6-Shogaol inhibits HSCs activation and liver fibrosis by regulating glycolytic reprogramming via targeting HIF-1α

BACKGROUND: Liver fibrosis is a representative scarring response that can ultimately lead to liver cancer. However, relevant antifibrotic drugs for the effective treatment of liver fibrosis in humans have not yet been identified. 6-Shogaol is derived from natural products and exhibits multiple biological activities, including anti-inflammatory and antioxidant properties; however, its efficacy and potential mechanism of action against liver fibrosis remains unclear. This study aimed to examine the anti-fibrotic properties and potential mechanisms of action of 6-Shogaol. METHODS: ) and bile duct ligation (BDL)) were constructed to evaluate the anti-fibrotic properties of 6-Shogaol in vivo. Transforming growth factor-β1 (TGF-β1)-induced human hepatic stellate cells (HSCs) LX-2 cells were used as in vitro models. Network pharmacology analysis was introduced to explore the key targets of 6-Shogaol regarding the mechanisms on liver fibrosis. Molecular docking, molecular dynamics simulations, drug affinity reactivity target stability (DARTS) and isothermal titration calorimetry (ITC) were used to detect the affinity and binding between 6-Shogaol and its target. Additionally, we invested the mechanism of 6-Shogaol through RNA sequencing combined with Western blotting, oxygen consumption rate (OCR), extracellular acidification rate (ECAR), immunofluorescence co-localization, histopathology, immunohistochemical staining and RT-qPCR. RESULTS: - and BDL-induced liver fibrosis in mice, including observations of improved liver function, decreased activity of HSCs, and decreased extracellular matrix (ECM) deposition. In an in vitro model, 6-Shogaol suppressed TGF-β1-induced LX-2 activation. Mechanistically, RNA sequence analysis revealed that the effect of 6-Shogaol on liver fibrosis is linked to glycolytic reprogramming. 6-Shogaol suppressed HSCs glycolysis by decreasing glycolytic enzymes (HK2, PKM2, and GLUT1) and glycolytic metabolite levels (lactic acid and pyruvic acid). Furthermore, network pharmacology suggested HIF-1α as a potential 6-Shogaol target. Molecular docking, molecular dynamics simulations, DARTS, and ITC confirmed that 6-Shogaol could directly bind to HIF-1α. Interestingly, we demonstrated that HIF-1α knockout significantly inhibited HSCs glycolysis and activation, whereas the overexpression of HIF-1α increased HSCs glycolysis and activation. Moreover, specific knockout of HIF-1α did not enhance the suppressive effect of 6-Shogaol on HSCs activation or fibrosis-associated protein expression in vivo. CONCLUSION: These findings showed that 6-Shogaol ameliorated liver fibrosis by modulating the expression of HIF-1α associated with glycolysis reprogramming and validate 6-Shogaol as a promising therapeutic strategy for liver fibrosis.