Alisol A 24-acetate protects against NASH-associated fibrosis via suppression of Kupffer cell-derived SPHK1/S1P axis

BACKGROUND: Non-alcoholic steatohepatitis (NASH), a progressive form of non-alcoholic fatty liver disease, is characterized by hepatic steatosis, oxidative stress, chronic inflammation, and fibrosis. These pathological features increase the risk of cirrhosis and hepatocellular carcinoma. However, effective pharmacological interventions for NASH remain limited. PURPOSE: This study aimed to investigate the therapeutic efficacy and underlying mechanisms of alisol A 24-acetate (AA), a triterpenoid derived from Alisma plantago-aquatica L., in a murine model of NASH. STUDY DESIGN: An experimental intervention study was conducted using a choline-deficient, L-amino acid-defined high-fat diet (CDAHFD)-induced NASH mouse model, complemented by in vitro assays with Kupffer cells (KCs) and hepatic stellate cells (HSCs). METHODS: Male C57BL/6 J mice were fed CDAHFD for 6 weeks with or without AA treatment. Histological and biochemical analyses were performed to evaluate hepatic steatosis, inflammation, and fibrosis. The expression of fibrosis markers and lipid peroxidation products was assessed via immunohistochemistry and ELISA. Mechanistic studies involved examining the SPHK1/S1P pathway in KCs and HSC activation in response to cytokine-stimulated KC-conditioned media, with and without AA or the SPHK1 inhibitor PF-543. RESULTS: AA significantly alleviated hepatic steatosis, reduced liver triglyceride levels, and suppressed oxidative stress markers, including malondialdehyde and reactive oxygen species. Proinflammatory cytokines (IL-1β, TNF-α) were markedly decreased. Fibrosis progression was attenuated, evidenced by reduced collagen deposition and α-smooth muscle actin expression. Mechanistically, AA inhibited SPHK1 phosphorylation at serine 225 in KCs, reducing S1P production and subsequent paracrine activation of HSCs. In vitro, AA pretreatment of KCs diminished their capacity to activate HSCs. Notably, co-treatment with PF-543 and AA showed no additive effect, confirming SPHK1 as the primary target. CONCLUSION: AA exerts potent anti-steatotic, anti-inflammatory, and antifibrotic effects in experimental NASH, primarily by targeting the SPHK1/S1P signaling axis in Kupffer cells. This study is the first to identify AA as a direct modulator of KC-derived SPHK1 signaling, offering a novel strategy to disrupt KC-HSC crosstalk and mitigate liver fibrosis in NASH.