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. IP, intraperitoneal injections. (C) Representative results of H&E and Oil Red O staining from the livers of mice in Ctrl, EtOH and EtOH+M 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, TG, and TC) in mice in three groups (n = 5). (G) Alterations in liver parameters (TG and TC) in mice in three groups (n = 5). (H) Alterations in mRNA expression of lipid metabolism genes (Srebp1, Srebp2, Acc1,Scd1, Lcn2, and Ldlr), systemic inflammation markers (Tnf-α, Il1b, 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 EOH group and the Ctrl group after RNA-seq in the liver of mice. (B) Volcano plot analysis showing differentially expressed genes between the EtOH+M and the EtOH group after RNA-seq in liver of mice. (C) The top 5 regulated pathways (both up- and down-regulated) and the top 3 genes within each pathway are shown (EtOH+M vs EtOH).. (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 (EtOH+M) (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)Quantification of triglyceride (TG) and total cholesterol (TC) levels in the in the culture supernatant of AML-12 cells treated with EtOH/PA in the presence or absence of increasing concentrations of MgIG (n = 4). (G-H) Changes in mRNA expression of lipid metabolism genes (Srebp1, Acc1,Scd1, Srebp2, Lcn2, and Ldlr) 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 (EtOH/PA and EtOH/PA+M) (n = 4). (B) Quantitative PCR results confirmed the knockdown or overexpression efficiency of Idi1 genes by siRNA or Idi1 plasmid in AML-12 cells. (C) The changes in AML-12 cell viability following Idi1 siRNA or Idi1 plasmid transfection (24 h) were also assessed (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 (EtOH/PA) and ethanol/PA + MgIG (EtOH/PA+M), with or without Idi1 knockdown/overexpression. Numbers above the lanes indicate the mean relative density normalized to the loading control for each group (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 EtOH+M group compared to the EtOH 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 (EtOH/PA and EtOH/PA+M) (n = 4). (B, C) Quantitative PCR confirmed the knockdown efficiency of Hsd11b1 (siRNA) and the overexpression efficiency of Hsd11b1(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 (EtOH/PA) and ethanol/PA + MgIG (EtOH/PA+M), with or without Hsd11b1 knockdown/overexpression. Numbers above the lanes indicate the mean relative density normalized to the loading control for each group (n = 3). (G,H) Impact of MgIG on Hsd11b1 Activity. 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. Numbers above the lanes indicate the mean relative density normalized to the loading control for each group (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. Numbers above the lanes indicate the mean relative density normalized to the loading control for each group (n = 3). Data are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bar: 50 μm.

The impact of 25 mg/kg MgIG on liver injury parameters in mice.

(A) Changes in body weight of mice with or without dietary and alcohol induction, and with or without MgIG treatment (n = 5). (B) Changes of NAS (NAFLD activity score), Oil Red O quantification (area%), with ALD and/or MgIG treatment (25 mg/kg; n = 5). (C) Representative liver H&E and Oil Red O staining results from mice with ALD and/or MgIG (25 mg/kg) treatment. (D) Changes of serum biochemical parameters (ALT, AST and TG) from mice with ALD and/or MgIG treatment (25 mg/kg; n = 5). *P < 0.05, **P < 0.01, ***P < 0.001.

Optimization tests of the concomitant treatment doses of ethanol and palmitic acid (PA) in mouse normal hepatocyte AML-12 cell line, to partially induce alcohol-associated liver disease (ALD) phenotypes.

(A) Changes of cell viability after a series of doses of ethanol PA treatment (n = 4). (B) Changes of cell viability after a series of doses of ethanol treatment (n = 4). (C) Changes of cell viability after different doses of PA and 250 mmol/L EtOH concurrent treatments (n = 4). (D) Representative low-magnification images of Nile Red staining in AML-12 cells treated with ethanol/PA, with or without co-treatment with different doses of MgIG. *P < 0.05, **P < 0.01, ***P < 0.001, ****P <0.0001. Scale bar: 20 μm.

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

(A) Densitometric analysis of inflammatory (TNF-α and IL-6) and apoptotic (Bax and Bcl-2) factors in AML-12 cells with Idi1 knockdown or overexpression following EtOH/PA treatment, with or without MgIG co-treatment. Data are expressed as mean ± SD (n = 3). (B) Representative low-magnification images of Nile Red staining in AML-12 cells with Idi1 knockdown or overexpression, treated with ethanol/PA, with or without co-treatment with different doses of MgIG.

Hsd11b1/Idi1 is involved in ethanol/palmitic acid (PA) concomitantly induced AML-12 hepatocyte damage and MgIG protection.

(A) Changes of cell viability ratio of AML-12 cells treated with ethanol/PA in the absence or presence of 0.25 mg/mL MgIG, and knock-down of specified genes (n = 4). (B) Quantitative PCR validation of RNA-seq exhibited top differentiated genes in AML-12 cells treated with ethanol/PA in the absence or presence of 0.25 mg/mL MgIG (n = 4). (C) Representative low-magnification images of Nile Red staining in AML-12 cells with Hsd11b1 knockdown or overexpression, treated with ethanol/PA, with or without co-treatment with different doses of MgIG. (D) Densitometric analysis of inflammatory (TNF-α and IL-6) and apoptotic (Bax and Bcl-2) factors in AML-12 cells with Hsd11b1 knockdown or overexpression following EtOH/PA treatment, with or without MgIG co-treatment. Data are expressed as mean ± SD (n = 3). (E) Western Blot after Co-IP was used to verify the binding of HSD11B1 with SREBP2. (F) The expression levels of Hsd11b1, Srebp2, and Idi1 in hepatocytes following the knockdown of HSD11B1, SREBP2, and IDI1, respectively (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001, ****P <0.0001.

MgIG directly binds to HSD11B1.

(A) Analysis of radius of gyration of three systems. (B) Solvent accessible surface area (SASA) analysis of the three systems. (C-D) Principal component analysis (PCA) of the three systems. (E) Cross-correlation analysis of three systems. (F) Binding free energy of Compound-HSD11B1 and EtOH-HSD11B1. APO-HSD11B1 represents unbound HSD11B1 in a physiological saline system, Compound-HSD11B1 represents HSD11B1 bound to MgIG in a physiological saline system, and EtOH-HSD11B1 represents HSD11B1 bound to MgIG in a 0.1 mg/mL ethanol solvent system.

(A) Immunofluorescent staining was conducted to visualize the distribution and expression of SREBP2 (red) in EtOH/PA-induced AML-12 cells, with or without 0.25 mg/mL MgIG. (B) Densitometric analysis of HSD11B1, SREBP2 and IDI1 protein levels (normalized to β-actin) in AML-12 cells with Hsd11b1 knockdown or overexpression following EtOH/PA treatment, with or without MgIG co-treatment. Data are expressed as mean ± SD (n = 3). Scale bar: 7.5 μm. EtOH, ethanol. PA, palmitic acid.

Knockdown and overexpression of Hsd11b1/Srebp2/Idi1 do not affect liver function in mice fed a normal diet.

(A) Representative liver H&E and Oil Red O staining results of normal diet mice with knockdown of Hsd11b1/Srebp2/Idi1 or overexpression of Hsd11b1/Idi1. (B) Changes in serum biochemical parameters (ALT and AST) of normal diet mice, with or without Hsd11b/Serep2/Idi1 knockdown or Hsd11b/Idi1 overexpression (n = 4). (C) Validation of knockdown efficiency of Hsd11b1/Srebp2/Idi1 and overexpression efficiency of Hsd11b1/Idi1 in mice fed a normal diet by using Western blot analysis. Scale bar: 50 μm. Si, siRNA knockdown. OE, overexpression.

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

(A-D) Changes of serum AST, TG, TC and TNF-α from ALD mice with Hsd11b1, Srebp2, or Idi1 knockdown or overexpression, with and/or without MgIG treatment (n = 5). (E) Changes in downstream gene expression after Hsd11b1 knockdown and overexpression (n = 5). (F) Changes in the expression of upstream and downstream genes after Srebp2 knockdown (n = 5). (G) Changes in upstream gene expression after Idi1 knockdown and overexpression (n = 5). (H) Densitometric analysis of HSD11B1, SREBP2, and IDI1 protein levels (normalized to β-actin) in livers from ethanol-induced liver injury mice with Hsd11b1 knockdown or overexpression, with or without MgIG co-treatment. Data are expressed as mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P <0.0001. KD, knockdown. OE, overexpression.

Primer sequences information for quantitative real-time PCR assay.