Limosilactobacillus reuteri alleviates intestinal oxidative damage by regulating gut microbiota- tryptophan metabolites-mitochondria axis-mediated oxidative stress and apoptosis

Limosilactobacillus reuteri alleviates intestinal oxidative damage by regulating gut microbiota- tryptophan metabolites-mitochondria axis-mediated oxidative stress and apoptosis | 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 Research Article Limosilactobacillus reuteri alleviates intestinal oxidative damage by regulating gut microbiota- tryptophan metabolites-mitochondria axis-mediated oxidative stress and apoptosis Shenao Zhan, Yujie Lv, Weichen Huang, Chaoyue Ge, Lianchi Wu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9415762/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Background: Intestinal oxidative stress poses significant challenges to gut health and production performance in laying hens. Limosilactobacillus reuteri ( L. reuteri ) has been demonstrated to mitigate oxidative stress and restore intestinal barrier function. However, the mechanisms underlying how L. reuteri alleviates intestinal oxidative stress remain unclear. This study investigated the protective effects and mechanisms of L. reuteri against diquat-induced intestinal oxidative injury in aged hens. Methods: 270 Jinbai laying hens (70 weeks old) were divided into control (CON), diquat (DQ), and L. reuteri + diquat (LRD) groups with 6 replicates each. Birds in CON and DQ groups are fed with basal diet, while birds in LRD group received basal diet with L. reuteri supplementation. After 10 weeks of pre-treatment, hens in DQ and LRD groups were injected intraperitoneally with diquat (10 mg/kg body weight), while hens in CON were injected intraperitoneally with an equivalent amount of 0.90% saline. Results: Diquat challenge impaired barrier integrity evidenced by the disrupted villus architecture, reduced villus height/crypt depth ratio, elevated serum permeability markers (LPS, DAO, and D-LA), and downregulated the mRNA expression of barrier genes ( MUC2 , ZO-1 , Occludin , Claudin-1 ). L. reuteri pretreatment significantly restored barrier function through a microbiota-metabolite-mitochondria axis. It reshaped gut microbiota by normalizing Firmicutes/Bacteroidota ratio and increasing the abundance of Lactobacillus genus. Metabolomics revealed enrichment of tryptophan metabolism with upregulated indole-3-propionic acid and indole-3-aceticacid. These changes mitigated mitochondrial dysfunction, which further inhibited apoptosis (downregulated TUNEL , BAX and Caspase-3 , upregulated BCL2 ). Furthermore, L. reuteri elevated SOD, GSH-Px and T-AOC activities via Nrf2/HO-1 pathway activation, and reduced pro-inflammatory cytokines IL-1β, TNF-α and IL-6 levels by suppressing TLR4/NF-κB signaling. Conclusion: Limosilactobacillus reuteri could alleviate intestinal oxidative damage induced by diquat exposure by regulating gut microbiota-tryptophan metabolites-mitochondria axis-mediated oxidative stress and apoptosis, and highlight its potential as a therapeutic probiotic for alleviating oxidative stress and mitochondrial dysfunction to prolong the gut health of aging poultry. Limosilactobacillus reuteri laying hens oxidative stress intestinal barrier mitochondria Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Oxidative stress is a prevalent issue in modern poultry production, particularly in laying hens subjected to environmental stressors, high-density farming, and dietary challenges [ 1 ]. In addition, oxidative stress often occurs in laying hens during the aging process, especially during post-peaking laying period[ 2 , 3 ]. Oxidative stress arises from an imbalance between reactive oxygen species (ROS) production and antioxidant defenses, which generally results in mitochondrial dysfunction, intestinal epithelial barrier function impairment, and increased intestinal permeability[ 4 ]. Accumulating evidence has shown that the elevated oxidative stress leads to inflammation and epithelium apoptosis during the aging process [ 5 , 6 ]. In laying hens, intestinal damages induced by oxidative stress can manifest as reduced egg production and quality, poor feed efficiency, and increased susceptibility to diseases, ultimately affecting economic outcomes [ 5 ]. Therefore, it is essential to explore ways to prolong the gut health of aging hens to ensure the durability of egg production. The administration of probiotic Lactobacillus strains has been shown to confer a range of antioxidant, antibacterial, anti-inflammatory, and immunomodulatory effects in the host [ 7 ]. Limosilactobacillus reuteri , a well-known probiotics of Lactobacillus strains, have emerged as promising interventions for mitigating oxidative stress in poultry [ 8 ]. L. reuteri colonizes the gut, reshapes microbiota composition (increasing Firmicutes/Bacteroidota ratio, enriching Lactobacillus), and modulates metabolic profiles [ 9 ]. Notably, it regulates tryptophan metabolism, restores the intestinal barrier, and promotes gut health [ 10 ]. Our laboratory previously isolated a probiotic strain, L. reuteri Y067, from the intestines of healthy laying hens, which had shown in vitro anti-inflammatory and antioxidant activities. In vivo study had revealed that L. reuteri Y067 could mitigate oxidative stress and restore intestinal barrier function and thus improving laying performance in aged laying hens (Table S2 ). However, the mechanisms underlying how L. reuteri Y067 alleviates intestinal oxidative stress remain unclear. Mitochondria serve as central hubs in oxidative stress responses, acting both as primary ROS generators via the electron transport chain and as targets of ROS-induced damage [ 11 ]. Under oxidative stress, mitochondrial dysfunction is characterized by elevated ROS, collapsed membrane potential (MMP), reduced ATP production, decreased mtDNA copy number, impaired biogenesis, dysregulated dynamics, and altered mitophagy [ 12 ]. These alterations amplify oxidative damage, activating downstream pathways such as Nrf2 for antioxidant defense and NF-κB for inflammation [ 13 , 14 ]. Consequently, inflammation exacerbates apoptosis and inhibits proliferation, further compromising barrier renewal [ 15 ]. However, the precise mechanisms by which L. reuteri integrate microbiota-metabolite remodeling with mitochondrial regulation to restore barrier function remain underexplored, especially in laying hens under oxidative stress. Diquat is commonly used as a model inducer of oxidative stress in animal studies, which exacerbates ROS generation through redox cycling, primarily targe ting the intestinal epithelium and disrupting mitochondrial function [ 16 ]. In this study, a diquat-induced intestinal impairment model in laying hens were used to investigate how L. reuteri alleviates diquat-induced intestinal injury through a microbiota-metabolite-mitochondria axis. By elucidating this axis, our work aims to provide insights into probiotic strategies for enhancing gut health in poultry production. 2. Methods 2.1. Experimental design All procedure of the animal experiments were approved by the Animal Care and Use Committee of Zhejiang University (Hangzhou, China; approval number ZJU20241152). 270 Jinghai laying hens (70 weeks old, laying rate 82.2% ± 1.2%) were randomly allocated to three groups, each with six replicates of 15 hens. Birds in control group (CON) and diquat-challenged group (DQ) received basal diets, while birds in L. reuteri supplementation with diquat-challenged group (LRD) received a basal diet supplemented with Limosilactobacillus reuteri Y067 (2.0 × 10 8 CFU/kg feed) for 10 weeks. The basal diet was formulated with maize and soybean (Table 1) to meet the nutritional standards of the National Research Council [ 17 ]. L. reuteri Y067 was isolated and maintained in our laboratory and deposited in the China Center for Type Culture Collection. After 10 weeks of dietary intervention, hens in the DQ and LRD groups received an intraperitoneal injection of diquat (1 mL/kg body weight, 10 mg/mL in 0.9% saline) to induce oxidative stress, as previously describe [ 18 , 19 ]. The CON group received an equivalent volume of 0.9% saline. Birds had unrestricted access to fresh water and mashed diets. The housing environment was maintained at 24°C, 50–60% humidity, with a 16-hour light/8-hour dark cycle. 2.2. Sample Collection Seven days after diquat injection, one hen per replicate was euthanized. Blood was drawn from the wing vein, centrifuged at 4,000 rpm for 15 minutes at 4°C, and serum was stored at -20°C, as previously described[ 20 ]. Jejunal samples were collected for histological, molecular, and biochemical analyses, and cecal contents were obtained for microbiota and metabolomics studies. 2.3. Laying performance For laying performance analysis, the number of eggs and total egg weight were monitored daily, and feed disappearance was recorded weekly on a replicate basis (n = 6) to calculate the laying rate (LR), average daily egg mass (ADEM), average daily feed intake (ADFI), and feed conversion ratio (FCR). 2.4. Serum Intestinal Permeability Markers Serum levels of diamine oxidase (DAO), D-lactate (DLA), and lipopolysaccharide (LPS) were measured to evaluate gut barrier integrity and endotoxemia. DAO and DLA were quantified using ELISA kits (Jiancheng Bioengineering Institute, Nanjing, China) following the manufacturer’s protocols. The level of LPS was measured using a chromogenic limulus amebocyte lysate assay kit (Xiamen Bioendo Technology, Xiamen, China). 2.5. Jejunal Morphology and Transmission Electron Microscopy (TEM) Jejunal segments were paraffin-embedded, sectioned at 5 µm, and stained with hematoxylin and eosin (H&E) to assess villus height (tip to crypt base), crypt depth (crypt base to submucosa), and villus-to-crypt ratio. Images were captured using an Olympus microscope (Tokyo, Japan), and 10 well-oriented villi per section were measured with ImageJ software, as previously described[ 21 ]. Periodic acid-Schiff (PAS) staining was used to quantify mucus layer thickness. Jejunal samples fixed in 2.5% glutaraldehyde were post-fixed in 1% osmium tetroxide, dehydrated, and embedded in epoxy resin. Ultrathin sections (70 nm) were stained with uranyl acetate and lead citrate and examined using a JEOL-JEM-1200EX transmission electron microscope (Peabody, MA, USA) to evaluate mitochondrial morphology and tight junction structures in enterocytes. 2.6. Immunohistochemistry Paraffin-embedded jejunal sections (5 µm) were deparaffinized, rehydrated, and antigen-retrieved in citrate buffer (pH 6.0) by microwave heating. Endogenous peroxidase was blocked with 3% H₂O₂, and non-specific sites with 5% BSA. Sections were incubated overnight at 4°C with anti-4-HNE primary antibody (rabbit polyclonal, 1:200; Abcam, ab46545), followed by HRP-conjugated secondary antibody (1:500) for 1 h at room temperature. Color was developed with DAB substrate, counterstained with hematoxylin, and imaged at 200× magnification. Negative controls omitted the primary antibody. Brown staining indicated positive 4-HNE expression. 2.7. Immunofluorescence and TUNEL Assays Jejunal tissues were fixed in 4% paraformaldehyde, dehydrated in 30% sucrose, embedded in OCT compound, and sectioned at 5–10 µm. Sections were permeabilized with 0.1% Triton X-100, blocked with goat serum, and incubated overnight at 4°C with primary antibodies. Sections were incubated with secondary antibodies for 1 h, washed, mounted with DAPI-containing medium, and visualized using a fluorescence microscope (BX-61, Olympus, Center Valley, PA, USA) ,as previously described [ 22 ]. Fluorescence intensity was quantified with ImageJ. TUNEL assays were performed using a TUNEL kit (Roche, Basel, Switzerland) to detect apoptotic cells, with signals quantified in five random fields per section. 2.8. Gene Expression Analysis Total RNA was extracted from jejunal mucosa using a FreeZol Reagent kit (Vazyme, China), and cDNA was synthesized with a cDNA synthesis kit (Vazyme, China). Quantitative real-time PCR (qRT-PCR) was performed on a Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA) using Taq Pro Universal SYBR qPCR Master Mix (Vazyme, China). Relative mRNA expression was calculated using the 2 −ΔΔCt method with β-actin as the reference gene (Table S1 )[ 23 ]. 2.9.Western blotting Jejunal tissues were lysed in RIPA buffer (Beyotime Biotechnology, P0013B). Protein concentration was determined by BCA assay (Beyotime Biotechnology, P0012). Equal protein amounts (30–50 µg) were separated by SDS-PAGE, transferred to PVDF membranes, and blocked with 5% non-fat milk in TBST for 1 h, as previously described[ 24 ]. Membranes were incubated overnight at 4°C with primary antibodies: anti-Nrf2 (1:1000; Proteintech, 16396-1-AP), anti-HO-1 (1:1000; Proteintech, 10701-1-AP), anti-GPX4 (1:1000; Proteintech, 67763-1-lg), anti-phospho-NF-κB p65 (1:1000; Thermo Fisher, 600-400-271), anti-IL-10 (1:1000; Proteintech, 82191-3-RR), and anti-β-actin (1:5000; Proteintech, 66009-1-lg). After TBST washes, HRP-conjugated secondary antibodies were applied for 1 h. Bands were visualized by an imaging system (Tanon, China). Band intensities were quantified with ImageJ and normalized to β-actin. 2.10. Jejunal Inflammatory Cytokine Quantification Jejunal mucosa was homogenized in PBS, and supernatants were collected after centrifugation at 4,000 rpm for 10 minutes at 4°C, as previously described[ 25 ]. Levels of IL-1β, TNF-α, IL-6, and IL-10 were measured using commercial ELISA kits (Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. 2.11. Jejunal Antioxidant Capacity Jejunal mucosa (0.1 g) was homogenized in 0.9 mL chilled PBS and centrifuged at 4,000 rpm for 10 minutes at 4°C. Supernatants and serum samples were analyzed for malondialdehyde (MDA; kit number: A003-1-2), total antioxidant capacity (T-AOC; kit number: A015-1-2), superoxide dismutase (SOD; kit number: A001-1-2), and glutathione peroxidase (GSH-Px; kit number: A005-1-2) using commercial kits (Jiancheng Bioengineering Institute, Nanjing, China), as previously described[ 26 ]. 2.12. Mitochondrial Analyses Jejunal mitochondria were isolated using a mitochondrial isolation kit (Beyotime Biotechnology, Shanghai, China). Tissue was homogenized in extraction buffer, centrifuged at 1,000–2,000 g to remove debris, and the supernatant was centrifuged at 10,000–12,000 g to pellet mitochondria. The level of reactive oxygen species (ROS) was measured using dichlorohydro-fluorescein diacetate (DCFH-DA) at 485 nm excitation and 530 nm emission, expressed as dichlorofluorescein (DCF) fluorescence intensity. Mitochondrial membrane potential (MMP) was assessed with a JC-1 assay kit (Beyotime Biotechnology), with MMP expressed as the red/green fluorescence ratio. Mitochondrial DNA (mtDNA) content was quantified via qPCR using primers for mtDNA-specific genes, normalized to nuclear DNA. ATP content was measured using an enhanced ATP assay kit based on luciferase luminescence (Beyotime Biotechnology, S0027). ATP content was expressed as nmol/mg protein. 2.13. 16S rRNA Sequencing DNA was extracted from cecal contents using a QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany). The V3–V4 region of the 16S rRNA gene was amplified and sequenced on an Illumina MiSeq platform (San Diego, CA, USA), as previously described[ 27 ]. Data were processed using QIIME2 to evaluate alpha diversity (Shannon and Simpson indices), beta diversity (principal coordinate analysis), and taxonomic profiles at phylum and Genus levels. 2.14. Metabolomics Analysis 2.14.1 Untargeted Metabolomics Analysis Cecal contents were analyzed for untargeted metabolomics using liquid chromatography-mass spectrometry (LC-MS). Samples were extracted in 80% methanol, and metabolites were separated on a Waters ACQUITY UPLC HSS T3 column (2.1 × 100 mm, 1.8 µm) using a Waters UPLC system coupled to a Q-Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA), as previously described[ 28 ]. Data were acquired in positive and negative ion modes, processed with XCMS software for peak detection, and analyzed using MetaboAnalyst for differential metabolite identification and pathway enrichment. 2.14.2. Targeted Metabolomics Analysis Cecal contents (approximately 200 mg) were homogenized in ice-cold methanol: acetonitrile: water (2:2:1, v/v/v) containing internal standards. After centrifugation (14,000 × g, 15 min, 4°C), the supernatant was evaporated and reconstituted in 50% methanol. Targeted quantification of tryptophan metabolites was performed using UHPLC-QqQ-MS/MS (Agilent 1290–6470) with a Waters ACQUITY UPLC BEH C18 column. Detection was conducted in positive ESI mode with multiple reaction monitoring (MRM). Quantification was achieved using external standard curves and internal standard normalization. Results were expressed as nmol/g wet weight. 2.15. Statistical Analysis Data were analyzed using SPSS 26.0 (IBM, Armonk, NY, USA) and expressed as mean ± standard deviation (SD). Data were compared using one-way ANOVA with Tukey’s post-hoc test. Graphs were generated using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA). Significance was set at P < 0.05. 3. Results 3.1. L. reuteri Restores Intestinal Barrier Integrity in Diquat-Challenged Laying Hens Histological examination of jejunal sections stained with HE revealed severe mucosal damages in the DQ group, including shortened villi, disrupted epithelial continuity, and inflammatory cell infiltration (Fig. 1 A). In contrast, the LRD group exhibited improved villus architecture with restored height and reduced structural disruption, approaching the intact morphology of the CON group (Fig. 1 A). Transmission electron microscopy (TEM) further confirmed barrier ultrastructural impairment in the DQ group, characterized by reduced tight junction density, widened intercellular spaces, and damaged microvilli (Fig. 1 B). L. reuteri pretreatment significantly alleviated these changes, evidenced by the increased tight junction structures and preserved microvilli morphology in the LRD group. Morphometric analysis showed that diquat exposure markedly reduced villus height and the villus height/crypt depth ratio ( P < 0.01), while increasing crypt depth compared to the CON group (Fig. 1 C–E). L. reuteri supplementation significantly restored villus height and the villus/crypt ratio ( P 0.05; Fig. 1 C–E). Serum permeability markers including LPS, DAO, and D-LA were significantly elevated in the DQ group ( P < 0.05; Fig. 1 F–H). L. reuteri pretreatment markedly reversed these indicators ( P < 0.01). qRT-PCR analysis demonstrated significant downregulation of barrier-related mRNA in the DQ group, including MUC2 , ZO-1 , Occludin , and Claudin-1 ( P < 0.05; Fig. 1 I–L). L. reuteri intervention significantly upregulated the mRNA expression of these genes (P < 0.05; Fig. 1 I–K). Immunofluorescence staining showed reduced and discontinuous distribution of Occludin and MUC2 in the DQ group, with weak fluorescence intensity along the apical membrane (Fig. 1 M, N). L. reuteri pretreatment restored continuous linear staining and increased fluorescence intensity for both proteins (Fig. 1 M, N). Quantitative analysis confirmed significantly lower Occludin and MUC2 fluorescence intensity in the DQ group compared to CON ( P < 0.01), while L. reuteri supplementation restoring these levels ( P < 0.05; Fig. 1 O, P). Alcian blue-periodic acid-Schiff (AB-PAS) staining revealed a thinned and disrupted mucus layer in the DQ group (Fig. 1 Q). L. reuteri pretreatment significantly increased mucus layer thickness ( P < 0.01), approaching CON levels (Fig. 1 R). 3.2. L. reuteri Reshapes the Gut Microbiota Composition in Diquat-Challenged Hens Alpha diversity analysis of cecal microbiota revealed significant alterations among groups. The ACE and Chao1 indices, reflecting community richness, were markedly decreased in the DQ group compared to CON group ( P < 0.05), while the Shannon and Simpson indices showed no significant differences (Fig. 2 A–D). L. reuteri pretreatment significantly restored ACE and Chao1 indices to levels comparable to the CON group ( P < 0.05; Fig. 2 A, B). Beta diversity analysis demonstrated distinct microbial community structures. Principal coordinate analysis (PCoA) and partial least squares discriminant analysis (PLS-DA) clearly separated the DQ group from the CON and LRD groups, with the LRD group clustering closer to the CON group (Fig. 2 E, F). These results indicate that diquat induced substantial microbiota dysbiosis, which was partially ameliorated by L. reuteri. As for microbiota composition, at the phylum level, diquat challenge increased the relative abundance of Firmicutes and decreased Bacteroidota compared to the CON group (Fig. 2 G). Quantitative analysis confirmed a significant elevation in Firmicutes ( P < 0.05) and reduction in Bacteroidota in the DQ group, with L. reuteri pretreatment restoring Bacteroidota abundance and normalizing the Firmicutes/Bacteroidota ratio ( P < 0.05; Fig. 2 H, I). At the genus level, the most abundant genera included Bacteroides , Rikenellaceae_RC9_gut_group , Lactobacillus , and Ruminococcus_torques_group (Fig. 2 J). Quantitative comparison showed that diquat exposure significantly decreased Bacteroides and Rikenellaceae_RC9_gut_group while increasing Ruminococcus_torques_group ( P < 0.05; Fig. 2 K). Notably, Lactobacillus abundance increased in the LRD group ( P < 0.01; Fig. 2 K). Linear discriminant analysis effect size (LEfSe) identified taxonomic biomarkers across groups. The cladogram illustrated enrichment of Spirochaetota phylum and beneficial genera such as Rikenellaceae and Spirochaetales in the CON group, Ruminococcus_torques_group and Colidextribacter in the DQ group, and Lactobacillus in the LRD group (Fig. 2 L). LDA score analysis (LDA > 3.5) confirmed Rikenellaceae_RC9_gut_group as the primary biomarker in the CON group, Ruminococcus_torques_group in the DQ group, and Lactobacillus in the LRD group (Fig. 2 M). These findings demonstrate that L. reuteri pretreatment effectively mitigates diquat-induced gut dysbiosis by restoring microbial diversity and enriching beneficial taxa, particularly Lactobacillus . 3.3. L. reuteri Remodels the Intestinal Metabolome with Significant Enrichment in Tryptophan Metabolism Non-targeted metabolomics analysis of jejunal tissues revealed distinct metabolic profiles among the three groups. Principal component analysis (PCA) showed clear separation between the DQ group and the CON group, while the LRD group clustered closer to the CON group (Fig. 3 A). Orthogonal partial least squares discriminant analysis (OPLS-DA) further confirmed significant differences in metabolic patterns, with good model fit and predictability (Fig. 3 B). Volcano plot analysis ( P < 0.05) identified a total of 582 upregulated and 415 downregulated metabolites in the DQ vs CON comparison, and 580 upregulated and 129 downregulated metabolites in the LRD vs DQ comparison (Fig. 3 C, D). These results indicate that diquat induced widespread metabolic disturbances, whereas L. reuteri pretreatment partially reversed these alterations. KEGG pathway enrichment analysis demonstrated that tryptophan metabolism was the most significantly enriched pathway in the LRD vs DQ group ( P 0.1; Fig. 3 E). This pathway showed the highest enrichment score and relevance to lipid remodeling under oxidative stress conditions. Hierarchical clustering heatmap of the top 30 differential metabolites revealed clear separation among groups, with multiple tryptophan metabolism-related metabolites showing upregulation in the LRD group compared to the DQ group (Fig. 3 F). Key metabolites enriched in this cluster included indole-3-acetic acid, trans-3-indoleacrylic acid, indole-3-aldehyde, indoleacetic acid, and 3-indolepropionic acid. Targeted metabolomics analysis of representative tryptophan metabolites confirmed indole-3-acetic acid, trans-3-indoleacrylic acid, indole-3-aldehyde, indoleacetic acid, and 3-indolepropionic acid were significant increases in the LRD group compared to the DQ group ( P < 0.05; Fig. 3 G).To further explore the relationship between gut microbiota and tryptophan metabolism, Spearman correlation analysis was performed between the top 10 abundant bacterial genera and five key tryptophan metabolites (Fig. 3 H). Lactobacillus exhibited significant positive correlations with multiple tryptophan metabolites, particularly indole-3-acetic acid and indole-3-propionic acid ( P < 0.05). In contrast, Ruminococcus_torques_group showed negative correlations with these metabolites, especially indole-3-propionic acid ( P < 0.05). Beneficial genera such as Faecalibacterium and Oscillospiraceae also displayed strong positive correlations with several tryptophan metabolites. 3.4. L. reuteri Alleviates Diquat-Induced Mitochondrial Dysfunction, Impaired Biogenesis, Dysregulated Dynamics, and Mitophagy Imbalance Transmission electron microscopy of jejunal tissue revealed severe mitochondrial ultrastructural damage in the DQ group, including mitochondrial swelling, disrupted cristae, and vacuolization (Fig. 4 A). In contrast, mitochondria in the CON and LRD groups exhibited intact morphology with well-organized cristae and normal matrix density (Fig. 4 A). Diquat challenge significantly increased mitochondrial ROS production ( P < 0.01), decreased mitochondrial membrane potential ( P < 0.01), reduced mtDNA copy number ( P < 0.01), and lowered ATP content ( P < 0.05) compared to the control group (Fig. 4 B–E). L. reuteri pretreatment markedly attenuated these changes, reducing ROS ( P < 0.05), restoring MMP ( P < 0.05), increasing mtDNA copy number ( P < 0.05), and elevating ATP levels ( P < 0.05) relative to the DQ group (Fig. 4 B–E). qRT-PCR analysis demonstrated that diquat exposure downregulated mitochondrial biogenesis-related mRNA expression, including PGC-1α , NRF1 , TFAM , and POLRMT ( P < 0.05; Fig. 4 F). L. reuteri supplementation significantly upregulated these genes ( P < 0.05; Fig. 4 F), indicating enhanced mitochondrial biogenesis. Regarding mitochondrial dynamics, diquat increased fission genes DRP1 and FIS1 ( P < 0.01) while decreasing fusion genes MFN1 and MFN2 ( P < 0.05; Fig. 4 G). L. reuteri reversed this imbalance by downregulating DRP1 and FIS1 ( P < 0.05) and upregulating MFN1 and MFN2 ( P < 0.05; Fig. 4 G). Immunofluorescence staining revealed disrupted mitophagy in the DQ group, with altered colocalization of LC3B, PINK1, and Parkin in jejunal (Fig. 4 H). L. reuteri restored the punctate distribution and colocalization of these markers, suggesting normalized mitophagic flux (Fig. 4 H). These mitochondrial improvements provide a foundation for enhanced antioxidant defense and reduced inflammation, which were further investigated. 3.5. L. reuteri Enhances Antioxidant Capacity and Activates the Nrf2 Pathway in Diquat-Challenged Laying Hens Immunohistochemical staining for 4-hydroxynonenal (4-HNE), a marker of lipid peroxidation, showed intense brown staining in the villi and crypts of the DQ group, accompanied by disrupted villus structure, indicating severe oxidative damage (Fig. 5 A). In the LRD group, 4-HNE staining intensity was markedly reduced, with improved villus morphology approaching the CON group (Fig. 5 A). In jejunal tissues, diquat challenge significantly decreased SOD, GSH-Px, and T-AOC activities, while increasing MDA content compared to the CON group ( P < 0.05; Fig. 5 B–E). L. reuteri pretreatment significantly restored GSH-Px and T-AOC activities ( P < 0.05) and reduced MDA levels ( P 0.05; Fig. 5 B–E). Serum antioxidant parameters followed a similar pattern: diquat exposure reduced SOD, GSH-Px, and T-AOC activities while elevating MDA content ( P < 0.05; Fig. 5 F–I). L. reuteri intervention significantly improved SOD, T-AOC and GSH-Px activities ( P 0.05; Fig. 5 F–I). qRT-PCR analysis of Nrf2 pathway-related genes revealed that diquat downregulated Nrf2 , NQO1 , GPX4 , and SOD1 mRNA expression. Notably, HO-1 mRNA levels showed no significant difference between CON and DQ groups ( P > 0.05), while Keap1 mRNA expression were significantly increased. L. reuteri pretreatment significantly upregulated Nrf2 , HO-1 , and NQO1 , with the restoration of GPX4 and SOD1 ( P < 0.05; Fig. 5 J–O).Immunofluorescence staining demonstrated reduced nuclear translocation of Nrf2 and cytoplasmic expression of HO-1 and GPX4 in the DQ group (Fig. 5 P–R). L. reuteri pretreatment enhanced Nrf2 nuclear localization and increased HO-1 and GPX4 fluorescence intensity (Fig. 5 P–R). Quantitative analysis confirmed significantly lower Nrf2, HO-1, and GPX4 fluorescence intensity in the DQ group compared to CON ( P < 0.05), with L. reuteri restoring these levels ( P < 0.05; Fig. 5 S). Western blot analysis revealed that diquat downregulated Nrf2, HO-1, and GPX4 protein expression. L. reuteri pretreatment significantly upregulated these proteins ( P < 0.01 vs DQ; Fig. 5 T-U), with Nrf2 and HO-1 levels restored to near CON values and GPX4 partially recovered. 3.6. L. reuteri Suppresses Diquat-Induced Inflammatory Response and NF-κB Signaling Enzyme-linked immunosorbent assay (ELISA) of jejunal tissues showed that DQ challenge significantly increased the protein levels of pro-inflammatory cytokines IL-1β, TNF-α, and IL-6 ( P < 0.05), while decreasing the anti-inflammatory cytokine IL-10 compared to the CON group ( P < 0.01; Fig. 6 A–D). LRD pretreatment markedly reduced IL-1β, TNF-α, and IL-6 levels ( P < 0.05) and elevated IL-10 content (P < 0.01) relative to the CON group (Fig. 6 A–D). Consistent with protein data, qRT-PCR analysis revealed that diquat significantly upregulated mRNA expression of IL-1β , TNF-α , and IL-6 ( P < 0.01), while downregulating IL-10 mRNA expression ( P < 0.05; Fig. 6 E–H). L. reuteri pretreatment significantly downregulated IL-1β , TNF-α , and IL-6 mRNA expression ( P < 0.05) and upregulated IL-10 mRNA expression ( P < 0.05; Fig. 6 E–H). Further qRT-PCR analysis of the TLR4/NF-κB pathway showed that diquat significantly upregulated TLR4 , MYD88 , IκBα , and NF-κB mRNA expression ( P < 0.01; Fig. 6 I–L). L. reuteri intervention significantly reduced TLR 4, MYD88 , IκBα , and NF-κB mRNA levels ( P < 0.05; Fig. 6 I–L), indicating suppression of the inflammatory signaling cascade. Immunofluorescence staining demonstrated increased macrophage infiltration (F4/80-positive cells) and nuclear translocation of phosphorylated p65 (p-p65 NF-κB) in the DQ group, with reduced IL-10 expression (Fig. 6 M–O). L. reuteri pretreatment decreased F4/80 and p-p65 fluorescence intensity while enhancing IL-10 staining (Fig. 6 M–O). Quantitative analysis confirmed significantly higher F4/80 and p-p65 intensity and lower IL-10 intensity in the DQ group compared to CON ( P < 0.01), with L. reuteri restoring these levels ( P < 0.05; Fig. 6 P). Western blot analysis showed increased phosphorylated p65 (p-p65 NF-κB) and decreased IL-10 protein expression in the DQ group ( P < 0.01 vs CON; Fig. 6 Q-R). L. reuteri pretreatment significantly reduced p-p65 and restored IL-10 protein levels ( P < 0.01 vs DQ; Fig. 6 Q-R). 3.7. L. reuteri Inhibits Apoptosis and Promotes Proliferation in Diquat-Challenged Laying Hens Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining revealed a significant increase in apoptotic cells in the jejunal epithelium of the DQ group, with intense green fluorescence in villi and crypts (Fig. 7 A). LRD pretreatment markedly reduced TUNEL-positive cells, with fluorescence intensity approaching the CON group (Fig. 7 A). Quantitative analysis confirmed significantly higher TUNEL fluorescence intensity in the DQ group compared to CON ( P < 0.01), with L. reuteri restoring levels ( P < 0.01; Fig. 7 B). qRT-PCR analysis of apoptosis-related genes showed that diquat significantly upregulated BAX and Caspase3 mRNA expression ( P 0.05; Fig. 7 C–E). The Bcl-2/Bax ratio was markedly decreased in the DQ group ( P < 0.05; Fig. 7 F). L. reuteri pretreatment significantly downregulated Caspase3 mRNA expression ( P < 0.05), upregulated BCL2 mRNA expression ( P < 0.01), and restored the Bcl-2/Bax ratio ( P < 0.01; Fig. 7 C–F). Immunofluorescence staining for proliferating cell nuclear antigen (PCNA), a marker of cell proliferation, showed reduced nuclear staining in the crypts and villi of the DQ group (Fig. 7 G). L. reuteri pretreatment increased PCNA-positive cells with strong nuclear fluorescence (Fig. 7 G). Quantitative analysis demonstrated significantly lower PCNA fluorescence intensity in the DQ group compared to CON ( P < 0.01), with L. reuteri restoring levels ( P < 0.01; Fig. 7 H). qRT-PCR analysis of proliferation-related genes revealed that diquat significantly downregulated LGR5 , PCNA , and β-catenin mRNA expression ( P < 0.01; Fig. 7 I–K). L. reuteri pretreatment significantly upregulated the mRNA expression of these genes ( P < 0.05; Fig. 7 I–K). 3.8. Integrative Correlation Analysis Reveals the Microbiota-Metabolite-Mitochondria-Barrier Axis Mediated by Lactobacillus reuteri To further elucidate the relationships among microbiota composition, tryptophan metabolites, and downstream intestinal parameters, spearman correlation network analysis was performed. As shown in Fig. 8 , Lactobacillus exhibited strong positive correlations with multiple protective indicators, including barrier proteins (MUC-2 and Occludin), mitochondrial ATP content, biogenesis factor NRF1, antioxidant markers (Nrf2, HO-1, and GSH-Px), anti-inflammatory cytokine IL-10, and proliferation marker PCNA. In contrast, Ruminococcus_torques_group showed significant negative correlations with these parameters and positive correlations with oxidative stress and inflammatory markers (Mt-ROS, NF-κB, IL-1β pathway). Among tryptophan metabolites, indole-3-acetic acid, indole-3-propionic acid, and dndoleacetic acid displayed strong positive associations with mitochondrial function (ATP, NRF1), antioxidant capacity (HO-1, GSH-Px), and barrier integrity (MUC-2, Occludin), while being negatively correlated with ROS, NF-κB signaling, and apoptosis-related markers (BCL2). These integrative correlations strongly support that L. reuteri -driven enrichment of beneficial microbiota and tryptophan metabolites serves as a central mediator linking microbial modulation to mitochondrial protection, antioxidant defense, inflammation suppression, and ultimately intestinal barrier restoration. 4. Discussion Intestinal oxidative stress represents a critical challenge in modern laying hen production, as aging, environmental and nutritional challenges trigger excessive ROS production, leading to barrier dysfunction, systemic inflammation, reduced egg production, deteriorated eggshell quality, and ultimately substantial economic losses to the poultry industry [ 29 , 30 ]. Therefore, alleviating intestinal barrier damages is the principal target in poultry production. L. reuteri , a well-characterized probiotic with remarkable colonization capacity and metabolic versatility, has demonstrated efficacy in alleviating intestinal oxidative stress and barrier injury [ 31 – 33 ]. The present study investigated how L. reuteri alleviates diquat-induced intestinal injury in laying hens through coordinated restoration of gut microbiota homeostasis, enrichment of tryptophan metabolites, recovery of mitochondrial function, rebalancing of Nrf2-NF-κB signaling crosstalk, and ultimately restoration of intestinal barrier integrity. Extensive studies have established that gut microbiota homeostasis is critical for maintaining intestinal barrier and metabolic regulation [ 34 ]. Previous research has consistently demonstrated that oxidative stress disrupts gut microbiota homeostasis, which are tendly accompanied by metabolic perturbations [ 35 ]. Consistently, our study observed that diquat-induced oxidative stress in laying hens disrupted cecal microbiota homeostasis, characterized by the reduced Lcatobacillus , elevated Firmicutes/Bacteroidota ratio and enhanced harmful bacteria such as Ruminococcus_torques_group . This microbiota dysbiosis was accompanied by the depletion of tryptophan metabolites. Studies have showed that L. reuteri can restore gut microbiota dysbiosis and modulates metabolic profiles in various intestinal inflammation and oxidative stress models [ 37 ]. Our study also found that L. reuteri pretreatment effectively restored the microbiota and metabolism in our diquat-challenged laying hens. Specifically, L. reuter increased the abundance of Lactobacillus , which is often related to intestinal barrier restoration. Lactobacillus has been reported to encourage intestinal epithelium regeneration [ 36 ], regulate tight junction protein [ 38 ] and maintain mucus layer thickness [ 39 ]. Meanwhile, our results also show that tryptophan metabolism is the top upregulated pathway, with multiple metabolites significantly elevated, including indole-3-acetic acid, trans-3-indoleacrylic acid and indole-3-aldehyde. Tryptophan metabolism is highly correlated with intestinal health[ 40 ]. Previous studies have demonstrated that indole metabolites alleviate intestinal barrier damage, promote epithelial repair, and inhibit inflammation [ 41 ]. Tryptophan metabolites can also regulate the expression of tight junction proteins to mitigate intestinal barrier [ 42 ]. Thus, the interaction of Lactobacillus reuteri with microbiota and metabolism may facilitate the intestinal barrier recovery. Mitochondria function both as major ROS generators and as primary targets of ROS-induced damage in oxidative stress pathophysiology [ 43 ]. Mitochondria dysfunction may hinder intestinal epithelium function, leading to intestinal barrier damage [ 44 ]. Studies have shown that Lactobacillus reuteri can mitigate oxidative stress, alleviate mitochondria dysfunction[ 45 , 46 ] and improve intestinal health [ 47 , 48 ]. Consistently, in our study L. reuteri intervention reduces ROS production and restores mitochondrial integrity. This may be because tryptophan metabolites of Lactobacillus reuteri improve mitochondrial function, reduce ROS accumulation, and protect mitochondrial integrity [ 49 ]. Meantime, we found L. reuteri recover ΔΨm and ATP content. Mitochondrial ATP production is essential for intestinal barrier maintenance, as they facilitate tight junction integrity, as well as influencing the expression of MUC-2 [ 50 ]. Our study also observed upregulated PGC-1α related biogenesis genes, rebalanced fission and fusion dynamics and normalized mitophagy. This may be because L. reuteri’s tryptophan metabolites, such as IAA, can boost mitochondrial biosynthesis, reduce oxidative stress [ 51 ], and promote a healthy mitophagy to maintain mitochondrial function [ 52 ]. Thus, upregulated PGC-1α promotes intestinal epithelial differentiation and mitochondrial biogenesis[ 53 ], rebalanced mitochondrial dynamics alleviates inflammation and epithelial damage [ 54 ] and moderate mitophagy clears damaged mitochondria and protect the intestinal barrier [ 55 ]. L. reuteri strongly regulates these functional pathways and restores mitochondrial health, thereby promoting intestinal barrier integrity. The Nrf2 and NF-κB pathways represent two master pathways that determine cellular responses to oxidative stress [ 56 ]. Nrf2 maintains the proliferation balance of intestinal stem cells [ 57 ] and NF-κB promotes apoptosis [ 58 ]. They collectively regulate downstream apoptosis and proliferation. Previous studies have shown that oxidative stress, while inhibiting Nrf2-mediated antioxidant defense, overactivates NF-κB-driven inflammatory responses[ 59 ], promotes apoptosis rather than proliferation, and ultimately leads to intestinal epithelial cell damage and barrier dysfunction [ 60 ]. Extensive evidence has demonstrated that L. reuteri alleviates intestinal damage by activating the Nrf2/HO-1 pathway [ 61 ], while inhibiting the NF-κB pathway to exert anti-inflammatory effects [ 62 ]. L. reuteri also improved the expression of intestinal TJs and maintained the integrity of the intestinal barrier by inhibiting apoptosis of intestinal epithelial cells [ 63 ] and stimulating the expansion of intestinal stem cells [ 64 ]. Consistently, L. reuteri intervention in our study rebalanced this molecular crosstalk by enhancing Nrf2 antioxidant defenses and suppressing NF-κB inflammatory signaling pathway. Furthermore, L. reuteri attenuated apoptosis, promoted proliferation and restored intestinal barrier. Mechanistically, Lactobacillus reuteri may influence upstream tryptophan metabolites to improve the oxidative-inflammatory balance, thereby alleviating intestinal damage. Studies have shown that tryptophan metabolites such as IAA and ILA can activate AhR/Nrf2 signaling to promote gut barrier [ 65 ], alleviate intestinal epithelial cell injury via regulation of the TLR4/NF-κB pathway to reduce mucosal damage and apoptosis [ 66 ], and modulate the proliferation of intestinal epithelial cell [ 67 ]. Meanwhile, restored mitochondrial function reduced ROS generation at the source, alleviating the oxidative trigger for both Nrf2 dissociation inhibition and TLR4-mediated NF-κB activation [ 68 ]. Finally, increased anti-inflammatory cytokine and restored mitochondrial function decreased intrinsic apoptosis signaling [ 69 ], while Nrf2 activation supported Wnt/β-catenin signaling to maintain intestinal stem cell populations and promote epithelial renewal. 5. Conclusion In conclusion, this study unveils the mechanisms by which L. reuteri protects against intestinal oxidative damages through tryptophan-mediated restoration of the microbiota-metabolite-mitochondria axis (Fig. 9 ). These findings not only advance our fundamental understanding of probiotic mechanisms but also provide a rational foundation for developing microbiome-targeted nutritional interventions to enhance intestinal health and productive performance in laying hens under oxidative stress conditions. Abbreviations 4-HNE 4-Hydroxynonenal ADEM Average daily egg mass ADFI Average daily feed intake β-catenin Beta-catenin BCL-2 B-cell lymphoma-2 CAS-3 Cysteinyl aspartate specific proteinase-3 CD Crypt depth Claudin-1 Claudin-1 DAO Diamine oxidase D-LA D-Lactic acid DQ Diquat DRP1 Dynamin-related protein 1 F4/80 F4/80 (macrophage marker) FCR Feed conversion ratio FIS1 Fission 1 GPX-4 Glutathione peroxidase-4 GSH-PX/GPX Glutathione peroxidase HO-1 Heme oxygenase-1 IκBα Inhibitor of kappa B alpha IL-1β Interleukin-1β IL-6 Interleukin-6 IL-10 Interleukin-10 Keap-1 Kelch-like ECH-associated protein-1 LC3B Microtubule-associated protein 1 light chain 3B LGR5 Leucine-rich repeat-containing G-protein coupled receptor 5 LPS Lpopolysaccharide LR Laying rate MDA Malondialdehyde MFN1 Mitofusin 1 MFN2 Mitofusin 2 MMP Mitochondrial membrane potential mtDNA Mitochondrial DNA MUC-2 Mucin-2 MyD88 Myeloid differentiation primary response 88 NF-κB Nuclear factor kappa B NF-κB p-p65 Phosphorylated p65 subunit of NF-κB NQO-1 NAD(P)H:quinone oxidoreductase-1 Nrf2 Nuclear factor erythroid 2-related factor 2 NRF1 Nuclear respiratory factor 1 Occludin Occludin Parkin Parkin (E3 ubiquitin-protein ligase) PCNA Proliferating cell nuclear antigen PINK1 PTEN-induced kinase 1 PGC-1α Peroxisome proliferator-activated receptor gamma coactivator 1-alpha POLRMT Mitochondrial RNA polymerase SOD Superoxide dismutase T-AOC Total antioxidant capacity TFAM Transcription factor A, mitochondrial TLR4 Toll-like receptor 4 TNF-α Tumor necrosis factor alpha TUNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling VH Villus height ZO-1 Zonula occludens Declarations Acknowledgments We would like to express our gratitude to all the members who participated in the sample collection and data counting process. Furthermore, we sincerely thank the reviewers and editors for all their valuable suggestions and revisions of our manuscript. Authors’ contributions SAZ: Investigation, Methodology, Data curation, Writing – original draft. YJL: Investigation. WCH: Investigation. CYG: Investigation. LCW: Investigation. DYY: Conceptualization, Funding, Supervision, Writing – review & editing. BL: Investigation, Formal analysis, Supervision, Writing – review & editing. The final draft of the manuscript has undergone a comprehensive review by all authors and received their full endorsement. Funding The research was financially supported by the National Natural Science Foundation of China (grant no. 32402779 and 32372892), the Zhejiang Provincial Natural Science Foundation of China (grant no. ZCLMS25C1701), and the Key Research and Development Program of Zhejiang Province (Grant no: 2024C02004). Availability of data and materials All data generated or analysed during this study are included in this published article and its supplementary information files. Additional raw data are available from the corresponding author on reasonable request. Declarations This work has received approval for research ethics from the Animal Care and Use Committee of Zhejiang University (Hangzhou, China, Protocol number ZJU20241152) and a proof/certificate of approval is available upon request. Consent for publication Not applicable. 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. 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Cell Mol Gastroenterol Hepatol. 2020;9:295–312. https://doi.org/10.1016/j.jcmgh.2019.10.002 Huang Q, Wang Z, Wu T, Ji S, Lu B. Taste receptor-mediated inflammation regulation: An anti-inflammatory strategy combining taste perception and metabolic sensing. Trends in Food Science & Technology. 2026;172:105718. https://doi.org/10.1016/j.tifs.2026.105718 Wen Z, Liu W, Li X, Chen W, Liu Z, Wen J, et al. A Protective Role of the NRF2-Keap1 Pathway in Maintaining Intestinal Barrier Function. Oxid Med Cell Longev. 2019;2019:1759149. https://doi.org/10.1155/2019/1759149 Xie W, Song L, Wang X, Xu Y, Liu Z, Zhao D, et al. A bovine lactoferricin-lactoferrampin-encoding Lactobacillus reuteri CO21 regulates the intestinal mucosal immunity and enhances the protection of piglets against enterotoxigenic Escherichia coli K88 challenge. Gut Microbes. 13:1956281. https://doi.org/10.1080/19490976.2021.1956281 Liu Y, Fatheree NY, Mangalat N, Rhoads JM. Lactobacillus reuteri strains reduce incidence and severity of experimental necrotizing enterocolitis via modulation of TLR4 and NF-κB signaling in the intestine. American Journal of Physiology-Gastrointestinal and Liver Physiology. American Physiological Society; 2012;302:G608–17. https://doi.org/10.1152/ajpgi.00266.2011 Zhou Q, Wu F, Chen S, Cen P, Yang Q, Guan J, et al. Lactobacillus reuteri improves function of the intestinal barrier in rats with acute liver failure through Nrf-2/HO-1 pathway. Nutrition. 2022;99–100:111673. https://doi.org/10.1016/j.nut.2022.111673 Ding X, Tang R, Zhao J, Xu Y, Fu A, Zhan X. Lactobacillus reuteri alleviates LPS-induced intestinal mucosal damage by stimulating the expansion of intestinal stem cells via activation of the Wnt/β-catenin signaling pathway in broilers. Poult Sci. 2024;103:104072. https://doi.org/10.1016/j.psj.2024.104072 Jia Y, He M, Wang F, Zhan Y, Deng Q, Shen J, et al. Indole-3-lactic acid protects the gut vascular barrier following intestinal ischemia injury through AhR/Nrf2/STAT3 mediated claudin 2 downregulation. Cell Commun Signal. 2025;23:447. https://doi.org/10.1186/s12964-025-02454-y Chen Y, Li Y, Li X, Fang Q, Li F, Chen S, et al. Indole‑3‑propionic acid alleviates intestinal epithelial cell injury via regulation of the TLR4/NF‑κB pathway to improve intestinal barrier function. Mol Med Rep. 2024;30:189. https://doi.org/10.3892/mmr.2024.13313 Ismael S, Rodrigues C, Santos GM, Castela I, Barreiros-Mota I, Almeida MJ, et al. IPA and its precursors differently modulate the proliferation, differentiation, and integrity of intestinal epithelial cells. Nutrition Research and Practice. 2023;17:616–30. https://doi.org/10.4162/nrp.2023.17.4.616 Morgan MJ, Liu Z. Crosstalk of reactive oxygen species and NF-κB signaling. Cell Res. Nature Publishing Group; 2011;21:103–15. https://doi.org/10.1038/cr.2010.178 He Z, Feng Y, Zhang Y, Gao X, Liu J, Liu S, et al. IL-10 alleviates ulcerative colitis by regulating mitochondrial function through reducing ISG15 expression. Cellular Signalling. 2025;134:111932. https://doi.org/10.1016/j.cellsig.2025.111932 Additional Declarations No competing interests reported. Supplementary Files 0414supplementarymaterials.docx Supplementary Information Table S1. Genes primer sequences for qPCR. Table S2. Effects of L. reuteri on the production performance of laying hens 0416WBsupplementaryfile.zip Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 15 May, 2026 Reviewers agreed at journal 14 May, 2026 Reviews received at journal 12 May, 2026 Reviewers agreed at journal 24 Apr, 2026 Reviewers invited by journal 21 Apr, 2026 Editor assigned by journal 21 Apr, 2026 Submission checks completed at journal 17 Apr, 2026 First submitted to journal 14 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-9415762","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":630712890,"identity":"a041b8d0-b114-4c21-bd2a-b1357268b4bd","order_by":0,"name":"Shenao Zhan","email":"","orcid":"","institution":"Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Shenao","middleName":"","lastName":"Zhan","suffix":""},{"id":630712891,"identity":"c41aaeec-43a3-45a7-a83c-11ec45808291","order_by":1,"name":"Yujie Lv","email":"","orcid":"","institution":"Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Yujie","middleName":"","lastName":"Lv","suffix":""},{"id":630712892,"identity":"1738dcf7-1317-49e1-8a6d-c969f2ab0963","order_by":2,"name":"Weichen Huang","email":"","orcid":"","institution":"Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Weichen","middleName":"","lastName":"Huang","suffix":""},{"id":630712893,"identity":"97a50c63-61de-4a14-bc49-8b5cbe24d39b","order_by":3,"name":"Chaoyue Ge","email":"","orcid":"","institution":"Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Chaoyue","middleName":"","lastName":"Ge","suffix":""},{"id":630712895,"identity":"fc5d9efe-c336-4774-9ebe-d2f4edc5b377","order_by":4,"name":"Lianchi Wu","email":"","orcid":"","institution":"Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Lianchi","middleName":"","lastName":"Wu","suffix":""},{"id":630712897,"identity":"bcd99a1d-930f-4a84-9cee-addb42aa17ed","order_by":5,"name":"Dongyou Yu","email":"","orcid":"","institution":"Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Dongyou","middleName":"","lastName":"Yu","suffix":""},{"id":630712899,"identity":"c83745af-f0fe-4af1-943d-ddf153d6effd","order_by":6,"name":"Bing Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIie3RPQrCMBTA8SeFdol2jUPvEAj0g14mQWgXEccOIoFC164eQyg4RwpOEVdHJ3HsCcS04lozCuY/5GXIDx4EwGb7wYjTn2sMPgDTN8eUEAxzYUw+J5HDzYBE3rR9AIl5c5F3DEXKhXeWoyQpZ1msF+MHKTMMKucCrdj4Yi0KyUCOIsOTquUCI2JGmhI0eZoReuvJ3u2JMCBJiUJgBNOdgkXMTjmt0HKcRL6iXVdsg7pW/Npt0qD21DjRuZgNE7HhM91v73VO956eNHhss9ls/9gLYm89dkioAdMAAAAASUVORK5CYII=","orcid":"","institution":"Zhejiang University","correspondingAuthor":true,"prefix":"","firstName":"Bing","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2026-04-14 12:56:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9415762/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9415762/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108803762,"identity":"32948d61-8d42-4222-941a-2a870f1976d5","added_by":"auto","created_at":"2026-05-08 15:06:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":821907,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLactobacillus reuteri restores intestinal structure and barrier function in diquat-challenged laying hens.\u003c/strong\u003e (A) Representative hematoxylin and eosin (H\u0026amp;E)-stained jejunal sections (scale bar = 200 μm). (B) Representative transmission electron microscopy (TEM) images of jejunal epithelial tight junctions and microvilli (scale bar = 0.5 μm). (C–E) Quantitative analysis of villus height (C), crypt depth (D), and villus height/crypt depth ratio (E). (F–H) Serum levels of lipopolysaccharide (LPS; F), diamine oxidase (DAO; G), and D-lactic acid (D-LA; H). (I–L) Relative mRNA expression of barrier-related genes MUC2 (I), ZO-1 (J), Occludin (K), and Claudin-1 (L) in jejunal tissues, determined by qRT-PCR. (M, N) Representative immunofluorescence staining of Occludin and MUC2 in jejunal sections (scale bar = 50 μm). (O, P) Quantitative analysis of Occludin (O) and MUC2 (P) immunofluorescence intensity. (Q) Representative Alcian blue-periodic acid-Schiff (AB-PAS) staining of jejunal mucus layer (scale bar = 200 μm). (R) Quantitative analysis of mucus layer thickness. Data are presented as mean ± SD (n = 6 per group). **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 vs DQ group; ns, no significance.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-9415762/v1/6060ee906efd084210bdecb5.png"},{"id":108248269,"identity":"b752955a-f760-43c7-a1a6-579fa08e57bb","added_by":"auto","created_at":"2026-05-01 01:11:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":515887,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLactobacillus reuteri reshapes the cecal microbiota composition in diquat-challenged laying hens.\u003c/strong\u003e (A–D) Alpha diversity indices of cecal microbiota, including Chao1 (A), Shannon (B), ACE (C), and Simpson (D). (E) Principal coordinate analysis (PCoA) at the OTU level. (F) Partial least squares discriminant analysis (PLS-DA) plot at the OTU level. (G) Stacked bar plot of microbial composition at the phylum level (top 10 phyla). (H, I) Relative abundance of Bacteroidota (H) and Firmicutes (I) at the phylum level. (J) Stacked bar plot of microbial composition at the genus level (top 15 genera). (K) Relative abundance of key genera: Bacteroides, Rikenellaceae_RC9_gut_group, Lactobacillus, and Ruminococcus_torques_group. (L) Cladogram showing taxonomic biomarkers (M) Linear discriminant analysis (LDA) score bar plot (LDA \u0026gt; 3.5). Data are presented as mean ± SD (n = 6 per group). **P \u0026lt; 0.01, *P \u0026lt; 0.05 vs DQ group; ns, no significance.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-9415762/v1/754bf8c842568e0170d53331.png"},{"id":108803546,"identity":"0f9cc0ad-1f3b-4e6c-bafe-27dac20a351c","added_by":"auto","created_at":"2026-05-08 14:59:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":466407,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLactobacillus reuteri remodels the cecal metabolome with significant enrichment in tryptophan metabolism in diquat-challenged laying hens.\u003c/strong\u003e (A) Principal component analysis (PCA) score plot of cecal metabolome profiles. (B) Orthogonal partial least squares discriminant analysis (OPLS-DA) score plot of cecal metabolome profiles. (C) Volcano plot of differential metabolites in DQ vs CON comparison (P \u0026lt; 0.05). (D) Volcano plot of differential metabolites in LRD vs DQ comparison (P \u0026lt; 0.05). (E) KEGG pathway enrichment analysis bubble plot showing significantly enriched pathways. (F) Hierarchical clustering heatmap of the top 30 differential metabolites across groups. (G) Targeted metabolomics analysis of representative tryptophan metabolites contents. (H) Spearman correlation heatmap between the top 10 abundant bacterial genera and five key tryptophan metabolites in the cecal contents. Data are presented as mean ± SD (n = 6 per group). **P \u0026lt; 0.01, *P \u0026lt; 0.05 vs DQ group.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-9415762/v1/f3f247dfc498646da65b04e6.png"},{"id":108248271,"identity":"dc601485-48de-4398-8419-664576556a09","added_by":"auto","created_at":"2026-05-01 01:11:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1167168,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLactobacillus reuteri alleviates mitochondrial dysfunction, impaired biogenesis, dysregulated dynamics, and mitophagy alterations in jejunum of diquat-challenged laying hens.\u003c/strong\u003e (A) Representative transmission electron microscopy (TEM) images of mitochondrial ultrastructure in jejunal epithelial cells (scale bar = 0.2 μm). (B–E) Quantitative analysis of mitochondrial reactive oxygen species (ROS) level (B), mitochondrial membrane potential (MMP; C), mtDNA copy number (D), and ATP content (E). (F) Relative mRNA expression of mitochondrial biogenesis-related genes PGC-1α, NRF1, TFAM, and POLRMT in jejunal tissues, determined by qRT-PCR. (G) Relative mRNA expression of mitochondrial dynamics-related genes MFN1, MFN2, DRP1, and FIS1 in jejunal tissues, determined by qRT-PCR. (H) Representative immunofluorescence staining of mitophagy markers LC3B, Parkin, PINK1, and merged images in jejunal sections (scale bar = 200 μm). Data are presented as mean ± SD (n = 6 per group). Different letters (a, b, c) indicate significant differences (P \u0026lt; 0.05); **P \u0026lt; 0.01, *P \u0026lt; 0.05 vs DQ group.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-9415762/v1/f84a8ee536bde45a99336a45.png"},{"id":108803698,"identity":"2ff8285b-1354-497f-a438-00480ce7a596","added_by":"auto","created_at":"2026-05-08 15:04:10","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":966202,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLactobacillus reuteri enhances antioxidant capacity and activates the Nrf2 pathway in diquat-challenged jejunum of laying hens.\u003c/strong\u003e (A) Representative immunohistochemical staining of 4-hydroxynonenal (4-HNE), a marker of lipid peroxidation (scale bar = 200 μm). (B–E) Antioxidant enzyme activities and lipid peroxidation in jejunal tissues: superoxide dismutase (SOD; B), glutathione peroxidase (GSH-Px; C), total antioxidant capacity (T-AOC; D), and malondialdehyde (MDA; E). (F–I) Serum antioxidant parameters: SOD (F), GSH-Px (G), T-AOC (H), and MDA (I). (J–O) Relative mRNA expression of Nrf2 pathway-related genes in jejunal tissues, determined by qRT-PCR: Keap1 (J), Nrf2 (K), HO-1 (L), NQO1 (M), GPX4 (N), and SOD1 (O). (P-R) Representative immunofluorescence staining of Nrf2, HO-1, GPX4 in jejunal sections (scale bar = 100 μm). (S) Quantitative analysis of Nrf2, HO-1, and GPX4 immunofluorescence intensity. (T-U) Representative Western blot bands and quantitative densitometry of Nrf2, HO-1, and GPX4 proteins in jejunal tissues, normalized to β-actin. Data are presented as mean ± SD (n = 6 per group). **P \u0026lt; 0.01, *P \u0026lt; 0.05 vs DQ group; ns, no significance.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-9415762/v1/b81415689660d876942f4614.png"},{"id":108248274,"identity":"b592f0aa-1f27-416c-b17a-5da4d9ddf4ab","added_by":"auto","created_at":"2026-05-01 01:11:43","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":626405,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLactobacillus reuteri suppresses diquat-induced inflammatory response and NF-κB signaling activation in jejunal tissues of laying hens.\u003c/strong\u003e (A–D) Jejunal protein levels of pro-inflammatory cytokines IL-1β (A), TNF-α (B), IL-6 (C), and anti-inflammatory cytokine IL-10 (D), determined by ELISA. (E–H) Relative mRNA expression of IL-1β (E), TNF-α (F), IL-6 (G), and IL-10 (H) in jejunal tissues, determined by qRT-PCR. (I–L) Relative mRNA expression of NF-κB pathway-related genes TLR4 (I), MYD88 (J), IκBα (K), and NF-κB (L) in jejunal tissues, determined by qRT-PCR. (M, N, O) Representative immunofluorescence staining of macrophage marker F4/80, phosphorylated p65 and IL-10 in jejunal sections (scale bar = 50 μm). (P) Quantitative analysis of F4/80, p-p65 NF-κB, and IL-10 immunofluorescence intensity. (Q) Representative Western blot bands of p-p65 NF-κB and IL-10 proteins in jejunal tissues, normalized to β-actin. (R) Quantitative densitometry of p-p65 NF-κB and IL-10 protein expression. Data are presented as mean ± SD (n = 6 per group). **P \u0026lt; 0.01, *P \u0026lt; 0.05 vs DQ group; ns, no significance.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-9415762/v1/0828384d0a37c20786a26b4b.png"},{"id":108491402,"identity":"4f1c0766-9540-477d-a4d9-238f2d745420","added_by":"auto","created_at":"2026-05-05 09:53:46","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":491472,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLactobacillus reuteri inhibits apoptosis and promotes proliferation of jejunal epithelium in diquat-challenged laying hens.\u003c/strong\u003e(A) Representative TUNEL immunofluorescence staining of apoptotic cells in jejunal sections (scale bar = 50 μm). (B) Quantitative analysis of TUNEL fluorescence intensity. (C–E) Relative mRNA expression of apoptosis-related genes BAX (C), BCL2 (D), and Caspase3 (E) in jejunal tissues, determined by qRT-PCR. (F) Bcl-2/Bax mRNA expression ratio. (G) Representative immunofluorescence staining of proliferating cell nuclear antigen (PCNA) in jejunal sections (scale bar = 50 μm; PCNA-positive nuclei in green/purple). (H) Quantitative analysis of PCNA fluorescence intensity. (I–K) Relative mRNA expression of proliferation-related genes LGR5 (I), PCNA (J), and β-catenin (K) in jejunal tissues, determined by qRT-PCR. Data are presented as mean ± SD (n = 6 per group). **P \u0026lt; 0.01, *P \u0026lt; 0.05 vs DQ group; ns, no significance.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-9415762/v1/8f9a5ebe0ec252bc15c6cb52.png"},{"id":108248277,"identity":"3be58b27-f7c8-43a9-8758-0f9dbd2cb28a","added_by":"auto","created_at":"2026-05-01 01:11:43","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":294392,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIntegrative correlation analysis reveals the microbiota-metabolite-mitochondria-barrier axis mediated by Lactobacillus reuteri.\u003c/strong\u003eSpearman correlation analysis was performed across all samples. Only significant correlations (|r| \u0026gt; 0.3, P \u0026lt; 0.05) are displayed. Red lines represent positive correlations, blue dashed lines represent negative correlations, and line thickness is proportional to the absolute value of the correlation coefficient. Nodes on the left represent bacterial genera and tryptophan metabolites; nodes on the right represent intestinal barrier markers (MUC-2, Occludin), permeability indicators (DAO), mitochondrial parameters (Mt-ROS, ATP), mitochondrial function-related genes (NRF1，DRP1), antioxidant markers (Nrf2, HO-1, GSH-Px), inflammatory markers (TLR4, NF-κB, IL-10), apoptosis marker (BCL2), and proliferation marker (PCNA).\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-9415762/v1/403ad10d0864af86937a8ed9.png"},{"id":108248278,"identity":"437e62ec-1968-4e04-a76a-ac6eaad082bb","added_by":"auto","created_at":"2026-05-01 01:11:44","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":287840,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMechanism by which Lactobacillus reuteri alleviates diquat-induced intestinal oxidative stress injury in laying hens via the microbiota-metabolite-mitochondria axis.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-9415762/v1/b96637e21574a9ba0053dbe4.png"},{"id":108809117,"identity":"6ff069ee-5d7d-454b-841a-04b402e7c451","added_by":"auto","created_at":"2026-05-08 15:50:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6148840,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9415762/v1/a7657216-8a79-4c84-b5af-8f26c00018d0.pdf"},{"id":108248268,"identity":"5cda10f7-0cd1-473d-a65d-d9416c506823","added_by":"auto","created_at":"2026-05-01 01:11:43","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":28217,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTable S1. Genes primer sequences for qPCR.\u003c/p\u003e\n\u003cp\u003eTable S2. Effects of \u003cem\u003eL. reuteri\u003c/em\u003e on the production performance of laying hens\u003c/p\u003e","description":"","filename":"0414supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-9415762/v1/f09de19cb2793aa90fea57c2.docx"},{"id":108248270,"identity":"e83dea99-ec4c-4945-9da3-bad54d419cbb","added_by":"auto","created_at":"2026-05-01 01:11:43","extension":"zip","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":6553816,"visible":true,"origin":"","legend":"","description":"","filename":"0416WBsupplementaryfile.zip","url":"https://assets-eu.researchsquare.com/files/rs-9415762/v1/fa443a5c0929aec33c0c0996.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"Limosilactobacillus reuteri alleviates intestinal oxidative damage by regulating gut microbiota- tryptophan metabolites-mitochondria axis-mediated oxidative stress and apoptosis","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eOxidative stress is a prevalent issue in modern poultry production, particularly in laying hens subjected to environmental stressors, high-density farming, and dietary challenges [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In addition, oxidative stress often occurs in laying hens during the aging process, especially during post-peaking laying period[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Oxidative stress arises from an imbalance between reactive oxygen species (ROS) production and antioxidant defenses, which generally results in mitochondrial dysfunction, intestinal epithelial barrier function impairment, and increased intestinal permeability[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Accumulating evidence has shown that the elevated oxidative stress leads to inflammation and epithelium apoptosis during the aging process [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In laying hens, intestinal damages induced by oxidative stress can manifest as reduced egg production and quality, poor feed efficiency, and increased susceptibility to diseases, ultimately affecting economic outcomes [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Therefore, it is essential to explore ways to prolong the gut health of aging hens to ensure the durability of egg production.\u003c/p\u003e \u003cp\u003eThe administration of probiotic \u003cem\u003eLactobacillus\u003c/em\u003e strains has been shown to confer a range of antioxidant, antibacterial, anti-inflammatory, and immunomodulatory effects in the host [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. \u003cem\u003eLimosilactobacillus reuteri\u003c/em\u003e, a well-known probiotics of \u003cem\u003eLactobacillus\u003c/em\u003e strains, have emerged as promising interventions for mitigating oxidative stress in poultry [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. \u003cem\u003eL. reuteri\u003c/em\u003e colonizes the gut, reshapes microbiota composition (increasing Firmicutes/Bacteroidota ratio, enriching Lactobacillus), and modulates metabolic profiles [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Notably, it regulates tryptophan metabolism, restores the intestinal barrier, and promotes gut health [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Our laboratory previously isolated a probiotic strain, \u003cem\u003eL. reuteri\u003c/em\u003e Y067, from the intestines of healthy laying hens, which had shown in vitro anti-inflammatory and antioxidant activities. In vivo study had revealed that \u003cem\u003eL. reuteri\u003c/em\u003e Y067 could mitigate oxidative stress and restore intestinal barrier function and thus improving laying performance in aged laying hens (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). However, the mechanisms underlying how \u003cem\u003eL. reuteri\u003c/em\u003e Y067 alleviates intestinal oxidative stress remain unclear.\u003c/p\u003e \u003cp\u003eMitochondria serve as central hubs in oxidative stress responses, acting both as primary ROS generators via the electron transport chain and as targets of ROS-induced damage [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Under oxidative stress, mitochondrial dysfunction is characterized by elevated ROS, collapsed membrane potential (MMP), reduced ATP production, decreased mtDNA copy number, impaired biogenesis, dysregulated dynamics, and altered mitophagy [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. These alterations amplify oxidative damage, activating downstream pathways such as Nrf2 for antioxidant defense and NF-κB for inflammation [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Consequently, inflammation exacerbates apoptosis and inhibits proliferation, further compromising barrier renewal [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. However, the precise mechanisms by which \u003cem\u003eL. reuteri\u003c/em\u003e integrate microbiota-metabolite remodeling with mitochondrial regulation to restore barrier function remain underexplored, especially in laying hens under oxidative stress.\u003c/p\u003e \u003cp\u003eDiquat is commonly used as a model inducer of oxidative stress in animal studies, which exacerbates ROS generation through redox cycling, primarily targe ting the intestinal epithelium and disrupting mitochondrial function [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In this study, a diquat-induced intestinal impairment model in laying hens were used to investigate how \u003cem\u003eL. reuteri\u003c/em\u003e alleviates diquat-induced intestinal injury through a microbiota-metabolite-mitochondria axis. By elucidating this axis, our work aims to provide insights into probiotic strategies for enhancing gut health in poultry production.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Experimental design\u003c/h2\u003e \u003cp\u003e All procedure of the animal experiments were approved by the Animal Care and Use Committee of Zhejiang University (Hangzhou, China; approval number ZJU20241152). 270 Jinghai laying hens (70 weeks old, laying rate 82.2% \u0026plusmn; 1.2%) were randomly allocated to three groups, each with six replicates of 15 hens. Birds in control group (CON) and diquat-challenged group (DQ) received basal diets, while birds in \u003cem\u003eL. reuteri\u003c/em\u003e supplementation with diquat-challenged group (LRD) received a basal diet supplemented with \u003cem\u003eLimosilactobacillus reuteri\u003c/em\u003e Y067 (2.0 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e CFU/kg feed) for 10 weeks. The basal diet was formulated with maize and soybean (Table\u0026nbsp;1) to meet the nutritional standards of the National Research Council [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. \u003cem\u003eL. reuteri\u003c/em\u003e Y067 was isolated and maintained in our laboratory and deposited in the China Center for Type Culture Collection. After 10 weeks of dietary intervention, hens in the DQ and LRD groups received an intraperitoneal injection of diquat (1 mL/kg body weight, 10 mg/mL in 0.9% saline) to induce oxidative stress, as previously describe [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The CON group received an equivalent volume of 0.9% saline. Birds had unrestricted access to fresh water and mashed diets. The housing environment was maintained at 24\u0026deg;C, 50\u0026ndash;60% humidity, with a 16-hour light/8-hour dark cycle.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Sample Collection\u003c/h2\u003e \u003cp\u003eSeven days after diquat injection, one hen per replicate was euthanized. Blood was drawn from the wing vein, centrifuged at 4,000 rpm for 15 minutes at 4\u0026deg;C, and serum was stored at -20\u0026deg;C, as previously described[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Jejunal samples were collected for histological, molecular, and biochemical analyses, and cecal contents were obtained for microbiota and metabolomics studies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Laying performance\u003c/h2\u003e \u003cp\u003eFor laying performance analysis, the number of eggs and total egg weight were monitored daily, and feed disappearance was recorded weekly on a replicate basis (n\u0026thinsp;=\u0026thinsp;6) to calculate the laying rate (LR), average daily egg mass (ADEM), average daily feed intake (ADFI), and feed conversion ratio (FCR).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Serum Intestinal Permeability Markers\u003c/h2\u003e \u003cp\u003eSerum levels of diamine oxidase (DAO), D-lactate (DLA), and lipopolysaccharide (LPS) were measured to evaluate gut barrier integrity and endotoxemia. DAO and DLA were quantified using ELISA kits (Jiancheng Bioengineering Institute, Nanjing, China) following the manufacturer\u0026rsquo;s protocols. The level of LPS was measured using a chromogenic limulus amebocyte lysate assay kit (Xiamen Bioendo Technology, Xiamen, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Jejunal Morphology and Transmission Electron Microscopy (TEM)\u003c/h2\u003e \u003cp\u003eJejunal segments were paraffin-embedded, sectioned at 5 \u0026micro;m, and stained with hematoxylin and eosin (H\u0026amp;E) to assess villus height (tip to crypt base), crypt depth (crypt base to submucosa), and villus-to-crypt ratio. Images were captured using an Olympus microscope (Tokyo, Japan), and 10 well-oriented villi per section were measured with ImageJ software, as previously described[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Periodic acid-Schiff (PAS) staining was used to quantify mucus layer thickness. Jejunal samples fixed in 2.5% glutaraldehyde were post-fixed in 1% osmium tetroxide, dehydrated, and embedded in epoxy resin. Ultrathin sections (70 nm) were stained with uranyl acetate and lead citrate and examined using a JEOL-JEM-1200EX transmission electron microscope (Peabody, MA, USA) to evaluate mitochondrial morphology and tight junction structures in enterocytes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Immunohistochemistry\u003c/h2\u003e \u003cp\u003eParaffin-embedded jejunal sections (5 \u0026micro;m) were deparaffinized, rehydrated, and antigen-retrieved in citrate buffer (pH 6.0) by microwave heating. Endogenous peroxidase was blocked with 3% H₂O₂, and non-specific sites with 5% BSA. Sections were incubated overnight at 4\u0026deg;C with anti-4-HNE primary antibody (rabbit polyclonal, 1:200; Abcam, ab46545), followed by HRP-conjugated secondary antibody (1:500) for 1 h at room temperature. Color was developed with DAB substrate, counterstained with hematoxylin, and imaged at 200\u0026times; magnification. Negative controls omitted the primary antibody. Brown staining indicated positive 4-HNE expression.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Immunofluorescence and TUNEL Assays\u003c/h2\u003e \u003cp\u003eJejunal tissues were fixed in 4% paraformaldehyde, dehydrated in 30% sucrose, embedded in OCT compound, and sectioned at 5\u0026ndash;10 \u0026micro;m. Sections were permeabilized with 0.1% Triton X-100, blocked with goat serum, and incubated overnight at 4\u0026deg;C with primary antibodies. Sections were incubated with secondary antibodies for 1 h, washed, mounted with DAPI-containing medium, and visualized using a fluorescence microscope (BX-61, Olympus, Center Valley, PA, USA) ,as previously described [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Fluorescence intensity was quantified with ImageJ. TUNEL assays were performed using a TUNEL kit (Roche, Basel, Switzerland) to detect apoptotic cells, with signals quantified in five random fields per section.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Gene Expression Analysis\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from jejunal mucosa using a FreeZol Reagent kit (Vazyme, China), and cDNA was synthesized with a cDNA synthesis kit (Vazyme, China). Quantitative real-time PCR (qRT-PCR) was performed on a Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA) using Taq Pro Universal SYBR qPCR Master Mix (Vazyme, China). Relative mRNA expression was calculated using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method with β-actin as the reference gene (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e)[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9.Western blotting\u003c/h2\u003e \u003cp\u003eJejunal tissues were lysed in RIPA buffer (Beyotime Biotechnology, P0013B). Protein concentration was determined by BCA assay (Beyotime Biotechnology, P0012). Equal protein amounts (30\u0026ndash;50 \u0026micro;g) were separated by SDS-PAGE, transferred to PVDF membranes, and blocked with 5% non-fat milk in TBST for 1 h, as previously described[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Membranes were incubated overnight at 4\u0026deg;C with primary antibodies: anti-Nrf2 (1:1000; Proteintech, 16396-1-AP), anti-HO-1 (1:1000; Proteintech, 10701-1-AP), anti-GPX4 (1:1000; Proteintech, 67763-1-lg), anti-phospho-NF-κB p65 (1:1000; Thermo Fisher, 600-400-271), anti-IL-10 (1:1000; Proteintech, 82191-3-RR), and anti-β-actin (1:5000; Proteintech, 66009-1-lg). After TBST washes, HRP-conjugated secondary antibodies were applied for 1 h. Bands were visualized by an imaging system (Tanon, China). Band intensities were quantified with ImageJ and normalized to β-actin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Jejunal Inflammatory Cytokine Quantification\u003c/h2\u003e \u003cp\u003eJejunal mucosa was homogenized in PBS, and supernatants were collected after centrifugation at 4,000 rpm for 10 minutes at 4\u0026deg;C, as previously described[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Levels of IL-1β, TNF-α, IL-6, and IL-10 were measured using commercial ELISA kits (Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11. Jejunal Antioxidant Capacity\u003c/h2\u003e \u003cp\u003eJejunal mucosa (0.1 g) was homogenized in 0.9 mL chilled PBS and centrifuged at 4,000 rpm for 10 minutes at 4\u0026deg;C. Supernatants and serum samples were analyzed for malondialdehyde (MDA; kit number: A003-1-2), total antioxidant capacity (T-AOC; kit number: A015-1-2), superoxide dismutase (SOD; kit number: A001-1-2), and glutathione peroxidase (GSH-Px; kit number: A005-1-2) using commercial kits (Jiancheng Bioengineering Institute, Nanjing, China), as previously described[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12. Mitochondrial Analyses\u003c/h2\u003e \u003cp\u003eJejunal mitochondria were isolated using a mitochondrial isolation kit (Beyotime Biotechnology, Shanghai, China). Tissue was homogenized in extraction buffer, centrifuged at 1,000\u0026ndash;2,000 g to remove debris, and the supernatant was centrifuged at 10,000\u0026ndash;12,000 g to pellet mitochondria. The\u003c/p\u003e \u003cp\u003elevel of reactive oxygen species (ROS) was measured using dichlorohydro-fluorescein diacetate (DCFH-DA) at 485 nm excitation and 530 nm emission, expressed as dichlorofluorescein (DCF) fluorescence intensity. Mitochondrial membrane potential (MMP) was assessed with a JC-1 assay kit (Beyotime Biotechnology), with MMP expressed as the red/green fluorescence ratio. Mitochondrial DNA (mtDNA) content was quantified via qPCR using primers for mtDNA-specific genes, normalized to nuclear DNA. ATP content was measured using an enhanced ATP assay kit based on luciferase luminescence (Beyotime Biotechnology, S0027). ATP content was expressed as nmol/mg protein.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13. 16S rRNA Sequencing\u003c/h2\u003e \u003cp\u003eDNA was extracted from cecal contents using a QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany). The V3\u0026ndash;V4 region of the 16S rRNA gene was amplified and sequenced on an Illumina MiSeq platform (San Diego, CA, USA), as previously described[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Data were processed using QIIME2 to evaluate alpha diversity (Shannon and Simpson indices), beta diversity (principal coordinate analysis), and taxonomic profiles at phylum and Genus levels.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14. Metabolomics Analysis\u003c/h2\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.14.1 Untargeted Metabolomics Analysis\u003c/h2\u003e \u003cp\u003eCecal contents were analyzed for untargeted metabolomics using liquid chromatography-mass spectrometry (LC-MS). Samples were extracted in 80% methanol, and metabolites were separated on a Waters ACQUITY UPLC HSS T3 column (2.1 \u0026times; 100 mm, 1.8 \u0026micro;m) using a Waters UPLC system coupled to a Q-Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA), as previously described[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Data were acquired in positive and negative ion modes, processed with XCMS software for peak detection, and analyzed using MetaboAnalyst for differential metabolite identification and pathway enrichment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.14.2. Targeted Metabolomics Analysis\u003c/h2\u003e \u003cp\u003eCecal contents (approximately 200 mg) were homogenized in ice-cold methanol: acetonitrile: water (2:2:1, v/v/v) containing internal standards. After centrifugation (14,000 \u0026times; g, 15 min, 4\u0026deg;C), the supernatant was evaporated and reconstituted in 50% methanol. Targeted quantification of tryptophan metabolites was performed using UHPLC-QqQ-MS/MS (Agilent 1290\u0026ndash;6470) with a Waters ACQUITY UPLC BEH C18 column. Detection was conducted in positive ESI mode with multiple reaction monitoring (MRM). Quantification was achieved using external standard curves and internal standard normalization. Results were expressed as nmol/g wet weight.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e2.15. Statistical Analysis\u003c/h2\u003e \u003cp\u003eData were analyzed using SPSS 26.0 (IBM, Armonk, NY, USA) and expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Data were compared using one-way ANOVA with Tukey\u0026rsquo;s post-hoc test. Graphs were generated using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA). Significance was set at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.1. \u003cem\u003eL. reuteri\u003c/em\u003e Restores Intestinal Barrier Integrity in Diquat-Challenged Laying Hens\u003c/h2\u003e \u003cp\u003eHistological examination of jejunal sections stained with HE revealed severe mucosal damages in the DQ group, including shortened villi, disrupted epithelial continuity, and inflammatory cell infiltration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In contrast, the LRD group exhibited improved villus architecture with restored height and reduced structural disruption, approaching the intact morphology of the CON group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Transmission electron microscopy (TEM) further confirmed barrier ultrastructural impairment in the DQ group, characterized by reduced tight junction density, widened intercellular spaces, and damaged microvilli (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). L. reuteri pretreatment significantly alleviated these changes, evidenced by the increased tight junction structures and preserved microvilli morphology in the LRD group. Morphometric analysis showed that diquat exposure markedly reduced villus height and the villus height/crypt depth ratio (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while increasing crypt depth compared to the CON group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC\u0026ndash;E). \u003cem\u003eL. reuteri\u003c/em\u003e supplementation significantly restored villus height and the villus/crypt ratio (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), although crypt depth remained comparable between the DQ and LRD groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC\u0026ndash;E). Serum permeability markers including LPS, DAO, and D-LA were significantly elevated in the DQ group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF\u0026ndash;H). \u003cem\u003eL. reuteri\u003c/em\u003e pretreatment markedly reversed these indicators (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). qRT-PCR analysis demonstrated significant downregulation of barrier-related mRNA in the DQ group, including \u003cem\u003eMUC2\u003c/em\u003e, \u003cem\u003eZO-1\u003c/em\u003e, \u003cem\u003eOccludin\u003c/em\u003e, and \u003cem\u003eClaudin-1\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI\u0026ndash;L). \u003cem\u003eL. reuteri\u003c/em\u003e intervention significantly upregulated the mRNA expression of these genes \u003cem\u003e(P\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI\u0026ndash;K).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eImmunofluorescence staining showed reduced and discontinuous distribution of Occludin and MUC2 in the DQ group, with weak fluorescence intensity along the apical membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eM, N). \u003cem\u003eL. reuteri\u003c/em\u003e pretreatment restored continuous linear staining and increased fluorescence intensity for both proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eM, N). Quantitative analysis confirmed significantly lower Occludin and MUC2 fluorescence intensity in the DQ group compared to CON (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while \u003cem\u003eL. reuteri\u003c/em\u003e supplementation restoring these levels (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eO, P). Alcian blue-periodic acid-Schiff (AB-PAS) staining revealed a thinned and disrupted mucus layer in the DQ group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eQ). \u003cem\u003eL. reuteri\u003c/em\u003e pretreatment significantly increased mucus layer thickness (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), approaching CON levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eR).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.2. \u003cem\u003eL. reuteri\u003c/em\u003e Reshapes the Gut Microbiota Composition in Diquat-Challenged Hens\u003c/h2\u003e \u003cp\u003eAlpha diversity analysis of cecal microbiota revealed significant alterations among groups. The ACE and Chao1 indices, reflecting community richness, were markedly decreased in the DQ group compared to CON group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while the Shannon and Simpson indices showed no significant differences (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u0026ndash;D). \u003cem\u003eL. reuteri\u003c/em\u003e pretreatment significantly restored ACE and Chao1 indices to levels comparable to the CON group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). Beta diversity analysis demonstrated distinct microbial community structures. Principal coordinate analysis (PCoA) and partial least squares discriminant analysis (PLS-DA) clearly separated the DQ group from the CON and LRD groups, with the LRD group clustering closer to the CON group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, F). These results indicate that diquat induced substantial microbiota dysbiosis, which was partially ameliorated by \u003cem\u003eL. reuteri.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs for microbiota composition, at the phylum level, diquat challenge increased the relative abundance of Firmicutes and decreased Bacteroidota compared to the CON group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). Quantitative analysis confirmed a significant elevation in Firmicutes (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and reduction in Bacteroidota in the DQ group, with \u003cem\u003eL. reuteri\u003c/em\u003e pretreatment restoring Bacteroidota abundance and normalizing the Firmicutes/Bacteroidota ratio (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH, I). At the genus level, the most abundant genera included \u003cem\u003eBacteroides\u003c/em\u003e, \u003cem\u003eRikenellaceae_RC9_gut_group\u003c/em\u003e, \u003cem\u003eLactobacillus\u003c/em\u003e, and \u003cem\u003eRuminococcus_torques_group\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ). Quantitative comparison showed that diquat exposure significantly decreased \u003cem\u003eBacteroides\u003c/em\u003e and \u003cem\u003eRikenellaceae_RC9_gut_group\u003c/em\u003e while increasing \u003cem\u003eRuminococcus_torques_group\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK). Notably, \u003cem\u003eLactobacillus\u003c/em\u003e abundance increased in the LRD group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK). Linear discriminant analysis effect size (LEfSe) identified taxonomic biomarkers across groups. The cladogram illustrated enrichment of Spirochaetota phylum and beneficial genera such as Rikenellaceae and Spirochaetales in the CON group, \u003cem\u003eRuminococcus_torques_group\u003c/em\u003e and Colidextribacter in the DQ group, and \u003cem\u003eLactobacillus\u003c/em\u003e in the LRD group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL). LDA score analysis (LDA\u0026thinsp;\u0026gt;\u0026thinsp;3.5) confirmed \u003cem\u003eRikenellaceae_RC9_gut_group\u003c/em\u003e as the primary biomarker in the CON group, \u003cem\u003eRuminococcus_torques_group\u003c/em\u003e in the DQ group, and \u003cem\u003eLactobacillus\u003c/em\u003e in the LRD group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eM). These findings demonstrate that \u003cem\u003eL. reuteri\u003c/em\u003e pretreatment effectively mitigates diquat-induced gut dysbiosis by restoring microbial diversity and enriching beneficial taxa, particularly \u003cem\u003eLactobacillus\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.3. \u003cem\u003eL. reuteri\u003c/em\u003e Remodels the Intestinal Metabolome with Significant Enrichment in Tryptophan Metabolism\u003c/h2\u003e \u003cp\u003eNon-targeted metabolomics analysis of jejunal tissues revealed distinct metabolic profiles among the three groups. Principal component analysis (PCA) showed clear separation between the DQ group and the CON group, while the LRD group clustered closer to the CON group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Orthogonal partial least squares discriminant analysis (OPLS-DA) further confirmed significant differences in metabolic patterns, with good model fit and predictability (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Volcano plot analysis (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) identified a total of 582 upregulated and 415 downregulated metabolites in the DQ vs CON comparison, and 580 upregulated and 129 downregulated metabolites in the LRD vs DQ comparison (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D). These results indicate that diquat induced widespread metabolic disturbances, whereas \u003cem\u003eL. reuteri\u003c/em\u003e pretreatment partially reversed these alterations. KEGG pathway enrichment analysis demonstrated that tryptophan metabolism was the most significantly enriched pathway in the LRD vs DQ group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, impact factor\u0026thinsp;\u0026gt;\u0026thinsp;0.1; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). This pathway showed the highest enrichment score and relevance to lipid remodeling under oxidative stress conditions. Hierarchical clustering heatmap of the top 30 differential metabolites revealed clear separation among groups, with multiple tryptophan metabolism-related metabolites showing upregulation in the LRD group compared to the DQ group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Key metabolites enriched in this cluster included indole-3-acetic acid, trans-3-indoleacrylic acid, indole-3-aldehyde, indoleacetic acid, and 3-indolepropionic acid. Targeted metabolomics analysis of representative tryptophan metabolites confirmed indole-3-acetic acid, trans-3-indoleacrylic acid, indole-3-aldehyde, indoleacetic acid, and 3-indolepropionic acid were significant increases in the LRD group compared to the DQ group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG).To further explore the relationship between gut microbiota and tryptophan metabolism, Spearman correlation analysis was performed between the top 10 abundant bacterial genera and five key tryptophan metabolites (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). \u003cem\u003eLactobacillus\u003c/em\u003e exhibited significant positive correlations with multiple tryptophan metabolites, particularly indole-3-acetic acid and indole-3-propionic acid (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In contrast, \u003cem\u003eRuminococcus_torques_group\u003c/em\u003e showed negative correlations with these metabolites, especially indole-3-propionic acid (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Beneficial genera such as \u003cem\u003eFaecalibacterium\u003c/em\u003e and \u003cem\u003eOscillospiraceae\u003c/em\u003e also displayed strong positive correlations with several tryptophan metabolites.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.4. \u003cem\u003eL. reuteri\u003c/em\u003e Alleviates Diquat-Induced Mitochondrial Dysfunction, Impaired Biogenesis, Dysregulated Dynamics, and Mitophagy Imbalance\u003c/h2\u003e \u003cp\u003eTransmission electron microscopy of jejunal tissue revealed severe mitochondrial ultrastructural damage in the DQ group, including mitochondrial swelling, disrupted cristae, and vacuolization (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In contrast, mitochondria in the CON and LRD groups exhibited intact morphology with well-organized cristae and normal matrix density (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Diquat challenge significantly increased mitochondrial ROS production (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), decreased mitochondrial membrane potential (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), reduced mtDNA copy number (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and lowered ATP content (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB\u0026ndash;E). \u003cem\u003eL. reuteri\u003c/em\u003e pretreatment markedly attenuated these changes, reducing ROS (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), restoring MMP (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), increasing mtDNA copy number (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and elevating ATP levels (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) relative to the DQ group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB\u0026ndash;E). qRT-PCR analysis demonstrated that diquat exposure downregulated mitochondrial biogenesis-related mRNA expression, including \u003cem\u003ePGC-1α\u003c/em\u003e, \u003cem\u003eNRF1\u003c/em\u003e, \u003cem\u003eTFAM\u003c/em\u003e, and \u003cem\u003ePOLRMT\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). \u003cem\u003eL. reuteri\u003c/em\u003e supplementation significantly upregulated these genes (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF), indicating enhanced mitochondrial biogenesis. Regarding mitochondrial dynamics, diquat increased fission genes \u003cem\u003eDRP1\u003c/em\u003e and \u003cem\u003eFIS1\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) while decreasing fusion genes \u003cem\u003eMFN1\u003c/em\u003e and \u003cem\u003eMFN2\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). \u003cem\u003eL. reuteri\u003c/em\u003e reversed this imbalance by downregulating \u003cem\u003eDRP1\u003c/em\u003e and \u003cem\u003eFIS1\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and upregulating \u003cem\u003eMFN1\u003c/em\u003e and \u003cem\u003eMFN2\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). Immunofluorescence staining revealed disrupted mitophagy in the DQ group, with altered colocalization of LC3B, PINK1, and Parkin in jejunal (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). \u003cem\u003eL. reuteri\u003c/em\u003e restored the punctate distribution and colocalization of these markers, suggesting normalized mitophagic flux (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). These mitochondrial improvements provide a foundation for enhanced antioxidant defense and reduced inflammation, which were further investigated.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.5. \u003cem\u003eL. reuteri\u003c/em\u003e Enhances Antioxidant Capacity and Activates the Nrf2 Pathway in Diquat-Challenged Laying Hens\u003c/h2\u003e \u003cp\u003eImmunohistochemical staining for 4-hydroxynonenal (4-HNE), a marker of lipid peroxidation, showed intense brown staining in the villi and crypts of the DQ group, accompanied by disrupted villus structure, indicating severe oxidative damage (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In the LRD group, 4-HNE staining intensity was markedly reduced, with improved villus morphology approaching the CON group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In jejunal tissues, diquat challenge significantly decreased SOD, GSH-Px, and T-AOC activities, while increasing MDA content compared to the CON group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB\u0026ndash;E). \u003cem\u003eL. reuteri\u003c/em\u003e pretreatment significantly restored GSH-Px and T-AOC activities (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and reduced MDA levels (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), although SOD activity showed no significant difference between DQ and LRD groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB\u0026ndash;E). Serum antioxidant parameters followed a similar pattern: diquat exposure reduced SOD, GSH-Px, and T-AOC activities while elevating MDA content (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF\u0026ndash;I). \u003cem\u003eL. reuteri\u003c/em\u003e intervention significantly improved SOD, T-AOC and GSH-Px activities (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with MDA levels showing improvement but no statistical significance between DQ and LRD groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF\u0026ndash;I).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eqRT-PCR analysis of Nrf2 pathway-related genes revealed that diquat downregulated \u003cem\u003eNrf2\u003c/em\u003e, \u003cem\u003eNQO1\u003c/em\u003e, \u003cem\u003eGPX4\u003c/em\u003e, and \u003cem\u003eSOD1\u003c/em\u003e mRNA expression. Notably, HO-1 mRNA levels showed no significant difference between CON and DQ groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), while \u003cem\u003eKeap1\u003c/em\u003e mRNA expression were significantly increased. \u003cem\u003eL. reuteri\u003c/em\u003e pretreatment significantly upregulated \u003cem\u003eNrf2\u003c/em\u003e, \u003cem\u003eHO-1\u003c/em\u003e, and \u003cem\u003eNQO1\u003c/em\u003e, with the restoration of \u003cem\u003eGPX4\u003c/em\u003e and \u003cem\u003eSOD1\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ\u0026ndash;O).Immunofluorescence staining demonstrated reduced nuclear translocation of Nrf2 and cytoplasmic expression of HO-1 and GPX4 in the DQ group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eP\u0026ndash;R). \u003cem\u003eL. reuteri\u003c/em\u003e pretreatment enhanced Nrf2 nuclear localization and increased HO-1 and GPX4 fluorescence intensity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eP\u0026ndash;R). Quantitative analysis confirmed significantly lower Nrf2, HO-1, and GPX4 fluorescence intensity in the DQ group compared to CON (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with \u003cem\u003eL. reuteri\u003c/em\u003e restoring these levels (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eS). Western blot analysis revealed that diquat downregulated Nrf2, HO-1, and GPX4 protein expression. \u003cem\u003eL. reuteri\u003c/em\u003e pretreatment significantly upregulated these proteins (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs DQ; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eT-U), with Nrf2 and HO-1 levels restored to near CON values and GPX4 partially recovered.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.6. \u003cem\u003eL. reuteri\u003c/em\u003e Suppresses Diquat-Induced Inflammatory Response and NF-κB Signaling\u003c/h2\u003e \u003cp\u003eEnzyme-linked immunosorbent assay (ELISA) of jejunal tissues showed that DQ challenge significantly increased the protein levels of pro-inflammatory cytokines IL-1β, TNF-α, and IL-6 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while decreasing the anti-inflammatory cytokine IL-10 compared to the CON group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u0026ndash;D). LRD pretreatment markedly reduced IL-1β, TNF-α, and IL-6 levels (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and elevated IL-10 content (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) relative to the CON group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u0026ndash;D). Consistent with protein data, qRT-PCR analysis revealed that diquat significantly upregulated mRNA expression of \u003cem\u003eIL-1β\u003c/em\u003e, \u003cem\u003eTNF-α\u003c/em\u003e, and \u003cem\u003eIL-6\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while downregulating \u003cem\u003eIL-10\u003c/em\u003e mRNA expression (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE\u0026ndash;H). \u003cem\u003eL. reuteri\u003c/em\u003e pretreatment significantly downregulated \u003cem\u003eIL-1β\u003c/em\u003e, \u003cem\u003eTNF-α\u003c/em\u003e, and \u003cem\u003eIL-6\u003c/em\u003e mRNA expression (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and upregulated \u003cem\u003eIL-10\u003c/em\u003e mRNA expression (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE\u0026ndash;H). Further qRT-PCR analysis of the TLR4/NF-κB pathway showed that diquat significantly upregulated \u003cem\u003eTLR4\u003c/em\u003e, \u003cem\u003eMYD88\u003c/em\u003e, \u003cem\u003eIκBα\u003c/em\u003e, and \u003cem\u003eNF-κB\u003c/em\u003e mRNA expression (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI\u0026ndash;L). \u003cem\u003eL. reuteri\u003c/em\u003e intervention significantly reduced \u003cem\u003eTLR\u003c/em\u003e4, \u003cem\u003eMYD88\u003c/em\u003e, \u003cem\u003eIκBα\u003c/em\u003e, and \u003cem\u003eNF-κB\u003c/em\u003e mRNA levels (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI\u0026ndash;L), indicating suppression of the inflammatory signaling cascade.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eImmunofluorescence staining demonstrated increased macrophage infiltration (F4/80-positive cells) and nuclear translocation of phosphorylated p65 (p-p65 NF-κB) in the DQ group, with reduced IL-10 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eM\u0026ndash;O). \u003cem\u003eL. reuteri\u003c/em\u003e pretreatment decreased F4/80 and p-p65 fluorescence intensity while enhancing IL-10 staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eM\u0026ndash;O). Quantitative analysis confirmed significantly higher F4/80 and p-p65 intensity and lower IL-10 intensity in the DQ group compared to CON (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), with \u003cem\u003eL. reuteri\u003c/em\u003e restoring these levels (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eP). Western blot analysis showed increased phosphorylated p65 (p-p65 NF-κB) and decreased IL-10 protein expression in the DQ group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs CON; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eQ-R). \u003cem\u003eL. reuteri\u003c/em\u003e pretreatment significantly reduced p-p65 and restored IL-10 protein levels (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs DQ; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eQ-R).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.7. \u003cem\u003eL. reuteri\u003c/em\u003e Inhibits Apoptosis and Promotes Proliferation in Diquat-Challenged Laying Hens\u003c/h2\u003e \u003cp\u003eTerminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining revealed a significant increase in apoptotic cells in the jejunal epithelium of the DQ group, with intense green fluorescence in villi and crypts (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). LRD pretreatment markedly reduced TUNEL-positive cells, with fluorescence intensity approaching the CON group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Quantitative analysis confirmed significantly higher TUNEL fluorescence intensity in the DQ group compared to CON (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), with \u003cem\u003eL. reuteri\u003c/em\u003e restoring levels (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). qRT-PCR analysis of apoptosis-related genes showed that diquat significantly upregulated \u003cem\u003eBAX\u003c/em\u003e and \u003cem\u003eCaspase3\u003c/em\u003e mRNA expression (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while slightly upregulating \u003cem\u003eBCL2\u003c/em\u003e mRNA expression (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC\u0026ndash;E). The Bcl-2/Bax ratio was markedly decreased in the DQ group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF). \u003cem\u003eL. reuteri\u003c/em\u003e pretreatment significantly downregulated \u003cem\u003eCaspase3\u003c/em\u003e mRNA expression (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), upregulated \u003cem\u003eBCL2\u003c/em\u003e mRNA expression (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and restored the Bcl-2/Bax ratio (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC\u0026ndash;F). Immunofluorescence staining for proliferating cell nuclear antigen (PCNA), a marker of cell proliferation, showed reduced nuclear staining in the crypts and villi of the DQ group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG). \u003cem\u003eL. reuteri\u003c/em\u003e pretreatment increased PCNA-positive cells with strong nuclear fluorescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG). Quantitative analysis demonstrated significantly lower PCNA fluorescence intensity in the DQ group compared to CON (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), with \u003cem\u003eL. reuteri\u003c/em\u003e restoring levels (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH). qRT-PCR analysis of proliferation-related genes revealed that diquat significantly downregulated \u003cem\u003eLGR5\u003c/em\u003e, \u003cem\u003ePCNA\u003c/em\u003e, and \u003cem\u003eβ-catenin\u003c/em\u003e mRNA expression (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI\u0026ndash;K). \u003cem\u003eL. reuteri\u003c/em\u003e pretreatment significantly upregulated the mRNA expression of these genes (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI\u0026ndash;K).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e3.8. Integrative Correlation Analysis Reveals the Microbiota-Metabolite-Mitochondria-Barrier Axis Mediated by \u003cem\u003eLactobacillus reuteri\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eTo further elucidate the relationships among microbiota composition, tryptophan metabolites, and downstream intestinal parameters, spearman correlation network analysis was performed. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, \u003cem\u003eLactobacillus\u003c/em\u003e exhibited strong positive correlations with multiple protective indicators, including barrier proteins (MUC-2 and Occludin), mitochondrial ATP content, biogenesis factor NRF1, antioxidant markers (Nrf2, HO-1, and GSH-Px), anti-inflammatory cytokine IL-10, and proliferation marker PCNA. In contrast, \u003cem\u003eRuminococcus_torques_group\u003c/em\u003e showed significant negative correlations with these parameters and positive correlations with oxidative stress and inflammatory markers (Mt-ROS, NF-κB, IL-1β pathway). Among tryptophan metabolites, indole-3-acetic acid, indole-3-propionic acid, and dndoleacetic acid displayed strong positive associations with mitochondrial function (ATP, NRF1), antioxidant capacity (HO-1, GSH-Px), and barrier integrity (MUC-2, Occludin), while being negatively correlated with ROS, NF-κB signaling, and apoptosis-related markers (BCL2). These integrative correlations strongly support that \u003cem\u003eL. reuteri\u003c/em\u003e-driven enrichment of beneficial microbiota and tryptophan metabolites serves as a central mediator linking microbial modulation to mitochondrial protection, antioxidant defense, inflammation suppression, and ultimately intestinal barrier restoration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIntestinal oxidative stress represents a critical challenge in modern laying hen production, as aging, environmental and nutritional challenges trigger excessive ROS production, leading to barrier dysfunction, systemic inflammation, reduced egg production, deteriorated eggshell quality, and ultimately substantial economic losses to the poultry industry [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Therefore, alleviating intestinal barrier damages is the principal target in poultry production. \u003cem\u003eL. reuteri\u003c/em\u003e, a well-characterized probiotic with remarkable colonization capacity and metabolic versatility, has demonstrated efficacy in alleviating intestinal oxidative stress and barrier injury [\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The present study investigated how \u003cem\u003eL. reuteri\u003c/em\u003e alleviates diquat-induced intestinal injury in laying hens through coordinated restoration of gut microbiota homeostasis, enrichment of tryptophan metabolites, recovery of mitochondrial function, rebalancing of Nrf2-NF-κB signaling crosstalk, and ultimately restoration of intestinal barrier integrity.\u003c/p\u003e \u003cp\u003eExtensive studies have established that gut microbiota homeostasis is critical for maintaining intestinal barrier and metabolic regulation [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Previous research has consistently demonstrated that oxidative stress disrupts gut microbiota homeostasis, which are tendly accompanied by metabolic perturbations [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Consistently, our study observed that diquat-induced oxidative stress in laying hens disrupted cecal microbiota homeostasis, characterized by the reduced \u003cem\u003eLcatobacillus\u003c/em\u003e, elevated Firmicutes/Bacteroidota ratio and enhanced harmful bacteria such as \u003cem\u003eRuminococcus_torques_group\u003c/em\u003e. This microbiota dysbiosis was accompanied by the depletion of tryptophan metabolites. Studies have showed that \u003cem\u003eL. reuteri\u003c/em\u003e can restore gut microbiota dysbiosis and modulates metabolic profiles in various intestinal inflammation and oxidative stress models [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Our study also found that \u003cem\u003eL. reuteri\u003c/em\u003e pretreatment effectively restored the microbiota and metabolism in our diquat-challenged laying hens. Specifically, \u003cem\u003eL. reuter\u003c/em\u003e increased the abundance of \u003cem\u003eLactobacillus\u003c/em\u003e, which is often related to intestinal barrier restoration. \u003cem\u003eLactobacillus\u003c/em\u003e has been reported to encourage intestinal epithelium regeneration [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], regulate tight junction protein [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] and maintain mucus layer thickness [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Meanwhile, our results also show that tryptophan metabolism is the top upregulated pathway, with multiple metabolites significantly elevated, including indole-3-acetic acid, trans-3-indoleacrylic acid and indole-3-aldehyde. Tryptophan metabolism is highly correlated with intestinal health[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Previous studies have demonstrated that indole metabolites alleviate intestinal barrier damage, promote epithelial repair, and inhibit inflammation [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Tryptophan metabolites can also regulate the expression of tight junction proteins to mitigate intestinal barrier [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Thus, the interaction of \u003cem\u003eLactobacillus reuteri\u003c/em\u003e with microbiota and metabolism may facilitate the intestinal barrier recovery.\u003c/p\u003e \u003cp\u003eMitochondria function both as major ROS generators and as primary targets of ROS-induced damage in oxidative stress pathophysiology [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Mitochondria dysfunction may hinder intestinal epithelium function, leading to intestinal barrier damage [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Studies have shown that \u003cem\u003eLactobacillus reuteri\u003c/em\u003e can mitigate oxidative stress, alleviate mitochondria dysfunction[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] and improve intestinal health [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Consistently, in our study \u003cem\u003eL. reuteri\u003c/em\u003e intervention reduces ROS production and restores mitochondrial integrity. This may be because tryptophan metabolites of \u003cem\u003eLactobacillus reuteri\u003c/em\u003e improve mitochondrial function, reduce ROS accumulation, and protect mitochondrial integrity [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Meantime, we found \u003cem\u003eL. reuteri\u003c/em\u003e recover ΔΨm and ATP content. Mitochondrial ATP production is essential for intestinal barrier maintenance, as they facilitate tight junction integrity, as well as influencing the expression of \u003cem\u003eMUC-2\u003c/em\u003e [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Our study also observed upregulated PGC-1α related biogenesis genes, rebalanced fission and fusion dynamics and normalized mitophagy. This may be because \u003cem\u003eL. reuteri\u0026rsquo;s\u003c/em\u003e tryptophan metabolites, such as IAA, can boost mitochondrial biosynthesis, reduce oxidative stress [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], and promote a healthy mitophagy to maintain mitochondrial function [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Thus, upregulated PGC-1α promotes intestinal epithelial differentiation and mitochondrial biogenesis[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], rebalanced mitochondrial dynamics alleviates inflammation and epithelial damage [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e] and moderate mitophagy clears damaged mitochondria and protect the intestinal barrier [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. \u003cem\u003eL. reuteri\u003c/em\u003e strongly regulates these functional pathways and restores mitochondrial health, thereby promoting intestinal barrier integrity.\u003c/p\u003e \u003cp\u003eThe Nrf2 and NF-κB pathways represent two master pathways that determine cellular responses to oxidative stress [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Nrf2 maintains the proliferation balance of intestinal stem cells [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e] and NF-κB promotes apoptosis [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. They collectively regulate downstream apoptosis and proliferation. Previous studies have shown that oxidative stress, while inhibiting Nrf2-mediated antioxidant defense, overactivates NF-κB-driven inflammatory responses[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e], promotes apoptosis rather than proliferation, and ultimately leads to intestinal epithelial cell damage and barrier dysfunction [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Extensive evidence has demonstrated that \u003cem\u003eL. reuteri\u003c/em\u003e alleviates intestinal damage by activating the Nrf2/HO-1 pathway [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e], while inhibiting the NF-κB pathway to exert anti-inflammatory effects [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. \u003cem\u003eL. reuteri\u003c/em\u003e also improved the expression of intestinal TJs and maintained the integrity of the intestinal barrier by inhibiting apoptosis of intestinal epithelial cells [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e] and stimulating the expansion of intestinal stem cells [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Consistently, \u003cem\u003eL. reuteri\u003c/em\u003e intervention in our study rebalanced this molecular crosstalk by enhancing Nrf2 antioxidant defenses and suppressing NF-κB inflammatory signaling pathway. Furthermore, \u003cem\u003eL. reuteri\u003c/em\u003e attenuated apoptosis, promoted proliferation and restored intestinal barrier. Mechanistically, \u003cem\u003eLactobacillus reuteri\u003c/em\u003e may influence upstream tryptophan metabolites to improve the oxidative-inflammatory balance, thereby alleviating intestinal damage. Studies have shown that tryptophan metabolites such as IAA and ILA can activate AhR/Nrf2 signaling to promote gut barrier [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e], alleviate intestinal epithelial cell injury via regulation of the TLR4/NF-κB pathway to reduce mucosal damage and apoptosis [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e], and modulate the proliferation of intestinal epithelial cell [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. Meanwhile, restored mitochondrial function reduced ROS generation at the source, alleviating the oxidative trigger for both Nrf2 dissociation inhibition and TLR4-mediated NF-κB activation [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. Finally, increased anti-inflammatory cytokine and restored mitochondrial function decreased intrinsic apoptosis signaling [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e], while Nrf2 activation supported Wnt/β-catenin signaling to maintain intestinal stem cell populations and promote epithelial renewal.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn conclusion, this study unveils the mechanisms by which \u003cem\u003eL. reuteri\u003c/em\u003e protects against intestinal oxidative damages through tryptophan-mediated restoration of the microbiota-metabolite-mitochondria axis (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). These findings not only advance our fundamental understanding of probiotic mechanisms but also provide a rational foundation for developing microbiome-targeted nutritional interventions to enhance intestinal health and productive performance in laying hens under oxidative stress conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e4-HNE 4-Hydroxynonenal\u003c/p\u003e\n\u003cp\u003eADEM Average daily egg mass\u003c/p\u003e\n\u003cp\u003eADFI Average daily feed intake\u003c/p\u003e\n\u003cp\u003e\u0026beta;-catenin Beta-catenin\u003c/p\u003e\n\u003cp\u003eBCL-2 B-cell lymphoma-2\u003c/p\u003e\n\u003cp\u003eCAS-3 Cysteinyl aspartate specific proteinase-3\u003c/p\u003e\n\u003cp\u003eCD Crypt depth\u003c/p\u003e\n\u003cp\u003eClaudin-1 Claudin-1\u003c/p\u003e\n\u003cp\u003eDAO Diamine oxidase\u003c/p\u003e\n\u003cp\u003eD-LA D-Lactic acid\u003c/p\u003e\n\u003cp\u003eDQ Diquat\u003c/p\u003e\n\u003cp\u003eDRP1 Dynamin-related protein 1\u003c/p\u003e\n\u003cp\u003eF4/80 F4/80 (macrophage marker)\u003c/p\u003e\n\u003cp\u003eFCR Feed conversion ratio\u003c/p\u003e\n\u003cp\u003eFIS1 Fission 1\u003c/p\u003e\n\u003cp\u003eGPX-4 Glutathione peroxidase-4\u003c/p\u003e\n\u003cp\u003eGSH-PX/GPX Glutathione peroxidase\u003c/p\u003e\n\u003cp\u003eHO-1 Heme oxygenase-1\u003c/p\u003e\n\u003cp\u003eI\u0026kappa;B\u0026alpha; Inhibitor of kappa B alpha\u003c/p\u003e\n\u003cp\u003eIL-1\u0026beta; Interleukin-1\u0026beta;\u003c/p\u003e\n\u003cp\u003eIL-6 Interleukin-6\u003c/p\u003e\n\u003cp\u003eIL-10 Interleukin-10\u003c/p\u003e\n\u003cp\u003eKeap-1 Kelch-like ECH-associated protein-1\u003c/p\u003e\n\u003cp\u003eLC3B Microtubule-associated protein 1 light chain 3B\u003c/p\u003e\n\u003cp\u003eLGR5 Leucine-rich repeat-containing G-protein coupled receptor 5\u003c/p\u003e\n\u003cp\u003eLPS Lpopolysaccharide\u003c/p\u003e\n\u003cp\u003eLR Laying rate\u003c/p\u003e\n\u003cp\u003eMDA Malondialdehyde\u003c/p\u003e\n\u003cp\u003eMFN1 Mitofusin 1\u003c/p\u003e\n\u003cp\u003eMFN2 Mitofusin 2\u003c/p\u003e\n\u003cp\u003eMMP Mitochondrial membrane potential\u003c/p\u003e\n\u003cp\u003emtDNA Mitochondrial DNA\u003c/p\u003e\n\u003cp\u003eMUC-2 Mucin-2\u003c/p\u003e\n\u003cp\u003eMyD88 Myeloid differentiation primary response 88\u003c/p\u003e\n\u003cp\u003eNF-\u0026kappa;B Nuclear factor kappa B\u003c/p\u003e\n\u003cp\u003eNF-\u0026kappa;B p-p65 Phosphorylated p65 subunit of NF-\u0026kappa;B\u003c/p\u003e\n\u003cp\u003eNQO-1 NAD(P)H:quinone oxidoreductase-1\u003c/p\u003e\n\u003cp\u003eNrf2 Nuclear factor erythroid 2-related factor 2\u003c/p\u003e\n\u003cp\u003eNRF1 Nuclear respiratory factor 1\u003c/p\u003e\n\u003cp\u003eOccludin Occludin\u003c/p\u003e\n\u003cp\u003eParkin Parkin (E3 ubiquitin-protein ligase)\u003c/p\u003e\n\u003cp\u003ePCNA Proliferating cell nuclear antigen\u003c/p\u003e\n\u003cp\u003ePINK1 PTEN-induced kinase 1\u003c/p\u003e\n\u003cp\u003ePGC-1\u0026alpha; Peroxisome proliferator-activated receptor gamma coactivator 1-alpha\u003c/p\u003e\n\u003cp\u003ePOLRMT Mitochondrial RNA polymerase\u003c/p\u003e\n\u003cp\u003eSOD Superoxide dismutase\u003c/p\u003e\n\u003cp\u003eT-AOC Total antioxidant capacity\u003c/p\u003e\n\u003cp\u003eTFAM Transcription factor A, mitochondrial\u003c/p\u003e\n\u003cp\u003eTLR4 Toll-like receptor 4\u003c/p\u003e\n\u003cp\u003eTNF-\u0026alpha; Tumor necrosis factor alpha\u003c/p\u003e\n\u003cp\u003eTUNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling\u003c/p\u003e\n\u003cp\u003eVH Villus height\u003c/p\u003e\n\u003cp\u003eZO-1 Zonula occludens\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to express our gratitude to all the members who participated in the sample collection and data counting process. Furthermore, we sincerely thank the reviewers and editors for all their valuable suggestions and revisions of our manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSAZ: Investigation, Methodology, Data curation, Writing \u0026ndash; original draft. YJL: Investigation. WCH: Investigation. CYG: Investigation. LCW: Investigation. DYY: Conceptualization, Funding, Supervision, Writing \u0026ndash; review \u0026amp; editing. BL: Investigation, Formal analysis, Supervision, Writing \u0026ndash; review \u0026amp; editing. The final draft of the manuscript has undergone a comprehensive review by all authors and received their full endorsement.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research was financially supported by the National Natural Science Foundation of China (grant no. 32402779 and 32372892), the Zhejiang Provincial Natural Science Foundation of China (grant no. ZCLMS25C1701), and the Key Research and Development Program of Zhejiang Province (Grant no: 2024C02004).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article and its supplementary information files. Additional raw data are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations\u0026nbsp;\u003c/strong\u003eThis work has received approval for research ethics from the Animal Care and Use Committee of Zhejiang University (Hangzhou, China, Protocol number ZJU20241152) and a proof/certificate of approval is available upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\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/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eShenao Zhan, Yujie Lv, Weichen Huang, Chaoyue Ge, Lianchi Wu, Dongyou Yu, and Bing Liu\u003c/p\u003e\n\u003cp\u003eCollege of Animal Sciences, Zhejiang University, Hangzhou 310058, China\u003c/p\u003e\n\u003cp\u003eYujie Lv, Weichen Huang, Chaoyue Ge, Lianchi Wu, Dongyou Yu, and Bing Liu\u003c/p\u003e\n\u003cp\u003eHainan Institute, Zhejiang University, Sanya 572000, China\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eDongyou Yu, and Bing Liu\u003c/p\u003e\n\u003cp\u003eZJU-Xinchang Joint Innovation Centre (TianMu Laboratory), Gaochuang Hi-Tech Park, Xinchang\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e465 312500, China\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSurai PF, Kochish II, Fisinin VI, Kidd MT. 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Cellular Signalling. 2025;134:111932. https://doi.org/10.1016/j.cellsig.2025.111932\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-animal-science-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jasb","sideBox":"Learn more about [Journal of Animal Science and Biotechnology](http://jasbsci.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jasb/default.aspx","title":"Journal of Animal Science and Biotechnology","twitterHandle":"@animalplantsci","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Limosilactobacillus reuteri, laying hens, oxidative stress, intestinal barrier, mitochondria","lastPublishedDoi":"10.21203/rs.3.rs-9415762/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9415762/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Intestinal oxidative stress poses significant challenges to gut health and production performance in laying hens. \u003cem\u003eLimosilactobacillus reuteri\u003c/em\u003e (\u003cem\u003eL. reuteri\u003c/em\u003e) has been demonstrated to mitigate oxidative stress and restore intestinal barrier function. However, the mechanisms underlying how \u003cem\u003eL. reuteri\u003c/em\u003ealleviates intestinal oxidative stress remain unclear. This study investigated the protective effects and mechanisms of \u003cem\u003eL. reuteri\u003c/em\u003e against diquat-induced intestinal oxidative injury in aged hens.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e 270 Jinbai laying hens (70 weeks old) were divided into control (CON), diquat (DQ), and \u003cem\u003eL. reuteri \u003c/em\u003e+ diquat (LRD) groups with 6 replicates each. Birds in CON and DQ groups are fed with basal diet, while birds in LRD group received basal diet with \u003cem\u003eL. reuteri\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003esupplementation. After 10 weeks of pre-treatment, hens in DQ and LRD groups were injected intraperitoneally with diquat (10 mg/kg body weight), while hens in CON were injected intraperitoneally with an equivalent amount of 0.90% saline.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Diquat challenge impaired barrier integrity evidenced by the disrupted villus architecture, reduced villus height/crypt depth ratio, elevated serum permeability markers (LPS, DAO, and D-LA), and downregulated the mRNA expression of barrier genes (\u003cem\u003eMUC2\u003c/em\u003e, \u003cem\u003eZO-1\u003c/em\u003e, \u003cem\u003eOccludin\u003c/em\u003e, \u003cem\u003eClaudin-1\u003c/em\u003e). \u003cem\u003eL. reuteri\u003c/em\u003e pretreatment significantly restored barrier function through a microbiota-metabolite-mitochondria axis. It reshaped gut microbiota by normalizing Firmicutes/Bacteroidota ratio and increasing the abundance of \u003cem\u003eLactobacillus \u003c/em\u003egenus. Metabolomics revealed enrichment of tryptophan metabolism with upregulated indole-3-propionic acid and indole-3-aceticacid. These changes mitigated mitochondrial dysfunction, which further inhibited apoptosis (downregulated \u003cem\u003eTUNEL\u003c/em\u003e, \u003cem\u003eBAX\u003c/em\u003e and \u003cem\u003eCaspase-3\u003c/em\u003e, upregulated \u003cem\u003eBCL2\u003c/em\u003e). Furthermore, \u003cem\u003eL. reuteri\u003c/em\u003e elevated SOD, GSH-Px and T-AOC activities via Nrf2/HO-1 pathway activation, and reduced pro-inflammatory cytokines IL-1β, TNF-α and IL-6 levels by suppressing TLR4/NF-κB signaling.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eLimosilactobacillus reuteri \u003c/em\u003ecould alleviate intestinal oxidative damage induced by diquat exposure by regulating gut microbiota-tryptophan metabolites-mitochondria axis-mediated oxidative stress and apoptosis, and highlight its potential as a therapeutic \u003ca href=\"https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/probiotic\"\u003eprobiotic\u003c/a\u003e for alleviating oxidative stress and mitochondrial dysfunction to prolong the gut health of aging poultry.\u003c/p\u003e","manuscriptTitle":"Limosilactobacillus reuteri alleviates intestinal oxidative damage by regulating gut microbiota- tryptophan metabolites-mitochondria axis-mediated oxidative stress and apoptosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-01 01:11:38","doi":"10.21203/rs.3.rs-9415762/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"275786494707344196691198122798422101649","date":"2026-05-15T04:24:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"60148136830538806143795307529633809701","date":"2026-05-14T11:15:02+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-13T00:25:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"327071413107144414970574150067336887329","date":"2026-04-24T16:22:30+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-22T02:45:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-22T01:55:54+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-17T12:27:02+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Animal Science and Biotechnology","date":"2026-04-14T12:47:34+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-animal-science-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jasb","sideBox":"Learn more about [Journal of Animal Science and Biotechnology](http://jasbsci.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jasb/default.aspx","title":"Journal of Animal Science and Biotechnology","twitterHandle":"@animalplantsci","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"dd5dc361-e7ce-44cd-aa5d-861186034bd1","owner":[],"postedDate":"May 1st, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"275786494707344196691198122798422101649","date":"2026-05-15T04:24:15+00:00","index":31,"fulltext":""},{"type":"reviewerAgreed","content":"60148136830538806143795307529633809701","date":"2026-05-14T11:15:02+00:00","index":29,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-13T00:25:00+00:00","index":23,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-01T01:11:38+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-01 01:11:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9415762","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9415762","identity":"rs-9415762","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}