沉默NOX4对百草枯诱导肺泡Ⅱ型上皮细胞氧化应激和凋亡的影响

NOX4 silence regulates oxidative stress and cell apoptosis in pulmonary alveolar type Ⅱ epithelial cells exposure to paraquat

目的通过siRNA沉默人肺泡Ⅱ型上皮样A549细胞中NOX4基因,探讨NOX4在百草枯诱导肺泡上皮细胞凋亡中的作用。方法根据GeneBank中NOX4全长cDNA序列,选取特异性序列为干扰作用的靶点,构建NOX4 siRNA表达载体,脂质体介导转染A549细胞,筛选出最佳干扰siRNA。实验设置正常对照组、PQ组、PQ+转染组及转染对照组。正常对照组仅给予培养基处理24 h,PQ组加入百草枯(700μM)处理24 h,PQ+转染组给予NOX4 siRNA转染细胞48 h后,用含百草枯(700μM)的DMEM培养液培养24 h,转染对照组仅加入NOX4 siRNA。结果与正常对照组相比,PQ组A549细胞活力下降,细胞凋亡率显著升高,NOX4表达明显增加,MDA含量显著增加,ROS水平明显升高;与正常对照组相比,转染对照组仅见NOX4表达显著降低;而与PQ组相比,PQ+转染组细胞活力上升,细胞凋亡率显著减低,NOX4表达明显降低,MDA含量显著减少,ROS水平亦显著下降。结论降低NOX4表达可抑制PQ诱导的A549细胞氧化应激和凋亡。这一结果可能为肺纤维化提供了新的治疗靶点。

Objective To explore the role of non-phagocytic oxidase 4(NOX4)in paraquat(PQ)induced oxidative stress and apoptosis in pulmonary alveolar type Ⅱ epithelial cells(A549).Methods NOX4 specific small interfering RNA(NOX4-siRNA)expression vectors were constructed according to its full-length cDNA sequence from GeneBank and transfected into A549 cells by lipofectamine to identify the optimal siRNA.A549 cells were divided into:control group,PQ group,PQ+NOX4 siRNA group and NOX4 siRNA group.Control group was pretreated with culture medium for 24 h.PQ group was cultured with paraquat(700μM)for 24 h.PQ+NOX4 siRNA group was transfected with NOX4 siRNA for 48 h and then cultured with paraquat(700μM)for 24 h.NOX4 siRNA group was transfected with NOX4 siRNA only.Results Paraquat exposure significantly reduced cell viability,promoted apoptotic rate and increased MDA and ROS production in the PQ group compared to those in the control group.Only NOX4 protein expression was reduced in the NOX4 siRNA group compared to that in the control group.The cell viability was notably increased,while the apoptotic rate,NOX4 expression and MDA and ROS production were significantly decreased in the PQ+NOX4 siRNA group compared to those in the PQ group.Results Compared with control group,NOX4 siRNA significantly inhibited the expression of NOX4 protein.PQ exposure could notably induce the cell apoptosis,MDA and ROS production in A549 cells.And NOX4 siRNA significantly reduced the apoptosis ratio,MDA and ROS level as compared with PQ group.Conclusion Reduction of NOX4 expression can inhibit PQ-induced cell apoptosis and oxidative stress in A549 cells,which may be a new target for therapy of pulmonary fibrosis.