The lignan compound matairesinol monoglucoside induces type I interferon production in HBV infection immunity by regulating STING signaling

Abstract

Background: the urgent need for effective prevention and treatment strategies for hepatitis B virus (HBV) has driven extensive research into natural compounds. This study aims to explore the therapeutic potential of matairesinol monoglucoside (MMG) in the treatment of HBV infection.

Methods: primary hepatocytes and Kupffer cells were isolated from wild-type (WT) or stimulator of interferon genes (STING) knockout mice and subsequently infected with AAV-HBV to establish an in vitro anti-HBV assay model. The anti-HBV effects of MMG were assessed by measuring HBV DNA, HBsAg, and HBeAg levels, as well as using qRT-PCR and ELISA to evaluate type I interferon markers (IFN-α and IFN-β), and a luciferase assay. In vivo anti-HBV effects were determined by pre-treating mice with MMG prior to AAV-HBV infection.

Results: MMG treatment significantly reduced the expression of HBV DNA, HBsAg, and HBeAg in both primary hepatocytes and Kupffer cells. Additionally, MMG enhanced the production of type I interferons (IFN-α and IFN-β) in both cell types. The knockout of STING diminished the effects of MMG on type I interferon production. Mechanistically, MMG was shown to modulate the STING-TBK1-IRF3 signaling axis, leading to increased IFN production.

Conclusions: MMG shows promise as a potential therapeutic agent against HBV by targeting the STING signaling pathway.

Fig. 1. Effects of MMG on the expression of HBV DNA, HBsAg and HBeAg. (A) The chemical structure of matairesinol monoglucoside (MMG). Mouse primary hepatocytes were treated with indicated concentrations of MMG together with AAV-HBV (10 MOI) for 24 hours, followed by analyzing the expression of HBV DNA (B), HBsAg (C) and HBeAg (D). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001.

Fig. 2. MMG enhances the production of type I interferon in mouse primary hepatocytes. Mouse primary hepatocytes were pretreated with 5 μM MMG for indicated time points, and then stimulated with AAV-HBV (10 MOI) for 12 hours. The expression of IFN-α (A) and IFN-β (B) was determined by qRT-PCR. Then, mouse primary hepatocytes were also pretreated with 5 μM MMG for 6 hours, and then stimulated with AAV-HBV (10 MOI) for indicated time points. The expression of IFN-α (C) and IFN-β (D) was determined by qRT-PCR. The supernatant of the cell culture was also prepared for ELISA assay to assess the expression of IFN-α (E) and IFN-β (F). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001.

Fig. 3. MMG upregulates the expression of type I interferon in mouse Kupffer cells. Mouse Kupffer cells were pretreated with 5 μM MMG for 6 hours, and then stimulated with AAV-HBV (10 MOI) for indicated time points. The expression of IFN-α (A) and IFN-β (B) was determined by qRT-PCR. The supernatant of the cell culture was also prepared for the ELISA assay to assess the expression of IFN-α (C) and IFN-β (D). ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001.

Fig. 4. MMG upregulates the transcriptional expression of STING. Luciferase assay of IFN-β (A) and ISRE (B) activation in HEK293T cells expressing STING, TBK1 or IRF3. Mouse primary hepatocytes (C) and mouse Kupffer cells (D) were treated with indicated concentrations of MMG together with AAV-HBV (10 MOI) for 24 hours, followed by qRT-PCR against STING. Mouse primary hepatocytes were treated with indicated concentrations of MMG together with AAV-HBV (10 MOI) for 24 hours, followed by western blot against STING and GAPDH, and (E) p-TBK1, TBK1, p-IRF3 and IRF3 (F). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001, ns means not significant.

Fig. 5. Knockout of STING attenuates the effects of MMG on type 1 interferon production. Primary mouse hepatocytes from wild-type or STING^−/− mice were pretreated with 5 μM MMG for 6 hours, and then stimulated with AAV-HBV (10 MOI) for 0 or 12 hours, followed by ELISA assay to assess the expression levels of IFN-α (A) and IFN-β (B). Mouse primary Kupffer cells from wild-type or STING^−/− mice were pretreated with 5 μM MMG for 6 hours, and then stimulated with AAV-HBV (10 MOI) for 0 or 12 hours, followed by ELISA assay to assess the expression levels of IFN-α (C) and IFN-β (D). ^****p < 0.0001, ns means not significant.

Fig. 6. MMG promotes AAV-HBV-induced antiviral activity in vivo. C57BL/6 mice were pretreated with 10 mg kg⁻¹ MMG for 24 hours, and then infected with AAV-HBV at 5 × 10¹⁰ viral genome equivalents through the tail vein injection. Two weeks later, blood samples were collected and serum HBsAg (A) and HBeAg (B) were measured by ELISA. Serum HBV DNA was measured by qRT-PCR (C). The serum was also prepared for the analysis of ALT expression (D). The expression levels of IFN-α (E) and IFN-β (F) were also determined by ELISA in the serum of mice. ^****p < 0.0001.