MgIG alleviates liver damage in a mouse model of chronic-binge alcoholic liver disease (the NIAAA model).

(A) Chemical structure of MgIG. (B) Flowchart illustrating the modeling process for the NIAAA model. (C) Representative results of H&E and Oil Red O staining from the livers of mice in Ctrl, A-Ctrl and MgIG groups (n = 5). (D) Alterations in NAS (NAFLD activity score) and Oil Red O quantification (a.v.: arbitrary value) (n = 5). (E) Ratios of liver weight to body weight (LW/BW) in mice (n = 5). (F) Alterations in serum biochemical parameters (ALT, AST and TG) in mice in three groups (n = 5). (G) Alterations in mRNA expression of lipid metabolism genes (Srebp-1c, Srebp2, Acc1, and Scd1), systemic inflammation markers (Tnf-α, Il-6, Il-6, and Il-16), and apoptosis-related genes (Bax and Bcl2) in the mice liver. The data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bar: 50 μm.

MgIG protects ethanol-induced hepatocyte injury in a cell model.

(A) Volcano plot analysis showing differentially expressed genes between the MgIG group (ALD+MgIG) and the A-Ctrl (ALD only) group after RNA-seq in liver of mice. (B) The top 5 regulated pathways (both up- and down-regulated) and the top 3 genes within each pathway are shown. (C) Volcano plot analysis showing differentially expressed genes between the A-Ctrl group and the Ctrl group after RNA-seq in liver of mice. (D) Changes in AML-12 cell viability, apoptosis, and Nile Red staining signals after ethanol/PA treatment, with or without co-treatment with different MgIG doses (0, 0.1, 0.25, 0.5, and 1 mg/ml) (n = 4). (E) Representative images of Nile Red staining in AML-12 cells treated with ethanol/PA, with or without co-treatment with different MgIG doses. (F) Changes in mRNA expression of lipid metabolism genes (Acc1 and Scd1) and systemic inflammation markers (Tnf-α and Il-6) in AML-12 cells treated with ethanol/PA, with or without co-treatment with different MgIG doses. Data are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar: 20 μm.

IDI1 is involved in MgIG-mediated hepatocyte protection against ethanol.

(A) Quantitative PCR validation of RNA-seq results revealed changes in Idi1 gene expression in AML-12 cells treated with ethanol/PA, with or without 0.25 mg/ml MgIG co-treatment (n = 4). (B) Quantitative PCR results confirmed the knockdown efficiency of Idi1 genes by siRNA in AML-12 cells. The changes in AML-12 cell viability following Idi1 siRNA transfection (24 h) were also assessed (n = 4). (C) Quantitative PCR results confirmed the overexpression efficiency of Idi1 genes via plasmid transfection in AML-12 cells. Changes in AML-12 cell viability following Idi1 plasmid transfection (24 h) were also evaluated (n = 4). (D) Changes in cell viability and apoptosis ratios were assessed in ethanol/PA-treated AML-12 cells, with or without 0.25 mg/ml MgIG, following Idi1 knockdown/overexpression (n = 4). (E, F) Western blot results for TNF-α, IL-6, Bax, and Bcl-2 and cell supernatant results for TNF-α and IL-6 in AML-12 cells treated with ethanol/PA and ethanol/PA + MgIG, with or without Idi1 knockdown/overexpression (n = 3). (G, H) Nile Red staining area (%) with corresponding representative cell staining images. Data are expressed as mean ± SD. For Western blot quantification: *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar: 20 μm.

HSD11B1 is the direct binding protein of MgIG for the protection of ethanol-induced hepatocytes injury.

(A) Bar chart displaying the top 10 upregulated and downregulated transcription factor activity scores in the MgIG (ALD+MgIG) group compared to the A-Ctrl (ALD only) group. (B) The glide score of MgIG binding to the protein structure as determined by molecular docking analysis. (C, D) RMSD and RMSF analysis of the three systems: APO-HSD11B1 (blue) represents unbound HSD11B1 in a physiological saline system, Compound-HSD11B1 (red) represents HSD11B1 bound to MgIG in a physiological saline system, and EtOH-HSD11B1 (green) represents HSD11B1 bound to MgIG in a 0.1 mg/mL ethanol solvent system. (E, F) Molecular modeling analysis of MgIG binding at the HSD11B1 domain in normal saline and ethanol systems. Left: Cartoon view of MgIG at the HSD11B1 binding site. Right: Close-up surface view of MgIG at the HSD11B1 binding sites. (G) Hydrogen bond analysis of HSD11B1-MgIG interactions in normal saline and ethanol systems. (H, I) The microscale thermophoresis (MST) assay demonstrated direct binding between varying doses of MgIG and human HSD11B1 protein at residues 187. WT: wild-type HSD11B1; M1, M2, M3: point mutations at Tyr177, Tyr183, and Lys187, respectively.

HSD11B1 is involved in MgIG-mediated hepatocyte protection against ethanol.

(A) Quantitative PCR validation of RNA-seq results revealed changes in Hsd11b1 gene expression in AML-12 cells treated with ethanol/PA, with or without 0.25 mg/ml MgIG co-treatment (n = 4). (B, C) Quantitative PCR confirmed the knockdown efficiency of Hsd11b1 (siRNA) and the overexpression efficiency of Idi1 (plasmid) in AML-12 cells, and cell viability changes were assessed 48 h after transfection (n = 4). (D) Nile Red staining area (%) with corresponding representative cell staining images (n=4). (E, F) Western blot results for TNF-α, IL-6, Bax, and Bcl-2 and cell supernatant results for TNF-α and IL-6 in AML-12 cells treated with ethanol/PA and ethanol/PA + MgIG, with or without Hsd11b1 knockdown/overexpression (n = 3). Data are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar: 20 μm.

MgIG exerts its protective effect via the HSD11B1-SREBP2-IDI1 axis in ALD model.

(A, B) Co-IP was used to verify the direct binding between HSD11B1 and SREBP2 with or without MgIG treatment. (C, D) Alterations in protein levels of HSD11B1, p-SREBP2, n-SREBP2, and IDI1 in AML-12 cells induced by EtOH/PA, following knockdown or overexpression of Hsd11b1, with and without MgIG treatment (n = 3). (E) Immunofluorescent staining was conducted to visualize the distribution and expression of SREBP2 (red) in EtoH/PA-induced AML-12 cells, with or without MgIG, following Hsd11b1 knockdown or overexpression. (F) Co-IP was used to verify the direct binding between HSD11B1 and IDI1. (G) Effects of Srebp2 on Idi1 transcriptional regulation were measured by luciferase assays in AML-12 and 293T cell lines. Idi1-wild-type (WT) or Idi1-mutant (Mut), plasmids with WT promoter cDNA clone of Idi1 or with mutant promoter cDNA clone plasmid; pRL-TK, an internal control reporter plasmid. (H, I) The expression levels of Hsd11b1, Srebp2, and Idi1 in normal liver were measured following the knockdown of Hsd11b1, Srebp2, and Idi1, or the overexpression of Hsd11b1 and Idi1, respectively (n = 4). (J, K) The expression levels of Hsd11b1, Srebp2, and Idi1 in ALD liver were measured following the knockdown of Hsd11b1, Srebp2, and Idi1, or the overexpression of Hsd11b1 and Idi1, respectively (n = 5). Data are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. ****P < 0.0001. Scale bar: 10 μm.

MgIG alleviates ALD-induced liver injury via the HSD11B1-SREBP2-IDI1 axis.

(A-D) Representative liver H&E and Oil Red O staining results from ALD mice with Hsd11b1, Srebp2, or Idi1 knockdown or overexpression, with and/or without MgIG co-treatment. Changes in quantitative NAS (NAFLD activity score) and Oil Red staining (area %) were calculated and analyzed (n = 5). (E-F) Changes of serum ALT and TNF-α from ALD mice with Hsd11b1, Srebp2, or Idi1 knockdown or overexpression, with and/or without MgIG co-treatment (n = 5). (G) Alterations in protein levels of HSD11B1, p-SREBP2, n-SREBP 2, and Idi1 in ALD mice, following knockdown or overexpression of Hsd11b1, with and without MgIG co-treatment (n = 3). Data are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bar: 50 μm.