He-He-Shu-Yang formula alleviates liver fibrosis by inhibiting hepatic stellate cell activation in vivo and in vitro

Abstract

BACKGROUND

Inhibition of liver fibrosis plays a crucial role in curbing the advancement of chronic disease to cirrhosis and even liver cancer. However, modern medicine currently lacks direct anti-fibrotic drugs. He-He-Shu-Yang formula (HHSY) is a renowned Chinese medicine for the treatment of liver fibrosis. However, its mechanism of action has not been fully unraveled.

AIM

To explore the efficacy and mechanism of action of HHSY through in vitro and in vivo experiments.

METHODS

A liver fibrosis rat model (carbon tetrachloride-induced) was treated with low- or high-dose HHSY (10.42 g/kg or 20.84 g/kg) or with colchicine (1 mg/kg) for 9 weeks. In vitro, LX-2 human hepatic stellate cells (HSCs) were activated using transforming growth factor-β1 and subsequently treated with HHSY-containing serum or a nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) inhibitor. Through high-performance liquid chromatography, histopathology (hematoxylin and eosin, Masson), immunohistochemistry, western blot, and quantitative reverse transcription polymerase chain reaction analyses, we demonstrated that HHSY inhibited HSC activation and suppressed the NOX4/reactive oxygen species (ROS)/nucleotide binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) pathway.

RESULTS

In vivo, HHSY improved liver function and alleviated liver pathology, including reducing inflammatory cell infiltration, and liver fibrosis in carbon tetrachloride rats. with more significant effects at higher doses. Immunohistochemistry revealed that HHSY could decrease alpha-smooth muscle actin, NOX4, and NLRP3 expression, as well as serum ROS levels (O₂^– and H₂O₂, P < 0.05). Western blot analysis confirmed HHSY also reduced NLRP3 protein levels (P < 0.05). In vitro, HHSY at 1.25% or 2.5% reduced the levels of ACTA2 mRNA, NOX4 protein and NOX4 mRNA, ROS production, and NLRP3 and IL-1β mRNA in activated LX-2 cells (P < 0.05).

CONCLUSION

HHSY effectively treats liver fibrosis, likely by inhibiting HSC activation through the NOX4/ROS/NLRP3 pathway. This underscores HHSY’s clinical relevance as a potential therapeutic option for liver fibrosis.

Keywords: He-He-Shu-Yang formula; Liver fibrosis; Mechanism; Hepatic stellate cells; Nicotinamide adenine dinucleotide phosphate oxidase 4 pathway; Reactive oxygen species pathway

Core Tip: High-performance liquid chromatography identified six key components in He-He-Shu-Yang formula (HHSY) that are effective in reducing liver fibrosis. HHSY has a definite therapeutic effect on liver fibrosis in vivo and in vitro models. HHSY’s mechanism may be through the inhibition of hepatic stellate cell activation via the nicotinamide adenine dinucleotide phosphate oxidase 4/reactive oxygen species/nucleotide binding oligomerization domain-like receptor family pyrin domain-containing 3 pathways.