T cell--associated immunoregulation and antiviral effect of oxymatrine in hydrodynamic injection HBV mouse model

Abstract

Although oxymatrine (OMT) has been shown to directly inhibit the replication of hepatitis B virus (HBV) in vitro, limited research has been done with this drug in vivo. In the present study, the antiviral effect of OMT was investigated in an immunocompetent mouse model of chronic HBV infection. The infection was achieved by tail vein injection of a large volume of DNA solution. OMT (2.2, 6.7 and 20 mg/kg) was administered by daily intraperitoneal injection for 6 weeks. The efficacy of OMT was evaluated by the levels of HBV DNA, hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg) and hepatitis B core antigen (HBcAg). The immunoregulatory activity of OMT was evaluated by serum ELISA and flow cytometry. Results shows that OMT at 20 mg/kg inhibited HBV replication, and it was more efficient than entecavir (ETV) in the elimination of serum HBsAg and intrahepatic HBcAg. In addition, OMT accelerated the production of interferon-γ (IFN-γ) in a dose-dependent manner in CD4⁺ T cells. Our findings demonstrate the beneficial effects of OMT on the enhancement of immunological function and in the control of HBV antigens. The findings suggest this drug to be a good antiviral therapeutic candidate for the treatment of HBV infection.

Keywords: ALT, alanine aminotransferase; AST, aspartate aminotransferase; CD4+ T cell; CHB, chronic hepatitis B; ETV, entecavir; HBV; HBV, hepatitis B virus; HBcAg, hepatitis B core antigen; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen; HE, hematoxylin and eosin; IFN-γ; IFN-γ, interferon-γ; IL-4, interleukin-4; Mouse; NAs, nucleoside and nucleotide analogs; OMT, oxymatrine; Oxymatrine; TCMs, traditional Chinese medicines; TNF-α, tumor necrosis factor-α.

Graphical abstract

Figure 1

Oxymatrine promotes serum HBV DNA clearance. (A) Proportion of animals whose serum HBV-DNA can be detected before and after treatment of saline (model group), entecavir (ETV group), or three doses of oxymatrine (OMT groups) for 1, 3, and 6 weeks (n = 10). Log-rank Mantel-cox test was used. The limit of detection of serum HBV DNA was 300 copies/mL. (B) Serum HBV DNA levels in C57BL/6 mice in different groups (n = 10). Data are presented as median [25th and 75th percentile]. ^*P < 0.05, ^**P < 0.01 compared with model group.

Figure 2

Oxymatrine accelerates the clearance of HBsAg and HBeAg. The levels of serum HBsAg (A) and HBeAg (B) in infected mice were measured. Data are presented as median [25th and 75th percentile]. ^*P < 0.05, ^**P < 0.01 compared with model group; ^#P < 0.05 compared with entecavir group (ETV); n = 10.

Figure 3

Oxymatrine influences the persistence of intrahepatic HBcAg. Immunohistochemistry staining (brown HBcAg-expressing hepatocytes) of liver sections from infected mice: (A) Model; (B) entecavir (ETV); (C)–(E) intraperitoneal injection of OMT at 2.2, 6.7 and 20 mg/kg, respectively. (F) Statistical analysis from each visual field (200× magnification). ^**P < 0.01 compared with the model group; ^##p < 0.01 compared with the ETV group; n = 6.

Figure 4

Cytokine revolved in HBV clearance in serum and spleen lymphocyte. Quantification of serum cytokine levels of IFN-γ (A) and TNF-α (B). The whole spleens were taken after 6-week treatment. Flow cytometry analysis showing the proportion of CD4⁺ and CD8⁺ T cells and the proportion of IFN-γ produced by CD4⁺ and CD8⁺ T cells in spleen lymphocytes (C)–(F) and IL-4 produced by CD4⁺ T cells (G). ^*P < 0.05, ^**P < 0.01 compared with model group; ^#P < 0.05, ^##P < 0.01 compared with the control group; n = 6.

Figure 5

IFN-γ is promoted in splenic T cells. Quantification of IFN-γ-positive cells by flow cytometry in CD4⁺ and CD8⁺ T cells from infected mice; n = 6.

Figure 6

Liver H&E staining and serum level of ALT and AST. H&E staining (200× magnification, n = 6): (A) Control; (B) Model; (C) Entecavir treatment (ETV); (D)–(F) Oxymatrine treatment at 2.2, 6.7 and 20 mg/kg, respectively. (G) and (H) Serum levels of ALT and AST; n = 10.