益气固表丸对慢性阻塞性肺疾病细胞模型线粒体中SIRT5表达的影响

Effect of pills for replenishi Qi and securing the external on the expression of SIRT5 in mitochondria of COPD cell models

目的探讨益气固表丸对慢性阻塞性肺疾病(COPD)支气管上皮样细胞线粒体中SIRT5表达的影响及对线粒体功能的调节作用。方法使用烟草提取物(CSE)刺激人支气管上皮样细胞16HBE建立COPD模型。实验设6组:正常组取人支气管上皮样细胞16HBE常规培养;COPD组取建模细胞常规培养;COPD+siRNA空转组取建模细胞,siRNA空转后培养;COPD+siRNA SIRT5组取建模细胞,siRNA转染后培养;COPD+益气固表丸组取建模细胞,加入益气固表丸含药血清培养;COPD+siRNA SIRT5+益气固表丸组取建模细胞,siRNA转染后加入益气固表丸含药血清培养。通过siRNA转染、RT-qPCR法筛选SIRT5最佳敲降靶序,显微镜下观察各组细胞形态和线粒体膜电位变化,流式细胞仪检测各组细胞线粒体膜电位及活性氧(ROS)含量,ELISA法测定各组细胞中ATP含量,RT-qPCR法检测各组细胞中SIRT5 mRNA相对表达量。结果与正常组相比,COPD各组细胞皱缩变圆,细胞密度低,细胞状态差;线粒体膜电位、ATP含量及SIRT5 mRNA相对表达量均明显降低(P均<0.05),ROS含量明显升高(P<0.05)。与COPD组比较,COPD+siRNA SIRT5组细胞状态更差,线粒体膜电位、ATP含量及SIRT5 mRNA相对表达量均明显降低(P均<0.05),ROS含量明显升高(P<0.05);与COPD组和COPD+siRNA SIRT5组比较,COPD+益气固表丸组和COPD+siRNA SIRT5+益气固表丸组细胞状态明显改善,线粒体膜电位、ATP含量及SIRT5 mRNA相对表达量均明显升高(P均<0.05),ROS含量均明显降低(P均<0.05)。结论益气固表丸能够通过调控CES诱导16HBE细胞线粒体中SIRT5的表达,从而改善线粒体功能障碍,起到治疗COPD的作用。

Objective It is to investigate the effect of pills for replenishi Qi and securing the external(PRQSE)on the expression of SIRT5 in mitochondria of bronchial epithelioid cells and its regulatory effect on mitochondrial function in chronic obstructive pulmonary disease(COPD).Methods The human bronchial epithelial-like cells 16HBE were stimulate by cigarette smoke extract(CSE)to establish COPD models.6 groups were set up in the experiment:the normal group in which the human bronchial epithelial-like cells 16HBE were normal cultured,the COPD group in which the modeled cells were normal cultured,the COPD+siRNA in which the modeled cells were cultured after siRNA sham-transfection,the COPD+siRNA SIRT5 group in which the modeled cells were cultured after siRNA transfection,the COPD+PRQSE group in which the modeled cells were cultured with medicated serum containing PRQSE,the COPD+siRNA SIRT5+PRQSE group in which the modeled cells were cultured with medicated serum containing PRQSE after siRNA transfection.The best SIRT5 knockdown sequence was screened by siRNA transfection and RT-qPCR,the changes of cell morphology and mitochondrial membrane potential in each group were observed under the microscope,the mitochondrial membrane potential and reactive oxygen species(ROS)content of cells in each group were detected by flow cytometry,the ATP content of cells in each group was determined by ELISA,and the relative expression of SIRT5 mRNA in cells of each group was detected by RT-qPCR.Results Compared with the normal group,the cells in each group of COPD were wrinkled and rounded,with low cell density and poor cellular status;the mitochondrial membrane potential,ATP content and relative expression of SIRT5 mRNA were significantly lower(all P<0.05),and the ROS content was significantly higher(P<0.05).Compared with the COPD group,the cellular status was worse in the COPD+siRNA SIRT5 group,and the mitochondrial membrane potential,ATP content and relative expression of SIRT5 mRNA were significantly lower(all P<0.05),and the ROS content was significantly higher(P<0.05);compared with the COPD group and COPD+siRNA SIRT5 group,the cellular status was significantly improved in the COPD+PRQSE group and COPD+siRNA SIRT5+PRQSE group,the mitochondrial membrane potential,ATP content and relative expression of SIRT5 mRNA were all significantly higher(all P<0.05),and the ROS content was significantly lower(P<0.05).Conclusion Pills for replenishi Qi and securing the external can improve mitochondrial dysfunction via regulating the expression of SIRT5 in mitochondria of 16HBE cells induced by CES,thus to play a role in the treatment of COPD.