Smilax glabra flavonoids ameliorate non-alcoholic steatohepatitis by regulating PKM2-dependent glycolysis and suppressing AIM2 inflammasome activation.

BACKGROUND: Nonalcoholic steatohepatitis (NASH) is a prevalent chronic liver disease characterized by hepatic steatosis, persistent inflammation and progressive liver damage. Smilax glabra flavonoids (SGF), the major bioactive constituents of Smilax glabra Roxb., exhibit potent antioxidant and anti-inflammatory activities. Nevertheless, their therapeutic role and molecular basis in NASH remain elusive. METHODS: This study aimed to investigate the protective effects of SGF against high-fat diet (HFD)-induced NASH in mice, with emphasis on PKM2-mediated glycolysis and AIM2 inflammasome activation. C57BL/6J mice were fed an HFD for 20 weeks to establish a NASH model and concurrently treated with SGF (30 or 90 mg/kg/day) or empagliflozin (EMPA). Comprehensive assessments, including biochemical detection, histological staining and molecular biological verification, were performed. In vitro, FFA-challenged AML-12 hepatocytes and LPS-stimulated RAW264.7 macrophages were adopted. siRNA-mediated knockdown, lentiviral overexpression, and cell co-culture models were further applied to validate the underlying mechanisms. RESULTS: SGF markedly attenuated HFD-induced obesity, dyslipidemia, hepatic steatosis, fibrosis and hepatocellular damage, mitigated hepatic oxidative stress and restored mitochondrial structure integrity. Mechanistically, SGF restrained glycolytic metabolism by downregulating the expression of HK2, p-PKM2, and LDHA and by reducing pyruvate and lactate production. Besides, SGF mitigated AIM2 inflammasome activation and decreased the levels of AIM2, ASC, caspase-1, and IL-1β. Mechanistic validation confirmed that PKM2 was abundantly expressed in macrophages, and PKM2 overexpression partially abrogated the inhibitory effects of SGF on glycolysis and AIM2 inflammasome signaling. Co-culture assays further demonstrated that PKM2 overexpression in RAW264.7 macrophages exacerbated lipid deposition, oxidative stress, glycolysis, and AIM2 inflammasome activation in AML-12 hepatocytes, thereby counteracting the hepatoprotective effects of SGF. CONCLUSION: SGF effectively ameliorates HFD-induced NASH by reversing metabolic disturbance, relieving oxidative stress and liver injury, and suppressing PKM2-dependent glycolysis and AIM2 inflammasome activation. Macrophage PKM2 acts as a key mediator of pathogenic macrophage-hepatocyte crosstalk, which is critical for the hepatoprotective effects of SGF against NASH. Overall, our study findings highlight SGF as a promising therapeutic option for this specific population.