Abstract:To investigate the optimal concentration of palmitic acid （PA） for inducing HepG2 cells to establish metabolic-associated fatty liver disease（MAFLD） and its effect on autophagy，this experiment adopted methods such as cell counting kit-8 （CCK8），oil red O staining，triglyceride （TG） content detection，immunofluorescence labeling of autophagy marker LC3，and immunoblotting for comprehensive evaluation. The results showed that PA at concentrations of 0.4-1.0 mmol/L significantly reduced cell viability. Moreover，PA at concentrations of 0.2-1.0 mmol/L significantly increased the number of lipid droplets and TG content in cells and reduced the number of LC3 fluorescent dots. PA at concentrations of 0.6-1.0 mmol/L significantly reduced the ratio of P-AMPK/AMPK，and PA at a concentration of 1.0 mmol/L increased the ratio of P-mTOR/mTOR. After comprehensive consideration of lipid accumulation，lipotoxicity，and autophagy，PA at a concentration of 0.6 mmol/L was finally selected for the construction of the MAFLD cell model. The water extracts of Ganoderma lucidum spore powder at concentrations of 50，200，and 800 μg/mL reduced the number of lipid droplets and TG content and increased the number of LC3 fluorescent dots. Therefore，the MAFLD model induced by PA at a concentration of 0.6 mmol/L could be used for the screening of drugs targeting autophagy in MAFLD.The water extracts of Ganoderma lucidum spore powder could improve cell death and lipid accumulation by restoring autophagy inhibited by PA.