摘要
目的:探讨没食子酸对人非小细胞肺癌A549细胞增殖的抑制作用,阐明其药物毒性的分子作用机制。方法:体外培养人非小细胞肺癌A549细胞,将A549细胞分为对照组及低、中和高剂量没食子酸组,没食子酸给药剂量分别为0、300、500和750μmol·L^(-1)。MTT法检测各组细胞存活率,瑞氏-姬姆萨染色液检测各组A549细胞形态表现,流式细胞术检测细胞凋亡率和活性氧(ROS)水平,Western blotting法检测各组A549细胞中Fas和FasL蛋白的表达水平;分光光度法检测各组A549细胞中过氧化氢酶(CAT)、超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)的活性。结果:与没食子酸处理6h比较,处理12和24h时各剂量没食子酸组A549细胞存活率明显降低(P<0.05),随着作用时间延长,细胞存活率逐渐降低;没食子酸处理24h后,与对照组比较,低、中和高剂量没食子酸组A549细胞存活率明显降低(P<0.05)。瑞氏-姬姆萨染色,各剂量没食子酸组细胞出现胞质皱缩,细胞体积变小、变圆等凋亡形态表现;随着没食子酸剂量增加,A549细胞凋亡率逐渐增加;随着没食子酸剂量的增加,A549细胞中Fas和FasL蛋白的表达水平有升高趋势,且Fas与FasL蛋白表达水平呈正相关关系(r=0.92,P<0.05)。与对照组比较,低、中和高剂量没食子酸组A549细胞中ROS水平升高(P<0.05);CAT活性升高,但组间比较差异无统计学意义(P>0.05),且CAT活性随没食子酸剂量增加而逐渐降低。低剂量没食子酸组A549细胞中SOD活性明显高于对照组(P<0.05);中和高剂量没食子酸组A549细胞中SOD活性虽高于对照组,但组间比较差异无统计学意义(P>0.05),且SOD活性随没食子酸剂量增加而逐渐降低。低剂量没食子酸组A549细胞中GSH-Px活性高于对照组,但组间比较差异无统计学意义(P>0.05);中和高剂量没食子酸组A549细胞中GSH-Px活性低于对照组,但组间比较差异亦无统计学意义(P>0.05);随着没食子酸剂量的不断增加,GSH-Px活性反而降低。低、中和高剂量没食子酸组间A549细胞中CAT、SOD和GSH-Px活性比较差异无统计学意义(P>0.05)。结论:没食子酸可能是通过诱导氧化损伤的方式抑制肺癌细胞增殖,Fas/FasL信号通路可能是其诱导细胞凋亡的重要作用机制之一。
Objective：To detect the inhibitory effect of gallic acid on the proliferation of human non-small cell lung cancer A549 cells,and to investigate the molecular mechanisms of its drug toxicity.Methods：The human nonsmall cell lung cancer A549 cells were cultured in vitro.The cells were divided into control group,low,middle,and high dosages of gallic acid groups（0,300,500 and 750μmol·L^（-1））.The survival rates of cells were tested by MTT method;the morphology of A549 cells were tested by Switzerland and Giemsa staining;the apoptotic rates of A549 cells and the levels of reactive oxygen species（ROS）in A549 cells were analyzed by FCM;the expression levels of Fas and FasL in the A549 cells in various groups were detected by Western blotting method;the activities of catalase（CAT）,superoxide dismutase（SOD）and glutathione peroxidase（GSH-Px）in the A549 cells in various groups were analyzed by spectrophotometry.Results：Compared with gallic acid treatment for 6h,the survival rates of A549 cells treated for 12 and 24hin different dosages of gallic acid groups were significantly decreased（P〈0.05）,and the cell survival rates were gradually decreased with the prolongation of treatment time.Compared with control group,the survival rates of A549 cells in different dosages of gallic acid groups were significantly decreased after treatment for 24h（P〈0.05）.The morphology of A549 cells in different dosages of gallic acid groups showed the typical apoptosis which were observed by Switzerland and Giemsa staining,such as the cytoplasmic shrinkage,small and round cell volume and so on.The results of and so on apoptotic examination showed that the apoptotic rates were increased with the increasing of dosage of gallic acid.The expression levels of Fas and FasL in A549 cells were increased with the increasing of gallic acid dosages.The expression levels of Fas were positively correlated with the expression levels of FasL（r=0.92,P〈0.05）.Compared with control group,the levels of ROS in A549 cells in low,middle and high dosages of gallic acid groups were increased（P〈0.05）.Compared with control group,the activities of CAT in A549 cells in different dosages of gallic acid groups were also increased,but there were no significant differences（P〉0.05）;the activities of CAT in A549 cells in different dosages of gallic acid groups were reduced with the increasing of gallic acid dosage.The activity of SOD in low dosage of gallic acid group was higher than that in control group（P〈0.05）.The activities of SOD in middle and high dosage of gallic acid groups were higher than that in control group（P〈0.05）,but there were no significant differences between various groups（P〈0.05）.The activities of SOD were reduced with the increasing of gallic acid dosages.The activity of GSH-Px in A549 cells in low dosage of gallic acid group was higher than that in control group,but there was no significant difference between various groups（P〉0.05）.The activities of GSH-Px in middle and high dosages of gallic acid groups were lower than that in control group,but there were also no significant differences between various groups（P〉0.05）.The activities of GSH-Px were also reduced with the increasing of gallic acid dosage.There were no significant differences of the activities of CAT,SOD,and GSH-Px in A549 cells between low,middle,and high dosages of gallic acid groups（P〉0.05）.Conclusion：Gallic acid can inhibit the proliferation of lung cancer cells by oxidative damages,and the Fas/FasL signal pathway might be one of the mechanisms for apoptosis-induced by gallic acid.
出处
《吉林大学学报（医学版）》
CAS
CSCD
北大核心
2016年第6期1092-1098,I0013,共8页
Journal of Jilin University：Medicine Edition
基金
吉林省教育厅科研项目资助课题(2015403)
吉林省吉林市科技局科研项目资助课题(201536113)
