以本產芸香萃取之肉桂芸香鹼作為緩解e抗原陰性B型肝炎病毒材料之研究

Harmine Purified from Indigenous Rue Neutralizes Proliferation of a Pre-core Mutant, HBeAg-negative Hepatitis B Virus In Vitro

10.6763/IJTCMHP.202311_7(2).0001

劉全喜(Liu, Chuan-Shi)；周淑玲(Chou, Shuling)；黃琳(Lin, Huang)；李明駿(Li, Ming-Jun)；盧世乾(Lu, Shih-Chien)；金立德(Chin, Li-Te)

本產芸香 ； 肉桂芸香鹼 ； e抗原陰性B型肝炎病毒 ； HepaRG細胞株 ； Indigenous rue ； Harmine ； HBeAg^- hepatitis B virus ； HepaRG cell line

中醫本草健康促進期刊

7卷2期（2023 / 11 / 15）

5 - 27

繁體中文;英文

慢性B型肝炎屬於全球嚴重的健康問題之一，特別慢性肝炎病患若帶有如e抗原陰性B型肝炎病毒等抗藥性病毒株，更成為B型肝炎防治的重大難題。尤其e抗原陰性慢性B型肝炎的盛行率一直在增加，可能反映了現有預防治療措施有其局限性，新的手段已刻不容緩。本研究透過特殊培養條件分化人類HepaRG細胞株後用於體外感染並擴增e抗原陰性B型肝炎病毒進行研究，結果發現HepaRG細胞感染B型肝炎病毒後出現慢性B型肝炎的肝細胞減少並脹大的病理特徵；序列分析證實所建構的B型肝炎病毒體外培養模型所擴增之e抗原陰性病毒屬於B基因型。使用此模型對以常見病毒藥物INFα2a、IL-1β及本產芸香萃取之肉桂芸香鹼進行敏感性測試，發現INFα2a僅產生微弱之抑制功效；IL-1β及本產芸香萃取之肉桂芸香鹼則能抑制近五成的B型肝炎病毒。故推論本產芸香萃取之肉桂芸香鹼（harmine）具有可用於治療慢性B型肝炎的潛力。

Chronic hepatitis B infection represents one of the serious health problems around the world. In particular, chronic hepatitis patients who carry drug-resistant viral strains such as HBe-antigen-negative (HBeAg^-) hepatitis B virus present a major hurdle in the prevention and/or treatment of the disease. As the prevalence of HBeAg^- chronic hepatitis B is increasing, which may be resulted from limitations of existing preventive and therapeutic measures, novel modalities are urgently needed. By using human HepaRG cell line, the present study differentiated the cells under special culture conditions and exploited them in an in vitro system to amplify HBeAg^- hepatitis B virus. After infection, number of cells with hepatic phenotype decreased and morphological swelling appearance developed characterizing the pathological changes. Furthermore, sequence analysis confirmed that the HBeAg^- virus amplified by the model belongs to the B genotype. In addition, drug sensitivity tests were performed with conventional anti-viral agents such as INF α2a, IL-1β, and harmine extracted from the indigenous rue. It was found that INF α2a had only a trivial inhibitory effect whereas IL-1β and harmine can inhibit nearly 50% of virus production. In conclusion, harmine has the potential to be studied and used to treat chronic hepatitis B.

1. Liu, C-S,Sun, L-T,Lai, Y-R(2022).Combination of natural ingredients based on indigenous rue simultaneously alleviates antimicrobial resistant (amr) bacteria and avian coronavirus.Int J TCM Health Pro,6,5-24.
   連結：
2. (1993).The History of Taiwan Agriculture.Taipei, Taiwan:Department of Cultural Publishing, Independent Evening News.
3. Alexopoulou, A,Karayiannis, P(2014).HBeAg negative variants and their role in the natural history of chronic hepatitis B virus infection.World J Gastroenterol,20,7644-7652.
4. Barbosa-Júnior, FV,Sanchez-Lermen, RLP,Vieira, KA(2015).Detection of hepatitis B activity in HBeAg-negative carriers with normal aminotransferase levels in central Brazil.Ann Hepatol,4,470-476.
5. Benzekri, R,Bouslama, L,Papetti, A(2018).Anti HSV-2 activity of Peganum harmala (L.) and isolation of the active compound.Microb. Pathog.,114,291-298.
6. Bonino, F,Hoyer, B,Nelson, J,Engle, R,Verme, G,Gerin, J(1981).Hepatitis B virus DNA inthe sera of HBsAg carriers: a marker of active hepatitis B virus replication in theliver.Hepatol,1,386-391.
7. Buti, M,Rodriguez-Frias, F,Jardi, R(2005).Hepatitis B virus genome variability and disease progression: the impact of pre-core mutants and HBV genotypes.J Clin Virol,34,S79-S82.
8. Chen, CJ,Yang, HI(2011).Natural history of chronic hepatitis B REVEALed.J. Gastroenterol. Hepatol,26,628-638.
9. Chen, D,Su, A,Fu, Y(2015).Harmine blocks herpes simplex virus infection through downregulating cellular NF-κB and MAPK pathways induced by oxidative stress.Antiviral Res,123,27-38.
10. Chen, D,Tian, X,Zou, X,Xu, S(2018).Harmine, a small molecule derived from natural sources, inhibits enterovirus 71 replication by targeting NF-κB pathway.Int Immunopharmacol,60,111-120.
11. Chin, L-T,Liu, K-W,Chen, Y-H(2021).Cell-based assays and molecular simulation reveal that the anti-cancer harmine is a specific matrix metalloproteinase-3 (MMP-3) inhibitor.Comput Biol Chem,94,107556.
12. Chu, CJ,Hussain, M,Lok, AS(2002).Hepatitis B virus genotype B is associated with earlier HBeAg seroconversion compared with hepatitis B virus genotype C.Gastroenterol,122,1756-1762.
13. Cornberg, M,Protzer, U,Petersen, J(2011).Prophylaxis, diagnosis and therapy of hepatitis B virus infection - the German guideline.Z Gastroenterol,49,871-930.
14. Domingo, E(2003).Quasispecies and the development of new antiviral strategies.Prog Drug Res,60,133-158.
15. Fernandez, M,Quiroga, JA,Carreno, V(2003).Hepatitis B virus downregulates the human interferon-inducible MxA promoter through direct interaction of precore/core proteins.J Gen Virol,84,2073-2082.
16. Ganem, D,Prince, AM(2004).Hepatitis B virus infection-natural history and clinical consequences.N Engl J Med,350,1118-1129.
17. Garson, JA,Grant, PR,Ayliffe, U(2005).Real-time PCR quantitation of hepatitis B virus DNA using automated sample preparation and murine cytomegalovirus internal control.J Virol Methods,126,207-213.
18. Hadziyannis, SJ,Lieberman, HM,Karvountzis, GG,Shafritz, DA(1983).Analysis of liver disease, nuclear HBcAg, viral replication, and hepatitis B virus DNA in liver and serum of HBeAg Vs. anti-HBe positive carriers of hepatitis B virus.Hepatol,3,656-662.
19. Hayes, CN,Chayama, K(2016).HBV culture and infectious systems.Hepatol Int,10,559-566.
20. Hudson, J,Graham, E,Towers, G(1986).Antiviral effect of harmine, a photoactive β‐carboline alkaloid.Photochem Photobiol,43,21-26.
21. Hutterer, C,Milbradt, J,Hamilton, S(2017).Inhibitors of dual-specificity tyrosine phosphorylation-regulated kinases (DYRK) exert a strong anti-herpesviral activity.Antiviral Res,143,113-121.
22. Isorce, N,Testoni, B,Locatelli, M(2016).Antiviral activity of various interferons and pro-inflammatory cytokines in non-transformed cultured hepatocytes infected with hepatitis B virus.Antiviral Res,130,36-45.
23. Jiang, X,Zou, J,Zhuang, Y(2017).The antiviral effects of harmine against BoHV-1 infection in vitro.Lett. Drug Des Discov,14,1303-1307.
24. Kao, J-H(2003).Hepatitis B virus genotypes and hepatocellular carcinoma in Taiwan.Intervirology,46,400-407.
25. Kumar, R(2022).Review on hepatitis B virus precore/core promoter mutations and their correlation with genotypes and liver disease severity.World J Hepatol,14,708-718.
26. Lee, H-w,Park, Y-k,Choi, Y(2020).Overview of anti-Hepatitis B virus agents.J Bacteriol Virol,50,141-149.
27. Liaw, YF,Jia, J-D,Chan, HLY(2011).Shorter durations and lower doses of peginterferon alfa-2a are associated with inferior hepatitis B e antigen seroconversion rates in hepatitis B virus genotypes B or C.Hepatol,54,1591-1599.
28. Liaw, YF,Sung, JJ,Chow, WC(2004).Lamivudine for patients with chronic hepatitis B and advanced liver disease.N Engl J Med,351,1521-1531.
29. Liu, K-W,Sun, L-T,Chin, L-T(2020).Establishment of a rue plant breeding system for harmine by using collected seeds and micropropagation.J Taiwan Agric Res,69,326-334.
30. Lok, AS,Heathcote, EJ,Hoofnagle, JH(2001).Management of hepatitis B: 2000-summary of a workshop.Gastroenterol,120,1828-1853.
31. Lok, AS,Zoulim, F,Locarnini, S(2007).Antiviral drug-resistant HBV: standardization of nomenclature and assays and recommendations for management.Hepatol,46,254-265.
32. Naito, H,Hayashi, S,Abe, K(2001).Rapid and specific genotyping system for hepatitis B virus corresponding to six major genotypes by PCR using type-specific primers.J Clin Microbiol,39,362-364.
33. Perrillo, R(2009).Benefits and risks of interferon therapy for hepatitis B.Hepatol,49,S103-S111.
34. Sarin, SK,Kumar, M,Lau, GK(2016).Asian-Pacific clinical practice guidelines on the management of hepatitis B: a 2015 update.Hepatol Int,10,1-98.
35. Sherlock, S,Thomas, HC(1983).Hepatitis B virus infection: the impact of molecular biology.Hepatol,3,455-456.
36. Shimakawa, Y,Veillon, P,Birguel, J,Pivert, A,Sauvage, V,Le Guillou-Guillemette, H,Roger, S,Njouom, R,Ducancelle, A,Amta, P(2022).Residual risk of mother-to-child transmission of hepatitis B virus infection despite timely birth-dose vaccination in Cameroon (ANRS 12303): a single-centre, longitudinal observational study.Lancet Glob Health,10,e521-e529.
37. Tacke, F,Kroy, DC(2016).Treatment for hepatitis B in patients with drug resistance.Ann Transl Med,4,334.
38. Tacke, F,Shirvani-Dastgerdi, E(2012).Impact of drug-resistance polymerase mutations on the replication of HBeAg-positive and HBeAg-negative hepatitis B virus strains in vitro.Hepat Mon,12,357-360.
39. Terrault, NA,Bzowej, NH,Chang, K-M(2016).ASLD guidelines for treatment of chronic hepatitis B.Hepatol,63,261-283.
40. Tolosa, L,Gómez-Lechón, MJ,Jiménez, N(2016).Advantageous use of HepaRG cells for the screening and mechanistic study of drug-induced steatosis.Toxicol. Appl Pharmacol,302,1-9.
41. Wang, Y-C,Yang, S-S,Su, C-W(2016).Predictors of response to pegylated interferon in chronic hepatitis B: A real-world hospital-based analysis.Sci Rep,6,29605.
42. Yum, JS,Ahn, BC,Jo, HJ(2012).Use of pre-S protein-containing hepatitis B virus surface antigens and a powerful adjuvant to develop an immune therapy for chronic hepatitis B virus infection.Clin Vaccine Immunol,19,120-127.