基于mTOR/STAT3信号轴探讨桑色素诱导非小细胞肺癌A549细胞自噬的机制  

Exploration of the Mechanism of Autophagy Induced by Morin in Non-small Cell Lung Cancer A549 Cells Based on mTOR/STAT3 Signaling Axis

作　　者：赵新月 田颖颖 刘闯 李依林 吕英楠 于尚玥 田时秋 裴海鸾 左泽平 王志斌[1,2] ZHAO Xinyue;TIAN Yingying;LIU Chuang;LI Yilin;LYU Yingnan;YU Shangyue;TIAN Shiqiu;PEI Hailuan;ZUO Zeping;WANG Zhibin(School of Chinese Materia Medica,Beijing University of Chinese Medicine,Beijing 102488,China;Beijing Tongrentang Technologies Co.,Ltd.Pharmaceutical Factory,Beijing 100079,China)

机构地区：[1]北京中医药大学中药学院,北京102488 [2]北京同仁堂科技发展股份有限公司制药厂,北京100079

出　　处：《中药新药与临床药理》2024年第3期317-324,共8页Traditional Chinese Drug Research and Clinical Pharmacology

基　　金：国家重点研发计划项目(2019YFC1711400)。

摘　　要：目的基于mTOR/STAT3信号轴探讨桑色素诱导非小细胞肺癌A549细胞自噬的机制。方法将A549细胞分为空白组及30、60、90、120、150μg·m L^(-1)桑色素组,培养24、48、72 h后采用CCK-8法检测细胞增殖活性,计算细胞抑制率。将A549细胞分为空白组及30、90、150μg·m L^(-1)桑色素组,培养细胞14 d后通过克隆形成实验检测细胞增殖情况;培养细胞24 h后,采用Beyo ClickTMEd U-488检测细胞增殖能力;流式细胞术检测细胞凋亡情况;吖啶橙染色检测细胞自噬情况;透射电镜观察细胞自噬体形成情况;Western Blot法检测细胞中凋亡、自噬及m TOR/STAT3信号轴相关蛋白的表达水平。将A549细胞分为空白组、空白+氯喹(10μg·m L^(-1))组、桑色素(30、150μg·m L^(-1))组、桑色素(30、150μg·m L^(-1))+氯喹(10μg·m L^(-1))组,干预48 h后采用CCK-8法检测细胞活性,计算细胞存活率。结果与空白组比较,干预24 h后60、90、120、150μg·m L^(-1)桑色素组的A549细胞抑制率显著升高(P<0.05,P<0.001);干预48、72 h后桑色素30、60、90、120、150μg·m L^(-1)组的A549细胞抑制率显著升高(P<0.001);30、90、150μg·m L^(-1)桑色素组的A549细胞集落数及绿色荧光的增殖阳性细胞数均显著减少(P<0.01,P<0.001),细胞凋亡率均显著升高(P<0.01,P<0.001),cleaved-PARP蛋白表达水平显著升高(P<0.001);90、150μg·m L^(-1)桑色素组A549细胞的p-P38/P38MAPK蛋白表达水平显著升高(P<0.01,P<0.001);30、90、150μg·m L^(-1)桑色素组A549细胞中出现不同程度的橙黄色荧光,以90、150μg·m L^(-1)桑色素组的橙黄色荧光显著;150μg·m L^(-1)桑色素组A549细胞胞浆中分别出现了自噬体和自噬溶酶体;150μg·m L^(-1)桑色素组A549细胞的LC3-Ⅱ蛋白表达明显上调(P<0.05),90、150μg·m L^(-1)桑色素组A549细胞的Atg16L1-Ⅱ蛋白表达显著上调(P<0.001),p-m TOR/m TOR及p-STAT3/STAT3蛋白表达显著下调(P<0.001)。与桑色素(150μg·m L^(-1))组比较,桑色素(150Objective To investigate the mechanism of morin-induced autophagy in non-small cell lung cancer A549 cells based on mTOR/STAT3 signaling axis.Methods A549 cells were divided into blank group and 30,60,90,120 and 150μg·mL^(-1)of morin groups.After 24,48 and 72 hours of culture,the cell proliferation activity was detected by CCK-8 method,and the cell inhibition rate was calculated.A549 cells were divided into blank group and 30,90,150μg·mL^(-1)morin groups.After 14 days of culture,the cell proliferation was detected by colony formation assay.After 24 hours of culture,the cell proliferation ability was detected by BeyoClickTM EdU-488.Apoptosis was detected by flow cytometry;acridine orange staining was used to detect cell autophagy;the formation of autophagosomes was observed by transmission electron microscopy.Western Blot was used to detect the expression levels of apoptosis,autophagy and mTOR/STAT3 signaling axis-related proteins in cells.A549 cells were divided into blank group,blank group+chloroquine(10μg·mL^(-1))group,morin(30,150μg·mL^(-1))group,morin(30,150μg·mL^(-1))+chloroquine(10μg·mL^(-1))group.After 48 hours of intervention,the cell activity was detected by CCK-8 method,and the cell survival rate was calculated.Results Compared with the blank group,the inhibition rate of A549 cells in 60,90,120,150μg·mL^(-1)of morin group was significantly increased after 24 hours of intervention(P<0.05,P<0.001).The inhibition rates of A549 cells in 30,60,90,120 and 150μg·mL^(-1)of morin groups were significantly increased after 48 and 72 hours of intervention(P<0.001).The number of A549 cell colonies and the number of green fluorescent proliferation positive cells in the 30,90,150μg·mL^(-1)of morin groups were significantly decreased(P<0.01,P<0.001),the apoptosis rate was significantly increased(P<0.01,P<0.001),and the protein expression level of cleaved-PARP was significantly increased(P<0.001).The protein expression levels of p-P38/P38 MAPK in A549 cells of 90 and 150μg·mL^(-1)of morin groups were

关 键 词：桑色素 非小细胞肺癌 A549细胞 增殖 凋亡 自噬 mTOR/STAT3信号轴 

分 类 号：R285.5[医药卫生—中药学]