Anti-HBV Activities of Polysaccharides from Thais clavigera (Küster) by In Vitro and In Vivo Study

Abstract

Hepatitis B virus (HBV) infection remains a major global health problem. It is therefore imperative to develop drugs for anti-hepatitis B with high-efficiency and low toxicity. Attracted by the observations and evidence that the symptoms of some patients from the Southern Fujian, China, suffering from hepatitis B were alleviated after daily eating an edible marine mollusk, Thais clavigera (Küster 1860) (TCK). Water-soluble polysaccharide from TCK (TCKP1) was isolated and characterized. The anti-HBV activity of TCKP1 and its regulatory pathway were investigated on both HepG2.2.15 cell line and HBV transgenic mice. The data obtained from in vitro studies showed that TCKP1 significantly enhanced the production of IFN-α, and reduced the level of HBV antigens and HBV DNA in the supernatants of HepG2.2.15 cells in a dose-dependent manner with low cytotoxicity. The result of the study on the HBV transgenic mice further revealed that TCKP1 significantly decreased the level of transaminases, HBsAg, HBeAg, and HBV DNA in the serum, as well as HBsAg, HBeAg, HBV DNA, and HBV RNA in the liver of HBV transgenic (HBV-Tg) mice. Furthermore, TCKP1 exhibited equivalent inhibitory effect with the positive control tenofovir alafenamide (TAF) on the markers above except for HBV DNA even in low dosage in a mouse model. However, the TCKP1 high-dose group displayed stronger inhibition of transaminases and liver HBsAg, HBeAg, and HBV RNA when compared with those of TAF. Meanwhile, inflammation of the liver was, by pathological observation, relieved in a dose-dependent manner after being treated with TCKP1. In addition, elevated levels of interleukin-12 (IL-12) and interferon γ (IFN-γ), and reduced level of interleukin-4 (IL-4) in the serum were observed, indicating that the anti-HBV effect of TCKP1 was achieved by potentiating immunocyte function and regulating the balance of Th1/Th2 cytokines.

Keywords: Thais clavigera (Küster); anti-HBV activities; in vitro; in vivo; polysaccharides.

Figure 1

Comparison of 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatives of standard monosaccharide and water-soluble polysaccharides from Thais clavigera (Küster 1860) (TCKP1) via the HPLC method. 1, mannose (Man); 2, glucosamine (GlcN); 3, glucuronic acid (GlcA); 4, GalA; 5, glucose (Glc); 6, galactose (Gal); 7, xylose (Xyl); and 8, arabinose (Ara).

Figure 2

ESI-MS spectrum of TCKP1. Conditions: 100 μg/mL TCKP1 with methanoic acid. A triple TOF mass spectrometer was used (100–40,000 Da).

Figure 3

Inhibitory effect of TCPK1 on HBsAg (A), HBeAg (B), and HBV DNA (C) in the supernatants of HepG2.2.15 cells and the effect on cell viability (D). Lamivudine (3TC) treated for 9 days was used as the positive control in A and B, and for 6 days in C. The promoting effect of TCKP1 on the level of interferon-α (IFN-α) in the culture supernatants (extracellular, (E)) and the cell lysates (intracellular, (F)) of HepG2.2.15 cells after the treatment for 3 days with 25 μg/mL lamivudine (3TC) as the positive control. All values are expressed as means ± S.D. from three separate experiments performed in triplicate. * p < 0.05, ** p < 0.01 vs. the negative control. ^# p < 0.05 vs. the 3TC positive control.

Figure 4

The level of DNA (A) and inhibitory effect of TCPK1 on HBsAg (B), HBeAg (C), AST (D), and ALT (E) in the serum of transgenic HBV mice. The effect of TCKP1 on liver weight of transgenic HBV mice (F). All values are expressed as means ± S.D. (n = 5). * p < 0.05, ** p < 0.01 vs. the negative control (NC); ^# p < 0.05 vs. the positive control (tenofovir alafenamide, TAF).

Figure 5

The effect of TCKP1 on liver morphological (A) and histopathological (B) changes of HBV transgenic mice. Liver tissue sections were subjected to histological examinations by staining with hematoxylin and eosin under a light microscope. Graphs shown are representative images from different groups of mice (H&E staining, ×200) (bar = 100 μm).

Figure 6

Inhibitory effect of TCPK1 on the production of HBV DNA (A), HBsAg (B), HBeAg (C), and HBV RNA (D) in livers of HBV transgenic mice. Effect of TCPK1 on level of IFN-γ (E), IL-4 (F), and IL-12 (G) in the serum of HBV-Tg mice. All values are expressed as means ± S.D. (n = 5). * p < 0.05, ** p < 0.05 vs. the negative control (NC); ^# p < 0.05 vs. the positive control (tenofovir aAlafenamide, TAF).