Application of Immunocompetent HBV cccDNA Mouse Model in the Efficacy Evaluation of Matrine and Artemisinin on Inhibiting HBV

doi:10.12300/j.issn.1674-5817.2021.016

Abstract

Abstract: Objective To evaluate the effects of matrine and artemisinin on immunocompetent hepatitis B virus (HBV) infected mouse model, and to lay a foundation for the application of the model. Methods HBV covalently closed circular DNA (HBV cccDNA) was synthesized in vitro. Male CBA/CaJ mice aged 8 to 10 weeks were injected with HBV cccDNA at high pressure through tail vein, and then intraperitoneally injected with matrine or artemisinin in acute or chronic infection periods, respectively. Quantitative PCR was used to detect HBV DNA and cccDNA in serum or liver tissues of mice. Enzyme-linked immunoadsorption assay (ELISA) was used to measure the content of HBV surface antigen (HBsAg) in serum. Immunohistochemistry was used to determine HBsAg and HBV core antigen (HBcAg) in liver tissues. Results In HBV acute infection period, matrine and artemisinin injection at week 4 significantly inhibited the secretion of HBsAg in serum of mice (both P<0.05), and reduced the expressions of HBV DNA (both P<0.01). In HBV chronic infection period, matrine and artemisinin had no significant inhibitory effects on HBsAg in serum and liver tissues (all P>0.05), but the decrease of HBsAg in serum in the group administered with matrine or artemisinin was significantly higher than that in the normal saline control group (both P<0.05). Moreover, matrine decreased the copy number of HBV DNA in serum and liver tissues (P<0.05 and P<0.01, respectively) and the copy number of HBV cccDNA in liver tissues (P<0.05). Artemisinin only inhibited the copy number of HBV DNA in serum of mice (P<0.01), but had no significant effect on HBV DNA and HBV cccDNA in liver tissues (both P>0.05). In addition, matrine had no significant effect on HBcAg in liver tissues (P>0.05), but artemisinin did the opposite (P<0.05). Conclusion The immunocompetent HBV cccDNA mouse model can be used to evaluate the efficacy of drugs for the treatment of acute and chronic hepatitis B.

Key words: Hepatitis B virus, Chronic infection, Efficacy evaluation, Matrine, Artemisinin, CBA/CaJ mice

CLC Number:

R-332
Q95-33

WU Yue, LÜ Xiaoqin, LIU Yang, XIANG Xia, ZHAO Zhonghua, XU Ruqing, PAN Shuohan, HE Mingzhong, ZHANG Huatang, LAI Guoqi. Application of Immunocompetent HBV cccDNA Mouse Model in the Efficacy Evaluation of Matrine and Artemisinin on Inhibiting HBV[J]. Laboratory Animal and Comparative Medicine, 2021, 41(2): 108-115.

References

[1] JIA L Y, GAO Y N, HE Y W, et al.HBV induced hepatocellular carcinoma and related potential immunotherapy[J]. Pharmacol Res, 2020, 159:104992. DOI:10.1016/j.phrs.2020.104992.
[2] European Association for the Study of the Liver. EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection[J]. J Hepatol, 2017, 67(2): 370-398. DOI:10.1016/j.jhep.2017.03.021.
[3] LIU J, LIANG W N, JING W Z, et al.Countdown to 2030: eliminating hepatitis B disease, China[J]. Bull World Health Organ, 2019, 97(3):230-238. DOI:10.2471/BLT.18.219469.
[4] SMITH S, HARMANCI H, HUTIN Y, et al.Global progress on the elimination of viral hepatitis as a major public health threat: an analysis of WHO Member State responses 2017[J]. JHEP Rep, 2019, 1(2):81-89. DOI:10.1016/j.jhepr.2019.04.002.
[5] CHEN Z C, ENGLE R E, SHEN C H, et al.Distinct disease features in chimpanzees infected with a precore HBV mutant associated with acute liver failure in humans[J]. PLoS Pathog, 2020, 16(8):e1008793. DOI:10.1371/journal.ppat.1008793.
[6] 王冠蕾, 富大智. 苦参碱与恩替卡韦联合介入治疗乙肝肝癌的临床疗效分析[J]. 贵州医药, 2018, 42(4):420-422. DOI:10.3969/j.issn.1000-744X.2018.04.013.
[7] CHOU H H, CHIEN W H, WU L L, et al.Age-related immune clearance of hepatitis B virus infection requires the establishment of gut microbiota[J]. Proc Natl Acad Sci USA, 2015, 112(7):2175-2180. DOI:10.1073/pnas.1424775112.
[8] CAO M, ZHAO Z H, TANG Y W, et al.A new hepatitis B virus e antigen-negative strain gene used as a reference sequence in an animal model[J]. Biochem Biophys Res Commun, 2018, 496(2):502-507. DOI:10.1016/j.bbrc.2018.01.081.
[9] LIU Y, ZHAO Z H, LV X Q, et al. Precise analysis of the effect of basal core promoter/precore mutations on the main phenotype of chronic hepatitis B in mouse models[J/OL]. J Med Virol (2020-05-19) [2021-01-02]. https://doi.org/10.1002/jmv.26025.
[10] WANG L, CAO M, WEI Q L, et al.A new model mimicking persistent HBV e antigen-negative infection using covalently closed circular DNA in immunocompetent mice[J]. PLoS One, 2017, 12(4):e0175992. DOI:10.1371/journal.pone.0175992.
[11] ROMERO M R, EFFERTH T, SERRANO M A, et al.Effect of artemisinin/artesunate as inhibitors of hepatitis B virus production in an “in vitro” replicative system[J]. Antiviral Res, 2005, 68(2):75-83. DOI:10.1016/j.antiviral.2005.07.005.
[12] SHI J F, HAN X, WANG J F, et al.Matrine prevents the early development of hepatocellular carcinoma like lesions in rat liver[J]. Exp Ther Med, 2019, 18(4):2583-2591. DOI:10.3892/etm.2019.7875.
[13] GONG X B, GAO Y, GUO G Q, et al.Effect of matrine on primary human hepatocytes in vitro[J]. Cytotechnology, 2015, 67(2):255-265. DOI:10.1007/s10616-013-9680-1.
[14] BLAZQUEZ A G, FERNANDEZ-DOLON M, SANCHEZ-VICENTE L, et al.Novel artemisinin derivatives with potential usefulness against liver/colon cancer and viral hepatitis[J]. Bioorg Med Chem, 2013, 21(14):4432-4441. DOI:10.1016/j.bmc.2013.04.059.
[15] 唐超, 马军伟, 刘辉, 等. 恩替卡韦联合苦参碱对慢性乙型肝炎的疗效及对炎症因子水平的影响[J]. 西北药学杂志, 2019, 34(5):673-676.
[16] 孔李远, 孔维环, 赵莉. 苦参碱联合恩替卡韦对慢性乙肝患者肝纤维化的影响[J]. 中国实用医刊, 2020, 47(8):108-110. DOI:10.3760/cma.j.cn115689-20191218-09088.
[17] 陈俊平. 苦参素注射液联合恩替卡韦治疗乙肝肝硬化患者的疗效评价[J]. 内蒙古医学杂志, 2020, 52(9):1095-1096. DOI:10.16096/J.cnki.nmgyxzz.2020.52.09.049.
[18] 王贵强, 王福生, 庄辉, 等. 慢性乙型肝炎防治指南(2019年版)[J]. 中国病毒病杂志, 2020, 10(1):1-25.DOI:10.16505/j.2095-0136.2019.0097.
[19] INDOLFI G, EASTERBROOK P, DUSHEIKO G, et al.Hepatitis B virus infection in children and adolescents[J]. Lancet Gastroenterol Hepatol, 2019, 4(6):466-476. DOI:10.1016/S2468-1253(19)30042-1.
[20] POLLICINO T, CAMINITI G.HBV-integration studies in the clinic: role in the natural history of infection[J]. Viruses, 2021, 13(3):368. DOI:10.3390/v13030368.
[21] YANG H C, KAO J H.Persistence of hepatitis B virus covalently closed circular DNA in hepatocytes: molecular mechanisms and clinical significance[J]. Emerg Microbes Infect, 2014, 3(9): e64. DOI:10.1038/emi.2014.64.
[22] AHMED M, WANG F, LEVIN A, et al.Targeting the Achilles heel of the hepatitis B virus: a review of current treatments against covalently closed circular DNA[J]. Drug Discov Today, 2015, 20(5):548-561. DOI:10.1016/j.drudis.2015.01.008.
[23] LAM W Y, LEUNG K T, LAW P T, et al.Antiviral effect of Phyllanthus Nanus ethanolic extract against hepatitis B virus (HBV) by expression microarray analysis[J]. J Cell Biochem, 2006, 97(4):795-812. DOI:10.1002/jcb.20611.
[24] CUI X, WANG Y, KOKUDO N, et al.Traditional Chinese medicine and related active compounds against hepatitis B virus infection[J]. Biosci Trends, 2010, 4(2):39-47.