基于TLR3补肾健脾方水提物抑制乙肝病毒复制机制研究  

Mechanism study on inhibition of hepatitis B virus replication by TLR3 based water extract of Bushen Jianpi Decoction

作　　者：朱晓骏[1] 郑超[1] 张鑫[1] 郑彦希 余卓[1] 孙学华[1] 高月求[1] ZHU Xiaojun;ZHENG Chao;ZHANG Xin;ZHENG Yanxi;YU Zhuo;SUN Xuehua;GAO Yueqiu(Hepatology Department,Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China)

机构地区：[1]上海中医药大学附属曙光医院肝病科,上海201203

出　　处：《上海中医药大学学报》2022年第S01期149-153,共5页Academic Journal of Shanghai University of Traditional Chinese Medicine

基　　金：国家自然科学基金资助项目(81673935,81774240,81774256,81904164,81973773);国家“艾滋病和病毒性肝炎等重大传染病防治”科技重大专项(2018ZX10725504);浦东新区中医联合体建设项目(PDZY-2019-0601);上海市中医药事业发展三年行动计划(ZY〔2018-2020〕-CCCX-2003-01);重大疑难疾病中西医临床协作试点项目(ZY〔2018-2020〕-FXTW-2004)

摘　　要：目的:通过pHBV1.3-pCR2.1质粒转染HepG2细胞探讨补肾健脾方水提物抑制乙肝病毒的免疫机制。方法:250μg/ml、500μg/ml补肾健脾方水提物干预pHBV1.3-pCR2.1质粒转染HepG2细胞,ELISA法检测细胞上清HBsAg、HBeAg含量;500μg/ml补肾健脾方水提物干预pHBV1.3-pCR2.1质粒转染HepG2细胞,Westen blot法检测Toll样受体3(TLR3)、p-IRF3蛋白表达,qRT-PCR法检测细胞内IFN-β mRNA表达;补肾健脾方干预HepG2细胞,pHBV1.3-pCR2.1质粒转染HepG2细胞,TLR13 siRNA下调TLR3表达,Westen blot法检测TLR3、p-IRF3蛋白表达,qRT-PCR法检测细胞内IFN-β mRNA表达。结果:500μg/ml补肾健脾方与空白对照组、250μg/ml补肾健脾方比较,HBsAg均明显下降(P<0.05,P<0.01);500μg/ml补肾健脾方与空白对照组、250μg/ml补肾健脾方比较,HBeAg均明显下降(P<0.05,P<0.01);补肾健脾方(500μg/ml)可以增强TLR3、p-IRF3蛋白表达,促进细胞内IFN-β mRNA表达(P<0.01);补肾健脾方干预pHBV1.3-pCR2.1质粒转染HepG2细胞,TLR3 siRNA抑制细胞TLR3蛋白表达,p-IRF3蛋白表达明显下降,同时细胞内IFN-β mRNA表达也明显下降(P<0.01)。结论:补肾健脾方水提物通过增强pHBV1.3-pCR2.1质粒转染HepG2细胞TLR3作用,促进细胞内IFN-β分泌,从而抑制乙型肝炎病毒的复制。Objective:To explore the immune mechanism on the water extract of Bushen Jianpi Decoction in inhibiting hepatitis B virus in HepG2 cells transfected with plasmid pHBV1.3-pCR2.1.Methods:After the water extract of Bushen Jianpi Decoction intervened in HepG2 cells transfected with plasmid pHBV1.3-pCR2.1 in two different concentrations(250μg/ml and 500μg/ml),HBsAg and HBeAg contents in cell supernatant were detected by ELISA.After the water extract of Bushen Jianpi Decoction intervened in HepG2 cells transfected with plasmid pHBV1.3-pCR2.1 in concentrations of 500μg/ml,the expressions of TRL3,p-IRF3 protein were detected by WB,and the expression of IFN-β mRNA was detected by qRT-PCR.Bushen Jianpi Decoction intervened in HepG2 cells led that HepG2 cells were transfected with plasmid pHBV1.3-pCR2.1 and TLR3 siRNA down regulated the expression of TLR3.The expressions of TLR3 and p-IRF3 protein were detected by Western blot,and the expression of IFN-β mRNA was detected by qRT-PCR.Results:HBsAg of Bushen Jianpi Decoction(500μg/ml)was significantly decreased compared with that in Bushen Jianpi Decoction(250μg/ml)and the model group(P<0.05,P<0.01).HBeAg of Bushen Jianpi Decoction(500μg/ml)was significantly decreased compared with that in Bushen Jianpi Decoction(250μg/ml)and the model group(P<0.05,P<0.01).Bushen Jianpi Decoction(500μg/ml)enhanced TLR3 and p-IRF3 protein expressions,and promoted IFN-β mRNA expression in cell(P<0.01).After Bushen Jianpi Decoction intervened in HepG2 cells transfected with plasmid pHBV1.3-pCR2.1,TLR3 siRNA inhibited TLR3 protein expression and p-IRF3 protein expression was decreased significantly and the expression of intracellular IFN-β mRNA expression was decreased significantly.Conclusion:The water extract of Bushen Jianpi Decoction can inhibit hepatitis B virus replication by enhancement of TLR3 effect in HepG2 cells transfected with plasmid pHBV1.3-pCR2.1 and promotion of intracellular IFN-β secretion.

关 键 词：补肾健脾方水提物 乙型肝炎病毒 TOLL样受体3 

分 类 号：R285[医药卫生—中药学]