清肺口服液通过ERK1/2通路调控RSV所致呼吸道炎症损伤的机制  

Mechanism of Qingfei Oral Liquid to Regulate Respiratory Inflammatory Damage Caused by RSV via ERK1/2 Pathway

作　　者：邹亚[1] 郭盛 景晓平[1] 何丽[1] 

机构地区：[1]上海交通大学附属儿童医院上海市儿童医院

出　　处：《中国实验方剂学杂志》2018年第2期86-91,共6页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金面上项目(81674020);上海交通大学“科技创新专项资金”多学科交叉项目(YG2014MS13)

摘　　要：目的：通过研究清肺口服液对呼吸道合胞病毒（respiratory syncytial virus，RSV）感染后气道上皮细胞胞外信号调节激酶（extracellular signal-regulated kinases，ERKl／2）磷酸化蛋白及相关炎症因子表达的影响，探讨清肺口服液抗RSV感染及其继发炎症损伤的调节机制。方法：RSV体外感染人支气管上皮细胞（human bronchial epithelial cells，16．HBE）建立气道上皮炎症损伤模型，设置空白组，模型组（RSV），利巴韦林组（利巴韦林），清肺口服液高、中、低质量浓度组，共6组。其中空白组给予含2％FBS的RPIM1640培养基进行培养，其余各组在空白组的基础上造模，模型组加入等量RSV（MOI=1），利巴韦林组加入等量利巴韦林注射液（0．2mg·L^-1），清肺口服液组给予清肺口服液高、中、低质量浓度（200，100，50mg·L^-1）进行干预。应用噻唑蓝（tetrazolium salt colorimetry，MTT）比色法检测清肺口服液对RSV感染16-HBE的毒性作用，实时荧光定量聚合酶链式反应（Real-timePCR）检测清肺口服液对细胞中RSV复制的影响，并用蛋白免疫印迹法（Westernblot）检验ERKl／2信号通路相关蛋白磷酸化表达变化，同时用酶联免疫吸附法（enzyme linked immunosorbentassay，ELISA）测定细胞上清中白细胞介素_6（interleukin-6，IL-6），白细胞介素．8（interleukin-8，IL-8）水平。结果：与空白组比较，模型组RSV大量增殖，16-HBE出现典型的炎症损伤表现，ERKl／2磷酸化蛋白水平显著提高（P〈0．01），同时细胞上清中释放大量的炎症因子IL-6，IL-8（P〈0．01）。加入清肺口服液或利巴韦林后，16-HBE中的RSV复制水平显著降低（P〈0．01），且清肺口服液（200，100mg·L^-1）可显著下调ERKl／2磷酸化蛋白的表达（P〈0．01），并显著降低细胞上清中炎症因子IL-6，IL-8的表达水平（P〈0．01）。结论：清肺口服液能够抑制RSV病毒复制Objective： By studying the impact on extracellular signal-regulated kinases （ERK1/2） phosphorylated protein and the related inflammatory factor in respiratory epithelial cells with respiratory syncytial virus （RSV） infection, this paper aims to investigate the anti-RSV effect of Qingfei oral liquid and its regulatory mechanism on secondary inflammatory damages. Method： RSV-infected human bronchial epithelial cells （16- HBE） were used to establish respiratory epithelial inflammatory damage models in vitro. The blank group, model group （RSV）, control group （ribavirin） and treatment groups （Qingfei oral liquid with high, middle and low dose）. The blank group received RPIM 1640 medium containing 2% FBS, while other groups were infected with RSV to make models on the basis of blank group. The model group received equal volume of RSV （MOI = 1 ） ; the control group received equal volume of Ribavirin （0.2 mg.L-1 ） ; and the treatment groups were intervened with Qingfei oral liquid （200, 100, 50 mg＇L-~）. Tetrazolium salt colorimetry （MTT） was used to detect the toxicity effect of Qingfei oral liquid on RSV-infected 16-HBE. Real-time PCR was used to detect the replication of RSV in 16-HBE, and Western blot was used to examine the changes in phosphorylation of ERK1/2 signaling pathway related proteins. In addition, enzyme linked immunosorbent assay （ELISA） was used to evaluate the levels of interleukin-6 （IL-6） and interleukin-8 （IL-8） in the supernatant. Result： As compared with blank group, RSV showed great proliferation in the model group; 16-HBE showed typical manifestation of inflammatory damages; ERK1/2 phosphorylated protein level was increased significantly （P 〈 0.01 ）, and a large amount of inflammatory factors （IL-6, IL-8） were released in the cellular supernatant （P 〈 0.01 ）. After adding Qingfei oral liquid or ribavirin, the replication of RSV was significantly inhibited in 16-HBE （P 〈 0.01）, and the Qingfei oral liquid （200, 100

关 键 词：清肺口服液 呼吸道合胞病毒 细胞外信号调节蛋白激酶 白细胞介素-6 白细胞介素-8 

分 类 号：R22[医药卫生—中医基础理论] R24[医药卫生—中医学]