毒热平注射液对甲型流感病毒H3N2体外感染细胞中TLR7信号通路的影响

Effects of Influenza virus H3N2 on TLR7 signal pathyway and intervention of Dureping Injection in vitro

目的:观察甲型流感病毒H3N2感染人胚肾上皮细胞(HEK-293T)细胞及人肺腺癌上皮细胞(A549)细胞后,对NF-κB转录活性以及TLR7、TNF-α、IL-8、IFN-βmRNA的影响,探讨H3N2对体外感染细胞中TLR7信号通路的影响及清热解毒中药毒热平注射液的干预作用。方法:采用MTT法体外检测各组药物对293T、A549细胞增殖的影响;H3N2感染293T细胞后,利用双荧光素酶报告系统,以利巴韦林、清开灵注射液为药物对照,检测毒热平注射液各浓度组细胞中NF-κB相对荧光素酶活性;H3N2感染A549细胞后,RT-PCR方法检测各组细胞中TLR7、TNF-α、IL-8、IFN-βmRNA水平。结果:MTT结果显示,毒热平、利巴韦林、清开灵注射液各浓度不影响细胞正常增殖(P>0.05)。报告基因结果显示,与细胞对照组相比,H3N2感染组细胞NF-κB转录活性显著增高(P<0.01);与H3N2感染组相比,利巴韦林0.5μg/mL,清开灵1/128稀释组,毒热平1μg/mL,10μg/mL,100μg/mL组均可不同程度下调NF-κB的转录活性(P<0.01)。RT-PCR结果显示,与细胞对照组比较,H3N2病毒组的TLR7、TNF-α、IL-8、IFN-βmRNA表达明显增高(P<0.01);与H3N2感染组相比,一定浓度的药物组TLR7、TNF-α、IL-8、IFN-βmRNA表达量明显降低(P<0.05或P<0.01)。结论:流感病毒H3N2攻击细胞可以显著上调TLR7受体的表达和核因子NF-κB转录活性,并引起下游相关靶基因的表达增多。毒热平注射液可以下调H3N2引起的TLR7信号通路的激活,抑制NF-κB的转录活性,减少下游炎性因子TNF-α、IL-8、IFN-β的表达。关键词:H3N2;毒热平注射液;NF-κB;双荧光素酶报告系统;TLR7;

Objective： To observe the relative transcriptional activity of reporter gene NF-κB in HEK-293T cells and expression of TLR7,TNF- α ,IL-8,IFN- β mRNA in A549 ceils infected by influenza virus H3N2 and intervention of Dureping injection in different concentrations, to find the effects of influenza virus H3N2 on TLR7 signal pathyway and intervention of Dureping Injection in vitro. Methods： To detect the cell proliferation of 293T cells and A549 cells exposed to Dureping, Ribavirin and Qingkailing injections in different concentrations by MTT; The relative luciferas of NF-κB in virus-infected 293T cells was detected by Dual-Luciferase cis-Reporting Systems; The expression of TLR7,TNF- α ,IL-8,IFN- β mRNA in virus-infected A549 cells were detected by RT-PCR. Results： Result of MTT showed that there was no significant difference between control cells and the ceils exposed to drugs （P〉0.05）. Compared with control cells, the relative luciferase activity of NF-κB in virus-infected cells was apparently up-regulated （P〈0.01）. Compared with the virus-infected cells, the relative luciferase activity of NF-κB in virus- infected cells treated by three injections in different concentrations was apparently down-regulated （P〈0.01）. And the expression of TLR7, TNF- α ,IL-8,IFN- β mRNA was apparently attenuated （P〈0.01）. Conclusion： Influenza virus H3N2 can up-regulate the TLR7 signal pathyway in vitro. Dureping injections can inhibit the inflammatory reaction induced by influenza virus H3N2 by down-regulating the TLR7 signal pathyway.