MgIG ameliorate acute alcohol induced-hepatic steatosis in zebrafish Larvae. a 5 dpf larvae were treated as indicated for 32 h and stained with whole-mount oil red O. The typical pictures of each group were shown. Arrows point to the liver. b Hepatic steatosis was defined as three or more lipid droplets deposition in liver. Bar chart indicates the percentage of larvae with steatosis (n = 97–223 each group; *P < 0.05 by one-way ANOVA).c H&E staining of paraffin sections through the livers of larvae treated as indicated. Clear cytoplasmic lipid droplets were seen in livers from larvae fed with alcohol. MgIG treatment decreased lipid droplets and ameliorated hepatic steatosis in alcohol-treated zebrafish larvae. d TC levels in the livers of zerafish larvae in each group. e TG levels in the livers of zerafish larvae in each group. (n = 50–60 each group; *P < 0.05, **P < 0.01, ***P < 0.001, by one-way ANOVA)

MgIG ameliorate acute alcohol induced-hepatic steatosis in zebrafish Larvae. a Oil red O staining of frozen sections through the livers of larvae treated as indicated. Clear lipid droplets were seen in livers from larvae fed with alcohol. MgIG and NAC treatment decreased lipid droplets and ameliorated hepatic steatosis in alcohol-treated zebrafish larvae. b Oil red O staining of frozen sections through the livers of zebrafish larvae. We categorized “Normal”, “Medium”, or “Severe” by varying degrees of lipid deposition in the livers of zebrafish by frozen liver sections. Representative images of “Normal”, “Medium”, or “Severe” were shown (× 400 magnification). c Quantification of steatosis is categorized “normal”, “medium”, or “severe”. Bar chart indicates the percentages of each category in each group, and the percentages of larvae in “normal” category is noted (n = 9–11 each group)

MgIG ameliorate acute alcohol induced-hepatomegaly in zebrafish Larvae. a Tg(lfabp10a:eGFP) zebrafish larvae were treated as indicated for 32 h and the typical pictures of each group were shown. Arrows point to the liver. Control group: 0% alcohol; EtOH group: 350 mM alcohol; EtOH + MgIG(0.1) group: 350 mM alcohol + 0.1 mg ml⁻¹ MgIG; EtOH + MgIG(0.05) group: 350 mM alcohol + 0.05 mg ml⁻¹ MgIG;EtOH + MgIG(0.01)group: 350 mM alcohol + 0.01 mg ml⁻¹ MgIG; EtOH + NAC(20) group: 350 mM alcohol + 20 µM NAC. b Images of Tg(lfabp10a:eGFP) zebrafish larvae treated with 0% alcohol, 350 mM alcohol, pre-treated with 0.1 mg ml⁻¹ MgIG, 0.05 mg ml⁻¹ MgIG, 0.01 mg ml⁻¹ MgIG, 20 µM NAC and co-exposed with 350 mM alcohol for 32 h. The morphological change of livers in zebrafish larvae were observed. Alcohol-treated larvae developed obvious hepatomegaly compared to the control group after 32 h of exposure, which was visibly ameliorated in MgIG-pretreated larvae. c The liver size was examined by image J software in zebrafish larvae exposed to 0% or 350 mM alcohol and larvae pre-treated with different concentrations of MgIG, 20 µM NAC and then co-exposed with 350 mM alcohol for 32 h. The liver size was normalized to control group. *P < 0.05, **P < 0.01, n.s.: no significant difference, by one-way ANOVA

Effect of MgIG on alcohol-induced ER stress. a The relative atf6, perk, irelα, bip and chop mRNA expression was analyzed by qRT-PCR in the livers of larvae treated with 0% or 350 mM alcohol, 0.1 mg ml⁻¹ MgIG, 350 mM alcohol + 0.1 mg ml⁻¹ MgIG. *P < 0.05, **P < 0.01, n.s.: no significant difference, by one-way ANOVA. b The expression of bip was detected by whole-mount in situ hybridization. The typical pictures of each group were shown. c Quantification of bip expression is categorized “strong”, “medium”, or “weak”. Bar chart indicates the percentages of each category in each group, and the percentages of larvae in “weak” category is noted. *P < 0.05, **P < 0.01, ***P < 0.001, n.s.: no significant difference, by Chi-Square test. d and e Protein expression of bip and chop was examined by western blot in zebrafish larvae exposed to 0% or 350 mM alcohol, 0.1 mg ml⁻¹ MgIG, or 350 mM alcohol + 0.1 mg ml⁻¹ MgIG for 32 h, the degree of protein expression was normalized to β-actin. *P < 0.05, **P < 0.01, ***P < 0.001, by one-way ANOVA

Effect of MgIG on alcohol-induced lipid metabolism dysfunction. a The relative acc1, fasn, hmgcs1, hmgcra, ppar-α, cpt-1, mtp, cd36 mRNA expression was analyzed by qRT-PCR in the livers of larvae treated with 0% or 350 mM alcohol, 0.1 mg ml⁻¹ MgIG, or 350 mM alcohol + 0.1 mg ml⁻¹ MgIG. *P < 0.05, **P < 0.01, ***P < 0.001, n.s.: no significant difference, by one-way ANOVA. b The expression of hmgcs1 was detected by whole-mount in situ hybridization. The typical pictures of each group were shown. c Quantification of hmgcs1 expression is categorized “strong”, “medium”, or “weak”. Bar chart indicates the percentages of each category in each group, and the percentages of larvae in “weak” category is noted. *P < 0.05, **P < 0.01, ***P < 0.001, n.s.: no significant difference, by Chi-Square test. d and e Protein expression of hmgcs1 was examined by western blot in larvae exposed to 0% or 350 mM alcohol, 0.1 mg ml⁻¹ MgIG, or 350 mM alcohol + 0.1 mg ml⁻¹ MgIG for 32 h, the degree of protein expression was normalized to β-actin. *P < 0.05, **P < 0.01, ***P < 0.001, by one-way ANOVA