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、150 μg·mL-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、150 μg·mL-1 桑色素组,培养细胞 14d后通过克隆形成实验检测细胞增殖情况;培养细胞 24h后,采用BeyoClickTM EdU-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mTOR/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(10 μg·mL-1)组、桑色素(30、150 μg·mL-1)组、桑色素(30、150 μg·mL-1)+氯喹(10 μg·mL-1)组,干预 48h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 24h 后 60、90、120、150 μ g·mL-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、150 μg·mL-1 组的A549 细胞抑制率显著升高(P＜0.001);30、90、150 μg·mL-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、150 μg·mL-1 桑色素组A549 细胞的p-P38/P38 MAPK蛋白表达水平显著升高(P＜0.01,P＜0.001);30、90、150 μg·mL-1 桑色素组A549 细胞中出现不同程度的橙黄色荧光,以 90、150 μg·mL-1 桑色素组的橙黄色荧光显著;150 μg·mL-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;150 μg·mL-1 桑色素组A549 细胞的LC3-Ⅱ蛋白表达明显上调(P＜0.05),90、150 μ g·mL-1 桑色素组 A549 细胞的 Atg16L1-Ⅱ蛋白表达显著上调(P＜0.001),p-mTOR/mTOR 及p-STAT3/STAT3 蛋白表达显著下调(P＜0.001).与桑色素(150 μg·mL-1)组比较,桑色素(150 μg·mL-1)+氯喹(10 μg·mL-1)组的A549 细胞存活率明显升高(P＜0.05).结论 桑色素能够抑制肺癌A549 细胞的增殖,促进其凋亡,并诱导自噬,其作用机制可能与mTOR/STAT3 信号轴有关.
Abstract
Objective 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 150 μg·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 30,90,150 μg·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(10 μg·mL-1)group,morin(30,150 μg·mL-1)group,morin(30,150 μg·mL-1)+ chloroquine(10 μg·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 60,90,120,150 μ g·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 150 μg·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 30,90,150 μg·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 150 μg·mL-1 of morin groups were significantly increased(P＜0.01,P＜0.001).Different degrees of orange fluorescence appeared in A549 cells of 30,90 and 150 μg·mL-1 of morin groups,and the orange fluorescence of 90 and 150 μg·mL-1 of morin groups was significant.Autophagosomes and autolysosomes appeared in the cytoplasm of A549 cells in 150 μg·mL-1 of morin group,respectively.The protein expression of LC3-Ⅱ in A549 cells of 150 μg·mL-1 of morin group was significantly up-regulated(P＜0.05).The protein expression of Atg16L1-Ⅱ in A549 cells of 90,150 μg·mL-1 of morin group was significantly up-regulated(P＜0.001),and the protein expressions of p-mTOR/mTOR and p-STAT3/STAT3 were significantly down-regulated(P＜0.001).Compared with the morin(150 μg·mL-1)group,the survival rate of A549 cells in the morin(150 μg·mL-1)+chloroquine(10 μg·mL-1)group was significantly increased(P＜0.05).Conclusion Morin can promote the apoptosis of A549 cells and induce autophagy in A549 cells,and the mechanism may be related to mTOR/STAT3 axis.
基金项目
国家重点研发计划项目(2019YFC1711400)
NETL
NSTL
万方数据