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.

GRAPHICAL ABSTRACT

Introduction

With the rising incidence of liver disease, liver transplantation (LT) has become the primary intervention for treating end-stage liver disease.¹ However, the ongoing shortage of donor organs has led to an increased use of marginal donor livers, such as from older adult donors, donations following circulatory death, livers infected with hepatitis viruses, and steatotic donor livers.^2,3 Among these, steatotic donor livers are of particular concern because the growing prevalence of metabolic dysfunction-associated steatohepatitis driven by shifts in dietary patterns underscores the need to optimize their use.^4,5 Unfortunately, owing to structural and functional abnormalities, steatotic livers are particularly vulnerable to ischemia-reperfusion injury (IRI), which increases the risk of perioperative complications during transplantation.^6,7 The effective control of IRI in transplanted livers, particularly in steatotic donor livers, is critical for reducing acute liver injury, preventing transplant failure, mitigating complications, prolonging graft survival, and could expand the pool of usable donor livers and help address the shortage of suitable organs.^8,9 Consequently, addressing IRI, particularly in steatotic donor livers, represents a significant clinical challenge that requires urgent solutions.

Numerous cellular and molecular mechanisms are involved in IRI. Various cell types, such as hepatic sinusoidal endothelial cells, hepatocytes, Kupffer cells, neutrophils, and platelets, contribute to these processes via interconnected molecular pathways, which include the activation of Toll-like receptor signaling, alterations in microRNA expression, production of reactive oxygen species, regulation of autophagy, and activation of hypoxia-inducible factors.^10,11 Recent studies have found that ferroptosis is a newly recognized form of regulated cell death and have confirmed its significant role in driving hepatic and renal IRI. Ferroptosis refers to iron- and lipid peroxidation-dependent cell death, characterized by increased iron accumulation, impaired lipid repair systems, and lipid peroxidation, which activate inflammatory responses and ultimately lead to membrane damage and cell death.^12,13 In LT, the donor liver undergoes both warm and cold ischemic phases, the longer the ischemic time, the more severe the IRI after reperfusion, particularly for steatotic donor livers. However, it remains unclear whether ferroptosis is closely associated with the extent of IRI in donor livers during LT. Currently, only a few studies have suggested that the ferroptosis mechanism may play an important role in LT, with inhibition of ferroptosis alleviating donor liver IRI.^14,15 However, due to the complexity of the donor liver tissue microenvironment, further research is needed to elucidate the mechanisms by which ferroptosis contributes to IRI, especially in steatotic donor livers, and to determine whether targeting ferroptosis could offer a therapeutic strategy for mitigating IRI in liver transplantation.

In recent years, the role of the gut microbiota in host health and disease has attracted significant attention. Studies have shown that dysbiosis of the gut microbiota is closely linked to the onset of various diseases, including inflammatory bowel disease, cancer, obesity, cardiovascular diseases, and diabetes.^16–18 Additionally, interventions targeting the gut microbiota alleviate IRI in the liver, heart and kidneys by reducing apoptosis, ferroptosis, lowering inflammatory responses, and decreasing immune cell recruitment.^19–21 Studies have found that oleanolic acid, produced by gut microbiota, can inhibit ferroptosis and thus prevent fatty liver IRI.²² Additionally, reducing the gut microbiota metabolite trimethylamine N-oxide has been shown to decrease ferroptosis in myocardial cells, alleviating myocardial cell IRI.²³ However, despite ongoing research, the specific effects and mechanisms by which different microbiota alleviate organ transplant-related IRI remain unclear. Lachnospiraceae, a core component of the gut microbiota that is known for its beneficial properties, can ferment various substrates and produce numerous metabolites.²⁴ It reportedly exhibits anti-inflammatory effects, induce immune responses, and helps maintain homeostasis. Moreover, Lachnospiraceae can indirectly influence peripheral organs through the activation of hormonal and neural pathways, presenting significant therapeutic potential.^25–27 Research has shown that supplementing Lachnospiraceae and its products can alleviate intestinal inflammation and rheumatoid arthritis in mice by inhibiting ferroptosis.^28,29 In liver injury studies, Lachnospiraceae has also been shown to suppress ferroptosis, thereby reducing acute liver injury in mice.²¹ Nonetheless, whether Lachnospiraceae can inhibit ferroptosis, inflammatory response and reduce donor livers especially in steatotic donor livers IRI in clinical LT remains to be determined and warrants further investigation.

In this study, we elucidated the mechanisms by which Lachnospiraceae mitigates IRI following LT. Our findings revealed that Lachnospiraceae-bacterium (Lachn.) can alleviate IRI in both normal and steatotic donor liver transplants. Specifically, pyruvate derived from Lachn. suppresses the expression of hepatic forkhead box O3 (FOXO3) and arachidonate 15-lipoxygenase (ALOX15), thereby reducing liver inflammation, susceptibility to ferroptosis, and the infiltration of inflammatory immune cells, ultimately alleviating IRI. Thus, preoperative modulation of Lachn. abundance is a potentially promising therapeutic strategy for mitigating IRI in LT.

Materials and methods

Animal models

Metabolic dysfunction-associated steatotic liver disease (MASLD) model

To establish the MASLD rat model, male rats (Sprague-Dawley), starting at 14 weeks of age, were fed a high-fat methionine-choline-deficient diet (HFD) for two weeks. Control animals received a normal standard diet (ND). All rats were housed in a specific pathogen-free environment, with a maximum of three rats per cage, and had ad libitum access to food and water throughout the experiments. Criteria for successful MASLD model: Each donor liver must be confirmed as having severe fatty liver through both H&E staining and Oil Red O staining. The rats were euthanized by an overdose of isoflurane followed by cervical dislocation prior to liver and serum sample collection.

Liver fibrosis model

The liver fibrosis rat model was induced using subcutaneous injections of CCl₄ in male rats. Each rat received a single injection dose of 2.5 ml/kg (20% concentration in olive oil), administered twice weekly for eight weeks, starting at eight weeks of age. Control animals were injected with saline. Criteria for a successful liver fibrosis model: H&E staining indicates significant hepatocyte damage, chronic inflammatory cell infiltration in adjacent liver tissues, and localized or extensive nodular lesions encapsulated by fibrous septa; Masson staining reveals extensive blue collagen fiber deposition, extending outward from the periportal area, with thick fibrous strands forming pseudo-lobules. The rats were euthanized by an overdose of isoflurane followed by cervical dislocation prior to sample collection.

Rat orthotopic liver transplantation (OLT) model

The OLT procedure was performed on rats (normal and CCl₄ treated rats) at 16 weeks of age. Buprenorphine was administered pre-operatively to both donor and recipient rats. Rats were anesthetized using 50 mg/kg of pentobarbital sodium, and the surgical site was sterilized with a 70% ethanol and betadine solution. The portal vein was skeletonized, and the livers were perfused with 4°C UW solution and excised. After 18 hours of cold storage, the donor livers were implanted into healthy recipient rats. Following reperfusion for six hours, blood samples were collected from the inferior vena cava. The livers were then perfused with normal saline from the portal vein and excised. Each group consisted of at least five rats. To avoid potential variations caused by surgical techniques, all liver transplantation procedures were performed by HC. All animal studies received approval from the Animal Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (No. 2023–3996).

Additional methods are provided in the Supplemental methods.

Results

Increased intestinal permeability and decreased Lachn. abundance in liver cirrhosis rats

Colonic and fecal tissues were collected from 12 pairs of normal and liver cirrhosis (LC) rats, and we observed increased intestinal permeability in LC rats compared to controls, as confirmed by western blotting, electron microscopy (Figure S1A-C), and FITC-dextran (FD4) assays (Figure S1D). Metagenomic analyses of fecal samples revealed significant microbial differences between the two groups at the phylum, genus, and species levels (Figure S1E-H). Notably, fluorescence in situ hybridization (FISH) further validated a reduced abundance of the core intestinal bacterium Lachnospiraceae (Lachn.) and an increase in Prevotella species in LC rats (Figure S1I, Figure S2A).

Further analysis revealed a negative correlation between Lachn. abundance and liver alanine transaminase (ALT) and aspartate transaminase (AST) levels, whereas Prevotella-sp. abundance was positively correlated (Figure S2B). Functional analysis of fecal microbiota revealed enrichment in metabolic pathways (Figure S2C). Untargeted metabolomics of feces identified significant differences in metabolites between groups, particularly those linked to metabolic pathways (Figure S2D, E). Notably, LC rat feces exhibited lower levels of short-chain fatty acids (SCFAs) and organic acids (valeric, pyruvic, and succinic acids), which are known to protect against intestinal permeability and are positively correlated with Lachn. abundance (Figure S2H). These findings suggest that changes in Lachn. abundance may influence intestinal permeability and merit further exploration, especially in the context of IRI following LT in LC recipients.

Lachnospiraceae improves intestinal permeability and alleviates steatotic donor liver IRI

Lachn. have been reported to play a crucial role in modulating host inflammatory responses and immune status.^24–27 To explore whether Lachn. can mitigate IRI post-LT, we administered Lachn. via gavage to LC rats (LC+Lachn.-R). Fecal samples metagenomic and untargeted metabolomic analysis revealed significant changes in microbial composition after Lachn. intervention, including increased Lachn. abundance and reduced Prevotella-sp species (Figures 1a, Figure S3A-E). Furthermore, gut microbiota functions were enriched in metabolic pathways (Figure S3F). FISH analysis confirmed the increased abundance of Lachn. and decreased Prevotella in colon tissues of LC+Lachn.-R rats (Figure 1b). Electron microscopy, western blotting, tissue and cellular polymerase chain reaction (PCR), and FITC-dextran (FD4) assays confirmed that intestinal epithelial cell alignment was tighter and permeability significantly improved in LC+Lachn.-R rats compared to LC rats (LC-R) (Figures 1c-e, Figure S3G, H).

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.

To evaluate IRI in LT, both normal and steatotic donor livers (normal diet-donor and high fat diet-donor, ND-D and HFD-D) were used. Hematoxylin and eosin (H&E) staining, TUNEL assay, and ELISA showed that Lachn. intervention significantly reduced ALT and AST levels, alleviating donor liver IRI, particularly in steatotic donor livers (Figures 1f, g, Figure S3I). Additionally, inflammatory cytokines were markedly reduced in transplanted livers (Figure S4A, B). Western blotting and PCR confirmed decreased levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6. Furthermore, significant downregulation of C-X-C motif chemokine ligand 2 (CXCL2), high mobility group box 1 (HMGB1), and monocyte chemoattractant protein-1 (MCP1) expression was observed in both the liver tissues and portal vein serum. Liver tissues immunofluorescence staining showed significant reduction in neutrophil (CD11b) and macrophage (CD68) infiltration, particularly in steatotic donor livers (Figures S4C, D). Finally, survival analysis showed that LC+Lachn.-R rats receiving either normal or steatotic donor livers had significantly higher survival rates 1 week post-LT compared to LC-R rats, with a more pronounced effect in transplants involving steatotic donor livers (Figure 1h, i). Therefore, these findings indicate that Lachn. enhances intestinal permeability, reduces inflammation in transplanted livers, and alleviates IRI in both normal and steatotic donor livers.

Validation of the mitigating effect of Lachn. in steatotic donor LT IRI in antibiotic-treated liver cirrhosis (LC+ABX) rats

To further explore the role of Lachn. in mitigating IRI in LT, we established an LC+ABX rat model and administered Lachn. intervention. Similar to the findings in LC+Lachn.-R rats, Lachn. treatment in LC-ABX rats (LC-ABX+Lachn.-R) altered gut microbiota composition, with increased Lachn. abundance and decreased Prevotella levels (Figure 2a, Figure S5A-D). Untargeted metabolomics revealed changes in microbial metabolic functions. Intestinal tissue analysis via FISH, electron microscopy, western blotting, FD4, and PCR showed that Lachn. intervention improved intestinal epithelial integrity and significantly enhanced gut permeability (Figure 2b-e, Figure S5 F).

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.

Regarding IRI in transplanted livers, H&E, TUNEL, and ELISA assays demonstrated Lachn. substantial mitigation of IRI, with a pronounced protective effect on steatotic donor livers (Figure 2f-h, Figure S5G, H). Inflammatory marker analysis in both liver tissues and portal vein serum revealed that Lachn. significantly reduced TNF-α, IL-6, CXCL2, HMGB1, and MCP-1 expression (Figure S6A-C). Additionally, Additionally, neutrophil and macrophage infiltration into transplanted livers was notably decreased following Lachn. treatment (Figures S6D, E). Survival analysis of LC-ABX rats subjected to LT revealed that Lachn.-treated rats, particularly those receiving steatotic donor livers, had significantly improved survival rates (Figures 2i,j). These results confirm that Lachn. can improve gut permeability and significantly attenuate IRI in transplanted livers.

Pyruvate acid from Lachn. enhances intestinal permeability and mitigates steatotic donor liver IRI

To identify the metabolites from Lachn. that alleviate IRI post-LT, we performed untargeted metabolomic analysis of fecal samples from normal (NC-R), LC-R, LC+Lachn.-R, LC-ABX-R, and LC-ABX+Lachn.-R rats. Following Lachn. intervention, increased Lachn. abundance was accompanied by elevated levels of butyrate, pyruvate, and valeric acid (Figure 3a, Figure S7A). Given the potential roles of these metabolites in intestinal barrier repair and anti-inflammatory responses, we treated LC rats with butyrate, pyruvate, and valeric acid to identify the active metabolite in Lachn.. FISH, electron microscopy, western blotting, FD4, and PCR assays revealed that pyruvic acid, but not butyrate or valeric acid, significantly restored epithelial tight junction integrity, improved intestinal permeability, and enhanced Lachn. abundance (Figure 3b-e, Figure S7B).

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.

In LT models using both normal and steatotic donor livers, pyruvic acid intervention significantly reduced the post-transplantation serum ALT and AST levels (Figure S7C). Histological analysis with H&E staining and TUNEL assays confirmed a substantial reduction in IRI, particularly in steatotic donor livers (Figure 3f-h, Figure S7D). Additionally, pyruvic acid also significantly reduced liver and serum levels of inflammatory markers, including TNF-α, IL-6, CXCL2, HMGB1, and MCP-1 (Figure S7E, Figure S8A-C). Furthermore, neutrophil and macrophage infiltration into the transplanted liver was significantly reduced following pyruvic acid treatment (Figure S8D). These results demonstrate that pyruvate from Lachn. improves gut permeability, reduces inflammation, and alleviates IRI in both normal and steatotic donor livers.

Validation of pyruvate acid from Lachn. in enhancing intestinal permeability and mitigated steatotic donor liver IRI

To further confirm that pyruvate acid derived from Lachn. improves intestinal permeability and mitigates IRI during LT, we administered butyrate, pyruvate, and valerate to LC-ABX rats. Intestinal permeability revealed that pyruvate acid significantly tightened epithelial cell junctions (Figure 4a-c, Figure S9A-C). In LT models, pyruvate treatment led to marked reductions in ALT and AST levels, along with decreased IRI severity, compared to butyrate and valerate interventions. Furthermore, pyruvate also significantly lowered the expression of inflammatory markers, including TNF-α, IL-6, CXCL2, HMGB1, and MCP-1, in both the transplanted liver and serum at the gene and protein levels (Figure 4d, e, Figure S9 D, E, Figure S10A-D). Additionally, pyruvate treatment significantly reduced neutrophil and macrophage infiltration in transplanted livers, particularly in steatotic donor livers (Figures 4e, Figures S10E). These findings suggest that pyruvate acid from Lachn. improves intestinal permeability and substantially alleviates IRI, with more pronounced effects observed during steatotic donor LT.

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.

Pyruvate acid mitigates ferroptosis in transplanted liver IRI by inhibiting ALOX15 expression

To investigate the mechanisms by which Lachn. alleviates IRI in LT, we performed transcriptomic and untargeted metabolomic analyses of both normal and steatotic donor livers post-transplantation. Lachn. intervention led to significant suppression of Alox15 expression in both normal and steatotic donor livers, while pyruvate levels were markedly elevated and inversely correlated with Alox15 expression (Figure 5a, Figure S11A, B, Figure S12A, B). Alox15 is known to play a pivotal role in inflammation and ferroptosis by regulating lipid metabolism and inducing oxidative stress. Further validation of the relationship between Alox15 and LT-induced IRI was performed using immunofluorescence analyses of transplanted livers from NC-R, LC-R, LC+pyruvate acid-R, LC-ABX-R, and LC-ABX+pyruvate acid-R. A significant reduction in 4-hydroxy 2-nonenal (4-HNE) levels and an increase in glutathione peroxidase 4 (GPX4) expression were noted in both normal and steatotic donor livers from rats treated with Lachn. and pyruvate acid, with the effects being particularly pronounced in steatotic livers (Figure 5b, c, Figure S12C-E).

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.

In vitro, pyruvate treatment in HepG2 cells, primary rat hepatocytes, and steatotic cell models confirmed its regulatory role on Alox15 expression and ferroptosis. Western blotting revealed that pyruvate treatment (0, 5, and 10 mm) upregulated GPX4 and ferroptosis suppressor protein 1 (FSP1), while inhibiting Alox15, lysophosphatidylcholine acyltransferase 3 (LPCAT3), and acyl-CoA synthetase long chain family member 4 (ACSL4) (Figure 5d, e, Figure S13A-D). Finally, manipulation of Alox15 expression through knockdown and overexpression in an IRI cell model confirmed the interaction between pyruvate acid and ALOX15 in regulating ferroptosis. Alox15 knockdown significantly reduced ferroptosis and inflammatory marker expression in both normal and steatotic hepatocytes, whereas Alox15 overexpression had the opposite effect. Notably, the biological effects of Alox15 overexpression were mitigated by exogenous pyruvate acid treatment (Figure 5f, g, Figure S13E-G). These findings suggest that pyruvate from Lachn. mitigates ferroptosis and inflammation in LT-induced IRI by inhibiting Alox15 expression.

Inhibition of ALOX15 reduces ferroptosis and mitigates IRI in steatotic donor liver

To validate the role of Alox15 in LT-induced IRI, LC rats were treated with the ALOX15 inhibitor ML351 (10 mg/kg/day) for 7 days before transplantation. H&E staining and TUNEL assay revealed a marked reduction in IRI in both normal and steatotic donor livers following ML351 treatment (Figure S14A, B). ELISA confirmed decreased serum ALT, AST, HMGB1, and IL-6 levels (Figure S14C, D). Additionally, ML351 treatment also reduced neutrophil and macrophage infiltration and suppressed ferroptosis in liver grafts (Figure S14E, F). Western blot analysis further corroborated these findings, demonstrating that ML351-mediated inhibition of ALOX15 effectively reduced ferroptosis and inflammation, thus mitigating IRI, particularly in steatotic donor livers (Figures S14G, H).

To further explore the role of ALOX15 in ferroptosis and IRI, prophylactic Alox15 knockdown was performed in both donor livers (normal and steatotic) and LC recipients before LT. H&E staining and TUNEL assay revealed significantly reduced graft injury in both normal and steatotic donor livers after prophylactic Alox15 knockdown. Notably, the recipient prophylactic Alox15 knockdown further attenuated graft damage (Figure S15A, B). ELISA results confirmed that Alox15 knockdown led to a significant decrease in postoperative ALT, AST, HMGB1, and IL-6 levels (Figure S15C, D). Reduced inflammatory cell infiltration and ferroptosis were observed in liver grafts with Alox15 knockdown, particularly in steatotic donor livers (Figures S15E, F). Western blot analysis further supported these findings, showing a substantial decrease in ferroptosis and inflammation, highlighting the enhanced protective effect in both normal and steatotic donor livers (Figure S15G, H). These results demonstrate that inhibition of Alox15, either through pharmacological intervention with ML351 or genetic knockdown, significantly mitigates IRI by reducing ferroptosis and inflammation. Pyruvate acid from Lachn. further amplifies this effect by downregulating ALOX15, providing effective protection in both normal and steatotic donor livers.

Reduced nuclear translocation of transcription factor FOXO3 inhibits ALOX15 expression

To elucidate the upstream regulatory mechanisms of Alox15, we observed coordinated changes in the transcription factors Foxo3 and Alox15 following intervention with Lachn.; and Foxo3 exhibited an inverse relationship with pyruvate acid levels (Figure S13 G). FOXO3 reportedly plays a complex dual role in IRI, potentially promoting cell death and exacerbating IRI in the liver.³⁰ To investigate the regulatory relationship between Foxo3 and Alox15, in vitro IRI cell model, pyruvate acid treatment significantly reduced the expression of both Foxo3 and Alox15 (Figure 6a, b). Moreover, knockdown of Foxo3 decreased ALOX15 expression in both normal and IRI conditions, whereas Alox15 knockdown did not influence FOXO3, indicating that FOXO3 regulates ALOX15 (Figure 6c, d).

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.

To further verify whether Foxo3 regulates Alox15 expression by binding to the Alox15 promoter, a dual-luciferase reporter assay demonstrated that FOXO3 activates the Alox15 promoter, particularly under IRI conditions, an effect attenuated by pyruvate acid treatment (Figure 6e). Using the JASPAR analysis, we identified four potential FOXO3 binding sites on the Alox15 promoter, two of which (P1 and P2) were validated using ChIP-qPCR (Figure 6f, g). Mutation of these binding sites further validated that FOXO3 directly binds and modulates Alox15 promoter activity (Figure 6h, i). To explore the mechanism by which FOXO3 regulates Alox15 expression, we analyzed the cytoplasmic and nuclear fractions of cells under both normal and IRI conditions. Pyruvic acid treatment reduced the nuclear translocation of FOXO3, and knockdown of Foxo3 also led to a significant decrease in its nuclear content (Figure 6j, k). These findings indicate that pyruvic acid derived from Lachn. inhibits the nuclear translocation of FOXO3, leading to the suppression of Alox15 activity. This regulatory mechanism reduces inflammation, lipid peroxidation, and ferroptosis, thereby mitigating IRI during liver transplantation.

Elevated Lachn. abundance associated with alleviated liver IRI in LT recipients

To clinically validate the association between Lachn. abundance and IRI in transplanted livers, we collected stool, serum, clinicopathological features, and LT specimens from 36 patients with LC undergoing LT (Table S1–2). Preoperative stool samples were analyzed via metagenomic sequencing and categorized into high and low Lachn. abundance groups (Relative Lachn. median abundance = 0.0000435). Patients with high Lachn. abundance exhibited significant reductions in postoperative ALT, AST, total bilirubin (TBIL), direct bilirubin (DBIL), white blood cell count, and neutrophil percentage on days 1, 3, and 7, especially in the steatotic donor liver (Figure 7a, Figure S16A). Untargeted fecal metabolomics and serum analysis revealed elevated pyruvate acid levels in both the stool and serum of those with high Lachn. abundance, which positively correlated with Lachn. levels (Figure 7b, c, Figure S16B).

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.

Furthermore, serum levels of inflammatory factors such as TNF-α, IL-6, CXCL2, HMGB1, and MCP-1 were significantly lower in high Lachn. abundance patients post-transplant, particularly in the steatotic donor liver recipients (Figure 7d, Figure S16C). H&E staining and TUNEL assay confirmed a marked reduction in IRI in both normal (high, n = 13; low, n = 14) and steatotic donor livers (high, n = 5; low, n = 4) from recipients with high Lachn. abundance (Figures 7e-g). These results were further supported by decreased hepatocyte ferroptosis and neutrophil and macrophage infiltration in transplanted livers from the high Lachn. group (Figure S16 D, E).

Protein analysis revealed significant reductions in ALOX15, FOXO3, ASCL4, TNF-α, and IL-1β levels in both normal and steatotic donor livers of recipients with high Lachn. abundance, suggest ing that Lachn. attenuates inflammation and ferroptosis by inhibiting FOXO3 and ALOX15 expression, thereby mitigating IRI, especially in steatotic donor livers (Figure 7h, i). Clinical outcomes further supported these findings, with high Lachn. abundance recipients experiencing fewer perioperative complications and a shorter hospital stay compared to those with low Lachn. abundance (Figure S16F, G). These results suggest that Lachn. can alleviate inflammation and IRI in LT recipients, improving clinical outcomes. Therefore, Lachn. has the potential to become a promising therapeutic target, particularly for the treatment of IRI in steatotic donor liver transplantation.

Discussion

To the best of our knowledge, this study is the first to systematically examine how the gut microbiota Lachn. influences intestinal permeability in LC and mitigates IRI in both normal and steatotic donor livers. The primary mechanism involves the suppression of FOXO3 and ALOX15 expression in transplanted livers by pyruvate produced by Lachn., which subsequently reduces inflammatory responses and ferroptosis. Notably, decreased FOXO3 expression reduced its nuclear translocation and binding to the Alox15 promoter, thereby inhibiting ALOX15 activity and alleviating donor liver IRI. These findings suggest that modulating Lachn. abundance in the gut microbiota of patients with LC could be a promising strategy for managing IRI post-LT and expanding the donor pool.

In this study, we observed significant differences in the abundance of gut microbiota related to intestinal permeability in the fecal samples of LC rats. Specifically, there was a decrease in Lachn. and an increase in Prevotella-sp. levels. Notably, supplementation with Lachn. not only reduced Prevotella-sp abundance but also significantly improved intestinal permeability. A recent study identified an increased abundance of Prevotella-sp. species in individuals with rheumatoid arthritis, periodontitis, metabolic disorders, and gut dysbiosis.³¹ The pathogenicity of Prevotella-sp. is associated with the transition of commensal bacteria into pathogens, facilitated by factors such as adhesins, hemolysins, secretion systems, exopolysaccharides, lipopolysaccharides, proteases, quorum-sensing molecules, and antibiotic resistance, all of which can compromise the intestinal barrier.^32,33 However, Prevotella-sp. is sometimes beneficial, particularly in those with fiber-rich diets, where it helps ferment plant fibers into short-chain fatty acids that promote gut health.³⁴ Our findings suggest that in rats with LC, Prevotella-sp. abundance is associated with increased intestinal permeability, whereas Lachn. supplementation reduces Prevotella-sp. levels and improves gut barrier function. This indicates a competitive interaction between Lachn. and Prevotella-sp. in maintaining the integrity of intestinal barrier.

Notably, in this study, we identified a wide variety of metabolites produced by Lachnospiraceae, such as alcohols, ketones, pyrazines, short (C2–C5) and long (C > 5) chain acids, phenols, aldehydes, and 30 other compounds including the well-known metabolite butyrate.^24,35,36 Lachnospiraceae and its metabolite butyrate and butyrate salts have been implicated in multiple diseases.^23,37,38 Wang et al.³⁹ found that melatonin can increase the levels of Lachnospiraceae_NK4A136 and butyrate, alleviating hippocampal inflammation and neuronal apoptosis through the crosstalk between the toll-like receptor 4/nuclear factor kappa B and monocarboxylate transporter 1/histone deacetylase 3 signaling pathways. Similarly, Huang et al.⁴⁰ demonstrated that increased Lachnospiraceae abundance and butyrate esters levels could enhance the gut barrier function and inhibit the translocation of bacteria-derived lipopolysaccharides, thereby reducing placenta-derived inflammation. However, our study is the first to show that pyruvate, not butyrate, derived from Lachnospiraceae exerts a protective effect in controlling hepatic IRI. Pyruvate is a 3-carbon compound found in human tissues and is physiologically used by cells as an energy substrate under anaerobic conditions; however, it has rarely been studied in the context of organ IRI. Sileri et al.⁴¹ reported that supplementing rats with pyruvate before liver ischemia-reperfusion (I/R) treatment reduced apoptosis and alleviated I/R injury. Additionally, Cicalese et al.⁴² evaluated the efficacy of pyruvate in organ preservation and transplantation, and found that oral pyruvate could reduce allograft rejection, inhibit perforin and granzyme-b expression, and decrease leukocyte infiltration. Nonetheless, the source of pyruvate and the mechanism by which it alleviates IRI after LT remain unclear. Our study is the first to show that pyruvate derived from Lachn. can inhibit Alox15 expression in the transplanted liver, suppress the inflammatory response and ferroptosis, and mitigate IRI.

Following Lachn. supplementation, we observed a significant reduction in Alox15 expression in both normal and steatotic donor livers. ALOX15 is a member of the lipoxygenase family and is involved in lipid metabolism and inflammatory responses. Its expression is closely associated with tissue inflammation and oxidative stress. ALOX15 primarily induces tissue inflammation and oxidative stress via ferroptosis.^43,44 Previous studies have highlighted the sensitivity of ALOX15 to ferroptosis during tissue IRI. For instance, Ma et al.⁴⁵ demonstrated that ALOX15 acts as a “burning point” during myocardial ischemia, igniting phospholipid oxidation and converting it into ferroptotic signals, thereby exacerbating myocardial cell IRI. Cai et al.⁴⁶ confirmed that ALOX15 expression is specifically increased in injured areas and that myocardial-specific knockout of ALOX15 can reduce the expression of the ferroptosis trigger 15-HpETE, thereby alleviating I/R injury in mice. Jia et al.⁴⁷ also confirmed that wogonin reduces lipid peroxidation and ferroptosis by regulating ALOX15 and iNOS, thereby alleviating hepatic IRI in rats and improving survival rates. In our study, Alox15 expression significantly increased after transplantation of both normal and steatotic donor livers, which contributed to ferroptosis. However, Lachn. supplementation reduced ferroptosis and IRI. Additionally, we found that, during IRI, Lachn. inhibited the expression of the transcription factor FOXO3, which worked synergistically with Alox15 to reduce injury. A previous study reported that FOXO3 promotes IRI by inducing inflammatory responses, apoptosis, autophagy, mitosis, pyroptosis, and oxidative damage.³⁰ However, FOXO3 expression and function vary across different tissue and organ IRIs. In the kidneys, enhanced FOXO3 expression is associated with reduced apoptosis and inflammation in renal tissue, whereas in the heart, liver, and brain tissues, increased FOXO3 expression is associated with increased apoptosis and inflammatory responses.^48–51 Our findings further support the notion that inhibiting FOXO3 can suppress Alox15 expression, thereby mitigating IRI in transplanted livers.

There are several limitations to our study. First, it primarily relies on a rat LT model. To enhance the reliability of our findings, validation using mouse LT models is necessary. Second, in the clinical validation of the mechanism, the sample size we included was insufficient. Considering variations in preoperative treatment regimens and gut microbiota among different populations, further studies with larger sample sizes are required. Lastly, our research is still at the experimental stage, and clinical trials are needed to facilitate its clinical translation. However, our study still has important clinical implications. In this study, we identified for the first time that pyruvate derived from Lachn. can suppress Foxo3 and Alox15 expression, inhibit the ferroptosis signaling pathway, and alleviate IRI in both normal and steatotic donor livers. Lachn. may represent a promising potential therapeutic target for preoperative strategies to mitigate IRI in LT.

In summary, this study demonstrated that supplementation with the gut bacterium Lachn. can restore intestinal barrier function. Additionally, Lachn.-derived pyruvate inhibited the expression of FOXO3 and ALOX15, resulting in reduced inflammation and ferroptosis in both normal and steatotic donor livers. Further reduction in FOXO3 expression amplified the inhibition of Alox15 activity. Thus, preoperative maintenance of Lachn. abundance may represent a promising therapeutic strategy to mitigate IRI in donor livers post-LT, particularly in those with steatosis. Nonetheless, the safety and efficacy of this approach warrants further clinical investigation.

Funding Statement

The study was supported by grants from the National Natural Science Foundation of China [No. 82130020, 82470689, 82403472 and 82072645], Hubei Provincial Natural Science Foundation Key Program [No. 2023AFA029], and Shanxi Basic Research Program (Free Exploration) [No. 202203021211042].

Disclosure statement

No potential conflict of interest was reported by the author(s).

Author contributions

SHD, HC, TXL and XLW contributed equally to this work. SHD, HC, TXL, XLW, DL and JYG conceived and designed the study. SHD, HC, TXL, XLW, DZ, JPM, TZ, SHZ, GLW, THZ, RL and MC search literature and collection of clinical samples. SHD, HC, TXL and XLW completed basic experiments and wrote the manuscript. DL and JYG reviewed and edited the manuscript. DL and JYG are the correspondent authors. All authors read and approved the final manuscript.

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request. The RNA and Metagenomic sequencing data generated in this study are publicly available in NCBI SRA database at PRJNA1209410, PRJNA1191191, and PRJNA1183580.

Ethics approval

The study protocol was reviewed and approved by the Ethics Committee and institutional Review Committee of Wuhan Union Hospital (No. UHCT-IEC-SOP-007-02-05).

All animal studies were conducted in accordance with the guidelines approved by the Animal Experimental Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology, China (No. 2023–3996).

Patient consent for publication

Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/19490976.2025.2460543.

Abbreviations

AST

Aspartate transaminase

ALOX15

Arachidonate 15-lipoxygenase

ALT

Alanine transaminase

CXCL2

C-X-C motif chemokine ligand 2

DBIL

Direct bilirubin

FD4

Fluorescein isothiocyanate dextran

FISH

Fluorescence in situ hybridization

HFD

High-fat methionine-choline-deficient diet

HFD-D

High fat diet-donor

HMGB1

High mobility group box 1

FOXO3

Forkhead box O3

GPX4

Glutathione peroxidase 4

IL6

Interleukin-6

IRI

Ischemia-reperfusion injury

Lachn

Lachnospiraceae-bacterium

LC

Liver cirrhosis

LT

Liver transplantation

LC+ABX

Antibiotic-treated liver cirrhosis

MASLD

Metabolic dysfunction-associated steatotic liver disease

MCP1

Monocyte chemoattractant protein-1

NC-R

Normal receptor

ND

Normal standard diet

ND-D

Normal diet-donor

OLT

Orthotopic liver transplantation

TBIL

Total bilirubin

TNF-α

Tumor necrosis factor-α