清肺通络方对肺炎支原体诱导A549细胞氧化应激损伤的影响及作用机制研究

Effect and mechanism of action of Qingfei Tongluo Formula on oxidative stress injury induced by Mycoplasma pneumoniae in A549 cells

目的探讨清肺通络方对肺炎支原体(MP)感染人类肺泡基底上皮细胞(A549细胞)后氧化应激损伤的影响及其可能的机制。方法取A549细胞分为空白组、MP感染组(MP浓度为1×10^(7)CFU·mL^(-1))、不同浓度清肺通络方组(清肺通络方浓度分别为0.5 mg·L^(-1)、1.0 mg·L^(-1)、2.0 mg·L^(-1)),于清肺通络方处理3 d时,以倒置显微镜观察各组细胞形态学改变,2',7'-二氯二氢荧光素二乙酸酯(DCFH-DA)荧光染色法检测不同组细胞内活性氧(ROS)含量,Western blot检测核转录因子E2相关因子2(Nrf2)、血红素加氧酶-1(HO-1)、胞浆伴侣分子(Keap1)、醌氧化还原酶(NQO1)表达水平。取A549细胞分为空白组、MP感染组、MP+siNC组、MP+清肺通络方组、MP+siNrf2组、MP+siNrf2+清肺通络方组,于清肺通络方处理3 d时进行细胞转染,Western blot检测Nrf2、Keap1、HO-1、NOQ1表达水平。结果适宜浓度的清肺通络方可上调MP感染后A549细胞内Nrf2、Keap1、HO-1和NOQ1蛋白表达,降低细胞内ROS水平,从而抑制细胞凋亡。抑制Nrf2表达后,清肺通络方的抗氧化效应降低。结论清肺通络方可减轻MP感染后A549细胞氧化损伤,其机制可能与调控Nrf2/抗氧化响应元件(ARE)信号通路有关。

Objective To investigate the effect and possible mechanism of Qingfei Tongluo Formula on oxidative stress injury after Mycoplasma pneumoniae(MP)infection of human alveolar basal epithelial cells(A549 cells).Methods A549 cells were divided into a blank group,an MP infected group(MP concentration was 1×10^(7)CFU·mL^(-1),and Qingfei Tongluo Formula groups at different concentrations(the concentrations of Qingfei Tongluo Formula were 0.5 mg·L^(-1),1.0 mg·L^(-1),2.0 mg·L^(-1),respectively).After the treatment of Qingfei Tongluo Formula for 3 days,the morphological changes of cells in each group were observed by inverted microscope,the contents of intracellular reactive oxygen species(ROS)were detected by 2',7'-dichlorofluorescein acetate(DCFH-DA)fluorescence staining,and the expression levels of nuclear transcription factor E2-related factor 2(Nrf2),heme oxygenase-1(HO-1),cytoplasmic chaperone molecule(Keap1)and quinone oxidoreductase(NQO1)were detected by Western blot.A549 cells were divided into a blank group,an MP infection group,an MP+siNC group,an MP+Qingfei Tongluo Formula group,an MP+siNrf2 group,and an MP+siNrf2+Qingfei Tongluo Formula group.The cells were transfected after being treated with Qingfei Tongluo Formula for 3 days,and the expression levels of Nrf2,Keap1,HO-1 and NOQ1 were detected by Western blot.Results A suitable concentration of Qingfei Tongluo Formula up-regulated the protein expression of Nrf2,Keap1,HO-1 and NOQ1 in A549 cells after MP infection and decreased the intracellular ROS level,which in turn inhibited cell apoptosis.After inhibiting the expression of Nrf2,the antioxidant effect of Qingfei Tongluo formula decreased.Conclusion Qingfei Tongluo Formula can attenuate oxidative injury in A549 cells after MP infection,and the mechanism may be related to the regulation of Nrf2/ARE signaling pathway.