基于TLR4-IRF3-IFN通路的清热利湿活血方治疗HBV转基因小鼠抗HBV作用机制研究

Anti-HBV mechanism of Qingrelishihuoxue Fang on HBV transgenic mice based on TLR4-IRF3-IFN pathway

目的:观察清热利湿活血方治疗HBV转基因小鼠抗乙肝病毒(HBV)及其机制作用。方法:HBV转基因小鼠随机分4组,苦参素组小鼠给予同体积含150mg/kg·d苦参素混悬液,清热利湿活血方组小鼠分别给予同体积含7、2g/kg·d混悬液,5周后,常规HE染色观察肝组织病理变化;ELISA法检测血清及肝组织中乙型肝炎病毒表面抗原(HBsAg);ELISA法检测血清干扰素α(IFNα)、干扰素β(IFNβ);定量PCR法检测血清中乙型肝炎病毒脱氧核糖核酸(HBVDNA)及肝组织中HBVDNA、Toll样受体4(TLR4)mRNA、干扰素调控因子3(IRF3)mRNA表达;Western bloting法检测肝组织TLR4、IRF3蛋白表达。结果:7g/kg·d清热利湿活血方可以减轻肝细胞轻度脂肪变性、肝细胞轻度水肿及Kuffer细胞增生等病理改变,可以显著降低HBV转基因小鼠肝脏和血清中的HBsAg、HBVDNA水平;清热利湿活血方(7g/kg·d)显著增强小鼠TLR4mRNA、IRF3mRNA及蛋白表达;清热利湿活血方(7g/kg·d)可显著升高血清IFNα、IFNβ。结论:清热利湿活血方具有抗HBV作用,其机制与激活TLR4-IRF3-IFN信号通路有关。

Objective： To observe the anti-HBV effect and the mechanism of Qingrelishihuoxue Fang in HBV transgenic mice. Methods： HBV transgenic mice were randomly divided into four groups,mice in Matrine group were fed the suspension containing 150 mg/kg · d Matrine,mice in high and low dose of Qingrelishihuoxue Fang groups were given the suspension containing 7 g/kg·d and 2 g/kg·d Qingrelishihuoxue Fang. After the treatment for five weeks,HE staining was used to observe pathological changes in liver tissues,ELISA was used to detect HBsAg in the serum and liver tissues,serum Interferon-α and Interferon-β,quantitative PCR was used to detect HBVDNA in serum,and HBVDNA、Toll-Like Receptor 4 mRNA、Interferon regulatory factor 3 mRNA in liver tissues. Western Bloting method was used to detect protein expressions of TLR4,IRF3 in liver tissues. Results： Qingrelishihuoxue Fang（7 g/kg·d） improved fatty degeneration of liver cells,liver cells edema and Kuffer cell hyperplasia. Qingrelishihuoxue Fang（7 g/kg·d） significantly reduced levels of HBsAg,HBVDNA in the serum and livers of HBV transgenic mice,differences between the drug and model groups were statistically significant（P〈0. 01,P〈0. 05）. Compared with the model group,Qingrelishihuoxue Fang（7 g/kg·d） significantly increased mRNA and protein expressions of TLR4 and IRF3,differences were statistically significant（P〈0. 01,P〈0. 05）. Qingrelishihuoxue Fang（7 g/kg·d） significantly increased levels of IFNα and IFNβ in the serum,differences were statistically significant（P〈0. 01,P〈0. 05）. Conclusion： Qingrelishihuoxue Fang has anti-HBV effect,the mechanism is related to activating TLR4-IRF3-IFN signaling pathway.