Suppression of the STAT3/MYC/MCL-1 pathway by the fixed Jianpi Huoxue Formula relieves early biochemical abnormalities in liver fibrosis

Abstract

BACKGROUND

Liver fibrosis (LF), a core pathological process driving chronic liver disease, has no ideal clinical intervention approaches. A fixed Jianpi Huoxue Formula (FJHF), a traditional Chinese medicine (TCM) prescription derived from the Sijunzi Decoction and Taohong Siwu Decoction, has shown clinical benefits against LF.

AIM

To investigate the mechanism by which FJHF attenuates LF via STAT3/MYC/MCL-1 pathway regulation.

METHODS

Three strategies were integrated: (1) Clinical single-cell RNA sequencing (scRNA-seq) was performed on liver tissues from three untreated cirrhosis patients and three FJHF-treated patients to characterize hepatic cell heterogeneity and FJHF-responsive gene expression; (2) A randomized controlled trial was conducted in 40 cirrhosis patients with hypersplenism (20 each in the Jianpi Huoxue Formula and control groups) to assess a hepatocellular injury indicator alanine aminotransferase (ALT), hepatic reserve function indicators [total bilirubin (TBIL) and prothrombin time], and fibrosis indicators [hyaluronic acid (HA), laminin, type III procollagen N-terminal peptide (PIIINP), and type IV collagen (CIV)]; and (3) In vitro experiments were performed in human hepatic stellate cells (HSCs) LX-2 using cell counting kit-8, transmission electron microscopy, western blotting, and quantitative real-time PCR to verify the regulatory role of FJHF in the STAT3/MYC/MCL-1 pathway.

RESULTS

FJHF modulated the proportion of HSC populations in a small-sample scRNA-seq cohort. According to clinical data, FJHF treatment significantly decreased ALT (P < 0.01), TBIL (P < 0.05), and the fibrosis markers HA, PIIINP, and CIV (P < 0.05) compared to the control group. In vitro, 20% FJHF-containing serum suppressed HSC activation, as indicated by reduced α-SMA, collagen I, and TIMP1 levels and elevated MMP1 levels. Notably, FJHF inhibits the STAT3/MYC/MCL-1 pathway to ameliorate LF.

CONCLUSION

Short-term FJHF intervention improves early biochemical abnormalities in LF by inhibiting the STAT3/MYC/MCL-1 pathway and suppressing HSC activation. This work provides a scientific basis for the application of FJHF and identifies the STAT3/MYC/MCL-1 axis as a potential target for TCM intervention in early LF metabolic abnormalities.

Keywords: Liver fibrosis; STAT3/MYC/MCL-1 pathway; Network pharmacology; Single-cell RNA sequencing; Hepatic stellate cells

Core Tip: This study comprehensively explored the pharmacological mechanism of a fixed Jianpi Huoxue Formula (FJHF). We integrated single-cell transcriptomics, clinical observations, and in vitro experiments to confirm that FJHF inhibits the STAT3/MYC/MCL-1 pathway, suppresses hepatic stellate cell activation, and ameliorates early biochemical abnormalities in liver fibrosis.