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目的观察清热解毒中药毒热平、痰热清注射液对流感病毒亚甲型鼠肺适应株(FM1)感染人胚肾上皮细胞株HEK-293T细胞中报告基因NF-κB相对转录活性以及靶基因肿瘤坏死因子(TNF-α)mRNA的影响,探讨清热解毒中药抗流感病毒FM1的作用机制。方法采用四唑盐(MTT)比色法体外检测毒热平、痰热清及利巴韦林注射液对HEK-293T细胞增殖的影响。FM1感染HEK-293T细胞后,利用双荧光素酶顺式报告系统,以利巴韦林对照,检测不同药物组中报告基因NF-κB-Luc相对荧光素酶活性;RT-PCR方法检测各组细胞中TNF-αmRNA水平。结果MTT结果显示,毒热平、痰热清、利巴韦林各药物组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,流感病毒FM1组细胞NF-κB转录活性显著增高(P<0.01),与FM1病毒组相比,毒热平1、10、100 mg/L,痰热清1/300、1/200、1/100倍稀释药液,利巴韦林50 mg/L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,FM1病毒组的TNF-αmRNA表达明显增高(P<0.05),各药物组与FM1病毒组相比,TNF-αmRNA表达量明显降低(P<0.01)。结论毒热平、痰热清治疗流感病毒的作用机制之一,可能是通过下调转录因子NF-κB的转录活性,从而影响下游靶基因TNF-α的表达,减少肺损伤和相应炎症反应。
Objective To observe the effects of TDP and Tanreqing Injection on the relative transcriptional activity of NF-κB and NF-κB in human embryo kidney cell line HEK-293T infected with influenza virus (FM1) Tumor necrosis factor (TNF-α) mRNA, to explore the mechanism of Qingrejiedu Chinese anti-influenza virus FM1. Methods MTT colorimetric method was used to detect the effect of TDP, Tanreqing and Ribavirin on the proliferation of HEK-293T cells. FM1 was transfected into HEK-293T cells, and the relative luciferase activity of NF-κB-Luc in different drug groups was tested by ribavirin control using dual luciferase cis-reporter system. RT-PCR was used to detect the expression of NF- TNF-α mRNA levels in cells. Results The results of MTT showed that the drug levels of TDP, Tanreqing and Ribavirin did not affect the normal proliferation of cells (P> 0.05). Compared with the control group, the NF-κB transcriptional activity of influenza virus FM1 group was significantly higher than that of the control group (P <0.01). Compared with FM1 group, the level of NF-κB 1, 10, 100 mg / L and phlegm Heat-clearing 1 / 300,1 / 200,1 / 100 times diluted solution, ribavirin 50 mg / L can down-regulate NF-κB transcriptional activity (P <0.01). The results of RT-PCR showed that compared with control group, the expression of TNF-αmRNA in FM1 group was significantly increased (P <0.05), and the expression of TNF-αmRNA in FM1 group was significantly lower than that in FM1 group (P <0.01) . Conclusion One of mechanisms of action of TDP and Tanreqing in treating influenza virus may be through down-regulating the transcriptional activity of NF-κB, thereby affecting the expression of downstream target gene TNF-α and decreasing lung injury and corresponding inflammatory reaction.