剑叶耳草多糖对乙型肝炎病毒的抑制作用及其机制研究

The Inhibitory Effects of Polysaccharides from Hedyotis Caudatifolia on Hepatitis B Virus

目的:观察剑叶耳草多糖对乙型肝炎病毒的抑制作用,并初步探究其分子机制。方法:(1)以乙型肝炎病毒转染的HepG2.2.15细胞为体外细胞模型,采用MTT法检测剑叶耳草多糖对HepG2.2.15细胞存活率的影响,在无毒浓度(50、100、200 mg/L)下作用HepG2.2.15细胞,并设置阳性药物恩替卡韦组(1 mg/L)和模型对照组,使用ELISA法测定上清液HBs Ag、HBe Ag的滴度,使用荧光定量PCR法测定上清液HBV DNA的含量。(2)颈静脉注射强阳性HBV DNA鸭血清建立广西麻鸭乙型肝炎模型,随机分为剑叶耳草多糖200、400、800 mg/kg、恩替卡韦1 mg/kg组和模型对照组,持续给药14天,鸭颈静脉采血,检测鸭血清中HBs Ag、HBe Ag及HBV DNA含量,HE染色观察肝损伤程度。(3)采用荧光定量PCR法检测HepG2.2.15细胞蛋白酪氨酸激酶1(JAK1)、信号传导及转录激活因子1(STAT1)、信号传导及转录激活因子2(STAT2)、干扰素刺激基因因子3(ISGF3)mRNA以及抗病毒蛋白寡聚腺营酸合成酶(OAS)和蛋白激酶R(PKR)mRNA表达水平。结果:(1)剑叶耳草多糖对HepG2.2.15细胞毒性较低。与模型对照组相比,50、100、200 mg/L剑叶耳草多糖能够明显抑制HepG2.2.15细胞分泌和表达HBV DNA,有效抑制HBs Ag和HBe Ag分泌。(2)与模型对照组广西麻鸭相比,剑叶耳草多糖200 mg/kg组给药后血清HBs Ag、HBe Ag及HBV DNA含量明显下降,肝病理损伤明显缓解。(3)100、200 mg/L剑叶耳草多糖能明显上调细胞因子JAK1、STAT1、STAT2、ISGF3 PKR mRNA以及抗病毒蛋白OAS的表达。结论:剑叶耳草多糖具有显著抑制乙型肝炎病毒的作用,其机制与激活JAK/STAT信号转导通路、促进抗病毒蛋白表达有关。

Objective:To investigate the inhibitory effect of polysaccharides from Hedyotis caudatifolia on hepatitis B virus(HBV),and its potential mechanisms.Methods:(1)The HepG 2.2.15 cells infected with HBV virus were served as the cell model and were randomly divided into the control group,the model group,the positive control entecavir group(1 mg/L)and Hedyotis caudatifolia polysaccharides groups(50,100,200 mg/L).MTT method was used to detect cells survival rate of HepG 2.2.15 cells after treatment.ELISA method was used to evaluate the levels of HBsA g,HBeA g in culture medium,fluorescent quantitative PCR method was used to determine the content of HBV DNA in culture medium.(2)Guangxi ducks were jugular injected with duck HBV DNA strong positive serum to establish the animal model of duck HBV.Model ducks were randomly divided into five groups:Hedyotis caudatifolia polysaccharides groups(200、400、800 mg/kg),the entecavir group(1 mg/kg/d)and the model group.All ducks were administered for 14 days.After administration,serum was collected to determine the levels of HBsA g,HBeA g and HBV DNA.The extent of liver injury was observed by HE staining.(3)Fluorescent quantitative PCR method was used to determine the mRNA expression levels of JAK1,STAT1,STAT2,ISGF3,OAS and PKR.Results:(1)The polysaccharides from Hedyotis caudatifolia showed the low cytotoxicity on HepG 2.2.15 cells.Compared with the model group,polysaccharides from Hedyotis caudatifolia(50,100,200 mg/L)significantly inhibited the secretion and expression of HBV DNA in HepG 2.2.15 cells,effectively inhibited the secretion of HBsA g and HBeA g.(2)Compared with the model group,the contents of HBsA g,HBeA g and HBV DNA in the serum were significantly decreased in 200 mg/kg Hedyotis caudatifolia polysaccharides group,and the liver lesions were significantly alleviated.(3)The polysaccharides from Hedyotis caudatifolia(100,200 mg/L)significantly inhibited the mRNA expression levels of cytokines JAK1,STAT1 STAT2 and ISGF3,as well as the mRNA expression of antiviral proteins OAS and PKR.Conclusion:The polysaccharides from Hedyotis lance can significantly inhibit HBV,its mechanism may be related to the activation of JAK/STAT signaling pathway and the promotion of antiviral protein expression.