Metformin significantly mitigates HIRI by reshaping gut microbiota in mice (a) Survival rate analysis (n = 10-12/group), Kaplan Meier was used to analyze statistical differences (Control vs IR, and IR+Met vs IR). (b) Morphological appearance, H&E and PAS staining of liver (n = 3/group). (c) Serum ALT and AST levels. (d) Levels of MDA and GSH in liver tissue. (e) Sobs index, PCoA (n = 4/group). (f) H&E staining of ileum and colon (n = 3/group). (g) Western blot of Occludin in colon (n = 3/group). (h) IFC of ZO-1 and Occludin in colon (n = 3/group). (i) qRT-PCR analysis of Ocln, Tjp1, Cldn7, F11r and Igsf5 in ileum. (j) Il6, Il1b and Il18 in ileum and colon. (n = 3–5/group). (k) FD-4 level in mice serum (n = 3). Data are expressed as mean ± standard deviation. One-way ANOVA was used to analyze statistical differences; *P < 0.05, **P < 0.01 and ***P < 0.001.

Metformin-reshaped fecal microbiota attenuates HIRI (a) Survival rate analysis (n = 10-12/group), Kaplan Meier was used to analyze statistical differences. (b) Serum ALT and AST levels. Levels of MDA (c) and GSH (d) in liver tissue. (e) H&E, PAS and DHE staining of liver (n = 3/group). (f) Alpha diversity and PCoA level of mice microbes. (g) H&E staining of ileum and colon and Western blot analysis of Occludin in colon (n = 3/group). (h) IFC of ZO-1 and Occludin in colon (n = 3/group). (i) qRT-PCR analysis of Ocln, Tjp1, Cldn7, F11r and Igsf5 in ileum, and Il6, Il1b and Il18 in ileum and colon. (n = 3– 5/group). (j) FD-4 level in mice serum (n = 3). (k) ALT, AST and MDA detection between Control and Control + Abx group; survival rate of IR + Met + Abx group (n = 10), Kaplan Meier was used to analyze statistical differences ; and the levels of serum ALT, AST, liver MDA, Fe, and GSH in IR + Met + Abx group. Data are expressed as mean ± standard deviation. One-way ANOVA was used to analyze statistical differences; *P < 0.05, **P < 0.01 and ***P < 0.001.

Metformin alleviates HIRI through inhibiting ferroptosis (a) Levels of Fe in liver tissue (n=6). qRT-PCR analysis of Acsl4 (b) Slc7a11 (c), Slc39a14 (d) and Ptgs2 (e) (n = 5-8/group). (f) Western blot analysis of ACSL4, FTH1, VDAC1, VDAC2, VDAC3, TFR1 and Xct in liver tissue (n = 3/group). (g) Lipid ROS staining and TEM analysis of liver tissues (n = 3/group). (h) Ferroptosis-related transcriptome cluster diagram heatmap. Data are expressed as mean ± standard deviation. One-way ANOVA was used to analyze statistical differences; *P < 0.05, **P < 0.01 and ***P < 0.001.

Metformin induces GABA-producing gut microbiota. (a) KEGG analysis of transcriptome. (b). HPLC-MS/MS of GABA content in liver, ELISA of GABA in liver (n = 6–9/group). (c) Community heatmap analysis on genus level. (d) qRT-PCR analysis of key metabolite enzyme glutamate decarboxylase 1 (Gad1), glutamic acid decarboxylase 2 (Gad2), 4-aminobutyrate aminotransferase (Abat), aldhehyde dehydrogenase family 5 (Aldh5a1) in liver (n = 5-8/group). (e) ELISA of GABA in feces (n = 7–10/group) (f) Western-blot analysis of GAD1 and GAD2 in liver. (g-h) qRT-PCR analysis of GAD and PAT in mice feces (n= 3-5/group). (i) Metagenomic sequencing analysis of human feces. (j-k) The level of GAD, PAT, GABA and Glu in human feces (n= 5-6/group) l) The level of GABA, Glu and putrescine in human feces vitro culture (n= 3/group). Data are expressed as mean ± standard deviation. One-way ANOVA was used to analyze statistical differences; *P < 0.05, **P < 0.01 and ***P < 0.001.

GABA is the critical metabolite of metformin-reshaped gut microbiota against HIRI-induced ferroptosis. (a) Diagram of GABA treatment experiment on mice model. (b) H&E and PAS staining of liver (n = 3/group). (c) Serum ALT and AST levels (n =6/group). Levels of MDA, GSH and Fe in liver tissues (d) (n =4/group). (e) Western blot analysis of ASCL4, FTH1, TFR1 and VADC1,2,3 in liver (n = 4/group). (f) Ferroptosis-related transcriptome cluster diagram heatmap (n = 3-5/group). Data are expressed as mean ± standard deviation. One-way ANOVA was used to analyze statistical differences; *P < 0.05, **P < 0.01 and ***P < 0.001.