Dopamine Inhibits the Expression of Hepatitis B Virus Surface and e Antigens by Activating the JAK/STAT Pathway and Upregulating Interferon-stimulated Gene 15 Expression

Abstract

Background and aims: Hepatitis B virus (HBV) infection is a major risk factor for cirrhosis and liver cancer, and its treatment continues to be difficult. We previously demonstrated that a dopamine analog inhibited the packaging of pregenomic RNA into capsids. The present study aimed to determine the effect of dopamine on the expressions of hepatitis B virus surface and e antigens (HBsAg and HBeAg, respectively) and to elucidate the underlying mechanism.

Methods: We used dopamine-treated HBV-infected HepG2.2.15 and NTCP-G2 cells to monitor HBsAg and HBeAg expression levels. We analyzed interferon-stimulated gene 15 (ISG15) expression in dopamine-treated cells. We knocked down ISG15 and then monitored HBsAg and HBeAg expression levels. We analyzed the expression of Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway factors in dopamine-treated cells. We used dopamine hydrochloride-treated adeno-associated virus/HBV-infected mouse model to evaluate HBV DNA, HBsAg, and HBeAg expression. HBV virus was collected from HepAD38.7 cell culture medium.

Results: Dopamine inhibited HBsAg and HBeAg expression and upregulated ISG15 expression in HepG2.2.15 and HepG2-NTCP cell lines. ISG15 knockdown increased HBsAg and HBeAg expression in HepG2.2.15 cells. Dopamine-treated cells activated the JAK/STAT pathway, which upregulated ISG15 expression. In the adeno-associated virus-HBV murine infection model, dopamine downregulated HBsAg and HBeAg expression and activated the JAK-STAT/ISG15 axis.

Conclusions: Dopamine inhibits the expression of HBsAg and HBeAg by activating the JAK/STAT pathway and upregulating ISG15 expression.

Keywords: Dopamine; HBV e antigen; HBV surface antigen; HepAD38 cell line; HepG2 cell line; HepG2.2.15 cell line; Hepatitis B virus; Human NTCP-expressing; JAK-STAT/ISG15 axis.

Fig. 1. Dopamine inhibits hepatitis B virus (HBV) amplification in a stable HBV-producing cell line.

(A) HepG2.2.15 cell viability following dopamine treatment for 12 days. (B, C) Hepatitis B virus surface and e antigen (HBsAg and HBeAg, respectively) levels after dopamine and entecavir (ETV, 20 nM, control) treatment, measured by enzyme-linked immunosorbent assay. (D, E) HBsAg and HBeAg expression following ETV (20 nM) and/or dopamine (125 µM) treatment. (F, G) Core- and extracellular particle-associated HBV DNA levels following dopamine and/or ETV treatment, determined by quantitative polymerase chain reaction. Data are presented as mean values with standard deviations relative to the control from at least three independent experiments. *p<0.05, **p<0.01, ***p<0.005 ****p<0.001. OD, optical density; DMSO, dimethyl sulfoxide; ETV, entecavir.

Fig. 2. Dopamine inhibits hepatitis B virus (HBV) amplification in an HBV infection system.

(A) Sodium taurocholate co-transporting polypeptide-expressing HepG2 (NTCP-G2) cell viability following dopamine treatment for 9 days. (B, C) Hepatitis B virus surface and e antigen (HBsAg and HBeAg, respectively) levels in NTCP-G2 cells infected with supernatant from HepAD38 cells and treated with dopamine or entecavir (ETV; 20 nM), measured by enzyme-linked immunosorbent assay. (D, E) HBsAg and HBeAg expression following ETV (20 nM) and/or dopamine (125 µM) treatment. (F, G) Core- and extracellular particle-associated HBV DNA levels in HB-infected NTCP-G2 cells following dopamine and/or ETV treatment, determined by quantitative polymerase chain reaction. Data are presented as mean values with standard deviations relative to the control from at least three independent experiments. *p<0.05, **p<0.01, ***p<0.005. OD, optical density; DMSO, dimethyl sulfoxide; Mock, uninfected negative control; ETV, entecavir.

Fig. 3. Dopamine does not inhibit covalently closed circular DNA (cccDNA) formation.

cccDNA, packaged pgRNA and HBV total RNA expression in (A, C and E) HepG2.2.15 cells and (B, D and F) HBV-infected sodium taurocholate co-transporting polypeptide-expressing HepG2 (NTCP-G2) cells following dopamine treatment for 12 and 9 days, respectively, as determined by quantitative polymerase chain reaction. Data are presented as mean values with standard deviations relative to the control from at least three independent experiments. *p<0.05. DMSO, dimethyl sulfoxide; HBV, hepatitis B virus.

Fig. 4. Dopamine regulated interferon-stimulated gene 15 (ISG15) expression.

(A) Volcano plots of differential gene expression between dimethyl sulfoxide (DMSO; control)- and dopamine-treated HepG2.2.15 cells. (B) Expression profiles of DMSO- and dopamine-treated HepG2.2.15 cells, determined by RNA sequencing. (C, D) ISG15 protein expression in HepG2.2.15 (left) and sodium taurocholate co-transporting polypeptide-expressing HepG2 (NTCP-G2, right) cells, determined by western blotting. Data are presented as mean values with standard deviations relative to the control from at least three independent experiments. ***p<0.005, ****p<0.001. FDR, false discovery rate; FC, fold change; DMSO, dimethyl sulfoxide.

Fig. 5. Interferon-stimulates gene 15 (ISG15) regulated hepatitis B virus surface and e antigen (HBsAg and HBeAg) expression.

(A, B) Cell culture and treatment in the knockdown and rescue experiments. (C, D) ISG15 protein expression in HepG2.2.15 cells with two ISG15-knockdown clones with (+) or without (−), determined by western blotting. (E, F) HBsAg and HBeAg expression in the ISG15-knockdown clones, determined by enzyme-linked immunosorbent assay (ELISA). (G, H) ISG15 gene and protein expression in the rescue experiment, in HepG2.2.15 cells treated siISG15-1 with (+) or without (−), dopamine with (+) or without (−), siControl with (+) or without (−), determined by western blotting. (I, J) HBsAg and HBeAg expression in the rescue experiment, in HepG2.2.15 cells treated siISG15-1 with (+) or without (−), dopamine with (+) or without (−), siControl with (+) or without (−), determined by ELISA. Data are presented as mean values with standard deviations relative to the control from at least three independent experiments. *p<0.05, **p<0.01, ***p<0.005, ****p<0.001. siRNA, small interfering RNA; OD, optical density.

Fig. 6. Dopamine upregulates the expression of Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway molecules.

STAT1, phosphorylated (p)-STAT1 (A-D), JAK1, and phosphorylated (p)-JAK1 (E- H) protein expressions in HepG2.2.15 cells treated with dopamine or dimethyl sulfoxide (DMSO, control) for 12 days, determined by western blotting. Data are presented as mean values with standard deviations relative to the control from at least three independent experiments. *p<0.05, **p<0.01. DMSO, dimethyl sulfoxide

Fig. 7. ISG15 activation with JAK-STAT pathway in cells.

(A) Cell culture and treatment in the ruxolitinib-treated experiments. (B) HepG2.2.15 cell viability following ruxolitinib treatment for 3 days. (C and D) JAK1, phosphorylated (p)-JAK1, STAT1, phosphorylated (p)-STAT1, and ISG15 expression in HepG2.2.15 cells treated ruxolitinib with (+) or without (−), dimethyl sulfoxide (DMSO, control) and combined dopamine with (+) or without (−) and ruxolitinib with (+) or without (−) for 3 days, determined by western blotting. Data are presented as mean values with standard deviations relative to the control from at least three independent experiments. *p<0.05, **p<0.01, *** p<0.005, **** p<0.001. OD, optical density; JAK, Janus kinase; STAT, signal transducer and activator of transcription.

Fig. 8. Dopamine inhibits hepatitis B virus (HBV) surface antigen (HBsAg) expression in a mouse model.

(A) Dopamine hydrochloride or placebo (normal saline; 10 µg/kg daily) treatment of adeno-associated virus (AAV)-HBV 1.3 mice. (B, C) Post-treatment HBsAg and hepatitis B virus e antigen (HBeAg) expression, determined by enzyme-linked immunosorbent assay. (D) Serum HBV DNA expression, determined by quantitative polymerase chain reaction. HBV DNA was quantified by qPCR (absolute quantification). The Y axis was adjusted to log10. (E) Mouse body weight. (F–J) HBsAg, interferon-stimulated gene 15 (ISG15), signal transducer and activator of transcription 1 (STAT1), phosphorylated (p)-STAT1, and Janus kinase 1 (JAK1) expression in mouse livers, determined by immunohistochemistry (IHC). 200× magnification, scale bar=100 µm. Data are presented as mean values with standard deviations relative to the control from at least five independent experiments. *p<0.05, **p<0.01, ***p<0.005, ****p<0.001. PBS, phosphate-buffered saline; IP, intraperitoneal; OD, optical density; IntDen, integrated density.

Fig. 9. Anti-hepatitis B virus mechanism of dopamine.

Dopamine inhibits hepatitis B virus surface and e antigen (HBsAg and HBeAg) expression through increasing ISG15 expression via JAK/STAT pathway activation. rcDNA, relaxed circular DNA; cccNDA, covalently closed circular DNA; pgRNA, pregenomic RNA; JAK, Janus kinase; STAT, signal transducer and activator of transcription.