Ba-Qi-Rougan formula alleviates hepatic fibrosis by suppressing hepatic stellate cell activation via the MSMP/CCR2/PI3K pathway

Abstract

Ethnopharmacological relevance: The Ba-Qi-Rougan formula (BQRGF) is a traditional and effective compound prescription from Traditional Chinese Medicine (TCM) utilized in treating hepatic fibrosis (HF).

Aim of the study: We aimed to evaluate the therapeutic efficacy of BQRGF on HF and explore the underlying mechanisms of action.

Materials and methods: UPLC-Q-TOF-MS technology was employed to identify the material basis of BQRGF. Mice with carbon tetrachloride (CCl₄)-induced HF received BQRGF at three doses (3.87, 7.74, and 15.48 g/kg per day). We examined serum and liver biochemical indicators and liver histology to assess the therapeutic impact. Primary mouse cells were isolated and utilized for experimental analysis. MSMP expression levels were examined in vitro and in vivo experimental models, including human and mouse tissue. Furthermore, lentivirus and small interfering RNA (siRNA) transfections were employed to manipulate microseminoprotein (MSMP) expression in LO2 cells (human normal liver cells). These manipulated LO2 cells were then co-cultured with LX2 human hepatic stellate cells (HSCs). Through the modulation of MSMP expression in co-cultured cells, administering recombinant MSMP (rMSMP) with or without BQRGF-medicated serum, and using specific pathway inhibitors or agonists in LX2 cells, we elucidated the underlying mechanisms.

Results: A total of 48 compounds were identified from BQRGF, with 12 compounds being absorbed into the bloodstream and 9 compounds being absorbed into the liver. Four weeks of BQRGF treatment in the HF mouse model led to significant improvements in biochemical and molecular assays and histopathology, particularly in the medium and high-dose groups. These improvements included a reduction in the level of liver injury and fibrosis-related factors. MSMP levels were elevated in human and mouse fibrotic liver tissues, and this increase was mitigated in HF mice treated with BQRGF. Moreover, primary cells and co-culture studies revealed that BQRGF reduced MSMP expression, decreased the expression of the hepatic stellate cell (HSC) activation markers, and suppressed critical phosphorylated protein levels in the CCR2/PI3K/AKT pathway. These findings were further validated using CCR2/PI3K/AKT signaling inhibitors and agonists in MSMP-activated LX2 cells.

Conclusions: Collectively, our results suggest that BQRGF combats HF by diminishing MSMP levels and inhibiting MSMP-induced HSC activation through the CCR2/PI3K/AKT pathway.

Keywords: Ba-Qi-Rougan formula; Hepatic fibrosis; Hepatic stellate cells; Microseminoprotein; PI3K/AKT pathway.