ShuXueNing injection alleviates liver fibrosis by inhibiting hepatic stellate cell activation and suppressing the EGFR/STAT3/HIF1A pathway

Abstracts: Background: ShuXueNing injection (SXN) is a commonly used traditional Chinese medicine preparation for dilating blood vessels and improving microcirculation. However, the efficacy and mechanism of SXN in treating liver fibrosis (LF) remain unclear. Methods: We intervened in the carbon tetrachloride-induced mouse LF model with SXN. After intervention, we collected mouse feces for 16S rRNA sequencing and serum samples to assess serological indices. We also collected mouse liver tissues for pathological and immunohistochemical (IHC) staining. We conducted network pharmacology analysis to comprehensively identify SXN target genes against LF. Hepatic stellate cell (HSC) line LX2 was exposed to TGF-β1 and subjected to various concentrations of SXN. Total tissue and cellular RNA were extracted and analyzed by transcriptome sequencing (RNA-seq) and real-time quantitative PCR (qRT-PCR). Results: Compared with the model group, SXN significantly increased the mouse body weight, reduced liver index, spleen index, and LF serum markers, alleviated inflammatory damage, decreased collagen deposition, and inhibited macrophage infiltration in the liver. SXN intervention significantly restored gut microbial diversity and selectively enriched populations of beneficial commensal bacteria. qRT-PCR results showed that SXN significantly down-regulated TGFB1, COL3A1, COL1A1 , and FN1 mRNA expression in activated HSCs. Network pharmacology and RNA-seq results showed that SXN's anti-LF function correlates with the JAK-STAT and HIF-1 signaling pathways. qRT-PCR and IHC results demonstrated that SXN significantly downregulated EGFR, STAT3, and HIF1A expression levels in the mouse liver tissues. Molecular docking results showed that Flavoxanthin, the main compound of SXN, had the strongest binding affinity for EGFR, STAT3, and HIF1A. Conclusion: SXN effectively inhibits LF progression by blocking HSC activation and suppressing JAK-STAT and HIF-1 signaling pathways. STAT3, HIF1A, and EGFR may be important SXN targets for treating LF.