Study design. (A) The chemical structure of GRb1. (B) Experimental process in zebrafish larvae. (C) Experimental procedure in L-O2 cells.

GRb1 alleviates alcohol-induced hepatic steatosis in zebrafish larvae. (A) H&E staining of zebrafish larvae. Figures are magnified as 400ⅹ. (B) Whole-mount staining with Oil Red O. Figures are magnified as 50ⅹ. (C) Quantitative analysis of the liver gray value of Oil Red O using ImageJ software. Data are shown as the mean ± SEM (n = 10 per group from two experiments). p < 0.05 (#), p < 0.01 (##), and p < 0.001 (###) compared with the control group; p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***) compared with the 350 mM EtOH group.

GRb1 significantly ameliorates hepatic steatosis caused by alcohol. (A) Nile Red staining of frozen liver sections from zebrafish larvae with liver-specific EGFP expression. Figures are magnified as 400ⅹ. (B) Quantitative analysis of the mean fluorescence intensity of Nile Red using ImageJ software. (C) Oil Red O staining of cryosections from zebrafish larvae. Figures are magnified as 400ⅹ. (D) Quantitative analysis of the area of lipid droplets in the liver based on Oil Red O staining using ImageJ software. Data are expressed as the mean ± SEM (n = 8–10 per group). p < 0.05 (#), p < 0.01 (##), and p < 0.001 (###) compared the control group; p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***) compared with the 350 mM EtOH group.

GRb1 reduces lipid accumulation stimulated by alcohol in L-O2 cells. (A) Oil Red O staining of L-O2 cells. Figures are magnified as 400?. (B) Quantitative analysis of the area of lipid droplets in L-O2 cells based on Oil Red O staining using ImageJ software. (C) Nile Red staining of L-O2 cells. Figures are magnified as 200?. (D) Quantitative analysis of the mean fluorescence intensity of Nile Red using ImageJ software. (E) Triglyceride levels in L-O2 cells. The data are displayed as the means ± SEM. p < 0.05 (#), p < 0.01 (##), and p < 0.001 (###) compared the control group; p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***) compared with the 100 mM EtOH group.

GRb1 protects zebrafish larvae against oxidative stress induced by alcohol exposure. (A) Fluorescence micrographs of DHE. Figures are magnified as 100ⅹ. (B) ROS quantification according to the mean intensity of red fluorescence using ImageJ software (n = 6–8 per group). (C) Fluorescence micrographs of NA-8. Figures are magnified as 100ⅹ. (D) GSH quantification according to the mean intensity of blue fluorescence using ImageJ software (n = 6–8 per group). Data are presented as the mean ± SEM. p < 0.05 (#), p < 0.01 (##), and p < 0.001 (###) compared the control group; p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***) compared with the 350 mM EtOH group.

GRB1 reduces the oxidative stress caused by alcohol in L-O2 cells. (A) Fluorescence micrographs of ROS in L-O2 cells. Figures are magnified as 200ⅹ. (B) Fluorescence micrographs of NA-8 in L-O2 cells. Figures are magnified as 200ⅹ. (C) Cellular fluorescence intensity of ROS as measured using a fluorescence microplate reader. (D) Cellular fluorescence intensity of GSH as measured using a fluorescence microplate reader. (E) MDA levels in L-O2 cells. (F) GSH levels in L-O2 cells. The data are displayed as the means ± SEM. p < 0.05 (#), p < 0.01 (##), and p < 0.001 (###) compared the control group; p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***) compared with the 100 mM EtOH group.

GRb1 alleviates neutrophil infiltration of the liver during acute alcohol injury. Neutrophil fluorescence micrographs and quantification of the number of neutrophils in the liver (within the cycle). After alcohol modeling and GRb1 administration, zebrafish larvae with neutrophil-specific EGFP expression were observed and imaged using an Olympus U-HGLGPS fluorescence microscope (Tokyo, Japan). Figures are magnified as 20 and 50ⅹ. The number of neutrophils in the liver was counted using particle analysis in ImageJ software. Data are presented as the mean ± SEM (n = 6–8 per group). p < 0.05 (#), p < 0.01 (##), and p < 0.001 (###) compared the control group; p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***) compared with the 350 mM EtOH group.

GRb1 downregulates the expression of proinflammatory cytokines. (A) NF-κB immunohistochemical staining of zebrafish larvae. Figures are magnified as 400ⅹ. (B) TNF-α immunohistochemical staining of zebrafish larvae. Figures are magnified as 400ⅹ. (C) Statistical analysis of the area of NF-κB expression in the liver of zebrafish larvae using ImageJ software (n = 6–8 per group). (D) Statistical analysis of the area of TNF-α expression in the liver of zebrafish larvae using ImageJ software (n = 6–8 per group). (E) and (F) Real-time PCR analysis of the mRNA levels of TNF-α and NF-κB in zebrafish larvae. The mRNA levels were normalized to β-actin mRNA levels and presented as fold change compared with the control group (n = 3 per group). (G) NF-κB immunofluorescence staining of L-O2 cells slides. Figures are magnified as 200ⅹ. (H) TNF-α immunofluorescence staining of L-O2 cell slides. Figures are magnified as 200ⅹ. (I) Quantitative analysis of NF-κB levels in L-O2 cells according to mean fluorescence intensity using ImageJ software. (J) Quantitative analysis of TNF-α levels in L-O2 cell according to mean fluorescence intensity using ImageJ software. Data are presented as the mean ± SEM. p < 0.05 (#), p < 0.01 (##), and p < 0.001 (###) compared with the control group; p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***) compared with the 350 mM EtOH group or 100 mM EtOH group.

Diagram of the protective mechanisms of GRb1 against alcohol-induced liver injury.