甘草内生菌联合顺铂对A549细胞的增殖及凋亡的影响

Effect of Glycyrrhiza uralensis Fisch. endophytes combined with cisplatin on proliferation and apoptosis of A549 cells

摘要:

目的 探讨甘草内生菌JTZB55联合顺铂对人肺癌A549细胞增殖和凋亡的影响及其可能机制。方法 MTT法检测JTZB55或/和顺铂对A549细胞存活率的影响，根据协同指数筛选出后续实验浓度，将A549细胞分为正常对照组、2 μg/mL顺铂组、800 μg/mL JTZB55组、2 μg/mL顺铂+800 μg/mL JTZB55组；流式细胞术检测各组药物对A549细胞凋亡的影响；Western blot法检测各组药物处理后细胞线粒体凋亡及内质网应激途径相关蛋白的表达水平。结果 MTT结果显示，顺铂及JTZB55作用于A549细胞后，细胞存活率显著降低；与顺铂组相比，联合组细胞存活率显著降低（P<0.05），且800 μg/mL JTZB55与2 μg/mL顺铂联用的协同指数最高；流式细胞术结果显示，JTZB55及顺铂均能促进A549细胞凋亡（P<0.01），两药联合组细胞凋亡率显著增高（P<0.01）；Western blot实验结果表明，顺铂联合JTZB55能显著降低B细胞淋巴瘤/白血病-2蛋白（B cell lymphoma/leukemia-2，Bcl-2）蛋白的表达，上调Bcl-2相关X蛋白（Bcl-2 associated X，Bax）、细胞色素C（Cytochrome C，CytC）、半胱氨酸天冬氨酸蛋白酶-3（cysteine aspartate protease-3，Caspase-3）、半胱氨酸天冬氨酸蛋白酶-8（cysteine aspartate protease-8，Caspase-8）、半胱氨酸天冬氨酸蛋白酶-9（cysteine aspartate protease-9，Caspase-9）、葡萄糖调节反应蛋白78（Glucose regulated protein 78，GRP78）、CCAAT-增强子结合蛋白同源蛋白（CCAAT/enhancer-binding protein-homologus protein，CHOP）蛋白的表达（P<0.05）。结论 JTZB55可以增强顺铂对A549细胞的增殖抑制作用，促进A549细胞的凋亡，其机制可能与下调Bcl-2和上调Bax、CytC、Caspase-3、Caspase-8、Caspase-9、GRP78、CHOP蛋白的表达相关。

Abstract:

Objective To investigate the effect of Glycyrrhiza uralensis Fisch. endophyte JTZB55 combined with cisplatin on proliferation and apoptosis of human lung cancer A549 cells and the possible mechanism.Methods MTT assay was used to determine the effect of JTZB55 and/or cisplatin on the survival rate of A549 cells. According to the synergistic index，the subsequent experimental concentration was screened out，and A549 cells were divided into normal control group，2 μg/mL cisplatin group，800 μg/mL JTZB55 group，and 2 μg/mL cisplatin + 800 μg/mL JTZB55 group. Flow cytometry was used to determine the effect of drugs in each group on the apoptosis of A549 cells. Western blot was used to measure the expression levels of mitochondrial apoptosis and endoplasmic reticulum stress pathway-related proteins in cells treated with drugs in each group.Results MTT results showed that the survival rate of A549 cells was decreased significantly after cisplatin and JTZB55 were administrated. Compared with the cisplatin group，the cell survival rate in the combination group was significantly decreased（P<0.05），and the synergistic index of 800 μg/mL JTZB55 combined with 2 μg/mL cisplatin was the highest. The results of flow cytometry showed that both JTZB55 and cisplatin could promote the apoptosis of A549 cells（P<0.01），and the apoptosis rate of A549 cells was significantly increased in the combination group（P<0.01）. Western blot results showed that cisplatin combined with JTZB55 could significantly decrease the expression of B cell lymphoma/leukemia-2（Bcl-2） protein and up-regulate the expression of Bcl-2 associated X（Bax），cytochrome C（CytC），cysteine aspartate protease-3（Caspase-3），cysteine aspartate protease-8（Caspase-8），cysteine aspartate protease-9（Caspase-9），glucose regulated protein 78（GRP78），and CCAAT/enhancer-binding protein-homologous protein（CHOP）（P<0.05）.Conclusion JTZB55 can enhance the inhibitory effect of cisplatin on the proliferation of A549 cells and promote the apoptosis of A549 cells. The mechanism may be related to the down-regulation of Bcl-2 and up-regulation of Bax，CytC，Caspase-3，Caspase-8，Caspase-9，GRP78，and CHOP.