Fig. 6

AKR1A1 knockdown in AML12 hepatocytes exacerbates lipid accumulation, inflammation, and fibrosis. AKR1A1 knockdown was conducted in AML12 hepatocytes by infecting cells with either a control (sh-control) or an AKR1A1-specific (sh-AKR1A1) shRNA lentiviral clone for 48 h. The shRNA-treated AML12 hepatocytes were then subjected to stimulation with a palmitic acid and oleic acid mixture (P/O) to induce steatosis in the absence or presence of 200 mM ethanol for 72 h. Afterward, western blotting and qRT‒PCR were performed to characterize AKR1A1 expression at both the protein (A) and mRNA (B) levels. ORO staining was performed to examine lipid accumulation in AML12 hepatocytes (D). The extent of lipid accumulation was quantified by extracting the bound ORO dye and detecting the absorbance at 450 nm (C). Furthermore, the related marker genes involved in FA transport and synthesis (Cd36, Fasn, Acaca, Cyp4a, Cyp2e1, Lipin1, Ppar-γ, and Srebp1) (E) and FA oxidation (Cpt1α, Acox1, and Pparα) (F) were examined at the mRNA level. In addition, shRNA-treated AML12 hepatocytes were stimulated with 1 µg/ml of LPS to induce inflammation for 24 h and stimulated with 5 ng/ml TGF-β1 to induce fibrosis for 48 h. After the treatments, qRT‒PCT was performed to characterize the mRNA levels of inflammation-related (Il-1b, Tnf-α) (G) and fibrosis-related (Fn1, α-Sma, Col1a1, and Timp-1) (H) marker genes. All values were normalized to the β-actin gene and expressed in relation to the sh-control group. Data are presented as the means ± SD (n ≥ 3). Statistical differences were marked as follows: ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 (compared with the sh-control Ctrl group); *p < 0.05, **p < 0.01, ***p < 0.001 (compared between the other groups)