内质网应激途径在百草枯诱导人肺Ⅱ型上皮样细胞A549凋亡过程中作用

The role of endoplasmic reticulum stress pathway in paraquat-induced apoptosis of human lung Type Ⅱ alveolar epithelial like cells A549

目的探讨百草枯(Paraquat,PQ)诱导人肺Ⅱ型上皮样细胞A549凋亡过程中内质网应激途径的发生机制。方法体外培养A549细胞,以PQ为处理因素,建立PQ诱导A549细胞发生ERS的模型:对照组(等量PBS)、PQ250μM组、PQ500μM组、PQ750μM组、PQ1000μM组。处理24h后利用MTT法测定细胞生存率,应用光学倒置显微镜和透射电镜观察细胞形态学变化,采用Annexin V-FITC/PI双染法检测细胞凋亡比例,在筛选出的凋亡率最高的PQ500μM组中,采用分光光度法测定Caspase-3的活化程度,并提取总蛋白,采用Western blotting试验方法检测内质网应激(ERS)通路相关蛋白GRP78、PERK、p-PERK、ATF6、c-ATF6、IRE1α、pIRE1α和CHOP的表达变化。结果 PQ处理细胞24h后,随PQ浓度增高,A549细胞生存率逐渐下降,细胞形态发生显著变化。在PQ(500μM)组,细胞凋亡率达到最高,Caspase-3活性显著增加,GRP78和CHOP蛋白表达量随诱导A549细胞时间延长显著增加,p-PERK,c-ATF6和p-IRE1α的表达量在6h时显著增加。结论 PQ可以通过激活内质网应激途径引发人肺Ⅱ型上皮样细胞A549凋亡。

Objective To study the role and mechanism of endoplasmic reticulum stress （ ERS ） pathway during Paraquat （ PQ ） -induced apoptosis of human type II alveolar epithelial like cells A549. Methods PQ-induced A549 cells apoptosis model： control group（equal PBS）, PQ 250IX M group, PQSOOIX M group, PQ750 IX M group and PQ100OIXM group were established. The cell survival rate was determined by MTI＇, the morphological changes were observed under optical inversion microscope and transmission electron microscope. The percentage of apoptosis was detected by Annexin V-FITC/PI double staining. The content of Caspase-3 was determined by spectrophotometric method, the protein expression of GRP78, PERK, p-PERK, ATF6, c-ATF6, IRE1 α and p-IRE1 α were detected by Western blotting. Results The viability of the cells decreased gradually with the increase of PQ concentration 24h after PQ-inducement, with the significant damaged cells morphology. The apoptotic rate of A549 cells reached the maximum. In PQ （500 μM） group, the activity of Caspase-3 was significantly increased and the expression level of GRP78 and CHOP proteins increased with time dependence, while the expression levels of p-PERK, c-ATF6 and p-IRE1 α were significantly increased at 6 hours. Conclusion The inducement of the apoptosis of human alveolar type II epithelial cells A549 by PQ might be related to activating the ERS pathway.