Lachnospiraceae-bacterium alleviates ischemia-reperfusion injury in steatotic donor liver by inhibiting ferroptosis via the Foxo3-Alox15 signaling pathway

Abstract

Ischemia-reperfusion injury (IRI) is a major obstacle in liver transplantation, especially with steatotic donor livers. Dysbiosis of the gut microbiota has been implicated in modulating IRI, and Lachnospiraceae plays a pivotal role in regulating host inflammatory and immune responses, but its specific role in liver transplantation IRI remains unclear. This study explores whether Lachnospiraceae can mitigate IRI and its underlying mechanisms. We found Lachnospiraceae-bacterium (Lachn.) abundance was significantly reduced in rats with liver cirrhosis. Lachn.-treated rats exhibited improved intestinal permeability, reduced IRI severity in both normal and steatotic donor livers, and decreased levels of neutrophil and macrophage infiltration, and inflammatory cytokines. Multi-omics analysis revealed elevated pyruvate levels in transplanted livers after Lachn. treatment, alongside reduced Alox15 and Foxo3 expression. Mechanistically, Lachn.-derived pyruvate inhibited Alox15 expression and reduced ferroptosis in normal and steatotic donor livers. Furthermore, reduced nuclear translocation of Foxo3 further suppressed Alox15 expression, alleviating IRI, especially in steatotic donor livers. Clinical samples confirmed reduced donor livers IRI in cirrhotic recipients with high Lachn. abundance after liver transplantation. In conclusion, Lachn. alleviates IRI in steatotic donor liver transplantation by inhibiting ferroptosis via the Foxo3-Alox15 axis, providing a potential therapeutic strategy to modulate gut microbiota to alleviate IRI following liver transplantation.

Keywords: Alox15; Lachnospiraceae-bacterium; ferroptosis; liver transplantation; steatotic donor livers.

Graphical abstract

Figure 1.

Lachn. improves intestinal permeability and alleviates steatotic donor liver IRI. (a) heatmap showing changes in the fecal microbiota of liver cirrhosis (LC) rats after treatment with Lachn. (LC+Lachn.). (b) fluorescence in situ hybridization (fish) experiments confirmed changes in Lachn. and Prevotella-sp. in the colon of normal (NC-R), LC-R, and LC+Lachn. rats (LC+Lachn.-R) (scale bar = 20 μm). (c – e) Electron microscopy (c), western blot (d), and FD4 assays (e) assessing intestinal permeability in normal rats, LC-R, and LC+Lachn.-R (scale bar = 2 μm). (f) Hematoxylin and eosin (H&E) staining of liver iri in normal, LC-R, and LC+Lachn.-R following transplantation with normal diet donor liver (ND-D) and high-fat diet donor liver (HFD-D) (scale bar = 20 μm). (g) terminal deoxynucleotidyl transferase dUTP nick end labeling (tunel) assay for liver iri in normal, LC-R, and LC+Lachn.-R post-transplantation with ND-D and HFD-D livers (scale bar = 20 μm). (H,I) survival analysis 1 week post-transplantation with ND-D and HFD-D livers in normal, LC-R, and LC+Lachn.-R. Statistical significance was determined using unpaired Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2.

Validation of the mitigating effect of Lachn. in steatotic donor LT IRI in antibiotic-treated liver cirrhosis (LC+ABX) rats. (a) heatmap illustrating changes in fecal intestinal microbiota following Lachn. intervention in abx-treated rats with liver cirrhosis (LC+ABX+Lachn.). (b) fluorescence in situ hybridization (fish) analysis confirmed the presence and variation of Lachn. and Prevotella-sp. in the colons of rats with LC, abx-treated rats with LC (LC+ABX), and LC+ABX+Lachn (scale bar = 20 μm). (c – e) Electron microscopy (c), Western blotting (d), and fluorescein isothiocyanate (FITC)-dextran assays (e) performed to assess intestinal permeability in LC-R, LC+ABX-R, and LC+ABX+Lachn.-R (scale bar = 2 μm). (f – h) Hematoxylin and eosin (H&E) and terminal deoxynucleotidyl transferase dUTP nick end labeling (tunel) assays to evaluate liver iri following transplantation of normal diet donor livers (ND-D) and high-fat diet steatotic donor livers (HFD-D) in LC-R, LC+ABX-R, and LC+ABX+Lachn.-R (scale bar = 20 μm). (i,j) one-week survival analysis after transplantation of ND-D and HFD-D livers in LC-R, LC+ABX-R, and LC+ABX+Lachn.-R. Statistical significance was determined using unpaired Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ns: not significant.

Figure 3.

Pyruvate acid from Lachn. enhances intestinal permeability and mitigates steatotic donor liver IRI. (a) venn diagram of differential metabolic products post-intervention with Lachn. in rats with liver cirrhosis (LC) and abx-treated rats with LC (LC+ABX). (b) fluorescence in situ hybridization (fish) analysis to assess changes in Lachn. and Prevotella-sp. in the colon of LC rats after intervention with pyruvic, valeric, and butyrate acids (scale bar = 20 μm). (c – e) Electron microscopy (c), western blot (d), and fluorescein isothiocyanate (FITC)-dextran assays (e) to evaluate changes in intestinal permeability in LC rats following intervention with pyruvic, valeric, and butyrate acids (scale bar = 2 μm). (f – h) Hematoxylin and eosin (H&E) and terminal deoxynucleotidyl transferase dUTP nick end labeling (tunel) assays to determine the degree of liver iri and semi-quantitative analyses post-transplantation of ND-D and HFD-D livers in LC rats treated with pyruvic, valeric, and butyrate acids (scale bar = 20 μm). Statistical significance was determined using unpaired Student’s t-test. **p < 0.01, ***p < 0.001, ns: not significant.

Figure 4.

Validation of pyruvate acid from Lachn. in enhancing intestinal permeability and mitigated steatotic donor liver IRI. (a – c) Electron microscopy (a), western blot (b), and fluorescein isothiocyanate (FITC)-dextran assays (c) evaluating changes in intestinal permeability in ABX-treated liver cirrhosis rats (LC+ABX) following intervention with pyruvic, valeric, and butyrate acids (scale bar = 2 μm). (d,e) Hematoxylin and eosin (H&E) and terminal deoxynucleotidyl transferase dUTP nick end labeling (tunel) assays to assess the level of liver iri and semi-quantitative analyses post-transplantation of ND-D and HFD-D liver in LC+ABX rats with pyruvic, valeric, and butyrate acids intervention (scale bar = 20 μm). (f) immunofluorescence analysis of neutrophil and macrophage infiltration in the transplant liver of LC+ABX rats after intervention with pyruvic, valeric, and butyrate acids, following transplantation of ND-D and HFD-D livers (scale bar = 20 μm). Statistical significance was determined using unpaired Student’s t-test. **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: no significant.

Figure 5.

Pyruvate acid mitigates ferroptosis in transplanted liver IRI by inhibiting ALOX15 expression. (a) venn diagram showing transcriptomic and untargeted metabolomic analyses in transplanted livers from normal and steatotic donor livers to normal rats (NC-R), rats with liver cirrhosis (LC-R), lc rats treated with Lachn. (LC+Lachn.-R), abx-treated rats with LC (LC+ABX-R), and Lachn. intervention in ABX-treated rats with lc (Lc+Abx+Lachn.-R). (b,c) tissue immunofluorescence detecting ferroptosis markers in livers from steatotic donors post-IT in NC-R, LC-R, LC+Lachn.-R, LC+ABX-R, and LC+ABX+Lachn.-R (scale bar = 20 μm). (d,e) expression of ALOX15 and ferroptosis markers in IRI model rat primary hepatocytes (RPH) and steatosis rph following pyruvate intervention. (f,g) examination of Alox15 overexpression, knockdown, and pyruvate intervention in RPH and steatosis RPH, assessing inflammatory markers and ferroptosis markers (GPX4, 4-HNE) in an IRI model.

Figure 6.

Reduced nuclear translocation of transcription factor FOXO3 inhibits ALOX15 expression. (a) Western blot analysis assessing the expression levels of FOXO3 and ALOX15 in HepG2 cells under normal culture conditions and in an IRI model following pyruvate intervention. (b) Western blot analysis to assess FOXO3 and ALOX15 expression levels following Foxo3 silencing and pyruvate intervention. (c,d) under normal culture conditions (c) and IRI model (d), examination of FOXO3 and ALOX15 expression levels in HepG2 cells with Foxo3, Alox15 knockdown, and pyruvate intervention (scale bar = 20 μm). (e) Dual-luciferase reporter assay to determine if Foxo3 can regulate Alox15 expression under normal culture conditions and in an iri model. (f) Potential binding sites of transcription factor Foxo3 on the Alox15 promoter. (g) ChIP-qPCR to verify the potential binding sites of Foxo3 on the Alox15 promoter. (H,I) Luciferase assays to revalidate the binding sites following mutation. (j) Western blot analysis of changes in cytoplasmic and nuclear FOXO3 expression levels in HepG2 cells under normal culture conditions and in an iri model with pyruvate intervention. (k) Western blot analysis to examine changes in cytoplasmic and nuclear FOXO3 expression levels following Foxo3 silencing and pyruvate intervention. **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: not significant.

Figure 7.

Elevated Lachn. abundance associated with alleviated liver IRI in LT recipients. (a) changes in alanine transaminase (alt), aspartate aminotransferase (ast), bilirubin, leukocytes, and neutrophil percentages within one week post-transplant in recipients with lc having high and low Lachnospiraceae abundance who received steatotic donor livers. (b) Volcano plot of differential fecal metabolites in liver cirrhosis recipients with varying Lachn. abundance. (c) Serum pyruvate levels in recipients with LC having different Lachn. abundance who received normal and steatotic donor livers. (d) serum inflammatory marker levels in recipients with LC having high and low Lachn. abundance who receive normal and steatotic donor livers. (e – g) Hematoxylin and eosin (H&E) and terminal deoxynucleotidyl transferase dUTP nick end labeling (tunel) assays to assess iri severity and perform semi-quantitative analysis in transplanted livers from normal and steatotic donors to recipients with different Lachn. abundance (scale bar = 20 μm). (h) Western blot evaluating changes in ALOX15 expression, ferroptosis markers, and inflammatory markers in transplanted livers from normal and steatotic donors to recipients with varying Lachn. abundance. Statistical significance was determined using one-way and two-way analysis of variance (anova). **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: not significance.