年第10期2566-2572,共7页Journal of Chinese Medicinal Materials

基　　金：国家自然科学基金面上项目(82172182);江苏省科技项目基础研究计划(自然科学基金)面上项目(BK20211136);徐州市科技局重点研发计划(社会发展)医药卫生面上项目(KC22136);徐州医科大学附属医院发展基金项目(XYFY202232);2022年北京协和医学基金会睿E急诊医学研究专项基金项目(22222012001)。

摘　　要：目的:研究参附注射液对脂多糖(LPS)诱导的小鼠肺上皮细胞MLE-12的保护作用,并进一步探究其对自噬和凋亡的作用及潜在的分子机制。方法:采用LPS处理小鼠肺上皮细胞MLE-12建立脓毒症急性肺损伤(ALI)细胞模型,分为空白对照组、模型组及参附注射液低、中、高浓度组。CCK-8法检测LPS和参附注射液对MLE-12细胞活力的影响;酶联免疫吸附试验检测细胞上清中TNF-α、IL-1β、IL-6含量;试剂盒检测细胞内ROS水平;蛋白免疫印迹法检测肺上皮细胞标记蛋白E-cadherin、Occludin、ZO-1,自噬相关蛋白LC3B、Beclin1,凋亡相关蛋白Bax、Bcl-2及PI3K-AKT信号通路相关蛋白的表达;细胞免疫荧光检测细胞内LC3B蛋白表达;流式细胞术检测参附注射液对LPS诱导下MLE-12细胞凋亡的影响。结果:与模型组比较,参附注射液各组MLE-12细胞活力显著升高,TNF-α、IL-1β、IL-6分泌及细胞内ROS生成显著降低,E-cadherin、Occludin、ZO-1、Beclin1蛋白表达及LC3BⅡ/LC3BⅠ水平显著升高,p-PI3K/PI3K、p-AKT/AKT水平显著降低,MLE-12细胞凋亡率显著降低(P<0.01)。结论:参附注射液可抑制LPS诱导的炎症因子分泌,减轻氧化应激,改善肺上皮细胞功能,增加细胞保护性自噬,抑制细胞凋亡,其机制可能与调控PI3K-AKT信号通路有关。Objective:To investigate the protective effect of Shenfu injection on lipopolysaccharide(LPS)induced mice lung epithelial cells MLE-12,to further explore its effect on autophagy and apoptosis and its potential molecular mechanism.Methods:The acute lung injury(ALI)cell model of sepsis was established by LPS treatment of mice lung epithelial cells MLE-12,which were divided into blank control group,model group and low,medium,high concentration groups of Shenfu injection.The effects of LPS and Shenfu injection on the viability of MLE-12 cells were detected by CCK-8 method.The contents of TNF-α,IL-1βand IL-6 in cell supernatant were detected by enzyme-linked immunosorbent assay.Intracellular ROS levels were detected by the kit.The expressions of labelled proteins E-cadherin,Occludin,ZO-1,autophagy-related proteins LC3B,Beclin1,apoptosis-related proteins Bax,Bcl-2 and PI3K-AKT signaling pathway-related protein were detected by Western Blot.LC3B protein expression was detected by cellular immunofluorescence.The effect of Shenfu injection on LPS-induced apoptosis of MLE-12 cells was detected by flow cytometry.Results:Compared with model group,MLE-12 cell viability significantly increased,TNF-α,IL-1β,IL-6 secretion and intracellular ROS production significantly decreased,E-cadherin,Occludin,ZO-1,Beclin1 protein expressions and LC3B-Ⅱ/LC3B-Ⅰlevel significantly increased in Shenfu injection each group.p-PI3K/PI3K and p-AKT/AKT levels were significantly decreased,and apoptosis rate of MLE-12 cells was significantly decreased(P<0.01).Conclusion:Shenfu injection can inhibit the secretion of LPS-induced inflammatory factors,relieve oxidative stress,improve the function of lung epithelial cells,increase cell protective autophagy and inhibit cell apoptosis,the mechanism may be related to the regulation of PI3K-AKT signaling pathway.

关 键 词：参附注射液 脓毒症 急性肺损伤 细胞自噬 细胞凋亡 PI3K-AKT信号通路 

分 类 号：R285.5[医药卫生—中药学]