疏风宣肺方和解表清里方体外干预甲型流感病毒H1N1诱导炎性细胞因子分泌作用的研究

摘要

Objective To investigate the effects of Shufeng -xuanfei and Jiebiao -qingli formulae on in-flammatory cytokines induced by virus H1N1 in human pulmonary carcinoma cell A549.Methods Human pulmona-ry epithelial cells A549 were cultured and infected by influenza virus H1N1,and were divided into cell control group , H1N1 -infected group,oseltamivir group,Shufeng -xuanfei group and Jiebiao -qingli group.The mRNA and protein expression of IL -1,TNF -α,IL -6,IL -10,MCP -1 and RANTES were detected by DNA microarray ,real -time PCR and western -blotting.Results Gene microarray showed that,as compared with cell control group ,the expres-sions of Il1β,Tnf,Ccl5,Il10,Il6,Ccl2 were obviously up -regulated in H1N1 -infected group.Compared with H1N1-infected group,the expressions of Il1β,Tnf,Ccl5,Il10,Il6,Ccl2 were obviously down -regulated in oseltamivir group, Shufeng -qingre group and Jiebiao -qingli group.RT -PCR showed that,as compared with cell control group,the mRNA expressions of IL -1,TNF -α,IL -6,IL -10,MCP -1 and RANTES were signifi-cantly increased in H1N1 -infected group (P <0.01). Compared with H1N1 -infected group,the mRNA ex-pressions of IL -1,TNF -α,IL -6,IL -10 and MCP -1 were significantly decreased in Shufeng -xuanfei group (P<0.01,P <0.05),and the mRNA expressions of IL -1、TNF -、IL -6、MCP -1 in Jiebiao -qingli group were sig-nificantly decreased (P <0.01,P <0.05).Western blotting showed that ,the levels of L -1,TNF -α,IL -6,IL -10,MCP -1 and RANTES in H1N1 -infected group were obviously increased as compared with those in cell control group (P <0.05).The levels of IL -1,TNF -α,IL -6,IL -10,MCP -1 and RANTES in Shufeng -qingre and Jiebiao -qingli group were decreased as compared with those in H 1N1 -infected group (P <0.05,P <0.01).Con-clusion Shufeng -xuanfei formula and Jiebiao -qingli formula can down -regulate the over -expressions of IL -1, TNF -α,IL -6,MCP -1 and RANTES mRNA and protein,reducing inflammation,restoring stability and balance of body's immune function.%目的：研究疏风宣肺方和解表清里方对甲型流感病毒H1 N1感染的人肺腺癌上皮细胞（A549）中炎性细胞因子的影响。方法培养 A549，甲型流感病毒 H1N1感染 A549后，分为细胞对照组、H1V1感染组、奥司他韦对照组、疏风宣肺组及解表清里组。采用实时定量聚合酶链式反应（RT －PCR）和Western blotting 法检测各组细胞中炎症相关的白细胞介素（IL）1、肿瘤坏死因子α（TNF －α）、IL －6、IL －10、单核细胞趋化蛋白1（MCP －1）、调节活化正常 T 细胞表达和分泌的趋化因子（RANTES）的 mRNA 及蛋白表达的变化。结果基因芯片结果提示，与细胞对照组比较，H1N1感染组差异表达基因 Il1β、Thf、Ccl5、Il10、Il6、Ccl2明显上调。与 H1N1感染组比较，奥司他韦组、疏风宣肺组和解表清里组差异基因 Thf、Il1β、Ccl5、Il10、Il6、Ccl2表达显著下调。 RT －PCR 结果显示，与细胞对照组比较，H1N1感染组 IL －1、TNF －α、IL －6、IL－10、MCP －1、RANTES 的 mRNA 表达均显著升高（P ＜0．01）。与 H1N1感染组比较，疏风宣肺组 IL －1、TNF－α、IL －6、IL －10、MCP －1的 mRNA 表达均明显降低（P ＜0．01，P ＜0．05），解表清里组 IL －1、TNF －α、IL －6、MCP －1的 mRNA 表达均明显降低（P ＜0．01，P ＜0．05）。 Western blotting 结果显示，H1N1感染组 IL －1、TNF －α、IL －6、IL －10、MCP －1、RANTES 蛋白表达较细胞对照组显著升高（P ＜0．05）；与 H1N1感染组比较，疏风宣肺组及解表清里组 IL －1、TNF －α、IL －6、IL －10、MCP －1、RANTES 蛋白表达均显著降低（P ＜0．05，P＜0．01）。结论疏风宣肺方和解表清里方均可抑制流感病毒感染后炎性细胞因子 IL －1、TNF －α、IL －6、MCP －1的 mRNA 过表达及蛋白分泌，减轻炎症反应，并恢复机体免疫功能的稳定和平衡。