清热利湿活血方对HBV转基因小鼠的抗病毒作用及对TKB1-IRF3表达的影响

Antiviral Effect of Heat-clearing,Dampness-draining and Blood-activating Recipe on HBV Transgenic Mice and Its Influence on Hepatic TKB1-IRF3 Expression

【目的】观察清热利湿活血方对乙型肝炎病毒(HBV)转基因小鼠的抗病毒作用及对肝组织TANK结合激酶1(TKB1)、干扰素调控因子3(IRF3)表达的影响,以阐明其作用机制。【方法】首先建立清热利湿活血方的HPLC指纹图谱。再将HBV转基因小鼠随机分为4组,即模型组,苦参素组,清热利湿活血方高、低剂量组,每组8只。苦参素组小鼠给予灌胃剂量为0.15 g·kg^(-1)·d^(-1)的苦参素混悬液,清热利湿活血方高、低剂量组小鼠分别给予灌胃剂量为7、2 g·kg^(-1)·d^(-1)的清热利湿活血方混悬液,给药共5周。治疗结束后,采用酶链免疫吸附法(ELISA)检测血清及肝组织乙型肝炎病毒表面抗原(HBsAg),采用常规苏木素—伊红(HE)染色观察肝组织病理变化,采用定量聚合酶链反应(qPCR)法检测血清中HBV DNA及肝组织中HBV DNA、TKB1 mRNA、IRF3 mRNA表达,采用蛋白免疫印迹(Western blot)法检测肝组织TKB1、IRF3的蛋白表达。【结果】标定14个特征峰构成清热利湿活血方的指纹图谱,其中1号峰为没食子酸,8号峰为柯里拉京,9号峰为虎杖苷,10号峰为鞣花酸,13号峰为甘草酸,14号峰为齐墩果酸。高剂量清热利湿活血方可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,显著降低HBV转基因小鼠肝脏和血清中的HBsAg、HBV DNA水平(P<0.05或P<0.01),显著增强小鼠TKB1、IRF3的mRNA及蛋白表达水平(P<0.05或P<0.01)。【结论】清热利湿活血方有抗HBV作用,其抗病毒机制可能与增强TKB1、IRF7 mRNA及蛋白表达,进而影响干扰素分泌有关。

Objective To observe the anti-virus effect of Heat-clearing,Dampness-draining and Blood-activatingRecipe（HDBR）on hepatitis B virus（HBV）transgenic mice and its influence on hepatic TANK-binding kinase 1（TKB1）,interferon regulatory factor 3（IRF3）expression,so as to reveal its therapeutic mechanism. Methods Firstly,We set up a HPLC fingerprint for HDBR. Secondly,HBV transgenic mice were randomly divided into 4 groups,namely model group,matrine group,and high-and low-dose HDBR groups,8 mice in each group.Matrine group was given introgastric administration of 0.15 g·kg^-1·d^-1 of matrine suspension,and high-and low-dose HDBR groups were given intragastric administration of 7,2 g·kg^-1·d^-1 of HDBR suspension,the treatmentlasting 5 weeks. After treatment,enzyme-linked immunosorbent assay（ELISA） was used to detect HBV surfaceantigen（HBs Ag）in the serum and liver tissue,hematoxylin eosin（HE）staining was used to observe pathologicalchanges of the liver tissue,quantitative polymerase chain reaction（q PCR）method was used to detect serum HBVDNA, hepatic HBV DNA, TKB1 m RNA and IRF3 m RNA, and Western blotting method was used to detectprotein expression of TKB1 and IRF3 in liver tissue. Results The 14 calibrated peaks constituted the characteristicfingerprints of HDBR, of which the 1 stpeak was gallic acid, the 8 thpeak was corilagin, the 9 thpeak waspolydatin,the 10 thpeak was ellagic acid,the 13 thpeak was glycyrrhizin,and the 14 thpeak was oleanolic acid.High-dose HDBR reduced fatty degeneration and edema in liver cells as well as the hyperplasia of Kuffer cells,significantly reduced HBs Ag and HBV DNA levels in the serum and liver of HBV transgenic mice（P〈0.05 or P〈0.01）,and increased mRNA and protein expression levels of TKB1 and IRF3（P〈0.05 or P〈0.01）. Conclusion HDBR has anti-HBV effect, and its therapeutic mechanism may be related to increasing m RNA and proteinexpression levels of TKB1 and IRF3,which results into the regulation of interferon secretion.