| Objective:This study aims to compare the anti-HBV activities of matrine type alkaloids including oxymatrine, matrine, sophocarpine and sophoridine, and the effect of their respective combination with entecavir on the HBV cells with anti-drug-resistance activity has also been compared, finally the mechanism of oxymatrine combining with entecavir to impact the anti-drug-resistance HBV cells has been further discussed. This study is about to provide the pharmacology basis for the resolution of drug-resistant HBV and the treatment of patients with chronic hepatitis B in clinics.Methods:1. The anti-HBV effects of oxymatrine, matrine, sophocarpine and sophoridine. Cell line HepG2.2.15, the acknowledged hepatitis B virus cell model, was applied to investigate the cytotoxicity of four alkaloids above. ELISA was used to test the secretion of HBsAg and HBeAg at 24 and 72h after drug administration. Real-time PCR was used to detect the copy number of the intracellular HBV and the one in supernatant liquor.2. Metabolomics study of sophoridine. UHPLC-Q-TOFMS/MS was applied to detect the endogenous metabolites after drug administration. PCA, PLS-DA and one-way variance were used to analyze were used to analyze the alteration of endogenous metabolites relative to anti-virus activity and screening characteristic biomarker for the exploration of the potential mechanism of anti-virus activity of sophoridine in vitro.3. The therapeutic effect of the combination of oxymatrine, matrine, sophocarpine and sophoridine with entecavir. Cell strain HepG2.A64 that was established solely by 302 hospital was used as the model. CPE and CCK-8 were used to test the cytotoxicity of oxymatrine, matrine, sophocarpine, sophoridine, entecavir and combination of alkaloids with entecavir respectively. ELISA and Real-time PCR were used to test the therapeutic effect of combination on HBsAg secretion of HepG2.A64 and HBV DNA copy.4. The mechanism of combination of oxymatrine and entecavir. HepG2.A64 was set as cell model and Real-time PCR was used to detect the influence of combination on p38 and NTCP mRNA expression. Western blot was applied to test the effect on p38 and p-p38 protein expression.Results:1. The therapeutic effects of oxymatrine, matrine, sophocarpine and sophoridine. No cytotoxicity was observed in 4 alkaloids under 400?g·mL-1?and lamivudine under 200?g·mL-1. So 100?g·mL-1,200?g·mL-1 and 400?g·mL-1 were selected for alkaloids while 100?g·mL-1 and 200?g·mL-1 were selected for lamivudine. 72h after administration, sophoridine at 100?g·mL-1 and 200?g·mL-1 had a stronger resisting effect on HBsAg and intracellular HBV DNA than lamivudine ?P<0.05?.2. Metabolomics study of sophoridine.34 biomarkers including C16 sphinganine, phytosphingosine, jervine and Cycloleucine were identified by analyzing the alteration of intracellular metabolites in HepG2.2.15 after drug administration. Compared with control group, the content of phytosphingosine in sophoridine group was lower, indicating that sophoridine might exert it anti-HBV activity by down-regulating the phosphorylation of p38.3. The therapeutic effect of the combination of oxymatrine, matrine, sophocarpine and sophoridine with entecavir. 72h after administration, the inhibitive ratio of combination of oxymatrine and entecavir at 100?g·mL-1 and 200?g·mL-1 were 27.58% and 57.23%, which was obviously higher than that of the single use. And the inhibitive ratio of the combination of oxymatrine and entecavir to HBV DNA was 76.35% ?P?0.05?.4. The mechanism of combination of oxymatrine and entecavir. Compared with control group and single use group, the mRNA and protein expression of p38 in combination group were both higher, while p-p38 expression was down-regulated. Besides, mRNA and protein expression of NTCP were down-regulated, which verified the results of metabolomics.Conclusions:Sophoridine exhibited the activity of anti-HBV. And the combination of oxymatrine and entecavir could improve the therapeutic effect on drug-resistant HBV. And the mechanism of this effect was speculated to be the influence on NTCP and p38 phosphorylation. |
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