Tianhuang formula ameliorates liver fibrosis by inhibiting CCL2-CCR2 axis and MAPK/NF-κB signaling pathway

Abstract

Ethnopharmacological relevance: In the progression of chronic liver diseases, liver fibrosis is a reversible pathophysiologic event for liver diseases prognosis and risk of cirrhosis. Liver injury factors of different etiologies mediate this process. There is still a lack of effective medications for treating liver fibrosis. Additionally, the ameliorative effects of traditional herbs on liver fibrosis have been commonly reported. Tianhuang formula (THF) is a drug combination consisting of 2 traditional Chinese herbs, which has been showing significant improvement in metabolic liver diseases. However, the hepatoprotective effect and mechanism of THF in ameliorating liver fibrosis are still unclear.

Aim of the study: This study aimed to investigate the effects of THF on carbon tetrachloride (CCl₄)-induced and methionine-choline-deficient (MCD) diet-induced liver fibrosis model and to reveal the potential mechanisms. It can provide experimental evidence for THF as a therapeutic candidate for liver fibrosis.

Materials and methods: In this study, CCl₄-induced mice were treated with THF (80 mg/kg, 160 mg/kg) or Fuzheng Huayu (FZHY) capsules (4.8 g/kg) for 6 weeks. MCD-induced mice received the same doses of THF or FZHY for 4 weeks. FZHY is used as a comparative study in these two models. Following that, using kit reagents detected changes in relevant serum and liver biochemical indicators. Histological changes in mouse liver were measured by staining of H&E and Sirius Red. The markers expression of liver fibrosis and inflammation were detected using qRT-PCR, western blotting and immunohistochemical staining analysis. The potential regulatory mechanism of THF to ameliorate liver fibrosis was performed by RNA-sequencing analysis. Finally, the analysis results were verified by immunofluorescence co-staining, qRT-PCR and western blotting.

Results: Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and hepatic triglyceride (TG) levels in CCl₄ and MCD-induced liver fibrosis mice were significantly improved after THF treatment. Meanwhile, the expression of fibrosis and inflammation markers were significantly suppressed. Furthermore, THF downregulated the expression of the macrophage marker CD68. According to RNA-sequencing analysis, we found the CCL2-CCR2 axis and MAPK/NF-κB as the potential signaling pathway for THF against liver fibrosis.

Conclusion: This study revealed that THF ameliorated liver injury, inflammation and fibrotic process by inhibiting CCL2-CCR2 axis and its downstream MAPK/NF-κB signaling pathway.

Keywords: CCL2-CCR2 axis; Inflammation; Liver fibrosis; MAPK/NF-κB; Tianhuang formula.