山药多糖通过p38 MAPK信号通路协同核苷酸类似物抗乙型肝炎病毒的机制研究

Study on mechanism of Chinese Yam polysaccharide synergizing nucleoside analogues against hepatitis B virus through p38 MAPK signaling pathway

探讨山药多糖(Chinese Yam polysaccharide,CYPS)协同核苷酸类似物(nucleoside analogues,NAs)抗乙型肝炎病毒(HBV)的分子机制。将不同浓度的山药多糖和恩替卡韦加入HepG2.2.15细胞,采用cell counting kit-8(CCK-8)法检测细胞毒性,筛选2种药物抑制HepG2.2.15细胞的最佳浓度及时间,细胞分为对照组、山药多糖组、恩替卡韦组和联合用药(山药多糖+恩替卡韦)组,采用酶联免疫吸附法(ELISA)检测各组药物对细胞上清表面抗原(hepatitis B virus surface antigen,HBsAg)和e抗原(hepatitis B virus e antigen,HBeAg)分泌量的影响;采用聚合酶链式反应(probe quantitative real-time PCR,probe qRT-PCR)检测药物对HepG2.2.15细胞HBV-DNA的影响;采用蛋白免疫印迹法(Western blot)检测药物对HepG2.2.15细胞p38 MAPK、p-p38 MAPK、Na^(+)-牛磺胆酸共转运多肽(Na^(+)-taurocholic cotransport polypeptide,NTCP)蛋白表达的影响;采用实时荧光定量聚合酶链式反应(quantitative real-time PCR,qRT-PCR)检测药物对HepG2.2.15细胞中p38 MAPK、NTCP mRNA表达的影响。结果显示,与对照组相比,山药多糖组、恩替卡韦组和联合用药组中HBeAg、HBsAg浓度均下降(P<0.01或P<0.001),均可抑制HepG2.2.15细胞HBV-DNA(P<0.01),其中联合用药组抑制HepG2.2.15细胞的HBV-DNA更明显(P<0.001),且p-p38 MAPK、NTCP蛋白表达水平均显著降低(P<0.05或P<0.01),p38 MAPK mRNA表达水平上升,NTCP mRNA表达水平下降(P<0.05或P<0.01)。综上,山药多糖可能通过p38 MAPK信号通路降低NTCP蛋白和mRNA的表达协同恩替卡韦抗HBV。

This study explore the molecular mechanism of the synergistic effect of Chinese Yam polysaccharides and nucleoside analogues(NAs) on hepatitis B virus(HBV) resistance.Different concentrations of Chinese Yam polysaccharide and entecavir were ad-ded to HepG2.2.15 cells.After the cytotoxicity was detected by cell counting kit-8(CCK-8),the optimal concentration and time of the two drugs to inhibit HepG2.2.15 cells were screened out.They were divided into control group,Chinese Yam polysaccharide group,entecavir group and combination drug group(Chinese Yam polysaccharide + entecavir).The drugs were added to HepG2.2.15 cells,ELISA was used to detect the effects of each group of drugs on the secretion of hepatitis B virus surface antigen(HBsAg) and hepatitis B virus e antigen(HBeAg) in cell supernatant,probe quantitative real-time PCR(probe qRT-PCR) was used to detect the effects of drugs on HBV-DNA in HepG2.2.15 cells,and Western blot was used to detect the effects of each group of drugs on the expression of p38 MAPK,p-p38 MAPK,NTCP proteins in HepG2.2.15 cells.The qRT-PCR was used to detect the effect of drugs on the expression of p38 MAPK and NTCP mRNA in HepG2.2.15 cells.The results showed that compared with control group,the concentrations of HBeAg and HBsAg in Chinese Yam polysaccharide group,entecavir group and combination group decreased(P<0.01 or P<0.001),and both of them inhibited HBV-DNA in HepG2.2.15 cells(P<0.01),and the HBV-DNA inhibition of HepG2.2.15 cells in the combination group was more obvious(P<0.001),and the protein expression levels of p-p38 MAPK and NTCP were significantly decreased(P<0.05 or P<0.01),the mRNA expression level of p38 MAPK increased,and the mRNA expression level of NTCP decreased(P<0.05 or P<0.01).To sum up,Chinese Yam polysaccharide can reduce the expression of NTCP protein and mRNA through p38 MAPK signaling pathway and cooperate with entecavir in anti-HBV.