Gut microbiota-derived 3-indoleacrylic acid mediates ferulic acid protection against aflatoxin B1 hepatotoxicity via AhR-ferroptosis inhibition in ducks

Gut microbiota-derived 3-indoleacrylic acid mediates ferulic acid protection against aflatoxin B1 hepatotoxicity via AhR-ferroptosis inhibition in ducks | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Gut microbiota-derived 3-indoleacrylic acid mediates ferulic acid protection against aflatoxin B1 hepatotoxicity via AhR-ferroptosis inhibition in ducks Qianqian Wang, Yanan Wang, Gaigai Wang, Xin Fang, Yutong Fu, Zixin Li, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9281710/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Aflatoxin B1 (AFB1) contamination is a persistent threat to feed safety and duck production because of its potent hepatotoxicity. Although cereal-derived bioactive compounds are considered promising interventions against AFB1-induced liver injury, the microbiome-dependent mechanisms underlying their protective effects remain poorly understood. Here, we aimed to determine whether the cereal-derived polyphenol ferulic acid could alleviate AFB1-induced liver injury in ducks in a gut microbiota-dependent manner and to elucidate the potential mechanism underlying its protective effects. The results show that ferulic acid exerts its hepatoprotective effect in a gut microbiota-dependent manner, as evidenced by antibiotic treatment and fecal microbiota transplantation in ducks. Specifically, dietary ferulic acid enriches Peptostreptococcus anaerobius , enhancing microbial conversion of tryptophan to 3-indoleacrylic acid both in vivo and in vitro . Mechanistically, by combining single-cell RNA sequencing and surface plasmon resonance, we demonstrate that 3-indoleacrylic acid functionally antagonizes aflatoxin B1-induced hyperactivation of the AhR pathway, thereby suppressing AhR-driven ferroptosis. This functional mechanism was validated through genetic knockdown and pharmacological interventions. Crucially, we reveal that 3-indoleacrylic acid ameliorates aflatoxin B1-induced liver inflammation by reprogramming the subset composition and function of macrophages, which was confirmed by macrophage-depletion models. Collectively, our findings unveil a precise molecular mechanism of host-microbe crosstalk, identifying the Peptostreptococcus anaerobius -derived tryptophan metabolite 3-indoleacrylic acid as a critical signaling mediator that inhibits Aflatoxin B1-induced hepatic AhR hyperactivation, ferroptosis, and macrophage reprogramming. This work provides a rationale for microbiome-targeted feed strategies to combat global aflatoxicosis and improve animal health. Biological sciences/Biochemistry Biological sciences/Microbiology Gut microbiota ducks liver injury ferulic acid Aflatoxin B1 3-indoleacrylic acid ferroptosis AhR Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Mycotoxin contamination is a persistent challenge to feed safety and farmed animal production worldwide. It is estimated that 60%–80% of feed ingredients are contaminated with mycotoxins during production or storage, and consumption of contaminated feed by farm animals compromises growth and health, resulting in substantial production losses 1 . Among the diverse mycotoxins, aflatoxin B1 (AFB1) is of particular concern because of its high toxicity and widespread occurrence 2 . Ducks are especially sensitive to AFB1 exposure, which is frequently associated with impaired growth performance, liver injury, and increased morbidity and mortality 3 . Mechanistic studies have confirmed that AFB1 is metabolized into the genotoxic compound aflatoxin-8,9-epoxide (AFBO) through the catalytic activity of cytochrome P450 (CYP450) enzymes in liver, leading to an excessive production of reactive oxygen species (ROS) 4 , 5 . This overproduction of ROS induces hepatic oxidative stress and ferroptosis 6 , 7 . Ferroptotic hepatocytes release damage-associated molecular patterns that activate hepatic macrophages and amplify inflammatory responses through pro-inflammatory cytokine production 8 . Beyond direct hepatic damage, AFB1 disrupts the gut-liver axis by compromising intestinal barrier integrity and microbial metabolic homeostasis including bile acid biotransformation, and short-chain fatty acid synthesis 9 – 11 . The gut microbiota and their metabolites are key mediators of gut-liver axis homeostasis and hepatic function, and their composition and metabolic activity are highly responsive to dietary regulation 12 . In broilers, supplementation with natural plant extracts has been shown to alleviate AFB1-induced liver injury by improving intestinal barrier function and modulating the gut microbiota 13 . Therefore, modulation of the gut microbiota by plant-derived compounds may offer a promising nutritional strategy for mitigating AFB1-induced hepatotoxicity. Whole grains are of particular interest because they are a rich dietary source of fiber and phenolic compounds closely associated with gut microbial metabolism 14 . Among these bioactive constituents, our previous work identified FA as a key compound in whole grains capable of protecting against AFB1-induced hepatic toxicity in mice using untargeted metabolomics and network pharmacology analyses 15 . However, FA exhibits poor oral bioavailability despite its profound systemic hepatoprotection, suggesting its therapeutic effects are likely mediated by indirect mechanisms rather than direct absorption 16 , 17 . While FA is known to modulate bacterial composition in metabolic disorders 18 , whether and how it leverages specific microbial metabolites to remotely orchestrate hepatic cell survival and immune responses remains a critical knowledge gap. In this study, we aimed to elucidate the mechanisms underlying the protective effects of FA against AFB1-induced liver injury in ducks, with a particular focus on the relationships among the gut microbiota, microbial metabolites, and host responses. Specifically, we sought to determine whether the hepatoprotective effects of FA are dependent on the gut microbiota and how FA influences microbial metabolic profiles under AFB1 exposure. We further aimed to identify candidate effector metabolites associated with FA treatment and to clarify how such metabolites regulate host signaling pathways and cellular responses involved in AFB1-induced liver injury. This study may provide mechanistic insight into the microbiota-mediated protective effects of FA and offer a theoretical basis for microbiome-targeted nutritional strategies to mitigate aflatoxicosis in poultry. Results FA alleviated AFB1-triggered liver injury in a gut microbe-dependent manner To study the impact of FA on AFB1-induced liver injury, ducks were administered AFB1 and FA either individually or in combination (AFB1 + FA) for four weeks (Figs. 1 A and 1 B). The results demonstrated that FA supplementation significantly ameliorated the AFB1-induced elevation of serum aminotransferase (AST) and aminotransferase (ALT), which are well-established biomarkers of hepatic damage (Fig. 1 C). Moreover, dietary FA markedly attenuated the AFB1-triggered liver inflammation, as evidenced by reduced infiltration of pro-inflammatory cells into hepatic tissue and decreased mRNA expression of pro-inflammatory cytokines, including IL-1β , IL-6 , and TNF-α (Figs. 1 D and 1 E). It has been reported that AFB1 can disrupt the intestinal barrier and alter gut microbiota composition, thereby facilitating the translocation of intestinal bacteria and their metabolites into the systemic circulation, which subsequently exerts adverse effects on hepatic health 19 . In this work, FA alleviated the AFB1-induced jejunal structural damage (Figures S1 A and S1B), and reversed downregulation of tight junction proteins ZO-1 and ZO-2 in jejunm (Figure S1 C). In addition, FA supplementation attenuated the AFB1-triggered intestinal inflammation by decreasing the expression of pro-inflammatory cytokines IL-6 , IL-8 , and TNF-α in the jejunum (Figure S1 D). The cecal microbiota composition was determined to examine the effects of FA or/and AFB1 on gut microbes by conducting 16S rRNA gene sequencing. Principal coordinates analysis (PCoA) showed a microbial separation among AFB1 group and other groups (Fig. 1 F). The predominant genus of gut microbiota was illustrated in Fig. 1 G. According to the Linear discriminant analysis Effect Size (LEfSe) analysis, Bacteroides , Megamonas , and Succinatimonas were enriched in FA+AFB1 group compared with AFB1 group (Fig. 1 H). Collectively, dietary FA effectively ameliorated hepatotoxicity, intestinal damage and gut microbiota dysbiosis induced by AFB1 in ducks. To further investigate the role of gut microbiota in FA alleviating AFB1-triggered liver injury, we carried out the gut microbiota depletion experiment with antibiotics and the FMT experiment (Figs. 1 A). The results indicated that antibiotics supplementation abolished the protective effects of FA on AFB1-induced elevations of serum ALT and AST (Figs. 1 I and 1 J). Moreover, antibiotic treatment prevented FA from mitigating AFB1-induced liver inflammation, as evidenced by elevating infiltration of pro-inflammatory cells into hepatic tissue and increasing expression of pro-inflammatory cytokines in the ANTI (FA+AFB1) group compared to the FA+AFB1 group (Figs. 1 K and 1 L). Consistent with the observations in the liver, FA had little protective effect on AFB1-induced jejunal damage in antibiotics-treated ducks. Specifically, FA failed to reverse AFB1-induced morphological damage (Figures S1 E and S1F), restore the expression of tight junction protein genes (Figure S1 G), or suppress the upregulation of pro-inflammatory cytokine genes following antibiotic treatment (Figure S1 H). In FMT experiment, the AFB1-originated microbiota induced hepatic damage, evidenced by increased serum AST and ALT activities, histopathological features, and higher hepatic mRNA expression of inflammatory cytokines. These negative effects were effectively attenuated in ducks honored with the FA+AFB1-originated microbiota (Fig. 1 M-P). In addition, AFB1-originated microbiota also impaired intestinal barrier integrity, as demonstrated by decreased villi height and a lower ratio of villi height to crypt depth. Notably, FA+AFB1-derived microbiota ameliorated these morphological alterations and upregulated mRNA expression of ZO-1 and ZO-2 in jejunum of ducks (Figures S1 I-K). Moreover, the lower jejunal mRNA expression of inflammatory genes were observed in FMT-FA+AFB1 group relative to FMT-AFB1 group (Figure S1 L). Distinct separation of cecal microbiota structure in the FMT-AFB1 group compared to other groups was revealed by PCoA analysis (Fig. 1 Q). The composition of the predominant gut microbiota at the genus level was presented in Fig. 1 R. In the FMT-FA+AFB1 group, there was a reduction in the relative abundance of potential pathogenic bacteria like Fusobacterium , Desulfovibrio , and Escherichia-Shigella compared to the FMT-AFB1 group (Fig. 1 S). These results indicated that the protective effects of FA against liver injury triggered by AFB1 were mediated by gut microbiota. FA alleviated AFB1-triggered liver injury by enriching of Peptostreptococcus anaerobius ( P. anaerobius ) and enhancing tryptophan (Trp) metabolism for IA production To investigate how FA restores AFB1-induced hepatic injury by the gut microbiota, we performed untargeted metabolomics analysis of cecal microbial metabolites to characterize functionally relevant microbial-derived metabolites. Principal component analysis (PCA) revealed that AFB1 treatment induced distinct metabolomic profiles in the cecum microbiota compared with CON group, whereas the FA+AFB1 group exhibited metabolomic profiles that largely overlapped with both the FA and CON groups (Fig. 2 A). Volcano plot analysis identified 160 significantly upregulated and 74 downregulated metabolites in FA+AFB1 group versus AFB1 group (Fig. 2 B). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the differential metabolites in the AFB1 and FA+AFB1 groups were mainly enriched in the Trp metabolic pathway, suggesting FA mitigated AFB1-induced hepatotoxicity via microbial Trp metabolic pathway modulation (Fig. 2 C). Follow-up targeted Trp metabolomics showed that FA addition upregulated 20 Trp metabolites and downregulated 9 Trp metabolites (Fig. 2 D). Notably, the contents of indole, IGA, and IA were markedly increased in the FA+AFB1 group versus the AFB1 group, accompanied by an increasing trend in KYNA and XA (Figures S2 A-E). To directly evaluate the protective effects of the five identified Trp metabolites against AFB1-induced liver injury, these metabolites were orally administered to mice daily for four weeks in combination with AFB1 treatment (Fig. 2 E). The results showed that all five metabolites significantly suppressed AFB1-induced body weight loss (Figure S3A) and elevation of serum AST (Fig. 2 F). Moreover, indole markedly decreased the serum ALT activity (Fig. 2 G), while kynurenic acid reduced mRNA expression of IL-1β and TNF-α induced by AFB1 (Fig. 2 H). Notably, IA exerted the most pronounced protective effects, as evidenced by the reduction in liver index and serum AST and ALT activities, together with marked suppression of IL-1β , IL-6 , and TNF-α expression (Figures S3B, 2F-H). Given the superior protective efficacy of IA observed in the screening model, we hypothesized that FA mitigates AFB1 hepatotoxicity primarily by promoting microbial IA production in ducks. To validate this, we quantified serum IA levels in ducks and found that FA supplementation markedly restored IA concentrations in AFB1-exposed ducks. Crucially, this elevation was confirmed to be microbiota-dependent, serum IA levels were significantly higher in ducks receiving the gut microbiota from the FA+AFB1 group compared to ducks receiving the gut microbiota from the AFB1 group, whereas antibiotic treatment abolished the FA-induced increase in serum IA (Figs. 2 I and 2 J). Taken together, these results provide compelling evidence that FA alleviates hepatic injury by facilitating the microbial biosynthesis of IA. To identify the specific bacterial species responsible for this increased IA production, we focused on known IA-producing candidates. Given that P. anaerobius , P. russellii , P. stomatis , and C. sporogenes are known to metabolize Trp into IA 20 , we analyzed their relative abundances in the cecal microbiota of ducks. Notably, P. anaerobius abundance was markedly higher in the FA+AFB1 group than in the AFB1 group (Fig. 2 K). Crucially, a parallel increase in both P. anaerobius abundance and serum IA levels was observed in FA+AFB1 mice relative to AFB1 mice (Figs. 2 L and 2 M). This consistent enrichment across both avian and mammalian models underscores the cross-species conservation of FA’s capacity to promote the P. anaerobius -IA axis. Next, we examined the effect of FA on P. anaerobius in vitro , revealing that FA supplementation significantly enhanced bacterial growth and upregulated the mRNA expression of key enzymes related to IA synthesis (aromatic amino acid aminotransferase (ArAT), phenylacetate dehydrogenase (fldH), phenyllactate dehydratase subunits (fldB, fldC), and acyl coenzyme a dehydrogenase (acdA)), thereby indicating its role in promoting the tryptophan-to-IA metabolic pathway (Figs. 2 N-Q). To directly validate this metabolic capability, we further assessed IA production in bacterial cultures. As anticipated, IA was clearly detected in the supernatants of P. anaerobius , and its production was significantly increased by the supplementation of Trp (Figs. 2 R and 2 S). Collectively, these results demonstrated that FA enhanced P. anaerobius -derived IA biosynthesis through coordinated transcriptional activation of the Trp metabolic pathway. Next, we evaluated the protective effects of oral P. anaerobius administration against AFB1-induced hepatotoxicity in mice (Fig. 2 T). Compared with the AFB1 group, P. anaerobius administration ameliorated liver injury, evidenced by improved liver histopathology, restored hepatic cord architecture, and reduced cellular vacuolization (Fig. 2 U). This was accompanied by decreased serum AST, ALT activities and reduced hepatic mRNA expression of inflammatory cytokines in mice (Figs. 2 V and 2 W). These data demonstrated that FA mitigated AFB1-induced liver injury by enriching intestinal P. anaerobius and enhancing Trp metabolism for IA production. IA alleviated AFB1-induced liver injury by inhibiting ferroptosis of hepatocyte To further explore the mechanism by which IA alleviated AFB1-induced liver injury, mice were allocated to CON, AFB1 or IA+AFB1 group (Figs. 3 A). IA administration ameliorated liver injury in AFB1-exposed mice, as evidenced by attenuated architectural distortion of histomorphology, along with decreased serum ALT and AST activities (Figs. 3 B and 3 C). Through integrated single-cell RNA sequencing analysis, nine major hepatic cell types were identified using uniform manifold approximation and projection (UMAP) dimensionality reduction (Figs. 3 D-F). Figure 3 G showed the composition of hepatic cell types among different groups. There were 341 upregulated genes and 357 downregulated genes in IA+AFB1-treated hepatocytes compared to those exposed solely to AFB1 (Fig. 3 H). KEGG enrichment analysis indicated that ferroptosis was the most significantly enriched pathway within the “Cell growth and death” subcategory of cellular processes (Fig. 3 I). These findings suggest that IA potentially mitigates AFB1-induced hepatotoxicity by suppressing ferroptosis signaling. Based on this evidence, we further investigated whether IA ameliorated AFB1-triggered liver injury by inhibiting ferroptosis. Transmission electron microscopy revealed mitochondrial shrinkage and outer membrane rupture in hepatocytes from AFB1-exposed mice, whereas these ultrastructural alterations were attenuated by dietary IA intervention (Figure. 3J). AFB1-exposed mice exhibited hepatic iron overload, elevated lipid peroxidation (serum malondialdehyde (MDA)), and glutathione (GSH) depletion-all of which are phenotypic hallmarks of ferroptosis. Notably, dietary IA administration reversed these changes (Figs. 3 K-M). IA upregulated mRNA expression of GPX4 (lipid peroxide scavenger) and CISD1 (mitochondrial iron-sulfur cluster protein) in the liver (Fig. 3 N). Consistently, western blot analysis indicated that dietary IA supplementation reversed the AFB1-induced downregulation of hepatic ferroptosis-inhibitory proteins (xCT, HO-1, and GPX4) in mice (Figs. 3 O and 3 P). Collectively, IA was found to inhibit AFB1-induced hepatocyte ferroptosis. Consistent with these results, dietary administration of P. anaerobius elevated serum IA levels compared with the AFB1 group (Figure S4A). Hepatic Fe²⁺ ions accumulation and serum MDA levels were reduced, while the serum concentration of GSH was increased in mice fed with P. anaerobius and AFB1 in comparison with those fed with AFB1 alone (Figures S4B-D). Dietary P. anaerobius administration upregulated mRNA expression of ferroptosis-suppressive genes, while enhancing GPX4 and HO-1 protein levels (Figures S4E-I). These findings showed that P. anaerobius may mitigate AFB1-induced hepatocyte ferroptosis by producing IA in mice. In vitro (Figs. 3 Q), the viability of AML12 cells was notably increased when it was treated with 50, 100 or 200 µM IA for 24 h compared with the untreated control and IA treatment reversed the AFB1-induced decline in cell viability (Figures S5A and S5B). Specifically, IA mitigated lipid peroxidation, activated GSH biosynthesis, cleared excess ROS, and suppressed Fe 2+ ions accumulation (Figs. 3 R-W). IA upregulated the levels of ferroptosis-inhibiting related proteins (Figs. 3 X and 3 Y). Taken together, these findings demonstrated that IA alleviated AFB1-induced liver injury by inhibiting ferroptosis of hepatocyte. To further verify the role of ferroptosis in IA-mediated hepatoprotection, the ferroptosis inhibitor Ferrostatin-1 (Fer-1) was used in the AFB1-exposed mice (Fig. 4 A). Relative to AFB1 exposure, Fer-1 intervention counteracted AFB1-induced elevations of serum AST and ALT, hepatic iron accumulation and lipid peroxidation, while enhanced GSH synthesis; co-treatment with IA and Fer-1 did not synergize these effects (Figs. 4 B-E). Histopathological assessment revealed that Fer-1 treatment suppressed AFB1-triggered hepatocellular vacuolization and disruption of cord architecture, with no additive protective effect observed when IA was co-administrated with Fer-1 (Fig. 4 G). Molecular analyses revealed that Fer-1 inhibited the downregulated expression of iron homeostasis genes ( GPX4 and CISD1 ) and the downregulated levels of xCT, HO-1, and GPX4 induced by AFB1; whereas IA co-administrated with AFB1 and Fer-1 failed to further enhance these regulatory responses (Figs. 4 F, 4 H-K). Collectively, AFB1 induced liver injury through ferroptosis activation, while IA exerted hepatoprotection by specifically inhibiting this cascade. IA mitigated AFB1-induced hepatocyte ferroptosis through an AhR-dependent pathway A recent study demonstrated that the aryl hydrocarbon receptor (AhR) is required for AFB1-induced toxicity 21 . Furthermore, emerging evidence indicates that AhR signaling is tightly linked to the regulation of ferroptosis 22 . Therefore, we investigated whether AFB1 induces hepatocyte ferroptosis via AhR and whether IA alleviates AFB1-induced liver injury by modulating this pathway. It was been observed that AFB1 exposure elevated hepatic mRNA expression of AhR and its downstream effectors ( CYP1A1 and CYP1A2 ), and increased protein levels of AhR and CYP1A1, these upregulations were effectively reversed by IA co-treatment in mice (Figs. 5 A-E). Given that AhR signaling modulates interleukin secretion profiles, particularly its dual regulation of anti-inflammatory IL-10, IL-22 and pro-inflammatory IL-17 23 , we next quantified serum and hepatic levels of these cytokines in mice. IA supplementation promoted the levels of anti-inflammatory cytokines IL-10 and IL-22, while suppressed the level of pro-inflammatory mediator IL-17 in the serum of mice (Fig. 5 F). IA also increased the level of IL-22 in the liver of mice (Fig. 5 G). Previous research has demonstrated both AFB1 and IA can also act as ligands of AhR 24 . In this study, we further compared their binding affinity to AhR using surface plasmon resonance (SPR) analysis, which revealed that AFB1 exhibited a significantly higher affinity than IA to AhR (Figs. 5 H and 5 J). Molecular docking analyses also revealed that AFB1 exhibits stronger binding affinity for AhR compared to IA, with respective -cdocker interaction energies of 34.76 kJ·mol⁻¹ (for AFB1) and 25.24 kJ·mol⁻¹ (for IA). For the AFB1-AhR complex, hydrogen bonds characterized the primary binding region within AhR. In contrast, hydrophobic interactions, classified as weak interaction forces, served as the dominant binding mechanism for the IA-AhR complex (Figs. 5 I and 5 K). This indicated that compared to IA, AFB1 acted as a full agonist of AhR, whereas IA functioned as an AhR antagonist when coexisting with AFB1. Collectively, despite exhibiting lower binding affinity for AhR, IA functionally antagonized AFB1-induced activation of the AhR pathway, ultimately alleviating hepatotoxicity in vivo . To validate the essential role of AhR in IA mitigating AFB1-induced liver injury, CH-223191 (CH) was administered to inhibit AhR signaling in mice (Fig. 5 L). Compared to AFB1 group, CH intervention suppressed AFB1-induced elevations of serum AST and ALT, rebalanced inflammatory homeostasis by downregulating the level of IL-17 in serum and upregulating levels of IL-10 and IL-22 in both serum and liver of mice, while there were no additional effects of IA on CH-treated mice with AFB1 exposure (Figs. 5 M-R). H&E staining revealed that CH treatment inhibited AFB1-triggered hepatic cord derangement and inflammatory infiltration, while IA and CH co-treatment did not enhance this protective phenotype on mice with AFB1 exposure (Fig. 5 S). Given the established role of AhR in ferroptosis regulation 25 , this study examined whether IA counteracted AFB1-induced ferroptosis via AhR blockade. Accumulated Fe 2+ ions deposition, increased serum MDA and depleted GSH levels in the liver of mice exposure to AFB1 were eliminated by CH intervention, but these protective effects did not augment when IA was co-administrated with CH (Figures S6A-C). CH-mediated AhR suppression rescued AFB1-triggered transcriptional repression of ferroptosis inhibitors, and upregulated the levels of xCT, HO-1, and GPX4; IA and CH co-treatment failed to further potentiated these regulatory effects on mice exposure to AFB1 (Figures S6D-L). These findings demonstrated that IA executed hepatoprotection through functional antagonism of AhR-driven ferroptotic signaling. In vitro cell assays demonstrated that CH treatment antagonized AFB1-induced cytotoxicity, whereas CH and IA co-administration conferred no additional viability restoration on cells exposure to AFB1 (Figs. 6 A and 6 B). CH intervention in AFB1 treatment suppressed the AFB1-induced elevation in intracellular MDA and the Fe 2+ ions levels without affecting GSH content; IA co-treatment with CH and AFB1 did not further augmented these changes (Figs. 6 C-F). In addition, The CH intervention inhibited the AFB1-induced upregulation of AhR protein level and downregulation of anti-ferroptotic proteins levels, including xCT, HO-1, and GPX4; IA co-treatment with CH and AFB1 failed to further potentiate these regulatory effects (Figs. 6 G-K). Genetic targeting of AhR validated its essential role in IA-conferred ferroptosis resistance (Fig. 6 L). siRNA screening secured siAhR2 as the optimal construct inducing effective AhR silencing in AML12 hepatocytes (Figs. 6 M and 6 N). AhR knockdown reversed AFB1-induced accumulation of ferroptotic biomarkers (ROS and Fe²⁺ ions), while combined IA treatment failed to potentiate this mitigation (Figs. 6 O-R). Additionally, AhR knockdown in AFB1-treated hepatocytes significantly downregulated AhR protein level and upregulated xCT, HO-1, and GPX4 protein levels compared to the AFB1-treated group; these changes were not further enhanced by IA co-administration (Figs. 6 S-W). Collectively, these results demonstrated that IA executed its hepatoprotective effects exclusively through competitive inhibition of AhR signaling. IA attenuated AFB1-induced hepatic inflammation via macrophage reprogramming Our previous results demonstrated that IA effectively inhibited the infiltration of pro-inflammatory cells into liver tissue induced by AFB1 exposure. Consistent with this finding, IHC staining further confirmed that IA significantly suppressed the AFB1-induced increase in hepatic macrophages in mice (Figs. 7 A and 7 B). Liver macrophages primarily comprise Kupffer cells (KCs) and monocyte-derived macrophages (MoMFs). To elucidate the specific effects of IA on these macrophage subsets, flow cytometry analysis was conducted. AFB1 exposure tended to increase the proportion of MoMFs (F4/80 lo CD11b hi ) compared to the CON group. Conversely, IA treatment significantly increased the KCs frequency and reduced the MoMFs ratio compared to the AFB1-exposed group (Figs. 7 C-E). Given the pivotal role of macrophage subset heterogeneity in modulating liver inflammatory responses and maintaining tissue homeostasis 26 , we further performed subclustering analysis on the KCs population. This analysis identified five distinct KCs subtypes (Fig. 7 F), which were annotated based on the expression of marker genes (Fig. 7 G). The differentially expressed genes in these subpopulations underwent reactome pathway enrichment analysis, the results revealed significant enrichment in the immune system and innate immune system pathways (Fig. 7 I). IA suppressed an AFB1-expanded pro-inflammatory subset (Subcluster4, highly expressed Marco, Cd38, Il1b), while enriching Subcluster0, highly expressed Mrc1, Il10ra, Tgfbr1 (Figs. 7 H, 7 J and 7 K). MoMFs resolved into six subclusters that manually annotated based on the expression of marker genes (Figs. 7 L and 7 M). Reactome pathway enrichment analysis showed that the immune system and innate immune system pathways were also enriched (Fig. 7 O). When we compared IA+AFB1 group with AFB1 group, IA suppressed a pro-inflammatory MoMFs subset (Subcluster0, highly expressed Cxcl9, Cxcl10, Nfkb1) and expanded an anti-inflammatory MoMFs subset (Subcluster1, highly expressed Mertk, C1qa, Abcg1) (Figs. 7 N, 7 P and 7 Q). Collectively, these results demonstrated that IA ameliorated AFB1-induced liver inflammation by reprogramming the subset composition and function of hepatic macrophages, specifically suppressing pro-inflammatory KCs and MoMFs subpopulations while enriching their anti-inflammatory counterparts, thereby restoring immune homeostasis. To directly verify the role of macrophages in the protective effects of IA, we employed a macrophage-depleted mice model using clodronate liposomes (CLP) to selectively deplete mononuclear phagocytes (Fig. 8 A). Hepatocyte injury, serum AST and ALT elevations and histopathological aberrations induced by AFB1 were alleviated in macrophage-depleted mice; while these effects were not augmented by co-administration IA and CLP into AFB1-treated mice (Figs. 8 B and 8 C). Critically, CLP inhibited the decrease of anti-inflammatory cytokines in the serum and liver of mice induced by AFB1, along with transcriptional upregulation of pro-inflammatory IL-1β, IL-6 , and TNF-α , while IA and CLP co-treatment did not produce additive effect on mice exposure to AFB1 (Figs. 8 D-F). Collectively, IA alleviated AFB1-induced hepatic inflammatory by rebalancing the secretion of pro-inflammatory cytokines and anti-inflammatory cytokines in macrophage. Discussion The gut microbiota plays pivotal roles in maintaining host physiological homeostasis, with dysbiosis in its composition and function being mechanistically linked to multiple disease states 27 . Substantial evidence indicates that the gut microbiome exerts multifaceted health-promoting effects through its involvement in diverse metabolic processes, such as short-chain fatty acids (SCFAs) production, Trp metabolism, and bile acid biotransformation 28 . Previous research has demonstrated that AFB1 exposure increased the abundance of potential pathogens such as Desulfovibrio and disrupted gut microbial metabolism, which subsequently led to indirect liver damage via the gut-liver axis 9 . Employing untargeted metabolomics, our study identified disruptions in intestinal microbial Trp metabolism as a potential key contributor to AFB1-induced hepatic injury. Maintenance of Trp metabolic homeostasis is essential for maintaining physiological hepatic function and supporting growth performance in farmed animals 29 , 30 .. This investigation further revealed that FA notably elevated levels of Trp-derived metabolites, aligning with existing evidence that FA modulated microbial composition and enhancing Trp metabolism to alleviate anxiety-like behaviors 31 . Moreover, supplementation with Trp-derived metabolites, particularly IA, effectively attenuated AFB1-induced liver inflammation, underscoring the functional significance of these metabolites in hepatoprotection. Studies have revealed that P. anaerobius , P. russellii , P. stomatis , and Clostridium sporogenes can metabolize Trp to produce IA 20 . The current investigation demonstrated for the first time that FA treatment directly enhanced its growth in vitro , and elevated the transcriptional levels of genes encoding enzymes related to Trp metabolism. This work comprehensively verifies the metabolic pathway by which FA enriched P. anaerobius and activates Trp-metabolism-related enzymes to produce IA, providing a theoretical basis for modulating gut microbiota structure and metabolism via dietary intervention. Previous research indicated that P. russellii (a commensal bacterium within the same genus as P. anaerobius ) enhanced intestinal epithelial barrier function and alleviated inflammation via IA production 20 . While some studies showed that P. anaerobius was enriched in colorectal cancer patients and promoted tumorigenesis through modulation of tumor immunity 32 , 33 . Thus, effects of P. anaerobius may depend on its site of action and dosage, requiring further validation for its practical applications. In the future, we may consider applying enzymes related to Trp metabolism or Trp metabolites for the prevention and alleviation of liver injury. AFB1 induces hepatic injury through a complex array of pathological processes, including inflammation, oxidative stress, apoptosis, and autophagy 6 , 34 . Recent evidence indicates that AFB1 also initiates hepatocyte ferroptosis, a regulated cell death pathway dependent on iron and driven by lipid peroxidation 35 . In this study, single-cell transcriptomics demonstrated that IA effectively alleviated AFB1-induced hepatic injury by inhibiting hepatocyte ferroptosis in vivo . This is supported by various data: transmission electron microscopy revealed that IA protected mitochondrial structure, and serum biomarker analysis indicated that IA decreased ferrous iron and MDA levels while restoring GSH content. Cellular assays further confirmed that IA restored mitochondrial function by reducing iron overload and scavenging ROS. Molecular analysis revealed that IA orchestrated two regulatory pathways: the reactivation of the GPX4-xCT antioxidant system through the upregulation of GPX4/xCT proteins; and the remodeling of iron homeostasis via the upregulation of ferroportin (FPN) to facilitate ferrous iron efflux, thereby inhibiting Fenton reactions. This is complemented by CISD1-mediated stabilization of mitochondrial iron-sulfur clusters to limit the release of free iron. Through combined intervention with the ferroptosis inhibitor Fer-1, this study further validated that the protective effect of IA was highly dependent on the suppression of the ferroptosis pathway. Single intervention with Fer-1 could partially reverse AFB1-induced liver injury, nevertheless, no further improvement was observed in the group receiving the combined treatment of IA and Fer-1. A similar trend was exhibited in the expression changes of ferroptosis-related molecular markers, including xCT, CISD1, and GPX4. These results confirm that ferroptosis functions as a critical mechanistic pathway in AFB1-induced hepatotoxicity and concurrently illustrate that the protective efficacy of IA is predominantly mediated through the inhibition of this pathway. The AhR, a ligand-activated transcription factor, exhibits dual roles in hepatic physiology. It regulates immune homeostasis through anti-inflammatory effects, whereas aberrant activation provokes metabolic disruption and inflammatory liver injury 36 . Endogenous metabolites like gut-derived indoles orchestrated anti-inflammatory programming via activation of the IL-22 axis, thereby modulating the functional maturation of intestinal intraepithelial lymphocytes and innate lymphoid cells 37 . Whereas exogenous ligand like dioxins induced hepatotoxic inflammation via CYP1A1/A2 upregulation 38 . Here, we identified AFB1 as an exogenous agonist that potently amplified hepatic AhR expression, eliciting inflammation. Crucially, IA reversed these AFB1-induced molecular changes and mitigated hepatic pathology. This antagonism is structurally elucidated by our biophysical characterization utilizing SPR and molecular docking. AhR ligands are functionally categorized into full agonists, partial agonists, and competitive antagonists, where efficacy is dictated by binding affinity and intrinsic activity 24 , 39 , AFB1 acted as a full agonist, engaging the AhR ligand-binding domain with high affinity predominantly via strong hydrogen bonds. In contrast, IA bound with lower affinity dominated by weaker hydrophobic interactions. Mechanistically, we identify IA as a partial agonist of AhR in vivo . Since IA triggers only a sub-maximal response compared to AFB1, it can inhibit the hyperactivation of AhR by AFB1. This dampening effect shifts the cytokine profile from a toxic, pro-inflammatory state (IL-17) to a restorative, homeostatic phenotype (IL-22/IL-10). This underscores the therapeutic advantage of endogenous partial agonists, which maintain basal physiological signaling while preventing toxic overstimulation 40 , 41 . Building upon existing evidence that ferroptosis is modulated by AhR via IA in HT29 cells 42 , this study demonstrates that AFB1 induces hepatic ferroptosis through AhR activation. Importantly, IA competitively inhibits AhR-driven ferroptosis, as demonstrated by both in vivo pharmacological AhR inhibition and in vitro siRNA-mediated AhR knockdown. This mechanistic insight positions the gut microbial metabolite IA as a potential therapeutic agent to counteract AFB1-triggered liver injury. Beyond hepatocyte-intrinsic mechanisms, macrophages, key orchestrators of innate immunity, critically modulate hepatic immune homeostasis 26 . Through flow cytometry and integrated single-cell RNA sequencing, this study demonstrated that IA ameliorated AFB1-induced liver inflammation by reprogramming the subset composition and function of hepatic macrophages, specifically suppressing pro-inflammatory KCs and MoMFs subpopulations while enriching their anti-inflammatory counterparts, thereby restoring immune homeostasis. Critically, this study found that macrophage depletion mediated by CLP significantly alleviated AFB1-induced liver injury. The absence of additive effects in the IA + CLP co-treatment group highlighted the critical role of functional macrophages was indispensable for IA-mediated anti-inflammatory effects. Although previous research has associated CLP with the alleviation of acetaminophen-induced injury 43 , this study pioneered the use of macrophage-depleted models to elucidate the mechanistic role of macrophages as crucial mediators of IA's protective effects against mycotoxin-induced inflammation. This provides a compelling rationale for developing macrophage-targeted therapeutic interventions. In summary, we found that FA alleviates AFB1-induced liver injury in ducks by remodeling the gut microbiota and promoting the production of P. anaerobius -derived IA. Mechanistically, IA confers hepatoprotection by restraining AhR-driven ferroptosis and inflammatory macrophage reprogramming. These findings provide a rationale for microbiome-based nutritional strategies to mitigate AFB1-induced injury and improve the health of farmed animals. Materials and methods Reagents The reagents and antibody information used in this study were listed in Table S1 and Table S2. Duck s One-day-old male Pekin ducks were housed in wire-floored cages, with eight ducklings per cage. Ducks were housed under conditions consistent with previous reports 3 . On day 28, ducks were euthanized to collect samples. Protective role of FA in ducks fed AFB1-contaminated diets: The 192 ducks that were randomly assigned to four groups, CON, FA, AFB1, and FA+AFB1 groups. Each group consisted of six replicate cages, with eight ducks per cage. The addition dosage of FA was 500 mg/kg of feed. The AFB1 concentration was 20 μg/kg, achieved by incorporating AFB1-contaminated peanut meal into the basal diet. To collect samples, one duck with an average body weight was euthanized from each replicate on the 28th day. Antibiotic intervention in FA+AFB1 ducks to assess gut microbiota dependency: The 24 ducks were randomly assigned into three groups: AFB1, FA+AFB1, and ANTI (FA+AFB1) group. The ducks of ANTI (FA+AFB1) group received antibiotics (1 g/L streptomycin, 1 g/L ampicillin, and 1 g/L neomycin) in their drinking water for a period of 28 days. All ducks were given a diet naturally contaminated with AFB1 (AFB1, 20 μg/kg), and FA (500 mg/kg) was included in the diets of the FA+AFB1 and ANTI (FA+AFB1) groups. FMT experiment investigate the role of gut microbiota in FA alleviating AFB1-induced liver injury: For eliminating the native gut microbiota, 32 recipient ducks received antibiotics in their drinking water for two weeks. After antibiotics treatment, recipient ducks were respectively separated into four groups (FMT-CON, FMT-FA, FMT-AFB1, and FMT-FA+AFB1) and received fecal microbiota transplants (10 mL per kg of body weight each day) from their respective donor ducks (FA supplementation for ducks fed with diet contaminated with AFB1) for 2 weeks. Give 10 mL per kg of body weight each day. The microbiota suspension was prepared according to previous studies 9 . Mice Four-week-old male Kunming mice were obtained from Beijing HFK Bioscience (Beijing, China). Mice were housed under conditions consistent with previous reports 15 . Efficacy of Trp metabolites efficacy in mice: Mice were randomly divided into 7 groups (n=8) and fed different candidate Trp metabolites: CON, AFB1, Indole+AFB1, indole-3-glyoxylic acid (IGA)+AFB1, IA+AFB1, kynurenic acid (KYNA)+AFB1, and xanthurenic acid (XA)+AFB1 groups. The addition dosage of AFB1 was 1.8 mg/kg of feed. The dosage of Indole, IGA and IA were 300 mg/kg of feed, the dosage of KYNA and XA were 600 mg/kg of feed. The duration of the experiment was 28 days. Evaluation of FA effects on P. anaerobius and IA production in AFB1-induced mice ： The 24 mice were divided randomly into three groups: CON, AFB1 and FA+AFB1 groups. AFB1 and FA were administered at dosages of 1.8 mg/kg feed and 300 mg/kg feed, respectively. The duration of the experiment was 28 days. Oral gavage of P. anaerobius in mice: The 24 mice were randomly assigned into three groups: CON, fed a basal diet and gavaged with 0.2 mL of phosphate-buffered saline (PBS); AFB1: fed AFB1 (1.8 mg/kg) diet and gavaged with 0.2 mL of PBS; PA+AFB1: fed AFB1 (1.8 mg/kg) diet and gavaged with 0.2 mL of fresh P. anaerobius suspension. The duration of the experiment was 28 days. The fresh P. anaerobius suspension was prepared. Briefly, P.anaerobius was cultured anaerobically in reinforced clostridial medium (RCM) at 37°C for 24 h. After cultivation, the cell of starin was centrifuged, washed twice, and resuspended in PBS to a final concentration of 1 × 10⁸ CFU/mL. Fresh suspensions were prepared daily prior to gavage. Mechanistic evaluation of IA against AFB1-induced liver injury: The 24 mice were divided randomly into three groups: CON, AFB1 and IA+AFB1 groups. To further investigate ferroptosis involvement in the mechanism, ferroptosis inhibitor Fer-1 was used and mice were divided into six groups: CON, AFB1, IA+AFB1, CON+Fer-1, AFB1+Fer-1, and IA+AFB1+Fer-1. Concurrently, to validate the involvement of the AhR in the protective effect of IA against AFB1-induced ferroptosis, mice were treated with the AhR inhibitor CH. Groups included CON, AFB1, IA+AFB1, CON+CH, AFB1+CH, and IA+AFB1+CH. Additionally, to assess the role of macrophages in IA-mediated protection against AFB1-induced hepatic injury, macrophage depletion was achieved through the administration of clodronate CLP. Mice were randomly divided into 6 groups: CON, AFB1, IA+AFB1, CON+CLP, AFB1+CLP, and IA+AFB1+CLP. The experiment lasted for 28 days. AFB1 and IA were administered at dosages of 1.8 mg/kg feed and 300 mg/kg feed, respectively. For Fer-1 administration, mice received daily intraperitoneal injections of Fer-1 (1 mg/kg body weight). For CH treatment, mice were subjected to daily oral gavage with CH (10 mg/kg body weight). For CLP-induced macrophage depletion, mice received weekly intraperitoneal injections of CLP (10 μL/g body weight). Effect of FA on the growth of P. anaerobius P. anaerobius (ATCC 27337) was inoculated into RCM medium supplemented with varying concentrations of FA (0, 10, 30, or 50 mg/L) and incubated anaerobically at 37 °C. Bacterial growth was monitored by sampling 500 μL of suspension every 2 h and measuring OD 600 with a ultraviolet (UV) spectrophotometer. After 24 hours of incubation, 4 mL of culture was centrifuged, and the resulting cell pellet was collected for qPCR analysis. Culture of P. anaerobius under Trp supplementation P. anaerobius was maintained anaerobically in RCM at 37 °C for 24h. The medium was supplemented as follows: PA, no additional supplement; PA+Trp, 50 μg/mL Trp. Cell culture AML-12 hepatocytes were acquired from the American Type Culture Collection (ATCC, VA, USA) and cultured as described previously 15 . Cells were treated with 200 μM IA and 8 μM AFB1 for 24h. To suppress AhR activation, cells were pre-treated for 2 h with the AhR antagonist CH (10 μM) prior to 24-h co-exposure with subsequent compounds. Cells were transfered with siAhR oligos (sense: 5'-CUUUCUCUCCCAUAAUGGAdTdT-3', antisense: 5'-UCCAUUAUGGGAGAGAAAGdTdT-3') using Lipofectamine 3000 reagent for AhR knockdown. Histopathology and transmission electron microscopy analysis of liver Liver or jejunum tissues underwent paraffin embedding, followed by sectioning at a thickness of 5 μm. After deparaffinization with xylene, the sections were rehydrated and mounted on glass slides. Haematoxylin and eosin (H&E) staining was then applied. High-resolution imaging was conducted utilizing a microscope slide scanner (3DHISTECH, Budapest, Hungary) in conjunction with a DP70 digital camera (Olympus, Tokyo, Japan). For ultrastructural analysis of mitochondria, liver tissues were treated with 2.5% glutaraldehyde for fixation, dehydrated using a graded series of ethanol, and embedded in epoxy resin. Ultrathin sections were mounted on transmission electron microscopy grids and examined using a transmission electron microscope (Hitachi HT7700, Tokyo, Japan). Biochemical analysis The activities/levels of aspartate AST, ALT, serum MDA, GSH, and hepatic iron concentrations were measured using commercial kits (Nanjing Jiancheng, Nanjing, China). Cytokine profiles (IL-10, IL-17, IL-22) in serum and liver of mice were analyzed by commercial ELISA kits (SLCY, Beijing, China). Q uantitative real-time PCR (qRT-PCR) analysis Total RNA extraction and cDNA synthesis were performed with a commercial kit (Vazyme, Nanjing, China). A two-step qRT-PCR was conducted using a Real-Time PCR Detection System from Bio-Rad, CA, USA. The mRNA expression of genes were determined based on the expression of the GAPDH. Specific primer sequences were listed in Table S3. 16S rRNA gene sequencing analysis Genomic DNA from the cecal microbiota was extracted employing a DNA extraction kit (Omega Bio-tek, GA, USA). The V3-V4 hypervariable region of the bacterial 16S rRNA gene was subsequently amplified on the PCR system (ABI, CA, USA). The resulting purified amplicons underwent sequencing on an Illumina MiSeq PE300 platform (Majorbio, Shanghai, China). The data was analyzed using the Majorbio Cloud Platform (https://www.majorbio.com). Untargeted metabolomics analysis Cecal content samples were homogenized in a methanol-water solution (4:1, v/v) utilizing a cryogenic grinder set at 50 Hz for 6 minutes. After centrifugation, the supernatants from the homogenates were collected. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was conducted using a UHPLC-Q Exactive™ HF-X system (Thermo Scientific, MA, USA). The raw data was analyzed using the Majorbio Cloud Platform (https://www.majorbio.com). Targeted Trp metabolomics analysis The cecal contents were homogenized at a ratio of 1:10 (w/v) in a solvent mixture of methanol, acetonitrile, and water (2:2:1, v/v/v). Following centrifugation, the supernatants of homogenates were collected for subsequent analysis. The separation of target compounds was achieved through ultra-high-performance liquid chromatography (Milford, MA, USA). The mass spectrometry analysis was executed on a SCIEX 6500 QTRAP triple quadrupole mass spectrometer (Framingham, MA, USA). Mass spectrometry data acquisition and quantitative analysis of the target compounds were performed using SCIEX Analyst workstation software (Version 1.6.3) and MultiQuant software (Version 3.0.3). Detection of IA content in serum and bacterial culture supernatant Serum samples were diluted in a 1:1 ratio with deionized water, and 50 μL aliquots were mixed with 300 μL of 1% (v/v) aqueous formic acid. Solid-phase extraction was performed using HLB cartridges (Thermo Scientific, MA, USA), which were sequentially conditioned with 2 mL methanol and 2 mL 1% aqueous formic acid. The pre-treated serum samples were loaded onto the cartridges, washed with 2 mL 1% aqueous formic acid, and eluted with 500 μL methanol. Then Eluates or bacterial culture supernatants were filtered through 0.22 μm membranes before high-performance liquid chromatography (HPLC) analysis. Chromatographic separation was performed using a C18 reversed-phase column. Isocratic elution were performed using a mobile phase of acetonitrile and 0.1% aqueous formic acid (3:7, v/v) at a flow rate of 1.0 mL/min. UV detection was conducted at a wavelength of 254 nm with 20 μL injection volumes over a 12-minute run time. Single-cell RNA sequencing Fresh mouse liver tissues were dissociated and filtered through 40 μm strainers, followed by enzymatic digestion and erythrocyte lysis. The resulting cell suspensions were resuspended in 0.04% BSA-PBS. Single-cell suspensions were then processed using the Chromium Single Cell 3ʹ Kit (10x Genomics, CA, USA). Library sequencing was performed on the Illumina NovaSeq 6000 platform (CA, USA) using 150-cycle paired-end reads as previously described 44 . Data processing utilized Cell Ranger (v7.1.0, https://www.10xgenomics.com/software) for alignment (mm10) and unique molecular identifier (UMI) counting. Seurat (v4.1.1, https://satijalab.org/seurat/) was employed to filter out low-quality cells based on nUMI/gene thresholds of ±2 standard deviations and a mitochondrial gene percentage of ≤5%. Variable genes were identified for integration (using FindIntegrationAnchors and IntegrateData in the seurat package), followed by PCA reduction to the top 30 principal components and t-distributed stochastic neighbor embedding (tSNE) visualization. Cell types were annotated using SingleR and marker genes. Differentially expressed genes (DEGs) were defined by |log2FC| > 0.25 and Q ≤ 0.05. Functional enrichment analyses for Gene Ontology (GO) terms and KEGG pathways were conducted using the Goato tool (https://github.com/tanghaibao/Goatools). Cell viability assay To assess cell viability, the CCK-8 assay was employed. In summary, cells were cultured in 96-well plates for 24 hours, after which they were exposed to different concentrations of reagents for an additional 24 hours. The CCK-8 assay was performed in accordance with the manufacturer's instructions (Beyotime, Shanghai, China). ROS determination Intracellular ROS generation in AML12 hepatocytes was quantified using a commercial detection kit (Beyotime, Shanghai, China) per standardized protocols. Briefly, the treated cells underwent 30-minute exposure to 10 μM DCF-DA at 37 °C, followed by image acquisition using fluorescence microscopy. FerroOrange staining Following 24-h treatment with various concentrations of reagents in 24-well plates, AML12 cells were incubated with FerroOrange fluorescent probe (1 μM) for 20 minutes in the dark. Subsequent confocal microscopy imaging was performed after incubation. Surface plasmon resonance (SPR) quantifies AhR–ligand binding The interactions of AhR ligands were investigated utilizing SPR on a Biacore S200 system (https://www.cytivalifesciences.com). AhR protein was immobilized on the activated surface of CM5 sensor chips via amine coupling. Serial dilutions of AFB1 (0.244-500 μM) and IA (7.8125-500 μM) were injected (30 μL/min), with 120-second association and dissociation phases. The binding kinetics were analyzed by employing double-referencing curve fitting using Biacore Evaluation Software to determine the equilibrium dissociation constants (KD). Molecular docking of AFB1 or IA with AhR Docking analysis was carried out by CDOCKER module of Discovery Studio 2019 (San Diego, America). The 3-dimensional (3D) structure of AhR was predicted by AlphaFold3 45 (https://alphafoldserver.com/). The 3D structure of IA (CID: 186907) and AFB1 (CID: 5375048) were downloaded from Pubchem (https://pubchem.ncbi.nlm.nih.gov/). The binding affinities of binding clusters were evaluated by -cdocker interaction energy. Isolation and flow cytometry analysis of mouse hepatic macrophages Hepatic tissue underwent intracardiac perfusion in situ using Hank's balanced salt solution containing 0.025% collagenase IV through left ventricular access as previously described 46 . The livers were mechanically dissociated through a 100 μm mesh and subsequently underwent differential centrifugation (50 g for 1 minute at 4 °C, followed by 500 g for 6 minutes at 4 °C). Leukocytes were enriched utilizing a discontinuous Percoll gradient centrifugation (40% over 70%) at 750 g for 22 minutes at 15 °C. Erythrocytes were lysed using Ammonium-Chloride-Potassium (ACK) buffer prior to surface staining with anti-CD45, anti-F4/80 and anti-CD11b. The primary antibodies were used at 1:1000 dilutions unless otherwise specified. Cellular viability was assessed using Zombie Aqua. Flow cytometric data acquisition was conducted on an LSR Fortessa platform (BD Biosciences, CA, USA) using the Flowlogic 600.0A analytical software (https://www.inivai.com). Statistic analysis Analyses were conducted using GraphPad Prism Version 9 software (San Diego, CA, USA). Comparisons between two groups were performed using a two-sided t-test; for more than three groups, the data underwent one-way ANOVA followed by multiple comparisons. Results are presented as mean ± SEM, with P < 0.05 considered significant. Declarations Acknowledgments This work was supported by the National Key Research and Development Program of China (Program No. 2023YFD1301005 ) and National Natural Science Foundation of China (31972604) . Ethics approval and consent to participate All animal procedures complied with the guidelines of the Institutional Animal Care and Use Committee of China Agricultural University ((Approval Nos: Aw41703202-1-4 and Aw32605202-1-4). Competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Availability of data and material The 16S rRNA gene sequencing datasets generated and analysed during the current study are available in the NCBI Sequence Read Archive (SRA) database under accession numbers PRJNA1367944 ( https://dataview.ncbi.nlm.nih.gov/object/PRJNA1367944?reviewer=l3dk4oagt6naotsngj24g6908d ). The raw metabolomics data have been deposited in Mendeley Data ( https://www.ebi.ac.uk/metabolights/reviewer1dfb8fa6-a5ff-4f79-80cd-b3c185762743 ; https://www.ebi.ac.uk/metabolights/reviewerd53a81e4-5478-4528-ac89-828c8d2045bc ). The single-cell sequencing data reported in this paper have been deposited in the OMIX, China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences ( https://ngdc.cncb.ac.cn/omix: accession no.OMIX015025 ). Author contributions Q ianqian Wang : Conceptualization, Data curation, Formal analysis, Writing – original draft. Yanan Wang : Data curation, Visualization. Gaigai Wang : Writing – review & editing. Xin Fang : Resources, Data curation. Yutong Fu : Writing – review & editing. Zixin Li : Writing – review & editing. Yongpeng Guo : Writing – review & editing. Jinglin Ma : Writing – review & editing. Lirong Hu : Writing – review & editing. Xiangfang Zeng : Writing – review & editing. Qiugang Ma : Writing – review & editing. Matthew Koci : Writing – review & editing. Lihong Zhao : Conceptualization, Resources, Writing – review & editing, Supervision, Funding acquisition. References Eskola, M. et al. Worldwide contamination of food-crops with mycotoxins: Validity of the widely cited 'FAO estimate' of 25. Crit Rev Food Sci Nutr 60 , 2773-2789 (2020). https://doi.org/10.1080/10408398.2019.1658570">https://doi.org/10.1080/10408398.2019.1658570 Liu, Y., Galani Yamdeu, J. H., Gong, Y. 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Nat Microbiol 9 , 1467-1482 (2024). https://doi.org/10.1038/s41564-024-01695-w">https://doi.org/10.1038/s41564-024-01695-w Xu, Q. et al. Critical role of caveolin-1 in aflatoxin B1-induced hepatotoxicity via the regulation of oxidation and autophagy. Cell Death Dis 11 , 6 (2020). https://doi.org/10.1038/s41419-019-2197-6">https://doi.org/10.1038/s41419-019-2197-6 Liang, D., Minikes, A. M. & Jiang, X. Ferroptosis at the intersection of lipid metabolism and cellular signaling. Mol Cell 82 , 2215-2227 (2022). https://doi.org/10.1016/j.molcel.2022.03.022">https://doi.org/10.1016/j.molcel.2022.03.022 Carambia, A. & Schuran, F. A. The aryl hydrocarbon receptor in liver inflammation. Semin Immunopathol 43 , 563-575 (2021). https://doi.org/10.1007/s00281-021-00867-8">https://doi.org/10.1007/s00281-021-00867-8 Hendrikx, T. et al. Bacteria engineered to produce IL-22 in intestine induce expression of REG3G to reduce ethanol-induced liver disease in mice. Gut 68 , 1504-1515 (2019). https://doi.org/10.1136/gutjnl-2018-317232">https://doi.org/10.1136/gutjnl-2018-317232 Pierre, S. et al. Aryl hydrocarbon receptor-dependent induction of liver fibrosis by dioxin. Toxicol Sci 137 , 114-124 (2014). https://doi.org/10.1093/toxsci/kft236">https://doi.org/10.1093/toxsci/kft236 Ondrova, K. et al. Monoterpenoid aryl hydrocarbon receptor allosteric antagonists protect against ultraviolet skin damage in female mice. Nat Commun 14 , 2728 (2023). https://doi.org/10.1038/s41467-023-38478-6">https://doi.org/10.1038/s41467-023-38478-6 Dvorak, Z., Sokol, H. & Mani, S. Drug Mimicry: Promiscuous Receptors PXR and AhR, and Microbial Metabolite Interactions in the Intestine. Trends Pharmacol Sci 41 , 900-908 (2020). https://doi.org/10.1016/j.tips.2020.09.013">https://doi.org/10.1016/j.tips.2020.09.013 Vyhlidalova, B. et al. Gut Microbial Catabolites of Tryptophan Are Ligands and Agonists of the Aryl Hydrocarbon Receptor: A Detailed Characterization. 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Nature 630 , 493-500 (2024). https://doi.org/10.1038/s41586-024-07487-w">https://doi.org/10.1038/s41586-024-07487-w Triantafyllou, E. et al. MerTK expressing hepatic macrophages promote the resolution of inflammation in acute liver failure. Gut 67 , 333-347 (2018). https://doi.org/10.1136/gutjnl-2016-313615">https://doi.org/10.1136/gutjnl-2016-313615 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9281710","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":630429959,"identity":"a57654be-c1ab-43aa-af66-cc3d9a00aeee","order_by":0,"name":"Qianqian Wang","email":"","orcid":"","institution":"China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Qianqian","middleName":"","lastName":"Wang","suffix":""},{"id":630429960,"identity":"8426883c-1c15-40a3-9326-38f3905d0846","order_by":1,"name":"Yanan Wang","email":"","orcid":"","institution":"China Agricultural 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15:08:54","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9281710/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9281710/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108621075,"identity":"1d406eb2-218b-4c4e-9d05-54831249cceb","added_by":"auto","created_at":"2026-05-06 14:50:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3828988,"visible":true,"origin":"","legend":"\u003cp\u003eFA alleviated AFB1-triggered liver injury in a gut microbe-dependent manner in ducks. (A) Schematic of experimental designs. (B) Experimental design for FA bioactivity assessment. (C) The activity of AST and ALT in the serum of ducks among CON, AFB1, FA, and FA+AFB1 groups, n=6. (D) Image showing H\u0026amp;E staining in the liver of ducks among CON, AFB1, FA, and FA+AFB1 groups, arrows indicate inflammatory cell infiltration, n=3. (E) The relative mRNA expression of \u003cem\u003eIL-1β\u003c/em\u003e,\u003cem\u003e IL-6, \u003c/em\u003eand \u003cem\u003eTNF-α\u003c/em\u003e in the liver of ducks among CON, AFB1, FA, and FA+AFB1 groups, n=6. (F) PCoA of the cecal microbiota in ducks among CON, AFB1, FA, and FA+AFB1 groups, n=6. (G) The ten most prevalent phyla of gut microbiota: among CON, AFB1, FA, and FA+AFB1 groups, n=6. (H) LEfSe analysis identified differentially abundant genera in the AFB1 and FA+AFB1 groups (LDA score \u0026gt;3, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05), n=6. (I) Antibiotics intervention experimental design. (J) The activity of AST and ALT in the serum of ducks among AFB1, FA+AFB1, and ANTI(FA+AFB1) groups, n=8. (K) Image showing H\u0026amp;E staining in the liver of ducks, arrows indicate inflammatory cell infiltration, n=3. (L) The relative mRNA expression of IL-1β, IL-6, and TNF-α in the liver of ducks among AFB1, FA+AFB1, and ANTI(FA+AFB1) groups, n=8. (M) FMT experimental design. (N) The activity of AST and ALT in the serum of ducks among FMT-CON, FMT-AFB1, FMT-FA, and FMT-FA+AFB1 groups, n=8. (O) Image showing H\u0026amp;E staining in the liver of ducks among FMT-CON, FMT-AFB1, FMT-FA, and FMT-FA+AFB1 groups, arrows indicate inflammatory cell infiltration, n=3. (P) The relative mRNA expression of \u003cem\u003eIL-1β\u003c/em\u003e,\u003cem\u003eIL-6, \u003c/em\u003eand \u003cem\u003eTNF-α\u003c/em\u003e in the liver of ducks among FMT-CON, FMT-AFB1, FMT-FA, and FMT-FA+AFB1 groups, n=8. (Q) PCoA of the gut microbiota in ducks among FMT-CON, FMT-AFB1, FMT-FA, and FMT-FA+AFB1 groups, n=8. (R) The ten most prevalent genera of gut microbiota among FMT-CON, FMT-AFB1, FMT-FA, and FMT-FA+AFB1 groups, n=8. (S) LEfSe analysis identified differentially abundant genera in the FMT-AFB1 and FMT-FA+AFB1 groups (LDA score \u0026gt;3, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, n=8).Note: The level of significance was set at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; *, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-9281710/v1/103b50590891d6adcfe49cd3.png"},{"id":108621079,"identity":"9adcc9d1-9511-414c-b0de-59a59f10dce9","added_by":"auto","created_at":"2026-05-06 14:50:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3395386,"visible":true,"origin":"","legend":"\u003cp\u003eFA alleviated AFB1-triggered liver injury by enriching of \u003cem\u003ePeptostreptococcus anaerobius\u003c/em\u003e(\u003cem\u003eP. anaerobius\u003c/em\u003e) and enhancing tryptophan metabolism for IA production. (A) PCA of cecal microbial metabolites in ducks, n=6. (B) Volcanic plot of differential metabolites between AFB1 and FA+AFB1 groups, n=6. (C) KEGG topological analysis between AFB1 and FA+AFB1 groups, n=6. (D) Volcano plot of differential Trp metabolites comparing AFB1 and FA+AFB1 groups, n=6. (E) Experimental design for validating Trp metabolite efficacy. (F, G) The activity of AST and ALT in the serum of mice, n=8. (H) The relative mRNA expression of \u003cem\u003eIL-1β\u003c/em\u003e, \u003cem\u003eIL-6\u003c/em\u003e, and \u003cem\u003eTNF-α\u003c/em\u003e in the liver of mice, n=8. (I) Experimental schematic illustration. (J) The level of IA in the serum of ducks, n=6-8. (K) Relative abundance of \u003cem\u003eP. anaerobius\u003c/em\u003e, \u003cem\u003eP. russellii\u003c/em\u003e, \u003cem\u003eP. stomatis\u003c/em\u003e, and \u003cem\u003eClostridium sporogenes\u003c/em\u003e in cecal microbiota of ducks, n=6. (L) Experimental design for evaluation of FA effects in mice. (M) Relative abundance of cecal \u003cem\u003eP. anaerobius\u003c/em\u003e (n=6) and serum IA concentration in mice (n=8). (N) Schematic diagram of cultivation of \u003cem\u003eP. anaerobius\u003c/em\u003e under different FA concentrations. (O) Effects of varying FA concentrations on \u003cem\u003eP. anaerobius\u003c/em\u003egrowth, n=3. (P) Schematic diagram of the Trp metabolic pathway. (Q) The relative mRNA expression of \u003cem\u003eArAT\u003c/em\u003e, \u003cem\u003efldH\u003c/em\u003e, \u003cem\u003efldB\u003c/em\u003e, \u003cem\u003efldC\u003c/em\u003e, and \u003cem\u003eacdA\u003c/em\u003e across treatment groups, n=6. (R) Schematic diagram of cultivation of \u003cem\u003eP. anaerobius\u003c/em\u003e and IA level detection. (S) IA content in fermentation supernatants, n=3. (T) Experimental schematic illustration. (U) Image showing H\u0026amp;E staining in the liver of mice, arrows indicate inflammatory cell infiltration, n=3. (V) The activity of AST and ALT in the serum of mice, n=8. (W) The relative mRNA expression of \u003cem\u003eIL-1β\u003c/em\u003e,\u003cem\u003e IL-6, \u003c/em\u003eand \u003cem\u003eTNF-α\u003c/em\u003ein the liver of mice, n=8.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-9281710/v1/52273c2385fc7d3aa6df0bf2.png"},{"id":108805838,"identity":"eae01d11-1d48-4274-8ee0-4a5aff79e2f6","added_by":"auto","created_at":"2026-05-08 15:27:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4658071,"visible":true,"origin":"","legend":"\u003cp\u003eIA alleviated AFB1-induced liver injury by inhibiting ferroptosis. (A) Experimental design for evaluation of IA effects in mice. (B) Image showing H\u0026amp;E staining in the liver of mice, arrows indicate inflammatory cell infiltration, n=3. (C) The activity of AST and ALT in the serum of mice, n=8. (D) Schematic of experimental design. (E) UMAP visualization of hepatic cell populations in mice, n=3. (F) Violin plots of cell-type-specific marker genes in mouse liver, n=3. (G) Composition of hepatic cell types among different groups, n=3. (H) Differentially expressed genes (DEGs) between IA+AFB1 and AFB1 groups in hepatocytes. (I) KEGG enrichment analysis between IA+AFB1 and AFB1 groups in hepatocytes. (J) Ultrastructure of hepatic mitochondria in mice，arrows indicate the outer mitochondrial membrane, n=3. (K) Hepatic iron content in mice, n=8. (L, M) The level of MDA and GSH in the serum of mice, n=8. (N) The relative mRNA expression of \u003cem\u003eFPN\u003c/em\u003e, \u003cem\u003eGPX4, \u003c/em\u003eand \u003cem\u003eCISD1 \u003c/em\u003ein the liver of mice, n=8. (O, P) The levels of HO-1, xCT, and GPX4 proteins in mice liver were assessed and quantified through Western blot analysis, n=6. (Q) Experimental design for evaluation of IA effects in cells. (R, S) The level of GSH and MDA in AML12 cells, n=3. (T) ROS generation visualized by DCFH-DA fluorescence, n=3. (U) Quantification of relative ROS fluorescence intensity, n=3. (V) Ferrous iron (Fe\u003csup\u003e2+\u003c/sup\u003e) accumulation detected by FerroOrange probe, n=3. (W) Quantification of relative Fe\u003csup\u003e2+\u003c/sup\u003e fluorescence, n=3. (X, Y) The levels of HO-1, xCT, and GPX4 proteins in AML12 cells were assessed and quantified through Western blot analysis, n=6.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-9281710/v1/54397129898a6f3873ba1b52.png"},{"id":108621082,"identity":"b9fb1d76-1083-4abb-85d2-83f2921fce08","added_by":"auto","created_at":"2026-05-06 14:50:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3782072,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Fer-1 and IA on ferroptosis of mouse liver cells induced by AFB1. (A) Experimental design for confirming the ferroptosis-dependent mechanism. (B) The activity of AST and ALT in the serum of mice, n=8. (C) Hepatic iron content in mice, n=8. (D, E) The level of MDA and GSH in the serum of mice, n=8. (F) The relative mRNA expression of \u003cem\u003eGPX4 \u003c/em\u003eand \u003cem\u003eCISD1 \u003c/em\u003ein the liver of mice, n=8. (G) Image showing H\u0026amp;E staining in the liver of mice, arrows indicate inflammatory cell infiltration, n=3. (H-K) The levels of HO-1, xCT, and GPX4 proteins in mice liver were assessed and quantified through Western blot analysis, n=6.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-9281710/v1/6c0f3abed975345bf712ceee.png"},{"id":108805767,"identity":"f1bef482-c08d-494f-b46d-8ba7e5040ccf","added_by":"auto","created_at":"2026-05-08 15:26:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4858559,"visible":true,"origin":"","legend":"\u003cp\u003eIA mitigated AFB1-induced hepatocyte ferroptosis through an AhR-dependent pathway in mice. (A) The relative mRNA expression of \u003cem\u003eAhR\u003c/em\u003e,\u003cem\u003e CYP1A1\u003c/em\u003e,\u003cem\u003e \u003c/em\u003eand \u003cem\u003eCYP1A2 \u003c/em\u003ein the liver of mice, n=8. (B, C) The levels of AhR protein in mice liver were assessed and quantified through Western blot analysis, n=6. (D) Depiction of IHC staining for CYP1A1 in the liver of mice. (E) Quantification of CYP1A1 IHC staining intensity, n=4. (F) The concentration of IL-10, IL-17, and IL-22 in the serum of mice, n=8. (G) The concentration of IL-10, IL-17, and IL-22 in the liver of mice, n=8. (H) The interaction of AFB1 with AhR by SPR. (I) Molecular docking analyses between AFB1 and AhR. (J) The interaction of IA with AhR by SPR. (K) Molecular docking analyses between IA and AhR. (L) Experimental design for assessing the role of AhR in IA-mediated protection in mice. (K) The activity of AST and ALT in the serum of mice, n=8. (L-N) The concentration of IL-10, IL-17, and IL-22 in the serum of mice, n=8. (O, P) The concentration of IL-10 and IL-22 in the liver of mice, n=8. (Q) Image showing H\u0026amp;E staining in the liver of mice, arrows indicate inflammatory cell infiltration, n=3.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-9281710/v1/a47ace0a94f6e954efa6ff05.png"},{"id":108621085,"identity":"978aa636-57f8-4d09-b574-aa2c2b040d5a","added_by":"auto","created_at":"2026-05-06 14:50:22","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3336908,"visible":true,"origin":"","legend":"\u003cp\u003eIA mitigated AFB1-induced hepatocyte ferroptosis through an AhR-dependent pathway in AML12 cells. (A) Experimental design for assessing the role of AhR in IA-mediated protection in cells. (B) Viability of AML12 cells treated with AFB1, IA, or CH-223191, n=6. (C, D) The level of GSH and MDA in AML12 cells, n=6. (E) Quantification of relative Fe\u003csup\u003e2+\u003c/sup\u003e fluorescence, n=3. (F) Fe\u003csup\u003e2+\u003c/sup\u003e accumulation detected by FerroOrange probe, n=3. (G-K) The levels of AhR, HO-1, xCT, and GPX4 proteins in AML12 cells were assessed and quantified through Western blot analysis, n=6. (L) Experimental design for assessing the role of AhR in IA-mediated protection in cells. (M) The relative mRNA expression of AhR in AML12 cells, n=4. (N) The levels of AhR protein in AML12 cells were assessed and quantified through Western blot analysis. (O) Quantification of relative ROS fluorescence intensity, n=3. (P) Quantification of relative Fe\u003csup\u003e2+\u003c/sup\u003e fluorescence, n=3. (Q) ROS generation visualized by DCFH-DA fluorescence, n=3. (R) Fe\u003csup\u003e2+\u003c/sup\u003e accumulation detected by FerroOrange probe, n=3. (S-W) The levels of AhR, HO-1, xCT, and GPX4 proteins in AML12 cells were assessed and quantified through Western blot analysis, n=3.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-9281710/v1/81ad0f2b7cde5db34371f877.png"},{"id":108805805,"identity":"1a9ea5be-8a4c-4b86-8ba9-46a1efc98d67","added_by":"auto","created_at":"2026-05-08 15:26:55","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3759398,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of IA on AFB1-induced macrophages reprogramming in the liver of mice. (A) Representative IHC staining image of F4/80 in the liver of mice, n=3. (B) Quantification of F4/80 IHC staining intensity, n=3. (C) Representative flow cytometry (FACS) dot plots of hepatic macrophages (CD45⁺F4/80⁺CD11b⁺), n=4. (D) Statistical results of FACS-based KCS (F4/80\u003csup\u003ehi\u003c/sup\u003eCD11b\u003csup\u003elo\u003c/sup\u003e) proportions, n=4. (E) Statistical results of FACS-based monocyte-derived macrophages (MoMFs) (F4/80\u003csup\u003elo\u003c/sup\u003eCD11b\u003csup\u003ehi\u003c/sup\u003e) proportions, n=4. (F) T-distributed stochastic neighbor embedding (T-SNE) projection of KCS in mice, n=3. (G) Heatmap of gene expression levels in different subpopulations of KCS, n=3. (H) The proportion of mouse KCS subpopulations among different groups, n=3. (I) Reactome pathway enrichment analysis, n=3. (J) Heatmap of pro-inflammatory gene expression in KCS subpopulations, n=3. (K) Heatmap of anti-inflammatory gene expression in KCS subpopulations, n=3. (L) T-SNE projection of MoMFs in mice, n=3. (M) Heatmap of gene expression levels in different subpopulations of MoMFs, n=3. (N) The proportion of mouse MoMFs subsets among different groups, n=3. (O) Reactome pathway enrichment analysis, n=3. (P) Heatmap of pro-inflammatory gene expression in MoMFs subpopulations, n=3. (Q) Heatmap of anti-inflammatory gene expression in MoMFs subpopulations, n=3.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-9281710/v1/db0aafd136c8958036b02638.png"},{"id":108805446,"identity":"87136f83-dd9c-49e3-8b60-b0e1838cecdb","added_by":"auto","created_at":"2026-05-08 15:26:00","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":3399205,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of IA on AFB1-induced liver damage in mice under macrophage depletion conditions. (A) Experimental design for evaluating the role of macrophages in IA-mediated protection. (B) The activity of AST and ALT in the serum of mice, n=8. (C) Image showing H\u0026amp;E staining in the liver of mice, arrows indicate inflammatory cell infiltration, n=3. (D) The concentration of IL-10, IL-17, and IL-22 in the serum of mice, n=8. (E) The concentration of IL-10, IL-17, and IL-22 in the liver of mice, n=8. (F) The relative mRNA expression of \u003cem\u003eIL-1β\u003c/em\u003e,\u003cem\u003e IL-6, \u003c/em\u003eand \u003cem\u003eTNF-α \u003c/em\u003ein the liver of mice, n=8.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-9281710/v1/655c1abf976e49088df405d6.png"},{"id":108810043,"identity":"a2cf4c0d-bd19-45cf-a231-5c4b0c6ac259","added_by":"auto","created_at":"2026-05-08 15:57:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":31467173,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9281710/v1/8396a489-ccf1-46b4-97e7-845400bdf287.pdf"},{"id":108621076,"identity":"1c509318-0784-4111-8f23-f0f5c906da58","added_by":"auto","created_at":"2026-05-06 14:50:21","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1821105,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-9281710/v1/e62f0093909c99c7068b0d77.docx"},{"id":108621077,"identity":"46628474-ab60-45f7-8340-5d06edab0f83","added_by":"auto","created_at":"2026-05-06 14:50:21","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":15129554,"visible":true,"origin":"","legend":"","description":"","filename":"fulluncroppedGelsandBlotsimages.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9281710/v1/e1dd588ecce775082b52322d.pdf"},{"id":108805730,"identity":"b514d173-e064-4219-b630-d72501fd34e8","added_by":"auto","created_at":"2026-05-08 15:26:44","extension":"jpeg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1363442,"visible":true,"origin":"","legend":"","description":"","filename":"graphicalabstract.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9281710/v1/526ff61586844f74fd290c02.jpeg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Gut microbiota-derived 3-indoleacrylic acid mediates ferulic acid protection against aflatoxin B1 hepatotoxicity via AhR-ferroptosis inhibition in ducks","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMycotoxin contamination is a persistent challenge to feed safety and farmed animal production worldwide. It is estimated that 60%\u0026ndash;80% of feed ingredients are contaminated with mycotoxins during production or storage, and consumption of contaminated feed by farm animals compromises growth and health, resulting in substantial production losses\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Among the diverse mycotoxins, aflatoxin B1 (AFB1) is of particular concern because of its high toxicity and widespread occurrence\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Ducks are especially sensitive to AFB1 exposure, which is frequently associated with impaired growth performance, liver injury, and increased morbidity and mortality\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMechanistic studies have confirmed that AFB1 is metabolized into the genotoxic compound aflatoxin-8,9-epoxide (AFBO) through the catalytic activity of cytochrome P450 (CYP450) enzymes in liver, leading to an excessive production of reactive oxygen species (ROS) \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. This overproduction of ROS induces hepatic oxidative stress and ferroptosis \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Ferroptotic hepatocytes release damage-associated molecular patterns that activate hepatic macrophages and amplify inflammatory responses through pro-inflammatory cytokine production \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Beyond direct hepatic damage, AFB1 disrupts the gut-liver axis by compromising intestinal barrier integrity and microbial metabolic homeostasis including bile acid biotransformation, and short-chain fatty acid synthesis \u003csup\u003e\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. The gut microbiota and their metabolites are key mediators of gut-liver axis homeostasis and hepatic function, and their composition and metabolic activity are highly responsive to dietary regulation\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. In broilers, supplementation with natural plant extracts has been shown to alleviate AFB1-induced liver injury by improving intestinal barrier function and modulating the gut microbiota\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Therefore, modulation of the gut microbiota by plant-derived compounds may offer a promising nutritional strategy for mitigating AFB1-induced hepatotoxicity.\u003c/p\u003e \u003cp\u003eWhole grains are of particular interest because they are a rich dietary source of fiber and phenolic compounds closely associated with gut microbial metabolism\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Among these bioactive constituents, our previous work identified FA as a key compound in whole grains capable of protecting against AFB1-induced hepatic toxicity in mice using untargeted metabolomics and network pharmacology analyses\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. However, FA exhibits poor oral bioavailability despite its profound systemic hepatoprotection, suggesting its therapeutic effects are likely mediated by indirect mechanisms rather than direct absorption \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. While FA is known to modulate bacterial composition in metabolic disorders \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, whether and how it leverages specific microbial metabolites to remotely orchestrate hepatic cell survival and immune responses remains a critical knowledge gap.\u003c/p\u003e \u003cp\u003eIn this study, we aimed to elucidate the mechanisms underlying the protective effects of FA against AFB1-induced liver injury in ducks, with a particular focus on the relationships among the gut microbiota, microbial metabolites, and host responses.\u003c/p\u003e \u003cp\u003eSpecifically, we sought to determine whether the hepatoprotective effects of FA are dependent on the gut microbiota and how FA influences microbial metabolic profiles under AFB1 exposure. We further aimed to identify candidate effector metabolites associated with FA treatment and to clarify how such metabolites regulate host signaling pathways and cellular responses involved in AFB1-induced liver injury. This study may provide mechanistic insight into the microbiota-mediated protective effects of FA and offer a theoretical basis for microbiome-targeted nutritional strategies to mitigate aflatoxicosis in poultry.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eFA alleviated AFB1-triggered liver injury in a gut microbe-dependent manner\u003c/h2\u003e \u003cp\u003eTo study the impact of FA on AFB1-induced liver injury, ducks were administered AFB1 and FA either individually or in combination (AFB1\u0026thinsp;+\u0026thinsp;FA) for four weeks (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The results demonstrated that FA supplementation significantly ameliorated the AFB1-induced elevation of serum aminotransferase (AST) and aminotransferase (ALT), which are well-established biomarkers of hepatic damage (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Moreover, dietary FA markedly attenuated the AFB1-triggered liver inflammation, as evidenced by reduced infiltration of pro-inflammatory cells into hepatic tissue and decreased mRNA expression of pro-inflammatory cytokines, including \u003cem\u003eIL-1β\u003c/em\u003e, \u003cem\u003eIL-6\u003c/em\u003e, and \u003cem\u003eTNF-α\u003c/em\u003e (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). It has been reported that AFB1 can disrupt the intestinal barrier and alter gut microbiota composition, thereby facilitating the translocation of intestinal bacteria and their metabolites into the systemic circulation, which subsequently exerts adverse effects on hepatic health \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. In this work, FA alleviated the AFB1-induced jejunal structural damage (Figures \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA and S1B), and reversed downregulation of tight junction proteins \u003cem\u003eZO-1\u003c/em\u003e and \u003cem\u003eZO-2\u003c/em\u003e in jejunm (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC). In addition, FA supplementation attenuated the AFB1-triggered intestinal inflammation by decreasing the expression of pro-inflammatory cytokines \u003cem\u003eIL-6\u003c/em\u003e, \u003cem\u003eIL-8\u003c/em\u003e, and \u003cem\u003eTNF-α\u003c/em\u003e in the jejunum (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD). The cecal microbiota composition was determined to examine the effects of FA or/and AFB1 on gut microbes by conducting 16S rRNA gene sequencing. Principal coordinates analysis (PCoA) showed a microbial separation among AFB1 group and other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). The predominant genus of gut microbiota was illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG. According to the Linear discriminant analysis Effect Size (LEfSe) analysis, \u003cem\u003eBacteroides\u003c/em\u003e, \u003cem\u003eMegamonas\u003c/em\u003e, and \u003cem\u003eSuccinatimonas\u003c/em\u003e were enriched in FA+AFB1 group compared with AFB1 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). Collectively, dietary FA effectively ameliorated hepatotoxicity, intestinal damage and gut microbiota dysbiosis induced by AFB1 in ducks.\u003c/p\u003e \u003cp\u003eTo further investigate the role of gut microbiota in FA alleviating AFB1-triggered liver injury, we carried out the gut microbiota depletion experiment with antibiotics and the FMT experiment (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The results indicated that antibiotics supplementation abolished the protective effects of FA on AFB1-induced elevations of serum ALT and AST (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ). Moreover, antibiotic treatment prevented FA from mitigating AFB1-induced liver inflammation, as evidenced by elevating infiltration of pro-inflammatory cells into hepatic tissue and increasing expression of pro-inflammatory cytokines in the ANTI (FA+AFB1) group compared to the FA+AFB1 group (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL). Consistent with the observations in the liver, FA had little protective effect on AFB1-induced jejunal damage in antibiotics-treated ducks. Specifically, FA failed to reverse AFB1-induced morphological damage (Figures \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eE and S1F), restore the expression of tight junction protein genes (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eG), or suppress the upregulation of pro-inflammatory cytokine genes following antibiotic treatment (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eH). In FMT experiment, the AFB1-originated microbiota induced hepatic damage, evidenced by increased serum AST and ALT activities, histopathological features, and higher hepatic mRNA expression of inflammatory cytokines. These negative effects were effectively attenuated in ducks honored with the FA+AFB1-originated microbiota (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eM-P). In addition, AFB1-originated microbiota also impaired intestinal barrier integrity, as demonstrated by decreased villi height and a lower ratio of villi height to crypt depth. Notably, FA+AFB1-derived microbiota ameliorated these morphological alterations and upregulated mRNA expression of \u003cem\u003eZO-1\u003c/em\u003e and \u003cem\u003eZO-2\u003c/em\u003e in jejunum of ducks (Figures \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eI-K). Moreover, the lower jejunal mRNA expression of inflammatory genes were observed in FMT-FA+AFB1 group relative to FMT-AFB1 group (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eL). Distinct separation of cecal microbiota structure in the FMT-AFB1 group compared to other groups was revealed by PCoA analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eQ). The composition of the predominant gut microbiota at the genus level was presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eR. In the FMT-FA+AFB1 group, there was a reduction in the relative abundance of potential pathogenic bacteria like \u003cem\u003eFusobacterium\u003c/em\u003e, \u003cem\u003eDesulfovibrio\u003c/em\u003e, and \u003cem\u003eEscherichia-Shigella\u003c/em\u003e compared to the FMT-AFB1 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eS). These results indicated that the protective effects of FA against liver injury triggered by AFB1 were mediated by gut microbiota.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eFA alleviated AFB1-triggered liver injury by enriching of\u003c/b\u003e \u003cb\u003ePeptostreptococcus anaerobius\u003c/b\u003e \u003cb\u003e(\u003c/b\u003e\u003cb\u003eP. anaerobius\u003c/b\u003e\u003cb\u003e) and enhancing tryptophan (Trp) metabolism for IA production\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate how FA restores AFB1-induced hepatic injury by the gut microbiota, we performed untargeted metabolomics analysis of cecal microbial metabolites to characterize functionally relevant microbial-derived metabolites. Principal component analysis (PCA) revealed that AFB1 treatment induced distinct metabolomic profiles in the cecum microbiota compared with CON group, whereas the FA+AFB1 group exhibited metabolomic profiles that largely overlapped with both the FA and CON groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Volcano plot analysis identified 160 significantly upregulated and 74 downregulated metabolites in FA+AFB1 group versus AFB1 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the differential metabolites in the AFB1 and FA+AFB1 groups were mainly enriched in the Trp metabolic pathway, suggesting FA mitigated AFB1-induced hepatotoxicity via microbial Trp metabolic pathway modulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Follow-up targeted Trp metabolomics showed that FA addition upregulated 20 Trp metabolites and downregulated 9 Trp metabolites (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Notably, the contents of indole, IGA, and IA were markedly increased in the FA+AFB1 group versus the AFB1 group, accompanied by an increasing trend in KYNA and XA (Figures \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA-E). To directly evaluate the protective effects of the five identified Trp metabolites against AFB1-induced liver injury, these metabolites were orally administered to mice daily for four weeks in combination with AFB1 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). The results showed that all five metabolites significantly suppressed AFB1-induced body weight loss (Figure S3A) and elevation of serum AST (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Moreover, indole markedly decreased the serum ALT activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG), while kynurenic acid reduced mRNA expression of \u003cem\u003eIL-1β\u003c/em\u003e and \u003cem\u003eTNF-α\u003c/em\u003e induced by AFB1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). Notably, IA exerted the most pronounced protective effects, as evidenced by the reduction in liver index and serum AST and ALT activities, together with marked suppression of \u003cem\u003eIL-1β\u003c/em\u003e, \u003cem\u003eIL-6\u003c/em\u003e, and \u003cem\u003eTNF-α\u003c/em\u003e expression (Figures S3B, 2F-H). Given the superior protective efficacy of IA observed in the screening model, we hypothesized that FA mitigates AFB1 hepatotoxicity primarily by promoting microbial IA production in ducks. To validate this, we quantified serum IA levels in ducks and found that FA supplementation markedly restored IA concentrations in AFB1-exposed ducks. Crucially, this elevation was confirmed to be microbiota-dependent, serum IA levels were significantly higher in ducks receiving the gut microbiota from the FA+AFB1 group compared to ducks receiving the gut microbiota from the AFB1 group, whereas antibiotic treatment abolished the FA-induced increase in serum IA (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ). Taken together, these results provide compelling evidence that FA alleviates hepatic injury by facilitating the microbial biosynthesis of IA. To identify the specific bacterial species responsible for this increased IA production, we focused on known IA-producing candidates. Given that \u003cem\u003eP. anaerobius\u003c/em\u003e, \u003cem\u003eP. russellii\u003c/em\u003e, \u003cem\u003eP. stomatis\u003c/em\u003e, and \u003cem\u003eC. sporogenes\u003c/em\u003e are known to metabolize Trp into IA \u003csup\u003e20\u003c/sup\u003e, we analyzed their relative abundances in the cecal microbiota of ducks. Notably, \u003cem\u003eP. anaerobius\u003c/em\u003e abundance was markedly higher in the FA+AFB1 group than in the AFB1 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK). Crucially, a parallel increase in both \u003cem\u003eP. anaerobius\u003c/em\u003e abundance and serum IA levels was observed in FA+AFB1 mice relative to AFB1 mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eM). This consistent enrichment across both avian and mammalian models underscores the cross-species conservation of FA\u0026rsquo;s capacity to promote the \u003cem\u003eP. anaerobius\u003c/em\u003e-IA axis. Next, we examined the effect of FA on \u003cem\u003eP. anaerobius in vitro\u003c/em\u003e, revealing that FA supplementation significantly enhanced bacterial growth and upregulated the mRNA expression of key enzymes related to IA synthesis (aromatic amino acid aminotransferase (ArAT), phenylacetate dehydrogenase (fldH), phenyllactate dehydratase subunits (fldB, fldC), and acyl coenzyme a dehydrogenase (acdA)), thereby indicating its role in promoting the tryptophan-to-IA metabolic pathway (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eN-Q). To directly validate this metabolic capability, we further assessed IA production in bacterial cultures. As anticipated, IA was clearly detected in the supernatants of \u003cem\u003eP. anaerobius\u003c/em\u003e, and its production was significantly increased by the supplementation of Trp (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eR and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eS). Collectively, these results demonstrated that FA enhanced \u003cem\u003eP. anaerobius\u003c/em\u003e-derived IA biosynthesis through coordinated transcriptional activation of the Trp metabolic pathway.\u003c/p\u003e \u003cp\u003eNext, we evaluated the protective effects of oral \u003cem\u003eP. anaerobius\u003c/em\u003e administration against AFB1-induced hepatotoxicity in mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eT). Compared with the AFB1 group, \u003cem\u003eP. anaerobius\u003c/em\u003e administration ameliorated liver injury, evidenced by improved liver histopathology, restored hepatic cord architecture, and reduced cellular vacuolization (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eU). This was accompanied by decreased serum AST, ALT activities and reduced hepatic mRNA expression of inflammatory cytokines in mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eV and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eW). These data demonstrated that FA mitigated AFB1-induced liver injury by enriching intestinal \u003cem\u003eP. anaerobius\u003c/em\u003e and enhancing Trp metabolism for IA production.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIA alleviated AFB1-induced liver injury by inhibiting ferroptosis of hepatocyte\u003c/h3\u003e\n\u003cp\u003eTo further explore the mechanism by which IA alleviated AFB1-induced liver injury, mice were allocated to CON, AFB1 or IA+AFB1 group (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). IA administration ameliorated liver injury in AFB1-exposed mice, as evidenced by attenuated architectural distortion of histomorphology, along with decreased serum ALT and AST activities (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Through integrated single-cell RNA sequencing analysis, nine major hepatic cell types were identified using uniform manifold approximation and projection (UMAP) dimensionality reduction (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-F). Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG showed the composition of hepatic cell types among different groups. There were 341 upregulated genes and 357 downregulated genes in IA+AFB1-treated hepatocytes compared to those exposed solely to AFB1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). KEGG enrichment analysis indicated that ferroptosis was the most significantly enriched pathway within the \u0026ldquo;Cell growth and death\u0026rdquo; subcategory of cellular processes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). These findings suggest that IA potentially mitigates AFB1-induced hepatotoxicity by suppressing ferroptosis signaling. Based on this evidence, we further investigated whether IA ameliorated AFB1-triggered liver injury by inhibiting ferroptosis. Transmission electron microscopy revealed mitochondrial shrinkage and outer membrane rupture in hepatocytes from AFB1-exposed mice, whereas these ultrastructural alterations were attenuated by dietary IA intervention (Figure. 3J). AFB1-exposed mice exhibited hepatic iron overload, elevated lipid peroxidation (serum malondialdehyde (MDA)), and glutathione (GSH) depletion-all of which are phenotypic hallmarks of ferroptosis. Notably, dietary IA administration reversed these changes (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK-M). IA upregulated mRNA expression of \u003cem\u003eGPX4\u003c/em\u003e (lipid peroxide scavenger) and \u003cem\u003eCISD1\u003c/em\u003e (mitochondrial iron-sulfur cluster protein) in the liver (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eN). Consistently, western blot analysis indicated that dietary IA supplementation reversed the AFB1-induced downregulation of hepatic ferroptosis-inhibitory proteins (xCT, HO-1, and GPX4) in mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eO and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eP). Collectively, IA was found to inhibit AFB1-induced hepatocyte ferroptosis. Consistent with these results, dietary administration of \u003cem\u003eP. anaerobius\u003c/em\u003e elevated serum IA levels compared with the AFB1 group (Figure S4A). Hepatic Fe\u0026sup2;⁺ ions accumulation and serum MDA levels were reduced, while the serum concentration of GSH was increased in mice fed with \u003cem\u003eP. anaerobius\u003c/em\u003e and AFB1 in comparison with those fed with AFB1 alone (Figures S4B-D). Dietary \u003cem\u003eP. anaerobius\u003c/em\u003e administration upregulated mRNA expression of ferroptosis-suppressive genes, while enhancing GPX4 and HO-1 protein levels (Figures S4E-I). These findings showed that \u003cem\u003eP. anaerobius\u003c/em\u003e may mitigate AFB1-induced hepatocyte ferroptosis by producing IA in mice. \u003cem\u003eIn vitro\u003c/em\u003e (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eQ), the viability of AML12 cells was notably increased when it was treated with 50, 100 or 200 \u0026micro;M IA for 24 h compared with the untreated control and IA treatment reversed the AFB1-induced decline in cell viability (Figures S5A and S5B). Specifically, IA mitigated lipid peroxidation, activated GSH biosynthesis, cleared excess ROS, and suppressed Fe\u003csup\u003e2+\u003c/sup\u003e ions accumulation (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eR-W). IA upregulated the levels of ferroptosis-inhibiting related proteins (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eX and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eY). Taken together, these findings demonstrated that IA alleviated AFB1-induced liver injury by inhibiting ferroptosis of hepatocyte.\u003c/p\u003e \u003cp\u003eTo further verify the role of ferroptosis in IA-mediated hepatoprotection, the ferroptosis inhibitor Ferrostatin-1 (Fer-1) was used in the AFB1-exposed mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Relative to AFB1 exposure, Fer-1 intervention counteracted AFB1-induced elevations of serum AST and ALT, hepatic iron accumulation and lipid peroxidation, while enhanced GSH synthesis; co-treatment with IA and Fer-1 did not synergize these effects (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-E). Histopathological assessment revealed that Fer-1 treatment suppressed AFB1-triggered hepatocellular vacuolization and disruption of cord architecture, with no additive protective effect observed when IA was co-administrated with Fer-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). Molecular analyses revealed that Fer-1 inhibited the downregulated expression of iron homeostasis genes (\u003cem\u003eGPX4\u003c/em\u003e and \u003cem\u003eCISD1\u003c/em\u003e) and the downregulated levels of xCT, HO-1, and GPX4 induced by AFB1; whereas IA co-administrated with AFB1 and Fer-1 failed to further enhance these regulatory responses (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH-K). Collectively, AFB1 induced liver injury through ferroptosis activation, while IA exerted hepatoprotection by specifically inhibiting this cascade.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eIA mitigated AFB1-induced hepatocyte ferroptosis through an AhR-dependent pathway\u003c/h3\u003e\n\u003cp\u003eA recent study demonstrated that the aryl hydrocarbon receptor (AhR) is required for AFB1-induced toxicity \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Furthermore, emerging evidence indicates that AhR signaling is tightly linked to the regulation of ferroptosis \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Therefore, we investigated whether AFB1 induces hepatocyte ferroptosis via AhR and whether IA alleviates AFB1-induced liver injury by modulating this pathway. It was been observed that AFB1 exposure elevated hepatic mRNA expression of \u003cem\u003eAhR\u003c/em\u003e and its downstream effectors (\u003cem\u003eCYP1A1\u003c/em\u003e and \u003cem\u003eCYP1A2\u003c/em\u003e), and increased protein levels of AhR and CYP1A1, these upregulations were effectively reversed by IA co-treatment in mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-E). Given that AhR signaling modulates interleukin secretion profiles, particularly its dual regulation of anti-inflammatory IL-10, IL-22 and pro-inflammatory IL-17 \u003csup\u003e23\u003c/sup\u003e, we next quantified serum and hepatic levels of these cytokines in mice. IA supplementation promoted the levels of anti-inflammatory cytokines IL-10 and IL-22, while suppressed the level of pro-inflammatory mediator IL-17 in the serum of mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). IA also increased the level of IL-22 in the liver of mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). Previous research has demonstrated both AFB1 and IA can also act as ligands of AhR \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. In this study, we further compared their binding affinity to AhR using surface plasmon resonance (SPR) analysis, which revealed that AFB1 exhibited a significantly higher affinity than IA to AhR (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ). Molecular docking analyses also revealed that AFB1 exhibits stronger binding affinity for AhR compared to IA, with respective -cdocker interaction energies of 34.76 kJ\u0026middot;mol⁻\u0026sup1; (for AFB1) and 25.24 kJ\u0026middot;mol⁻\u0026sup1; (for IA). For the AFB1-AhR complex, hydrogen bonds characterized the primary binding region within AhR. In contrast, hydrophobic interactions, classified as weak interaction forces, served as the dominant binding mechanism for the IA-AhR complex (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eK). This indicated that compared to IA, AFB1 acted as a full agonist of AhR, whereas IA functioned as an AhR antagonist when coexisting with AFB1. Collectively, despite exhibiting lower binding affinity for AhR, IA functionally antagonized AFB1-induced activation of the AhR pathway, ultimately alleviating hepatotoxicity \u003cem\u003ein vivo\u003c/em\u003e. To validate the essential role of AhR in IA mitigating AFB1-induced liver injury, CH-223191 (CH) was administered to inhibit AhR signaling in mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eL). Compared to AFB1 group, CH intervention suppressed AFB1-induced elevations of serum AST and ALT, rebalanced inflammatory homeostasis by downregulating the level of IL-17 in serum and upregulating levels of IL-10 and IL-22 in both serum and liver of mice, while there were no additional effects of IA on CH-treated mice with AFB1 exposure (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eM-R). H\u0026amp;E staining revealed that CH treatment inhibited AFB1-triggered hepatic cord derangement and inflammatory infiltration, while IA and CH co-treatment did not enhance this protective phenotype on mice with AFB1 exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eS). Given the established role of AhR in ferroptosis regulation \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, this study examined whether IA counteracted AFB1-induced ferroptosis via AhR blockade. Accumulated Fe\u003csup\u003e2+\u003c/sup\u003e ions deposition, increased serum MDA and depleted GSH levels in the liver of mice exposure to AFB1 were eliminated by CH intervention, but these protective effects did not augment when IA was co-administrated with CH (Figures S6A-C). CH-mediated AhR suppression rescued AFB1-triggered transcriptional repression of ferroptosis inhibitors, and upregulated the levels of xCT, HO-1, and GPX4; IA and CH co-treatment failed to further potentiated these regulatory effects on mice exposure to AFB1 (Figures S6D-L). These findings demonstrated that IA executed hepatoprotection through functional antagonism of AhR-driven ferroptotic signaling.\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e cell assays demonstrated that CH treatment antagonized AFB1-induced cytotoxicity, whereas CH and IA co-administration conferred no additional viability restoration on cells exposure to AFB1 (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). CH intervention in AFB1 treatment suppressed the AFB1-induced elevation in intracellular MDA and the Fe\u003csup\u003e2+\u003c/sup\u003e ions levels without affecting GSH content; IA co-treatment with CH and AFB1 did not further augmented these changes (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-F). In addition, The CH intervention inhibited the AFB1-induced upregulation of AhR protein level and downregulation of anti-ferroptotic proteins levels, including xCT, HO-1, and GPX4; IA co-treatment with CH and AFB1 failed to further potentiate these regulatory effects (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG-K). Genetic targeting of AhR validated its essential role in IA-conferred ferroptosis resistance (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eL). siRNA screening secured siAhR2 as the optimal construct inducing effective AhR silencing in AML12 hepatocytes (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eM and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eN). AhR knockdown reversed AFB1-induced accumulation of ferroptotic biomarkers (ROS and Fe\u0026sup2;⁺ ions), while combined IA treatment failed to potentiate this mitigation (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eO-R). Additionally, AhR knockdown in AFB1-treated hepatocytes significantly downregulated AhR protein level and upregulated xCT, HO-1, and GPX4 protein levels compared to the AFB1-treated group; these changes were not further enhanced by IA co-administration (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eS-W). Collectively, these results demonstrated that IA executed its hepatoprotective effects exclusively through competitive inhibition of AhR signaling.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eIA attenuated AFB1-induced hepatic inflammation via macrophage reprogramming\u003c/h3\u003e\n\u003cp\u003eOur previous results demonstrated that IA effectively inhibited the infiltration of pro-inflammatory cells into liver tissue induced by AFB1 exposure. Consistent with this finding, IHC staining further confirmed that IA significantly suppressed the AFB1-induced increase in hepatic macrophages in mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Liver macrophages primarily comprise Kupffer cells (KCs) and monocyte-derived macrophages (MoMFs). To elucidate the specific effects of IA on these macrophage subsets, flow cytometry analysis was conducted. AFB1 exposure tended to increase the proportion of MoMFs (F4/80\u003csup\u003elo\u003c/sup\u003eCD11b\u003csup\u003ehi\u003c/sup\u003e) compared to the CON group. Conversely, IA treatment significantly increased the KCs frequency and reduced the MoMFs ratio compared to the AFB1-exposed group (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC-E). Given the pivotal role of macrophage subset heterogeneity in modulating liver inflammatory responses and maintaining tissue homeostasis \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, we further performed subclustering analysis on the KCs population. This analysis identified five distinct KCs subtypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF), which were annotated based on the expression of marker genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG). The differentially expressed genes in these subpopulations underwent reactome pathway enrichment analysis, the results revealed significant enrichment in the immune system and innate immune system pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI). IA suppressed an AFB1-expanded pro-inflammatory subset (Subcluster4, highly expressed Marco, Cd38, Il1b), while enriching Subcluster0, highly expressed Mrc1, Il10ra, Tgfbr1 (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eK). MoMFs resolved into six subclusters that manually annotated based on the expression of marker genes (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eL and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eM). Reactome pathway enrichment analysis showed that the immune system and innate immune system pathways were also enriched (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eO). When we compared IA+AFB1 group with AFB1 group, IA suppressed a pro-inflammatory MoMFs subset (Subcluster0, highly expressed Cxcl9, Cxcl10, Nfkb1) and expanded an anti-inflammatory MoMFs subset (Subcluster1, highly expressed Mertk, C1qa, Abcg1) (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eN, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eP and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eQ). Collectively, these results demonstrated that IA ameliorated AFB1-induced liver inflammation by reprogramming the subset composition and function of hepatic macrophages, specifically suppressing pro-inflammatory KCs and MoMFs subpopulations while enriching their anti-inflammatory counterparts, thereby restoring immune homeostasis. To directly verify the role of macrophages in the protective effects of IA, we employed a macrophage-depleted mice model using clodronate liposomes (CLP) to selectively deplete mononuclear phagocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). Hepatocyte injury, serum AST and ALT elevations and histopathological aberrations induced by AFB1 were alleviated in macrophage-depleted mice; while these effects were not augmented by co-administration IA and CLP into AFB1-treated mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). Critically, CLP inhibited the decrease of anti-inflammatory cytokines in the serum and liver of mice induced by AFB1, along with transcriptional upregulation of pro-inflammatory \u003cem\u003eIL-1β, IL-6\u003c/em\u003e, and \u003cem\u003eTNF-α\u003c/em\u003e, while IA and CLP co-treatment did not produce additive effect on mice exposure to AFB1 (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD-F). Collectively, IA alleviated AFB1-induced hepatic inflammatory by rebalancing the secretion of pro-inflammatory cytokines and anti-inflammatory cytokines in macrophage.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe gut microbiota plays pivotal roles in maintaining host physiological homeostasis, with dysbiosis in its composition and function being mechanistically linked to multiple disease states \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Substantial evidence indicates that the gut microbiome exerts multifaceted health-promoting effects through its involvement in diverse metabolic processes, such as short-chain fatty acids (SCFAs) production, Trp metabolism, and bile acid biotransformation \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Previous research has demonstrated that AFB1 exposure increased the abundance of potential pathogens such as \u003cem\u003eDesulfovibrio\u003c/em\u003e and disrupted gut microbial metabolism, which subsequently led to indirect liver damage via the gut-liver axis \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Employing untargeted metabolomics, our study identified disruptions in intestinal microbial Trp metabolism as a potential key contributor to AFB1-induced hepatic injury. Maintenance of Trp metabolic homeostasis is essential for maintaining physiological hepatic function and supporting growth performance in farmed animals\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.. This investigation further revealed that FA notably elevated levels of Trp-derived metabolites, aligning with existing evidence that FA modulated microbial composition and enhancing Trp metabolism to alleviate anxiety-like behaviors \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Moreover, supplementation with Trp-derived metabolites, particularly IA, effectively attenuated AFB1-induced liver inflammation, underscoring the functional significance of these metabolites in hepatoprotection.\u003c/p\u003e \u003cp\u003eStudies have revealed that \u003cem\u003eP. anaerobius\u003c/em\u003e, \u003cem\u003eP. russellii\u003c/em\u003e, \u003cem\u003eP. stomatis\u003c/em\u003e, and \u003cem\u003eClostridium sporogenes\u003c/em\u003e can metabolize Trp to produce IA \u003csup\u003e20\u003c/sup\u003e. The current investigation demonstrated for the first time that FA treatment directly enhanced its growth \u003cem\u003ein vitro\u003c/em\u003e, and elevated the transcriptional levels of genes encoding enzymes related to Trp metabolism. This work comprehensively verifies the metabolic pathway by which FA enriched \u003cem\u003eP. anaerobius\u003c/em\u003e and activates Trp-metabolism-related enzymes to produce IA, providing a theoretical basis for modulating gut microbiota structure and metabolism via dietary intervention. Previous research indicated that \u003cem\u003eP. russellii\u003c/em\u003e (a commensal bacterium within the same genus as \u003cem\u003eP. anaerobius\u003c/em\u003e) enhanced intestinal epithelial barrier function and alleviated inflammation via IA production \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. While some studies showed that \u003cem\u003eP. anaerobius\u003c/em\u003e was enriched in colorectal cancer patients and promoted tumorigenesis through modulation of tumor immunity \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Thus, effects of \u003cem\u003eP. anaerobius\u003c/em\u003e may depend on its site of action and dosage, requiring further validation for its practical applications. In the future, we may consider applying enzymes related to Trp metabolism or Trp metabolites for the prevention and alleviation of liver injury.\u003c/p\u003e \u003cp\u003eAFB1 induces hepatic injury through a complex array of pathological processes, including inflammation, oxidative stress, apoptosis, and autophagy \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Recent evidence indicates that AFB1 also initiates hepatocyte ferroptosis, a regulated cell death pathway dependent on iron and driven by lipid peroxidation \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. In this study, single-cell transcriptomics demonstrated that IA effectively alleviated AFB1-induced hepatic injury by inhibiting hepatocyte ferroptosis \u003cem\u003ein vivo\u003c/em\u003e. This is supported by various data: transmission electron microscopy revealed that IA protected mitochondrial structure, and serum biomarker analysis indicated that IA decreased ferrous iron and MDA levels while restoring GSH content. Cellular assays further confirmed that IA restored mitochondrial function by reducing iron overload and scavenging ROS. Molecular analysis revealed that IA orchestrated two regulatory pathways: the reactivation of the GPX4-xCT antioxidant system through the upregulation of GPX4/xCT proteins; and the remodeling of iron homeostasis via the upregulation of ferroportin (FPN) to facilitate ferrous iron efflux, thereby inhibiting Fenton reactions. This is complemented by CISD1-mediated stabilization of mitochondrial iron-sulfur clusters to limit the release of free iron. Through combined intervention with the ferroptosis inhibitor Fer-1, this study further validated that the protective effect of IA was highly dependent on the suppression of the ferroptosis pathway. Single intervention with Fer-1 could partially reverse AFB1-induced liver injury, nevertheless, no further improvement was observed in the group receiving the combined treatment of IA and Fer-1. A similar trend was exhibited in the expression changes of ferroptosis-related molecular markers, including xCT, CISD1, and GPX4. These results confirm that ferroptosis functions as a critical mechanistic pathway in AFB1-induced hepatotoxicity and concurrently illustrate that the protective efficacy of IA is predominantly mediated through the inhibition of this pathway.\u003c/p\u003e \u003cp\u003eThe AhR, a ligand-activated transcription factor, exhibits dual roles in hepatic physiology. It regulates immune homeostasis through anti-inflammatory effects, whereas aberrant activation provokes metabolic disruption and inflammatory liver injury \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Endogenous metabolites like gut-derived indoles orchestrated anti-inflammatory programming via activation of the IL-22 axis, thereby modulating the functional maturation of intestinal intraepithelial lymphocytes and innate lymphoid cells \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Whereas exogenous ligand like dioxins induced hepatotoxic inflammation via CYP1A1/A2 upregulation \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Here, we identified AFB1 as an exogenous agonist that potently amplified hepatic AhR expression, eliciting inflammation. Crucially, IA reversed these AFB1-induced molecular changes and mitigated hepatic pathology. This antagonism is structurally elucidated by our biophysical characterization utilizing SPR and molecular docking. AhR ligands are functionally categorized into full agonists, partial agonists, and competitive antagonists, where efficacy is dictated by binding affinity and intrinsic activity \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, AFB1 acted as a full agonist, engaging the AhR ligand-binding domain with high affinity predominantly via strong hydrogen bonds. In contrast, IA bound with lower affinity dominated by weaker hydrophobic interactions. Mechanistically, we identify IA as a partial agonist of AhR \u003cem\u003ein vivo\u003c/em\u003e. Since IA triggers only a sub-maximal response compared to AFB1, it can inhibit the hyperactivation of AhR by AFB1. This dampening effect shifts the cytokine profile from a toxic, pro-inflammatory state (IL-17) to a restorative, homeostatic phenotype (IL-22/IL-10). This underscores the therapeutic advantage of endogenous partial agonists, which maintain basal physiological signaling while preventing toxic overstimulation \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Building upon existing evidence that ferroptosis is modulated by AhR via IA in HT29 cells \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, this study demonstrates that AFB1 induces hepatic ferroptosis through AhR activation. Importantly, IA competitively inhibits AhR-driven ferroptosis, as demonstrated by both \u003cem\u003ein vivo\u003c/em\u003e pharmacological AhR inhibition and \u003cem\u003ein vitro\u003c/em\u003e siRNA-mediated AhR knockdown. This mechanistic insight positions the gut microbial metabolite IA as a potential therapeutic agent to counteract AFB1-triggered liver injury.\u003c/p\u003e \u003cp\u003eBeyond hepatocyte-intrinsic mechanisms, macrophages, key orchestrators of innate immunity, critically modulate hepatic immune homeostasis \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Through flow cytometry and integrated single-cell RNA sequencing, this study demonstrated that IA ameliorated AFB1-induced liver inflammation by reprogramming the subset composition and function of hepatic macrophages, specifically suppressing pro-inflammatory KCs and MoMFs subpopulations while enriching their anti-inflammatory counterparts, thereby restoring immune homeostasis. Critically, this study found that macrophage depletion mediated by CLP significantly alleviated AFB1-induced liver injury. The absence of additive effects in the IA\u0026thinsp;+\u0026thinsp;CLP co-treatment group highlighted the critical role of functional macrophages was indispensable for IA-mediated anti-inflammatory effects. Although previous research has associated CLP with the alleviation of acetaminophen-induced injury \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, this study pioneered the use of macrophage-depleted models to elucidate the mechanistic role of macrophages as crucial mediators of IA's protective effects against mycotoxin-induced inflammation. This provides a compelling rationale for developing macrophage-targeted therapeutic interventions.\u003c/p\u003e \u003cp\u003eIn summary, we found that FA alleviates AFB1-induced liver injury in ducks by remodeling the gut microbiota and promoting the production of \u003cem\u003eP. anaerobius\u003c/em\u003e-derived IA. Mechanistically, IA confers hepatoprotection by restraining AhR-driven ferroptosis and inflammatory macrophage reprogramming. These findings provide a rationale for microbiome-based nutritional strategies to mitigate AFB1-induced injury and improve the health of farmed animals.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eReagents\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe reagents and antibody information used in this study were listed in Table S1 and Table S2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDuck\u003c/strong\u003e\u003cstrong\u003es\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOne-day-old male Pekin ducks were housed in wire-floored cages, with eight ducklings per cage. Ducks were housed under conditions consistent with previous reports \u003csup\u003e3\u003c/sup\u003e. On day 28, ducks were euthanized to collect samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtective role of FA in ducks fed AFB1-contaminated diets:\u003c/strong\u003eThe 192 ducks that were randomly assigned to four groups, CON, FA, AFB1, and FA+AFB1 groups. Each group consisted of six replicate cages, with eight ducks per cage. The addition dosage of FA was 500 mg/kg of feed. The AFB1 concentration was 20 \u0026mu;g/kg, achieved by incorporating AFB1-contaminated peanut meal into the basal diet. To collect samples, one duck with an average body weight was euthanized from each replicate on the 28th day.\u003c/p\u003e\n\u003cp\u003eAntibiotic intervention in FA+AFB1 ducks to assess gut microbiota dependency: The 24 ducks were randomly assigned into three groups: AFB1, FA+AFB1, and ANTI (FA+AFB1) group. The ducks of ANTI (FA+AFB1) group received antibiotics (1 g/L streptomycin, 1 g/L ampicillin, and 1 g/L neomycin) in their drinking water for a period of 28 days. All ducks were given a diet naturally contaminated with AFB1 (AFB1, 20 \u0026mu;g/kg), and FA (500 mg/kg) was included in the diets of the FA+AFB1 and ANTI (FA+AFB1) groups.\u003c/p\u003e\n\u003cp\u003eFMT experiment investigate the role of gut microbiota in FA alleviating AFB1-induced liver injury: For eliminating the native gut microbiota, 32 recipient ducks received antibiotics in their drinking water for two weeks. After antibiotics treatment, recipient ducks were respectively separated into four groups (FMT-CON, FMT-FA, FMT-AFB1, and FMT-FA+AFB1) and received fecal microbiota transplants (10 mL per kg of body weight each day) from their respective donor ducks (FA supplementation for ducks fed with diet contaminated with AFB1) for 2 weeks. Give 10 mL per kg of body weight each day. The microbiota suspension was prepared according to previous studies \u003csup\u003e9\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eMice\u003c/p\u003e\n\u003cp\u003eFour-week-old male Kunming mice were obtained from Beijing HFK Bioscience (Beijing, China). Mice were housed under conditions consistent with previous reports \u003csup\u003e15\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEfficacy of Trp metabolites efficacy in mice:\u0026nbsp;\u003c/strong\u003eMice were randomly divided into 7 groups (n=8) and fed different candidate Trp metabolites: CON, AFB1, Indole+AFB1, indole-3-glyoxylic acid (IGA)+AFB1, IA+AFB1, kynurenic acid (KYNA)+AFB1, and xanthurenic acid (XA)+AFB1 groups. The addition dosage of AFB1 was 1.8 mg/kg of feed. The dosage of Indole, IGA and IA were 300 mg/kg of feed, the dosage of KYNA and XA were 600 mg/kg of feed. The duration of the experiment was 28 days.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEvaluation of FA effects on\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eP. anaerobius\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and IA production in AFB1-induced mice\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003eThe 24 mice were divided randomly into three groups: CON, AFB1 and FA+AFB1 groups. AFB1 and FA were administered at dosages of 1.8 mg/kg feed and 300 mg/kg feed, respectively. The duration of the experiment was 28 days.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOral gavage of \u003cem\u003eP. anaerobius\u003c/em\u003e in mice:\u0026nbsp;\u003c/strong\u003eThe 24 mice were randomly assigned into three groups: CON, fed a basal diet and gavaged with 0.2 mL of phosphate-buffered saline (PBS); AFB1: fed AFB1 (1.8 mg/kg) diet and gavaged with 0.2 mL of PBS; PA+AFB1: fed AFB1 (1.8 mg/kg) diet and gavaged with 0.2 mL of fresh \u003cem\u003eP. anaerobius\u003c/em\u003e suspension. The duration of the experiment was 28 days. The fresh \u003cem\u003eP. anaerobius\u003c/em\u003e suspension was prepared. Briefly, \u003cem\u003eP.anaerobius\u003c/em\u003e was cultured anaerobically in reinforced clostridial medium (RCM) at 37\u0026deg;C for 24 h. After cultivation, the cell of starin\u003cu\u003e\u0026nbsp;\u003c/u\u003ewas centrifuged, washed twice, and resuspended in PBS to a final concentration of 1 \u0026times; 10⁸ CFU/mL. Fresh suspensions were prepared daily prior to gavage.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMechanistic evaluation of IA against AFB1-induced liver injury:\u0026nbsp;\u003c/strong\u003eThe 24 mice were divided randomly into three groups: CON, AFB1 and IA+AFB1 groups. To further investigate ferroptosis involvement in the mechanism, ferroptosis inhibitor Fer-1 was used and mice were divided into six groups: CON, AFB1, IA+AFB1, CON+Fer-1, AFB1+Fer-1, and IA+AFB1+Fer-1. Concurrently, to validate the involvement of the AhR in the protective effect of IA against AFB1-induced ferroptosis, mice were treated with the AhR inhibitor CH. Groups included CON, AFB1, IA+AFB1, CON+CH, AFB1+CH, and IA+AFB1+CH. Additionally, to assess the role of macrophages in IA-mediated protection against AFB1-induced hepatic injury, macrophage depletion was achieved through the administration of clodronate CLP. Mice were randomly divided into 6 groups: CON, AFB1, IA+AFB1, CON+CLP, AFB1+CLP, and IA+AFB1+CLP. The experiment lasted for 28 days. AFB1 and IA were administered at dosages of 1.8 mg/kg feed and 300 mg/kg feed, respectively. For Fer-1 administration, mice received daily intraperitoneal injections of Fer-1 (1 mg/kg body weight). For CH treatment, mice were subjected to daily oral gavage with CH (10 mg/kg body weight). For CLP-induced macrophage depletion, mice received weekly intraperitoneal injections of CLP (10 \u0026mu;L/g body weight).\u003c/p\u003e\n\u003cp\u003eEffect of FA on the growth of \u003cem\u003eP. anaerobius\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eP. anaerobius\u003c/em\u003e (ATCC 27337) was inoculated into RCM medium supplemented with varying concentrations of FA (0, 10, 30, or 50 mg/L) and incubated anaerobically at 37 \u0026deg;C. Bacterial growth was monitored by sampling 500 \u0026mu;L of suspension every 2 h and measuring OD\u003csub\u003e600\u003c/sub\u003e with a ultraviolet (UV) spectrophotometer. After 24 hours of incubation, 4 mL of culture was centrifuged, and the resulting cell pellet was collected for qPCR analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCulture of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eP. anaerobius\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eunder Trp supplementation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eP. anaerobius\u003c/em\u003e was maintained anaerobically in RCM at 37 \u0026deg;C for 24h. The medium was supplemented as follows: PA, no additional supplement; PA+Trp, 50 \u0026mu;g/mL Trp.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAML-12 hepatocytes were acquired from the American Type Culture Collection (ATCC, VA, USA) and cultured as described previously \u003csup\u003e15\u003c/sup\u003e. Cells were treated with 200 \u0026mu;M IA and 8 \u0026mu;M AFB1 for 24h. To suppress AhR activation, cells were pre-treated for 2 h with the AhR antagonist CH (10 \u0026mu;M) prior to 24-h co-exposure with subsequent compounds. Cells were transfered with siAhR oligos (sense: 5\u0026apos;-CUUUCUCUCCCAUAAUGGAdTdT-3\u0026apos;, antisense: 5\u0026apos;-UCCAUUAUGGGAGAGAAAGdTdT-3\u0026apos;) using Lipofectamine 3000 reagent for AhR knockdown.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistopathology and transmission electron microscopy\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;analysis\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eof liver\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLiver or jejunum tissues underwent paraffin embedding, followed by sectioning at a thickness of 5 \u0026mu;m. After deparaffinization with xylene, the sections were rehydrated and mounted on glass slides. Haematoxylin and eosin (H\u0026amp;E) staining was then applied. High-resolution imaging was conducted utilizing a microscope slide scanner (3DHISTECH, Budapest, Hungary) in conjunction with a DP70 digital camera (Olympus, Tokyo, Japan). For ultrastructural analysis of mitochondria, liver tissues were treated with 2.5% glutaraldehyde for fixation, dehydrated using a graded series of ethanol, and embedded in epoxy resin. Ultrathin sections were mounted on transmission electron microscopy grids and examined using a transmission electron microscope (Hitachi HT7700, Tokyo, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiochemical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe activities/levels of aspartate AST, ALT, serum MDA, GSH, and hepatic iron concentrations were measured using commercial kits (Nanjing Jiancheng, Nanjing, China). Cytokine profiles (IL-10, IL-17, IL-22) in serum and liver of mice were analyzed by commercial ELISA kits (SLCY, Beijing, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQ\u003c/strong\u003e\u003cstrong\u003euantitative real-time PCR (qRT-PCR)\u003c/strong\u003e\u003cstrong\u003eanalysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA extraction and cDNA synthesis were performed with a commercial kit (Vazyme, Nanjing, China). A two-step qRT-PCR was conducted using a Real-Time PCR Detection System from Bio-Rad, CA, USA. The mRNA expression of genes were determined based on the expression of the GAPDH. Specific primer sequences were listed in Table S3.\u003c/p\u003e\n\u003cp\u003e16S rRNA gene sequencing analysis\u003c/p\u003e\n\u003cp\u003eGenomic DNA from the cecal microbiota was extracted employing a DNA extraction kit (Omega Bio-tek, GA, USA). The V3-V4 hypervariable region of the bacterial 16S rRNA gene was subsequently amplified on the PCR system (ABI, CA, USA). The resulting purified amplicons underwent sequencing on an Illumina MiSeq PE300 platform (Majorbio, Shanghai, China). The data was analyzed using the Majorbio Cloud Platform (https://www.majorbio.com).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUntargeted metabolomics analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCecal content samples were homogenized in a methanol-water solution (4:1, v/v) utilizing a cryogenic grinder set at 50 Hz for 6 minutes. After centrifugation, the supernatants from the homogenates were collected. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was conducted using a UHPLC-Q Exactive\u0026trade; HF-X system (Thermo Scientific, MA, USA). The raw data was analyzed using the Majorbio Cloud Platform (https://www.majorbio.com).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTargeted Trp metabolomics analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cecal contents were homogenized at a ratio of 1:10 (w/v) in a solvent mixture of methanol, acetonitrile, and water (2:2:1, v/v/v). Following centrifugation, the supernatants of homogenates were collected for subsequent analysis. The separation of target compounds was achieved through ultra-high-performance liquid chromatography (Milford, MA, USA). The mass spectrometry analysis was executed on a SCIEX 6500 QTRAP triple quadrupole mass spectrometer (Framingham, MA, USA). Mass spectrometry data acquisition and quantitative analysis of the target compounds were performed using SCIEX Analyst workstation software (Version 1.6.3) and MultiQuant software (Version 3.0.3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of IA content in serum and bacterial culture supernatant\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSerum samples were diluted in a 1:1 ratio with deionized water, and 50 \u0026mu;L aliquots were mixed with 300 \u0026mu;L of 1% (v/v) aqueous formic acid. Solid-phase extraction was performed using HLB cartridges (Thermo Scientific, MA, USA), which were sequentially conditioned with 2 mL methanol and 2 mL 1% aqueous formic acid. The pre-treated serum samples were loaded onto the cartridges, washed with 2 mL 1% aqueous formic acid, and eluted with 500 \u0026mu;L methanol. Then Eluates or bacterial culture supernatants were filtered through 0.22 \u0026mu;m membranes before high-performance liquid chromatography (HPLC) analysis. Chromatographic separation was performed using a C18 reversed-phase column. Isocratic elution were performed using a mobile phase of acetonitrile and 0.1% aqueous formic acid (3:7, v/v) at a flow rate of 1.0 mL/min. UV detection was conducted at a wavelength of 254 nm with 20 \u0026mu;L injection volumes over a 12-minute run time.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSingle-cell RNA sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFresh mouse liver tissues were dissociated and filtered through 40 \u0026mu;m strainers, followed by enzymatic digestion and erythrocyte lysis. The resulting cell suspensions were resuspended in 0.04% BSA-PBS. Single-cell suspensions were then processed using the Chromium Single Cell 3ʹ Kit (10x Genomics, CA, USA). Library sequencing was performed on the Illumina NovaSeq 6000 platform (CA, USA) using 150-cycle paired-end reads as previously described \u003csup\u003e44\u003c/sup\u003e. Data processing utilized Cell Ranger (v7.1.0, https://www.10xgenomics.com/software) for alignment (mm10) and unique molecular identifier (UMI) counting. Seurat (v4.1.1, https://satijalab.org/seurat/) was employed to filter out low-quality cells based on nUMI/gene thresholds of \u0026plusmn;2 standard deviations and a mitochondrial gene percentage of \u0026le;5%. Variable genes were identified for integration (using FindIntegrationAnchors and IntegrateData in the seurat package), followed by PCA reduction to the top 30 principal components and t-distributed stochastic neighbor embedding (tSNE) visualization. Cell types were annotated using SingleR and marker genes. Differentially expressed genes (DEGs) were defined by |log2FC| \u0026gt; 0.25 and Q \u0026le; 0.05. Functional enrichment analyses for Gene Ontology (GO) terms and KEGG pathways were conducted using the Goato tool (https://github.com/tanghaibao/Goatools).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell viability assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess cell viability, the CCK-8 assay was employed. In summary, cells were cultured in 96-well plates for 24 hours, after which they were exposed to different concentrations of reagents for an additional 24 hours. The CCK-8 assay was performed in accordance with the manufacturer\u0026apos;s instructions (Beyotime, Shanghai, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eROS determination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIntracellular ROS generation in AML12 hepatocytes was quantified using a commercial detection kit (Beyotime, Shanghai, China) per standardized protocols. Briefly, the treated cells underwent 30-minute exposure to 10 \u0026mu;M DCF-DA at 37 \u0026deg;C, followed by image acquisition using fluorescence microscopy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFerroOrange staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing 24-h treatment with various concentrations of reagents in 24-well plates, AML12 cells were incubated with FerroOrange fluorescent probe (1 \u0026mu;M) for 20 minutes in the dark. Subsequent confocal microscopy imaging was performed after incubation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSurface plasmon resonance (SPR) quantifies AhR\u0026ndash;ligand binding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe interactions of AhR ligands were investigated utilizing SPR on a Biacore S200 system (https://www.cytivalifesciences.com). AhR protein was immobilized on the activated surface of CM5 sensor chips via amine coupling. Serial dilutions of AFB1 (0.244-500 \u0026mu;M) and IA (7.8125-500 \u0026mu;M) were injected (30 \u0026mu;L/min), with 120-second association and dissociation phases. The binding kinetics were analyzed by employing double-referencing curve fitting using Biacore Evaluation Software to determine the equilibrium dissociation constants (KD).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular docking of AFB1 or IA with AhR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDocking analysis was carried out by CDOCKER module of Discovery Studio 2019 (San Diego, America). The 3-dimensional (3D) structure of AhR was predicted by AlphaFold3\u003csup\u003e45\u003c/sup\u003e(https://alphafoldserver.com/). The 3D structure of IA (CID: 186907) and AFB1 (CID: 5375048) were downloaded from Pubchem (https://pubchem.ncbi.nlm.nih.gov/). The binding affinities of binding clusters were evaluated by -cdocker interaction energy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation and flow cytometry analysis of mouse hepatic macrophages\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHepatic tissue underwent intracardiac perfusion in situ using Hank\u0026apos;s balanced salt solution containing 0.025% collagenase IV through left ventricular access as previously described \u003csup\u003e46\u003c/sup\u003e. The livers were mechanically dissociated through a 100 \u0026mu;m mesh and subsequently underwent differential centrifugation (50 g for 1 minute at 4 \u0026deg;C, followed by 500 g for 6 minutes at 4 \u0026deg;C). Leukocytes were enriched utilizing a discontinuous Percoll gradient centrifugation (40% over 70%) at 750 g for 22 minutes at 15 \u0026deg;C. Erythrocytes were lysed using Ammonium-Chloride-Potassium (ACK) buffer prior to surface staining with anti-CD45, anti-F4/80 and anti-CD11b. The primary antibodies were used at 1:1000 dilutions unless otherwise specified. Cellular viability was assessed using Zombie Aqua. Flow cytometric data acquisition was conducted on an LSR Fortessa platform (BD Biosciences, CA, USA) using the Flowlogic 600.0A analytical software (https://www.inivai.com).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistic analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnalyses were conducted using GraphPad Prism Version 9 software (San Diego, CA, USA). Comparisons between two groups were performed using a two-sided t-test; for more than three groups, the data underwent one-way ANOVA followed by multiple comparisons. Results are presented as mean \u0026plusmn; SEM, with \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 considered significant.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThis work was supported by the National Key Research and Development Program of China (Program No.\u0026nbsp;\u003c/strong\u003e2023YFD1301005\u003cstrong\u003e) and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eNational Natural Science Foundation of China (31972604)\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal procedures complied with the guidelines of the Institutional Animal Care and Use Committee of China Agricultural University ((Approval Nos: Aw41703202-1-4 and Aw32605202-1-4).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 16S rRNA gene sequencing datasets generated and analysed during the current study are available in the NCBI Sequence Read Archive (SRA) database under accession numbers PRJNA1367944 ( https://dataview.ncbi.nlm.nih.gov/object/PRJNA1367944?reviewer=l3dk4oagt6naotsngj24g6908d ). The raw metabolomics data have been deposited in Mendeley Data ( https://www.ebi.ac.uk/metabolights/reviewer1dfb8fa6-a5ff-4f79-80cd-b3c185762743 ; https://www.ebi.ac.uk/metabolights/reviewerd53a81e4-5478-4528-ac89-828c8d2045bc ). The single-cell sequencing data reported in this paper have been deposited in the OMIX, China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences ( https://ngdc.cncb.ac.cn/omix: accession no.OMIX015025 ).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQ\u003cstrong\u003eianqian Wang\u003c/strong\u003e: Conceptualization, Data curation, Formal analysis, Writing \u0026ndash; original draft.\u003cstrong\u003e\u0026nbsp;Yanan Wang\u003c/strong\u003e: Data curation, Visualization.\u003cstrong\u003e\u0026nbsp;Gaigai Wang\u003c/strong\u003e: Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eXin Fang\u003c/strong\u003e: Resources, Data curation. \u003cstrong\u003eYutong Fu\u003c/strong\u003e: Writing \u0026ndash;\u0026nbsp;review \u0026amp; editing.\u003cstrong\u003e\u0026nbsp;Zixin Li\u003c/strong\u003e: Writing \u0026ndash; review \u0026amp; editing.\u0026nbsp;\u003cstrong\u003eYongpeng Guo\u003c/strong\u003e: Writing \u0026ndash; review \u0026amp; editing.\u003cstrong\u003e\u0026nbsp;Jinglin Ma\u003c/strong\u003e: Writing \u0026ndash; review \u0026amp; editing.\u0026nbsp;\u003cstrong\u003eLirong Hu\u003c/strong\u003e: Writing \u0026ndash; review \u0026amp; editing.\u0026nbsp;\u003cstrong\u003eXiangfang Zeng\u003c/strong\u003e: Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eQiugang Ma\u003c/strong\u003e: Writing \u0026ndash; review \u0026amp; editing.\u0026nbsp;\u003cstrong\u003eMatthew Koci\u003c/strong\u003e:\u0026nbsp;Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eLihong Zhao\u003c/strong\u003e: Conceptualization, Resources, Writing \u0026ndash; review \u0026amp; editing, Supervision, Funding acquisition.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eEskola, M.\u003cem\u003e et al.\u003c/em\u003e Worldwide contamination of food-crops with mycotoxins: Validity of the widely cited \u0026apos;FAO estimate\u0026apos; of 25. \u003cem\u003eCrit Rev Food Sci Nutr\u003c/em\u003e \u003cstrong\u003e60\u003c/strong\u003e, 2773-2789 (2020). https://doi.org/10.1080/10408398.2019.1658570\u0026quot;\u0026gt;https://doi.org/10.1080/10408398.2019.1658570\u003c/li\u003e\n\u003cli\u003eLiu, Y., Galani Yamdeu, J. 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Although cereal-derived bioactive compounds are considered promising interventions against AFB1-induced liver injury, the microbiome-dependent mechanisms underlying their protective effects remain poorly understood. Here, we aimed to determine whether the cereal-derived polyphenol ferulic acid could alleviate AFB1-induced liver injury in ducks in a gut microbiota-dependent manner and to elucidate the potential mechanism underlying its protective effects. The results show that ferulic acid exerts its hepatoprotective effect in a gut microbiota-dependent manner, as evidenced by antibiotic treatment and fecal microbiota transplantation in ducks. Specifically, dietary ferulic acid enriches \u003cem\u003ePeptostreptococcus anaerobius\u003c/em\u003e, enhancing microbial conversion of tryptophan to 3-indoleacrylic acid both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e. Mechanistically, by combining single-cell RNA sequencing and surface plasmon resonance, we demonstrate that 3-indoleacrylic acid functionally antagonizes aflatoxin B1-induced hyperactivation of the AhR pathway, thereby suppressing AhR-driven ferroptosis. This functional mechanism was validated through genetic knockdown and pharmacological interventions. Crucially, we reveal that 3-indoleacrylic acid ameliorates aflatoxin B1-induced liver inflammation by reprogramming the subset composition and function of macrophages, which was confirmed by macrophage-depletion models. Collectively, our findings unveil a precise molecular mechanism of host-microbe crosstalk, identifying the \u003cem\u003ePeptostreptococcus anaerobius\u003c/em\u003e-derived tryptophan metabolite 3-indoleacrylic acid as a critical signaling mediator that inhibits Aflatoxin B1-induced hepatic AhR hyperactivation, ferroptosis, and macrophage reprogramming. This work provides a rationale for microbiome-targeted feed strategies to combat global aflatoxicosis and improve animal health.\u003c/p\u003e","manuscriptTitle":"Gut microbiota-derived 3-indoleacrylic acid mediates ferulic acid protection against aflatoxin B1 hepatotoxicity via AhR-ferroptosis inhibition in ducks","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-06 14:50:15","doi":"10.21203/rs.3.rs-9281710/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"167215333961138634062563940893370224455","date":"2026-05-15T03:25:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"171572280843978278992904989529510564181","date":"2026-05-11T02:15:46+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-27T12:31:04+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-23T17:16:46+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-07T05:12:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Biofilms and Microbiomes","date":"2026-03-31T15:01:23+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-biofilms-and-microbiomes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjbiofilms","sideBox":"Learn more about [npj Biofilms and Microbiomes](http://www.nature.com/npjbiofilms/)","snPcode":"41522","submissionUrl":"https://submission.springernature.com/new-submission/41522/3","title":"npj Biofilms and Microbiomes","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4206b0fb-c26b-49d5-a93d-f9f13aebb885","owner":[],"postedDate":"May 6th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"167215333961138634062563940893370224455","date":"2026-05-15T03:25:50+00:00","index":43,"fulltext":""},{"type":"reviewerAgreed","content":"171572280843978278992904989529510564181","date":"2026-05-11T02:15:46+00:00","index":40,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":67099513,"name":"Biological sciences/Biochemistry"},{"id":67099514,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-05-06T14:50:15+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-06 14:50:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9281710","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9281710","identity":"rs-9281710","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}