Abstract
Objective: To screen and analyze key genes of ferroptosis and autophagy using bioinformatics and to validate them correspondingly in order to provide a basis for identifying key therapeutic targets for alcoholic liver disease (ALD). Methods: Bioinformatics analysis was used to screen key genes of ferroptosis and autophagy in ALD based on the GEO, FerrDb, KEGG, and HAMdb databases. Liver tissues from 12 ALD cases after liver transplantation were collected for immunohistochemistry to verify the expression of key genes. Different concentrations of ethanol were used to intervene in the human liver cell line L02 for 24 hours. Cell counting kit-8 (CCK8), lactate dehydrogenase (LDH), oil red O staining, reactive oxygen species (ROS) levels, real-time fluorescence quantitative PCR, and Western blot were used to detect the expression of key genes. The gene and protein expression changes of key genes were detected after intervention with the ferroptosis inhibitor ferrostatin-1 (Fer-1) or the autophagy inhibitor 3-methyladenine (3-MA).The t-test was used for comparison between the two groups, and one-way analysis of variance was used for comparison between multiple groups. Result: Bioinformatics analysis screened out the key ferroptosis gene ACSL4, the key autophagy gene CXCR4, and the key ferroptosis and autophagy genes PRKAA2 and CDKN2A. The expression of the four key genes was significantly upregulated in the liver tissues of ALD patients (ALD vs. control, t=9.132～15.240, P<0.01 or P<0.001). The LDH release increased (200 mmol/L vs. conrtol, F=10.51, P<0.01) at ethanol concentration of 200 mmol/L in the ALD in vitro hepatocyte model. Cell viability was significantly inhibited (100, 200 mmol/L vs. conrtol, F=177.30, P<0.001) at ethanol concentration of 100 mmol/L and 200 mmol/L. Lipid deposition and ROS accumulation were observed in the cells (100, 200 mmol/L vs. conrtol, F=27.65～245.40, P<0.01 or P<0.001). The expression of four key genes and their proteins was significantly upregulated (100, 200 mmol/L vs. conrtol, F=5.092～81.770, P<0.05). The gene and protein expressions of long-chain acyl-CoA synthetase 4(ACSL4), protein kinase AMP-activated catalytic subunit alpha 2 (PRKAA2), and cyclin dependent kinase inhibitor 2a (CDKN2A) were significantly downregulated (200 mmol/L+Fer-1 vs. 200 mmol/L, F=6.40～930.10, P<0.05) following intervention with the ferroptosis inhibitor Fer-1, while C-X-C chemokine receptor type 4 (CXCR4) protein expression was inhibited (200 mmol/L+Fer-1 vs. 200 mmol/L, F=18.60, P<0.01). The expressions of all four key genes and their proteins were significantly downregulated (200 mmol/L+3-MA vs. 200 mmol/L, F=10.66～116.40, P<0.05) after intervention with the autophagy inhibitor 3-MA. Conclusion: ACSL4 is a key gene in ferroptosis in ALD. CXCR4 is a key gene in autophagy in ALD. PRKAA2 and CDKN2A are key genes to ferroptosis and autophagy in ALD, and they are expected to become therapeutic targets for ALD in the future.