Betulinic acid ameliorates lipopolysaccharide-induced intestinal barrier dysfunction in broilers by remodeling gut microbiota homeostasis and inhibiting the LYG2/NOD1/NF-κB signaling axis

Abstract

Betulinic acid (BA), a natural pentacyclic triterpenoid obtained from agroforestry by-products, possesses potent antioxidant, anti-inflammatory and intestinal-protective bioactivities. Our prior work verified that BA alleviated intestinal inflammatory damage in mouse and piglet models. Nevertheless, its protective effects and underlying molecular mechanisms against lipopolysaccharide (LPS)-evoked intestinal barrier dysfunction in broilers remain poorly understood. Under basal feeding conditions, dietary BA supplementation improved ileal mucosal integrity by elevating villus height and crypt depth, upregulating tight junction genes (Occludin, ZO-1) expression, boosting antioxidant capacity, and inhibiting pro-inflammatory gene transcription. In the LPS-challenged inflammatory model, BA pretreatment rescued LPS-reduced feed intake, restored goblet cell density and epithelial microvillus ultrastructure, decreased serum DAO and LPS leakage, and relieved intestinal inflammatory overactivation. Transcriptomics screened LYG2 as a pivotal hub gene modulated by BA. Follow-up RT-qPCR and immunofluorescence verified BA blunted LPS-triggered overactivation of the LYG2/NOD1/NF-κB cascade at transcriptional and protein levels. 16S rRNA sequencing illustrated BA reversed LPS-induced gut dysbiosis via enriching beneficial Ruminococcus_torques and Enterococcus. Correlation assays indicated these two taxa positively correlated with intestinal barrier markers and negatively correlated with inflammatory genes in the LYG2/NOD1/NF-κB axis. This study unveils a dual protective mechanism of BA against LPS-mediated intestinal barrier injury in broilers: BA remodels dysregulated gut microbiota and inhibits the LYG2/NOD1/NF-κB inflammatory cascade to sustain intestinal mucosal homeostasis. BA represents a safe, green phytogenic feed additive to alleviate intestinal inflammation and cut antibiotic usage in poultry farming. Our findings provide novel insights into dietary BA as a potential nutritional strategy to mitigate ileal injury in poultry, with implications for improving gut health and productivity in broilers.

Introduction

Intensive poultry farming has rapidly expanded to meet the rising global demand for animal-derived products. However, high-density rearing increases broiler susceptibility to pathogen invasion and immune stress, which disturbs intestinal homeostasis and impairs growth performance (Chen et al., 2014). The intestinal barrier maintains structural integrity and functional stability via intact tight junctions, balanced gut microbiota, and coordinated mucosal immune responses (Chen et al., 2014). Inflammatory insults disrupt tight junction architecture and microbial equilibrium, triggering intestinal barrier dysfunction and hyperpermeability (Cheng et al., 2019), which facilitate the translocation of luminal pathogens and endotoxin. This pathological process exacerbates systemic inflammatory cascades and causes considerable economic losses in commercial poultry production (He et al., 2025). Lipopolysaccharide (LPS), a major endotoxin derived from Gram-negative bacteria, is a well-validated inflammatory inducer that closely mimics bacterial infection-associated intestinal lesions by inducing aberrant innate immune activation, excessive pro-inflammatory cytokine secretion and intestinal homeostatic disruption (He et al., 2025; Zhao et al., 2024). Growing evidence indicates that crosstalk between gut microbiota and mucosal innate immunity is essential for maintaining intestinal barrier stability under inflammatory stress (Zheng et al., 2020). Lysozyme G2 (LYG2) is a core antimicrobial peptide that regulates microbial homeostasis through bacterial recognition and lysis; peptidoglycan released from lysed bacteria further triggers the downstream nucleotide-binding oligomerization domain containing 1 (NOD1) / nuclear factor kappa-B (NF-κB) inflammatory signaling cascade to modulate intestinal barrier function (Nile et al., 2004; Ragland and Criss, 2017). Current anti-stress strategies relying on zinc oxide or plant essential oils have obvious drawbacks, including heavy metal accumulation, microbial dysbiosis, ingredient volatility and mucosal irritation, limiting their large-scale and safe application. In this context, exploring novel, natural, low-residue and multi-targeted functional bioactive components has become a pivotal research direction for green antibiotic replacement and sustainable poultry farming.

Natural phytogenic bioactive compounds are increasingly recognized for intestinal health modulation and antibiotic substitution due to their high biosafety (Yu et al., 2024), low toxicity, and potent anti-inflammatory and microecological regulatory capacities, which align well with the core scope of functional food and feed research (Gadde et al., 2017). Typical phytochemicals, including curcumin (Ruan et al., 2022), quercetin and ursolic acid (Wei et al., 2025; Zhao et al., 2025), have been shown to alleviate intestinal inflammation and restore barrier function by remodeling gut microbiota and regulating host immune responses, providing fundamental support for the development of high-performance functional feed additives (Zhao et al., 2025). Betulinic acid (BA), a naturally occurring pentacyclic triterpenoid isolated from renewable agroforestry by-products (white birch bark and Lycopus lucidus) (Cîntă Pînzaru et al., 2002; Yun et al., 2003), possesses unique advantages over conventional antibiotic alternatives, including low toxicity and multi-target regulatory potential, making it a promising green additive for sustainable poultry production (Cîntă Pînzaru et al., 2002; Yi et al., 2010). Accumulating studies have confirmed the versatile biological activities of BA (Ou et al., 2021), encompassing anti-inflammatory, antioxidant, antibacterial and immunomodulatory functions (Luo et al., 2020; Ou et al., 2021). BA protected against intestinal damage induced by mycotoxins and chemotherapeutic stress by sustaining redox homeostasis and suppressing overactive inflammatory responses (Luo et al., 2020; Ou et al., 2021). Our previous work also verified that BA supplementation effectively reversed LPS-induced gut microbiota dysbiosis in piglets, highlighting its powerful microecological regulatory ability (Zhao et al., 2024; He et al., 2025). Nevertheless, current studies on BA-mediated intestinal protection are largely restricted to rodent models. Whether BA alleviates LPS-triggered intestinal barrier dysfunction in broilers via gut microbiota remodeling and subsequent regulation of the LYG2/NOD1/NF-κB innate immune axis remains undefined, which greatly hinders the translational application of BA as an innovative natural functional ingredient in intensive poultry production.

Accordingly, the present study systematically explored the protective effects of dietary BA supplementation against LPS-induced intestinal barrier dysfunction in broilers, with a particular focus on clarifying the underlying mechanism from the perspective of gut microbiota–innate immune crosstalk. The results demonstrate that BA attenuates LPS-stimulated intestinal inflammatory injury and barrier impairment by restoring gut microbiota homeostasis and modulating the LYG2/NOD1/NF-κB signaling cascade. This study provides solid experimental evidence supporting natural BA as an efficient, safe and sustainable functional feed additive for improving poultry intestinal health, and offers innovative insights into the development and utilization of green antibiotic alternative bioresources

Materials and methods

Animals and treatments

Arbor Acres (AA) broilers were acquired from Changsha Xindacheng poultry Ltd (Changsha, China). All animal experimental procedures were approved by the Animal Care and Use Committee of Hunan Agricultural University (approval code: 2021032) and complied with national guidelines for laboratory animal welfare in China. Two consecutive broiler trials were conducted in this study. Trial 1 served as a dose-screening experiment to determine the optimal dietary supplementation dose of natural triterpenoid BA, while Trial 2 further explored the protective effects and underlying mechanisms of BA against LPS-induced intestinal barrier dysfunction in broilers.

Trial 1: Dose screening of dietary betulinic acid supplementation in broilers. A total of 100 healthy one-day-old male AA broilers with consistent initial body weight (BW) were randomly assigned to five treatment groups (n = 5 replicates per group; 4 chicks per replicate): basal diet control group (CON), low-dose BA group (4 mg kg⁻¹ diet, LBA), medium-dose BA group (24 mg kg⁻¹ diet, MBA), high-dose BA group (144 mg kg⁻¹ diet, HBA), and antibiotic positive control group (75 mg kg⁻¹ chlortetracycline, CTC). The whole feeding period lasted 42 days. The basal diet was formulated according to the broiler nutritional standards recommended by the National Research Council (NRC), with detailed dietary composition presented in Table S1. Broilers were housed in environmentally controlled rooms with ad libitum access to feed and water throughout the experiment. The room relative humidity was maintained at 60-70%. The ambient temperature was set at 30–32°C in the first week and reduced by 3°C per week thereafter. The lighting schedule was artificially controlled as 23 h light per day in the first week, and adjusted to a 19 h light/5 h dark cycle from day 8 to day 42. After a 12 h overnight fast on days 21 and 42, 10 broilers per group (2 birds per replicate cage) were randomly selected and humanely euthanized for the collection of blood and ileal tissues (Zhao et al., 2024).

Trial 2: Protective effect and mechanism of BA against LPS-induced intestinal inflammatory injury in broilers. A total of 192 one-day-old AA broilers were randomly distributed into four experimental groups (n = 6 replicates per group; 8 chicks per replicate), including the control group (CON), single BA supplementation group (optimal medium dose screened from Trial 1), LPS challenge group, and BA pretreatment combined with LPS challenge group (BA+LPS). To establish an intestinal inflammatory injury model, broilers in the LPS and BA+LPS groups received intraperitoneal injection of LPSat a dose of 1 mg kg⁻¹ BW on days 17, 19 and 21. At 3 h post the final LPS injection on day 21, two broilers per replicate were anesthetized and euthanized to harvest blood, full-length ileum segments and cecal digesta for subsequent molecular detection and multi-omics analysis. All feeding and environmental management conditions were consistent with those described in Trial 1 (Zhao et al., 2024).

Determination of growth performance indices

Mortality and daily feed intake were recorded throughout the feeding period. Individual BW was recorded on days 1, 21 and 42 to calculate average daily gain (ADG), average daily feed intake (ADFI) and feed conversion ratio (FCR).

$$\text{ADG}=(\text{Final}\text{body}\text{weight}-\text{Initial}\text{body}\text{weight})/\text{Number}\text{of}\text{experimental}\text{days}$$

$$\text{ADFI}=\text{Total}\text{feed}\text{intake}/\left(\text{Number}\text{of}\text{days}\times \text{Number}\text{of}\text{broilers}\right)$$

$$\text{FCR}=\text{ADFI}/\text{ADG}$$

Histomorphological analysis of ileal tissue

Fresh ileal tissue segments were fixed in 4% neutral buffered formalin for 24 h, subjected to gradient ethanol dehydration and paraffin embedding. Continuous 4 μm tissue slices were prepared for two separate staining procedures: haematoxylin and eosin (H&E) staining for the evaluation of intestinal villus morphology and crypt structure, and alcian blue-periodic acid-schiff (AB-PAS) staining for the quantitative analysis of mucus-secreting goblet cells.

Transmission electron microscopy (TEM) observation of intestinal tight junctions

The ileum specimens were fixed in 2.5% glutaraldehyde solution, followed by standard dehydration, resin infiltration and embedding steps. Ultrathin sections with a thickness of 60-70 nm were prepared, stained with uranyl acetate and lead citrate, and subsequently imaged under a TEM to visualize the ultrastructure of epithelial tight junctions.

Real-time quantitative PCR (RT-qPCR) analysis of target gene expression

Total RNA was extracted from ileal mucosal tissues, and complementary DNA (cDNA) was synthesized via reverse transcription following the kit manufacturer’s standard protocols. The relative mRNA expression levels of barrier-related, innate immune and inflammatory genes (including LYG2, NOD1 and NF-κB cascade-related genes) were quantified using the 2^⁻ΔΔCt method, with GAPDH selected as the internal reference gene. All target primer sequences are listed in Table S2.

Transcriptome RNA sequencing and functional enrichment of differentially expressed genes (DEGs)

Fresh ileal mucosal samples were snap-frozen immediately after dissection and stored in liquid nitrogen prior to RNA extraction. Total RNA isolation, quality control, library construction and Illumina NovaSeq 6000 paired-end sequencing were conducted per our previously published protocol (He et al., 2025), and detailed bioinformatic pipelines are supplied in supplementary materials.

Protein-protein interaction (PPI) network analysis

A PPI network was constructed based on DEGs derived from ileal transcriptome profiling. Interaction data were retrieved from the STRING database (version 11.5; https://string-db.org/), and network visualization was implemented via cytoscape software (Li et al., 2025).

16S rRNA gene sequencing of cecal gut microbiota

Approximately 100 mg of fresh aseptically collected cecal digesta was immediately frozen at −80°C for microbial DNA extraction. The full experimental workflow for 16S rRNA high-throughput sequencing and subsequent bioinformatic analysis was performed according to established methods (He et al., 2025), with complete operational details provided in supplementary materials. Correlation analysis between differential microbial taxa and the expression of intestinal barrier/innate immune genes was conducted to clarify the microbiota-mediated regulatory pathway of BA.

Immunofluorescence staining for intestinal NOD1 and phosphorylated p65 protein

Paraffin-embedded ileal tissue slices were deparaffinized and rehydrated via a xylene–ethanol gradient series. Immunofluorescence staining was carried out to detect the protein abundance and localization of NOD1 and phosphorylated NF-κB p65 (p-P65). After three 5 min PBS washes, tissue sections were incubated with diluted goat anti-rabbit IgG secondary antibody (1:1000 dilution) at room temperature for 2 h. Nuclei were counterstained with DAPI in the dark for 15 min, and fluorescence signals were captured under a laser scanning fluorescence microscope for semi-quantitative analysis.

Statistical analyses

All data were subjected to the Shapiro-wilk test on SPSS software (IBM, New York, USA). Data conforming to normal distribution were tested for homogeneity of variance and one-way ANOVA analysis. Duncan’s multiple range test was used for post hoc comparison, and Tamhane's T2 test was used for post-hoc test for heterogeneity of variance. Analyzed data were recorded as means ± standard error of the mean (SEM) and probability values. P < 0.05 represented a significant difference, P < 0.01 represented a highly significant difference. Clustering correlation heatmap was performed using the ChiPlot (https://www.chiplot.online/).

Results

BA supplementation improves growth performance and serum biochemical parameters in broilers

As shown in Fig. S1, dietary BA supplementation effectively elevated the BW of broilers across the experimental period. Specifically, the MBA group exhibited a 207 g higher BW than the control group on day 42 (Fig. S1D). During the growth phases of days 1-42 and days 21-42, the average daily gain (ADG) increased by 4.94 g and 7.49 g, respectively (Fig. S1E, G), while the feed conversion ratio (FCR) decreased from 1.75 to 1.60 and from 1.90 to 1.69, respectively (Fig. S1F, H). These findings demonstrated that dietary BA supplementation could improve the growth performance of broilers. To further evaluate the physiological effects of BA on broilers, serum biochemical indices were subsequently determined. Compared with the CON group, BA supplementation markedly decreased serum total protein (TP) and low-density lipoprotein cholesterol (LDL-C) levels, while increasing high-density lipoprotein cholesterol (HDL-C) content (Fig. S1K–S1M). Given the essential role of immune organs in mediating host immune function and disease resistance, we further assessed immune organ indices of broilers. Notably, no significant differences in immune organ indices were observed among the CON, BA-treated, and CTC groups (Fig. S2). Collectively, these results suggest that appropriate dietary BA supplementation can moderately improve the growth performance of broilers, with the MBA dosage exhibiting the optimal efficacy. In addition, BA supplementation exerted no adverse toxic effects or immune-related side effects on broilers, indicating the safety of its application in broiler feeding.

BA supplementation enhances intestinal barrier integrity, antioxidant capacity and anti-inflammatory responses in broilers

To characterize the protective effects of BA on intestinal barrier integrity, ileal histological morphology was evaluated. On day 21, BA supplementation significantly increased ileal villus height and crypt depth relative to the CON group (Fig. 1A–D). On day 42, the villus height/crypt depth ratio (VCR) was notably elevated in the MBA group (Fig. 1E–H). Consistent with the morphological improvements, the mRNA expression of intestinal tight junction proteins (Occludin and Zonula Occludens-1(ZO-1)) was substantially upregulated after BA intervention (Fig. 1I–J). In terms of intestinal antioxidant status, dietary BA supplementation increased the levels of reduced glutathione (GSH) and the activities of superoxide dismutase (SOD) and catalase (CAT), while decreasing malondialdehyde (MDA) accumulation in the ileum (Fig. 2A–D). Furthermore, BA supplementation suppressed the transcriptional levels of key inflammatory and pyroptotic genes, including cysteinyl aspartate specific proteinase-1(Caspase-1), Interleukin-18(IL-18) and IL-6 (Fig. 2E, 2F). These findings demonstrate that dietary BA supplementation efficiently enhances intestinal antioxidant capacity, restrains excessive inflammatory responses, and strengthens intestinal barrier integrity in broilers, with BA exerting more prominent protective effects at the early growth stage (day 21).

Fig. 1.

Effects of BA supplementation on intestinal barrier function of broilers. (A and E) Histopathological observation and of ileum on day 21 and 42 (Scale bar: 400μm and 100 μm). (B-D and F-H) The villus height, crypt depth, the VCR on day 21 and 42. (I-J) RT-qPCR analysis of ZO-1 and Occludin mRNA expression in the ileum on days 21 and 42 (n=3). The values are shown as mean ± SEM. *P < 0.05, **P < 0.01, compared with the CON group.

Fig. 2.

Effects of BA supplementation on intestinal redox system and inflammatory cytokine genes expressions. (A-D) The levels of GSH, SOD, CAT and MDA in the ileum on day 21and 42 (n=6). (E-F) RT-qPCR analysis of the mRNA levels of NLRP3, Caspase-1, IL-1β, IL-18, IL-6 and TNF-α in the ileum of broilers on day 21 and 42 (n=3) The values are shown as mean ± SEM. *P < 0.05, **P < 0.01, compared with the CON group.

BA supplementation alleviates LPS-induced intestinal barrier dysfunction in broilers

An LPS-challenged intestinal injury model was established to further validate the protective effect of BA against intestinal damage in broilers. First, the adverse impacts of LPS stress on broiler growth and intestinal barrier function were evaluated. During the LPS induction period (days 17-21), compared with the CON group, LPS challenge reduced broiler BW by 5 g (Fig. 3B), and significantly decreased ADF and ADG (p<0.05) (Fig. 3C, D). Consistent with the suppressed growth performance, LPS stimulation triggered severe intestinal barrier damage, characterized by impaired intestinal morphology, disrupted intestinal ultrastructure, and elevated intestinal permeability. Specifically, LPS exposure markedly increased serum diamine oxidase (DAO) and LPS concentrations (Fig. 4G, H), inhibited the protein expression of intestinal tight junction Occludin (Fig. 4I), and upregulated the transcription levels of pro-inflammatory genes (tumor necrosis factor-alpha (TNF-α) and inducible nitric oxide synthase(iNOS)) (Fig. 4J–L), collectively confirming that LPS successfully induced intestinal barrier dysfunction and inflammatory injury in broilers.

Fig. 3.

Effects of BA supplementation on LPS-induced impairment of growth performance in broilers. (A) Experimental grouping and treatment. (B) The body weight of broilers on days 1-21. (C) The average daily feed intake on days 1-17 and 17-21. (D) The ADG on days 1-17 and 17-21. (E) The FCR on days 1-17 and 17-21 (n=6). The values are shown as mean ± SEM. *P < 0.05, **P < 0.01, compared with the CON group. ^#P < 0.05, ^##P < 0.01 compared with the LPS group.

Fig. 4.

Effects of BA supplementation on intestinal barrier dysfunction caused by LPS in broilers. (A) Histopathological observation of ileum (scale bar: 500 μm and 200 μm). (B-D) The villus height, crypt depth and the VCR in ileum (n=3). (E) The number of goblet cells in the of broilers (scale bar: 200 μm and 50 μm). (F) Ul-trastructural changes of ileum (scale bar: 10 μm and 2 μm, red arrow: broken intestinal microvilli). (G-H) The serum LPS and DAO contents (n=6). (M) RT-qPCR analysis of ZO-1, Occludin,TNF-α, IL-6 and iNOS mRNA expression levels in the ileum (n=3). The values are shown as mean ± SEM. *P < 0.05, **P < 0.01, compared with the CON group. ^#P < 0.05, ^##P < 0.01 vs. the LPS group.

Subsequently, the protective effects of dietary BA supplementation on LPS-induced intestinal damage were investigated. Compare to the LPS-challenged group, BA supplementation effectively rescued LPS-induced growth inhibition, increasing the ADG by 0.31 g and significantly improving ADF of broilers during days 17–21 (p < 0.05) (Fig. 3B–E). Intestinal morphological staining results further demonstrated that BA pretreatment alleviated LPS-caused intestinal structural damage in the ileum, as reflected by reduced crypt depth and elevated villus/crypt ratio (VCR) (Fig. 4A–D). AB-PAS staining revealed that BA supplementation restored the number of ileal goblet cells which were depleted by LPS challenge (Fig. 4E). TEM ultrastructural observation showed that LPS-induced pathological lesions, including broken microvilli, disordered microvillus arrangement and widened intercellular gaps, were substantially ameliorated by BA pretreatment (Fig. 4F). In terms of intestinal barrier function and inflammatory response, BA pretreatment significantly upregulated the expression of tight junction protein Occludin, reduced serum DAO and LPS leakage levels, and suppressed the transcription of pro-inflammatory TNF-α and iNOS (Fig. 4G–L). Taken together, these results demonstrate that BA supplementation can effectively mitigate LPS-triggered growth retardation, intestinal structural damage, permeability elevation and inflammatory responses, thereby alleviating intestinal barrier dysfunction in broilers.

Transcriptomic profiling reveals key DEGs and immune-related pathways modulated by BA under LPS stress

RNA-seq transcriptomic analysis was performed to uncover the underlying mechanism by which BA protects against LPS-induced intestinal damage. Principal component analysis (PCA) showed distinct transcriptional profiles among the CON, BA, LPS and BA+LPS groups, indicating reliable grouping and significant transcriptomic alterations induced by LPS and BA intervention (Fig. 5A). Differential gene screening identified 137 upregulated and 124 downregulated genes in the LPS group relative to the CON group. Compared with the LPS group, BA pretreatment reversed the expression of 163 upregulated and 128 downregulated genes (Fig. 5B). Protein–protein interaction (PPI) network analysis further screened core hub genes, including C-C motif chemokine ligand 4 (CCL4), fibrinogen gamma chain (FGC), lysozyme g2 (LYG2), which were closely involved in intestinal immune regulation (Fig. 5C). KEGG functional enrichment analysis indicated that these DEGs were primarily enriched in peptidoglycan metabolism, mucosal immune response and lysosomal signaling pathways (Fig. 5D, E). Accumulated evidence has demonstrated that LYG2 acts as a critical innate immune effector that mediates bacterial lysis and peptidoglycan release, thereby activating the downstream NOD1/NF-κB inflammatory cascade. Consistent with these reports, our transcriptomic data strongly suggest that BA may alleviate LPS-induced intestinal injury by targeting the LYG2-mediated innate immune axis.

Fig. 5.

Effect of BA supplementation on the ileum transcriptome induced by LPS in broilers. (A) The principle component analysis plotting PC1 versus PC2. (B) The bar plot of differential expression. (C) PPI analysis network graph of all DEGs. (D-E) The go enrichment analysis of DEGs in ileum.

BA inhibits the LPS-activated LYG2/NOD1/NF-κB innate immune signaling pathway in ileum of broilers

To validate the transcriptomic findings, key DEGs and core signaling molecules were verified by RT-qPCR. The results showed that LPS challenge significantly upregulated the mRNA levels of FGG, LYG2, TC2N and CCL4, whereas BA pretreatment markedly reversed these upregulated expression patterns (Fig. 6A, 6B). Pearson’s correlation analysis confirmed high consistency between RNA-seq and RT-qPCR data, with LYG2 exhibiting the strongest correlation coefficient, verifying the reliability of the transcriptomic results (Fig. 6C). In addition, LPS-induced upregulation of NOD1 and receptor-interacting serine/threonine-protein kinase 2 (RIPK2) was significantly suppressed by BA supplementation (Fig. 6D). Immunofluorescence staining further demonstrated that LPS exposure increased the protein abundance of NOD1 and phosphorylated p65 (p-P65), while BA intervention effectively inhibited the activation and nuclear translocation of NOD1 and p65 (Fig. 6E–G). Taken together, these results confirm that BA ameliorates LPS-induced intestinal inflammatory injury by suppressing the LYG2/NOD1/NF-κB innate immune signaling cascade.

Fig. 6.

BA modulates the LPS-induced LYG2/NOD1/NF-κB pathway in broilers (A-B) RT-qPCR validation of mRNA expressions of FGG, LYG2, TC2N and CCL4 mRNA expressions of ileum. (C) Pearson’s correlation analysis between RNA-Seq and RT-qPCR. (D) The RT-qPCR analysis of theNOD1, NF-κB, RIPK2 mRNA expressions of ileum (n=3). (E-G) IF results for NOD1 and p-P65 in the ileum (n = 3, scale bar, 200 µm). The values are shown as mean ± SEM. *P < 0.05, **P < 0.01, compared with the CON group. ^#P < 0.05, ^##P < 0.01 vs. the LPS group.

BA remodels gut microbiota dysbiosis induced by LPS challenge in broilers

16S rRNA gene sequencing was performed to explore the modulatory effect of BA on gut microbial composition under inflammatory stress. Rank-abundance curves indicated sufficient sequencing depth and adequate species coverage across all samples (Fig. 7A). Principal coordinate analysis (PCoA) revealed distinct microbial community structures among different groups, indicating that LPS and BA intervention significantly reshaped gut microbial ecology (Fig. 7B). Microbial composition analysis at the phylum and genus levels further confirmed the differential microbial distribution caused by LPS and BA treatment (Fig. 7C, D). Kruskal–Wallis difference analysis showed that BA supplementation significantly enriched the relative abundances of Ruminococcus_torques, Colidextribacter and Enterococcus at the genus level compared with the LPS group (Fig. 7E). Correlation analysis revealed that Ruminococcus_torques and Enterococcus were positively correlated with the expression of intestinal tight junction genes (ZO-1 and Occludin) and negatively correlated with serum DAO and LPS levels (Fig. 7F). Furthermore, these beneficial microbial genera were negatively correlated with the expression of inflammatory signature genes (FGG, LYG2, TC2N, CCL4, NOD1, NF-κB and RIPK2) (Fig. 7G). In summary, BA effectively remodels LPS-induced gut microbiota dysbiosis, enriches beneficial intestinal bacteria, and further suppresses intestinal inflammatory responses, thereby maintaining intestinal barrier homeostasis.

Fig. 7.

Effect of BA supplementation on gut microbiota disorder induced by LPS in broilers. (A) Rank-Abundance curves. (B) PCoA plots at the species level. (C-D) Bar plot of community composition at the phylum level and genus level. (E) Kruskal-Wallis H test bar plot at the genus level. (F) Pearson’s correlation analysis between gut microbiota and intestinal barrier function. (G) Pearson’s correlation analysis between RNA-Seq and RT-qPCR related parameters, gut microbiota and mRNA levels of FGG, LYG2, TC2N, CCL4, NOD1, NF-κB and RIPK2 related parameters.

Discussion

Intensive high-density poultry rearing inevitably increases broiler susceptibility to immune stress and pathogenic infection, which readily triggers intestinal oxidative injury, inflammatory overactivation and gut microbiota dysbiosis, ultimately destroying intestinal barrier homeostasis and restricting healthy growth of broilers (Zhao et al., 2024). Plant-derived functional bioactive compounds have emerged as promising antibiotic alternatives in poultry production, owing to their excellent antioxidant, anti-inflammatory and microecological regulatory properties, which can effectively improve intestinal histomorphology, immune function and nutrient absorption efficiency (Abd El-Hack et al., 2022). BA, a natural pentacyclic triterpenoid extracted from agroforestry by-products, has been verified to alleviate multiple types of intestinal damage by maintaining redox homeostasis, inhibiting inflammatory responses and modulating gut microbial composition (He et al., 2025; Ou et al., 2021). Nevertheless, the protective effects and molecular mechanisms of BA against LPS-induced intestinal barrier dysfunction in broilers remain poorly defined. Accordingly, the present study systematically explored the protective efficacy of dietary BA supplementation on intestinal morphology, inflammatory injury and gut microbiota homeostasis in LPS-challenged broilers, aiming to provide experimental evidence for the application of BA as a novel and safe functional feed additive for poultry intestinal health protection (Fig. 8).

Fig. 8.

Dietary BA supplementation significantly ameliorated LPS-induced intestinal barrier dysfunction, inflammatory injury, and gut microbiota dysbiosis, consequently maintaining intestinal barrier homeostasis.

In the present study, preliminary phenotypic detection of growth performance, serum biochemical indices and immune organ coefficients confirmed that routine dietary BA supplementation exerted no toxic or adverse effects on broilers, demonstrating the high biosafety of BA in poultry application. Immune organ indices (thymus, spleen and bursa of Fabricius) are classic intuitive indicators for evaluating the non-specific immune status of poultry (Zhu et al., 2023; Li et al., 2023). Previous pharmacological studies have reported that BA exerts dose- and species-dependent immunomodulatory effects in mice, with distinct regulatory trends on different immune organs (Kong et al., 2021; Yi et al., 2010). Consistent with these findings, our results showed no significant changes in immune organ indices in BA-treated broilers, further indicating that BA exhibits mild and safe immunomodulatory activity rather than immune hyperactivation or immunosuppression in broilers, which is conducive to maintaining steady-state immune function. Intestinal morphological structure is the core basis for nutrient digestion and absorption and intestinal barrier function (Oladokun and Adewole, 2023). Longer intestinal villi and appropriate crypt depth expand the intestinal absorption area, while a VCRreflects vigorous intestinal epithelial cell renewal and intact mucosal structure, representing superior intestinal health status (Huang et al., 2022). Our results demonstrated that medium-dose BA supplementation significantly increased ileal villus height, crypt depth and VCR in broilers at the early growth stage, accompanied by significant upregulation of the tight junction proteins ZO-1 and Occludin. Moreover, BA supplementation effectively enhanced intestinal antioxidant levels (SOD, CAT, GSH), reduced MDA accumulation, and suppressed the transcription of inflammatory genes (Caspase-1, IL-18, IL-6). These findings are consistent with previous studies reporting that BA alleviates mycotoxin- and chemotherapy-induced intestinal injury by improving tight junction integrity, and restraining oxidative and inflammatory responses (He et al., 2025; Ou et al., 2021). Collectively, these results validate that optimal dietary BA supplementation can effectively enhance intestinal antioxidant capacity, inhibit excessive inflammatory responses, and strengthen intestinal barrier structural integrity under normal feeding conditions.

To further verify the protective effect of BA against pathological intestinal damage, a classic LPS-induced intestinal inflammatory injury model was established in broilers. In consistent with previous studies on plant natural extracts (Ilyina et al., 2014), dietary BA supplementation did not alter the basal growth performance of broilers, but effectively rescued the reduction in average daily feed intake caused by continuous LPS stimulation. Morphological and ultrastructural observations further confirmed that BA pretreatment restored LPS-induced reductions in ileal goblet cell number and microvillus integrity, improved intestinal histopathological damage, and increased intestinal VCR. As key markers of intestinal barrier injury, elevated serum DAO and LPS levels reflect increased intestinal permeability and barrier leakage (Zhang et al., 2025). In this study, BA intervention significantly reduced serum DAO and LPS contents, upregulated tight junction gene expression, and downregulated the transcription of pro-inflammatory genes (TNF-α, iNOS) in LPS-challenged broilers. These comprehensive phenotypic results fully demonstrate that BA can effectively ameliorate LPS-triggered intestinal barrier structural damage and functional dysfunction in broilers, verifying its prominent intestinal protective activity.

To clarify the potential mechanism of BA-mediated intestinal protection, transcriptomic profiling was performed on broiler ileal tissues. Transcriptome analysis screened out core differential genes closely associated with intestinal innate immunity, among which LYG2 was identified as a key hub gene responding to BA intervention. LYG2 is a critical antimicrobial peptide that mediates bacterial lysis and peptidoglycan release; released peptidoglycans further activate the NOD1/RIPK2 downstream cascade, thereby triggering NF-κB nuclear translocation and massive pro-inflammatory cytokine secretion, ultimately exacerbating intestinal inflammatory injury (Ragland and Criss, 2017; Bi et al., 2017). Subsequent RT-qPCR and immunofluorescence validation confirmed that LPS challenge significantly upregulated the expression of LYG2 and its downstream inflammatory pathway genes (NOD1, RIPK2, NF-κB), while BA pretreatment significantly suppressed the activation of this signaling axis. Combined with previous reports that inhibition of the NOD1/NF-κB pathway effectively relieves intestinal inflammatory damage (Li et al., 2017; Gadde et al., 2017), our findings solidly confirm that BA alleviates LPS-induced intestinal inflammatory injury by negatively regulating the LYG2/NOD1/NF-κB innate immune signaling cascade.

Gut microbiota homeostasis is a key determinant of intestinal barrier function and immune balance, and microbiota dysbiosis is a core driver of inflammatory intestinal diseases (Chopyk and Grakoui, 2020). In the present study, 16S rRNA sequencing revealed that LPS-induced gut microbiota dysbiosis was significantly remodeled by BA supplementation. Specifically, BA notably enriched the relative abundances of Ruminococcus_torques and Enterococcus. Existing studies have demonstrated that Ruminococcus_torques is closely correlated with intestinal metabolic homeostasis and barrier repair, and its enrichment is conducive to alleviating intestinal metabolic disorders and inflammatory damage (Wu et al., 2021). Although partial Enterococcus strains are considered conditional pathogens, multiple probiotic-derived Enterococcus strains possess definite immunomodulatory and intestinal protective functions (Zhou et al., 2020), which can inhibit inflammatory factor secretion and repair intestinal barrier damage (Yang et al., 2023; Zhou et al., 2020). Further correlation analysis in this study verified that Ruminococcus_torques and Enterococcus were positively correlated with intestinal tight junction gene expression and negatively correlated with serum barrier leakage indices (DAO, LPS). More importantly, these two beneficial genera were significantly negatively correlated with the expression of core genes in the LYG2/NOD1/NF-κB inflammatory axis. Collectively, these results indicate that BA can remodel LPS-induced gut microbiota dysbiosis by enriching beneficial intestinal bacteria, and further inhibit the overactivation of the LYG2/NOD1/NF-κB inflammatory pathway, thereby maintaining intestinal barrier homeostasis in broilers.

Conclusions

The present study systematically demonstrates that dietary BA supplementation improves intestinal morphological structure, enhances antioxidant capacity, maintains immune homeostasis, and reduces basal intestinal inflammatory levels in broilers. Furthermore, BA pretreatment effectively alleviates LPS-induced intestinal barrier dysfunction, inflammatory injury and gut microbiota dysbiosis. Mechanistically, BA exerts intestinal protective effects by enriching beneficial intestinal bacterial genera (Ruminococcus_torques and Enterococcus) and suppressing the activation of the LYG2/NOD1/NF-κB innate immune signaling axis. This study clarifies the multi-target protective mechanism of BA against intestinal inflammatory injury in broilers, providing solid experimental evidence and a novel theoretical basis for the application of BA as a safe and green functional feed additive to improve poultry intestinal health, replace antibiotics in sustainable poultry production.

Ethics declaration

All animal experimental procedures were approved by the Animal Care and Use Committee of Hunan Agricultural University (approval code: 2021032) and complied with national guidelines for laboratory animal welfare in China.

Author contribution

Zhaoping Ou contributed to the conception and design of the study, formal analysis, data curation, visualization, and preparation of the initial manuscript draft. Wenjiang Yang contributed to animal management, experimental measurements, sample collection, formal analysis, data curation, visualization, and the preparation of the original manuscript draft. Wengyu Ba contributed to the analysis of the experimental data, the preparation of the original manuscript, and the scientific discussion. Ziming Wang contributed to the analysis of the experimental data, the preparation of the original manuscript, and the scientific discussion. Hongyi Ding contributed to the analysis of the experimental data, the preparation of the original manuscript. Li Kong contributed to data acquisition and curation and critically reviewed the manuscript. Jiajing Zhou contributed to the analysis and interpretation of the experimental findings. Zhuliang Tan contributed to the conception and design of the study, methodology development, and critical revision of the manuscript. Muliang Li contributed to study conception, provision of resources, funding acquisition, supervision, and critical revision of the manuscript. Wei Tang contributed to study conception, provision of resources, funding acquisition, supervision, and critical revision of the manuscript. Quanwei Li contributed to the conception and design of the study, data curation, the major findings, and the reviewed the paper. Jine Yi contributed to the conception of the study and the design of the overall experimental scheme, the scientific guidance throughout the research, the development and refinement of the scientific conclusions, and funding acquisition and reviewed the paper. Shuiping Liu contributed to the conception of the study and the design of the overall experimental scheme, the scientific guidance throughout the research, the development and refinement of the scientific conclusions, and funding acquisition and reviewed the paper. Zhaoping Ou and Wenjiang Yang contributed equally to this work.

Disclosures

The authors declare no competing interests.

Data availability

Data will be made available on request.