Activation of AMPK by triptolide alleviates nonalcoholic fatty liver disease by improving hepatic lipid metabolism, inflammation and fibrosis

Triptolide is naturally isolated from Tripterygium wilfordii Hook F., possessing multiple biological activities. Hepatotoxicity is one of the main side effects of triptolide. However, the effect of triptolide on nonalcoholic fatty liver disease remains unknown (NAFLD). This study aimed to observe the amelioration of triptolide against NAFLD and investigate the engaged mechanism. Two typical animal models of NAFLD, obese db/db mice and methionine/choline-deficient (MCD) diet-fed mice, were used. Hepatic steatosis, inflammation, and fibrosis were evaluated by H&E and Masson staining. Oil red O staining and lipid extraction analysis were used to detect fat content in mice livers. Expression of lipid metabolism, inflammatory and fibrogenic genes was also detected by Real-time PCR and Western blotting, respectively. Phosphoproteomics, molecular docking, and TR-FRET assay were performed to provide further insight into how triptolide improved NAFLD. Intraperitoneal injection of triptolide at a daily dose of 50 μg/kg significantly alleviated MCD diet-induced nonalcoholic steatohepatitis (NASH), but 100 μg/kg triptolide caused severe hepatotoxicity. Pathological staining confirmed low-dose triptolide treatment reducing hepatic lipid deposition, inflammation, and fibrosis in NASH. Serum biochemical analysis revealed a reduction in the level of liver enzymes and bilirubin. MCD also induced rising expression of typical genes and proteins related to fibrosis (fibronectin, α-SMA, collagens, TGF-β) and inflammation (ILs, TNF-α, MCP-1), which was suppressed by low-dose triptolide. Data from the proteomics/phosphoproteomics and TR-FRET assay indicated triptolide was a potential allosteric AMPK agonist to increase the phosphorylation on Thr172 residue, with the EC50 of 277.78 μM and 231.02 μM for AMPKα1 and AMPKα2, respectively. Moreover, triptolide exhibited an ability to activate AMPK and further led to increasing ACC1 phosphorylation in the liver. The positive results that triptolide ameliorated hepatic lipogenesis, fatty acid oxidation, and fibrosis of NAFLD via activating AMPK were further confirmed in db/db mice with 10-week intervention (50 μg/kg, i.v., twice a week). This study demonstrates that dose-related triptolide as an allosteric AMPK agonist has the potential to alleviate NAFLD without hepatotoxicity.