Abstract: Objective This experiment aimed to investigate the protective effects and mechanisms of berberine (BBR) on hepatic steatosis by activation of KLF4 via targeting HDAC/H3K9ac in palmitic acid (PA)-induced HepG2 cells in vitro. Methods HepG2 cells were induced by PA to construct hepatocyte steatosis model. The fat deposition was determined by oil red O staining. The cellular HDAC1, H3K9ac, KLF4, SREBP-1c and PPARγ expression was assayed by Western blot. The H3K9Ac in the KLF4 promoter region was determined by chromatin immunoprecipitation (ChIP). The supernatant cytokine levels were identified by ELISA kits. The KLF4 gene was silenced by siRNA to verify the protective effects of BBR by targeting HDAC/H3K9Ac/KLF4 in PA-induced HepG2 cells. Results The BBR treatment significantly improved lipid deposition in PA-induced HepG2 cells compared to that in model cells, and the supernatant TNF-α, IL-1β and IL-6 levels were (514.7±46.4) pg/ml, (241.5±37.7) pg/ml and (362.7±19.9) pg/ml, significantly lower than [(1162.0±110.8) pg/ml, (635.8±73.4) pg/ml and (1110.0±85.1) pg/ml, respectively, P < 0.001] in the model; the expression of HDAC1 protein in BBR-intervened group decreased by 19.0%, while the expression of H3K9ac and KLF4 protein increased by 1.53 and 1.52 times, the level of H3K9ac in KLF4 promoter region increased by 3.97 times, and the expression of lipid synthesis related gene SREBP-1c protein decreased by 49.1%, the expression of lipid decomposition related gene PPARγ protein increased 1.84 times compared to in the model cells; compared with empty plasmid transfection, the expression level of HDAC1 protein in cells transfected with eukaryotic plasmids was increased by 2.02 times, and the expression level of H3K9Ac protein was decreased by 57.1%; compared with SiRNA-NC transfection, the KLF4 protein expression level in HepG2 cells after SiRNA-KLF4 transfection was significantly decreased (P < 0.01), lipid deposition was significantly increased, the levels of TNF-α, IL-1β and IL-6 were (887.9±89.9) pg/ml, (471.9±38.4) pg/ml and (793.1±59.3) pg/ml, significantly higher than [(534.2±46.1) pg/ml, (260.3±26.9) pg/ml and (372.0±30.6) pg/ml, P < 0.01] in SiRNA-NC transfected cells, and the expression of lipid synthesis related gene SREBP-1c protein was increased by 1.77 times, while the expression of PPARγ protein associated with lipid decomposition decreased by 41.6%. Conclusion Taking together, the findings in the present study indicate that BBR protects from cell steatosis and inflammatory reactions by activation of KLF4 via regulating HDAC1/H3K9ac in PA-induced HepG2 cells, which provides the insight into new therapeutic approaches for patients with non-alcoholic fatty liver disease management.

Key words: HepG2 cells, Berberine, Histone deacetylase, Histone H3 lysine residue 9 acetylation, Krüppel-like factor4, In vitro