HA-20 prevents hepatocyte steatosis in metabolic-associated fatty liver disease via regulating Ca2+ relative signalling pathways

Metabolic-associated fatty liver disease (MAFLD) is caused by hepatocyte steatosis and is associated with obesity, type II diabetes, and heart disease. There are currently no effective drugs to treat MAFLD. This study explored the effect of HA-20, an oleanolic acid derivative, on hepatocyte steatosis in MAFLD. HepG2, L02, and AML12 cells were developed using oleic acid for in vitro MAFLD cell assays, and a high-fat diet + high-fructose diet-induced (HFHF) MAFLD mouse model was established for in vivo studies. The results demonstrated that HA-20 prevented hepatocyte steatosis in cell assays and caused 26.3, 57.7 and 70.0% inhibition of triglyceride (TG) levels in the 5.0, 10.0 and 20.0 μM HA-20 groups, respectively. The EC50 values of HA-20 treatment in HepG2, L02 and AML12 cells were 9.7 ± 0.6 μM, 42.4 ± 3.5 μM and 71.0 ± 14.7 μM, respectively. HA-20 also prevented hepatocyte steatosis in the MAFLD mouse model, the liver triglyceride contents were 2.3 ± 0.4 and 1.5 ± 0.2 mmol/L in the 2.5 and 5.0 mg/kg/day HA-20 groups, lower than 6.2 ± 0.7 mmol/L in the HFHF group and 3.3 ± 0.4 mmol/L in the metformin group. Further mechanistic investigation revealed that HA-20 increased the phosphorylation of calmodulin-dependent protein kinase kinase (p-CaMKK) and the phosphorylation of AMP-activated protein kinase (p-AMPK), at least partially by increasing intracellular Ca2+ concentration, which suppressed lipogenesis and enhanced β-oxidation. Our findings provide new insight into preventing MAFLD by increasing Ca2+ and suggest that HA-20 possesses therapeutic potential for MAFLD management.