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. 2026 Sep 21;105(12):107815. doi: 10.1016/j.psj.2026.107815

Mechanism of tea saponin regulating the Nrf2/HO-1/NQO1 pathway to alleviate Salmonella Enteritidis-induced intestinal pathological injury in chicks

Bo Zhang a, Ke Li b, Jianyun Feng c, Jiangnan Ni a, Junjie Liu a, Ao Dai a, Hongya Yan b, Rong Zhao b, Zhenju Yuan b, Aiguo Xin b,⁎
PMCID: PMC13625794  PMID: 42777368

Abstract

Previous studies have demonstrated that Salmonella Enteritidis triggers intestinal pyroptosis and pathological damage in hosts. Tea saponin (Ts) exhibits favorable anti-inflammatory, antioxidant and intestinal barrier-protective activities, whereas its protective role and molecular mechanism against Salmonella Enteritidis-induced intestinal injury in chicks remain poorly elucidated. This study explored the protective effects of Ts on Salmonella Enteritidis-caused intestinal damage in chicks and whether the Nrf2/HO-1/NQO1 signaling pathway mediates its function. A total of 60 7-day-old specific pathogen-free (SPF) chicks were randomly assigned to six groups: control, Salmonella Enteritidis-infected model, low, medium and high-dose Ts intervention groups, and florfenicol positive control group. Except the control group, all chicks received intraperitoneal Salmonella Enteritidis injection for model establishment, followed by 7 consecutive days of corresponding treatments. Diarrhea score, histopathological changes in the ileum and liver, serum liver function parameters (ALT, AST, ALP, TBIL), oxidative stress markers (SOD, GSH‑PX, CAT, MDA), and inflammatory cytokines (TNF‑α, IL‑6, IL‑1β, IL‑10) were measured, and the expression levels of Nrf2/HO‑1/NQO1 pathway components were evaluated at both mRNA and protein levels. Network pharmacology combined with molecular docking further validated the binding relationship between Ts and pathway key proteins. The untreated infected group presented aggravated diarrhea, severe intestinal and hepatic lesions, disordered liver function, oxidative stress and inflammation, along with suppressed Nrf2/HO‑1/NQO1 pathway expression. Ts intervention reversed these damages dose‑dependently, with high‑dose Ts showing the optimal efficacy. In conclusion, Ts mitigates diarrhea, inflammation and visceral pathological injury in Salmonella Enteritidis‑infected chicks, and its effects are associated with activation of the Nrf2/HO‑1/NQO1 pathway, improved antioxidant defense and inhibited inflammatory responses.

Keywords: Tea saponin, Salmonella Enteritidis, Intestinal pathological injury, Nrf2/HO-1/NQO1 pathway, Molecular mechanism

Introduction

Salmonella spp., as a prototypical Gram-negative foodborne pathogen, is widely distributed in the natural environment. It can invade chicks via contaminated feed, drinking water, and rearing environments, and is well-recognized as one of the core pathogens responsible for enteric diseases in poultry (Adetunji et al., 2022). Given that the immune system and intestinal mucosal barrier of chicks are not yet fully developed, Salmonella can extensively colonize and proliferate in the intestinal epithelium upon invasion (Zhang et al., 2023). It modulates host inflammatory signaling pathways through its virulence factors, triggers excessive inflammatory responses and cell death, and compromises intestinal mucosal integrity, resulting in clinical manifestations in chicks including diarrhea, anorexia, and growth retardation. In severe cases, it can even induce systemic infection and mortality, which inflicts enormous economic losses on the poultry farming industry in China (Ma et al., 2024). Meanwhile, Salmonella can persistently colonize chicks and enter the food chain via animal-derived foods such as poultry meat and eggs, causing foodborne illnesses in humans including acute gastroenteritis and sepsis (Chousalkar and Willson, 2022). This poses a severe threat to the health of infants, the elderly, and immunocompromised individuals, and has emerged as a critical challenge for prevention and control in the field of public health (Gast et al., 2024). Currently, antibiotics remain the mainstay for the clinical prevention and control of Salmonella infection in poultry. However, many countries have restricted or banned the use of antibiotics as growth promoters and for treatment of salmonellosis due to concerns over resistance and food safety (Castello et al., 2023). Moreover, long‑term irrational use and overuse of antibiotics readily drive the development of multidrug resistance in Salmonella, leading to a sharp decline in therapeutic efficacy (Castello et al., 2023). Furthermore, antibiotics disrupt intestinal microbiota homeostasis in livestock and poultry, trigger intestinal microecological dysbiosis, further exacerbate the difficulty of pathogenic bacterial control, and severely restrict the green and sustainable development of the poultry farming industry (Zhan et al., 2025). Against this backdrop, the identification of safe, highly effective natural antibacterial and anti-inflammatory active substances with no risk of inducing drug resistance, coupled with the development of antibiotic alternatives, has become a prominent research hotspot and core direction in the fields of poultry farming and animal epidemic prevention and control (Mudasir Ahmad et al., 2024).

Ts is a natural triterpenoid saponin compound extracted from the seeds, leaves, and other tissues of Camellia plants. Its molecular structure consists of an aglycone backbone covalently linked to glycosyl moieties, which endows it with intrinsic amphiphilic physicochemical properties featuring both hydrophilic and lipophilic characteristics. Ts possesses a wide spectrum of biological activities, including anti-inflammatory, antioxidant, and immunomodulatory effects. Coupled with its wide natural availability, low toxicity, and negligible residual risk in edible animal products, Ts has emerged as a key research target for the development of antibiotic alternatives in the livestock and poultry farming industry (Makiej et al., 2025). In recent years, accumulating studies have further characterized the biological functions of Ts in livestock and poultry. Chi et al. demonstrated that Ts supplementation effectively ameliorated oxidative stress and immunosuppression in chickens without inducing observable adverse effects (Chi et al., 2017). Similarly, Wang et al. reported that Ts alleviated oxidative stress and enhanced immune function in dairy cows (Wang et al., 2017). Our previous studies confirmed that Ts effectively mitigated intestinal injury induced by pathogenic Escherichia coli in mice via targeted regulation of the NF-κB/NLRP3 signaling pathway, as evidenced by its ability to suppress inflammatory responses and repair the impaired intestinal mucosal barrier (Zhang et al., 2026a). These findings verified the protective effect of Ts against pathogen-induced intestinal injury and its regulatory activity on the NLRP3 signaling axis, which are highly consistent with previous reports on the anti-inflammatory mechanisms of tea-derived extracts, further supporting the practical application potential of Ts (Zhang et al., 2026a). Nevertheless, although the antibacterial and anti-inflammatory activities of Ts and its underlying signaling regulatory mechanisms have been preliminarily validated, and Ts has exhibited promising prospects in improving intestinal health and systemic immunity in livestock and poultry, several critical knowledge gaps remain to be addressed. Specifically, the ameliorative effect of Ts on Salmonella-induced intestinal pathological injury in chicks has not yet been fully elucidated, and systematic investigations on the optimal intervention dosage and in vivo action duration of Ts in chicks are still lacking. In addition, no relevant studies have explored the protective effect of Ts against secondary injury in visceral organs (such as the liver) of chicks after Salmonella infection (Tian et al., 2025). These unresolved issues have greatly restricted the development and application of Ts as an anti-infective feed additive for poultry production.

The nuclear factor erythroid 2‑related factor 2 (Nrf2) is a master transcriptional regulator of cellular antioxidant defense. Under physiological conditions, Nrf2 is sequestered in the cytoplasm by Keap1, upon oxidative or electrophilic stimulation, Nrf2 translocates into the nucleus and drives the transcription of antioxidant response element (ARE)‑dependent genes, including heme oxygenase‑1 (HO‑1) and NAD(P)H:quinone oxidoreductase 1 (NQO1) (Chen et al., 2017). The Nrf2/HO‑1/NQO1 cascade plays a pivotal role in counteracting oxidative stress, mitigating inflammation, and maintaining intestinal epithelial integrity in various pathological settings, including bacterial infections (Loboda et al., 2016). However, whether Ts exerts its protective effects against Salmonella‑induced intestinal injury through modulation of this pathway remains to be elucidated.

Building on the aforementioned critical knowledge gaps and our prior research findings, the present study is designed to establish a chick model of Salmonella infection-induced intestinal pathological injury, and to investigate the ameliorative effect of Ts on intestinal and hepatic pathological injury in Salmonella-challenged chicks. In conjunction with a panel of molecular biological techniques, we will elucidate the regulatory effects of Ts on the intestinal mucosal barrier, inflammatory responses, oxidative stress, and cellular pyroptosis, and further validate the association between core therapeutic targets and key signaling pathways via network pharmacology and molecular docking. This study aims to provide solid experimental evidence and theoretical support for the development and practical application of Ts in poultry anti-infective research, and ultimately facilitate the green and sustainable development of the poultry farming industry.

Materials and methods

Experimental animals

A total of 60 7-day-old SPF White Leghorn chicks with a body weight of 180±10 g were purchased from Guizhou Forster Biotechnology Co., Ltd. The birds were housed in stainless steel cages (10 chicks per cage) with autoclaved wood shavings as bedding. Room temperature was maintained at 32±1°C for the first week and then gradually reduced to 28±1°C until the begin of the experiment (day 14). Relative humidity was kept at 55±5%, and a 12 h light/12 h dark cycle was provided. Feed (a corn‑soybean meal‑based starter diet, sterilized by gamma irradiation, containing 20% crude protein and 12.5MJ/kg metabolizable energy) and autoclaved drinking water were provided ad libitum. Sterility of feed and water was verified by aerobic plate counts (no bacterial growth on tryptic soy agar at 37°C for 48 h). The formal experiment was initiated after a 7-day acclimation period, at which point the chicks were 14 days of age. All animal experiments were approved by the Experimental Animal Ethics Committee of Kunming University (Approval No. SYXKK2025-0004) and conducted in accordance with the Guidelines for the Ethical Review of Animal Welfare (EAE-GZU-2022-E021).

Bacterial strains and reagents

The Salmonella Enteritidis strain used in this study was preserved in our laboratory and validated in our previous research (Zhang et al., 2026b). Ts was purchased from Shanghai Yuanye Biotechnology Co., Ltd. Florfenicol, hematoxylin and eosin (HE) staining kit, immunohistochemistry (IHC) kit, and assay kits for ALT, AST, ALP, and TBIL were purchased from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd. Assay kits for SOD, GSH-Px, CAT activities, and MDA content were purchased from Nanjing Jiancheng Bioengineering Institute. ELISA kits for tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), and interleukin-10 (IL-10) were purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd. RT-PCR kit and reagents for Western blot analysis were purchased from Takara Bio (Dalian) Co., Ltd. Primary antibodies against ZO-1, Occludin, Claudin-1, TNF-α, IL-1β, Nrf2, HO-1, NQO1, and corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Wanlei Biotechnology Co., Ltd. RT-PCR primers were synthesized by Shanghai Sangon Biotech Co., Ltd. All other chemical reagents used in this study were of domestic analytical grade.

Experimental design

After the 7-day acclimation period, 60 chicks were randomly divided into 6 groups with 10 chicks per group. The grouping details were as follows:

Control group: Chicks received daily oral gavage of 0.2 mL PBS for 7 consecutive days. Salmonella infection group (Untreated infected): Chicks were inoculated with Salmonella Enteritidis (The infection dose was 0.2 mL per chick, using a bacterial suspension with a concentration of 3.27 × 108CFU/mL) (Zhang et al., 2026b) and received neither Ts nor antibiotic intervention, but were administered daily oral gavage of 0.2 mL PBS for 7 consecutive days. Ts low-dose intervention group (Ts-L): Chicks were inoculated with Salmonella Enteritidis and received daily oral gavage of Ts at a dose of 50 mg/kg body weight for 7 consecutive days. Ts medium-dose intervention group (Ts-M): Chicks were inoculated with Salmonella Enteritidis and received daily oral gavage of TS at a dose of 100 mg/kg body weight for 7 consecutive days. Ts high-dose intervention group (Ts-H): Chicks were inoculated with Salmonella Enteritidis and received daily oral gavage of Ts at a dose of 200 mg/kg body weight for 7 consecutive days. Positive group (Florfenicol): Chicks were inoculated with Salmonella Enteritidis and received daily oral gavage of florfenicol at a dose of 50 mg/kg body weight for 7 consecutive days (Schematic representation of experimental protocol shown in Fig. 1A).

Fig. 1.

Fig. 1

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Ts alleviates the pathological damage caused by Salmonella infection in chicks. Data are presented as mean±SD (n = 10 per group). (## P > 0.05; * P<0.05; ** P<0.01).

(A) Experimental protocol.

(B) Diarrhea scores of chicks in different groups.

(C) HE staining of the ileum.

(D) Pathology scores of the ileum.

(E) MPO activity in the ileum.

(F) HE staining of the liver.

(G) Pathology scores of the liver.

After Salmonella Enteritidis challenge, the general condition, and defecation characteristics of chicks were closely monitored daily. Diarrhea severity was assessed daily using a 4‑point scale: 0=normal formed feces. 1=soft but formed feces. 2=pasty or semi‑liquid feces. 3=severe watery diarrhea. The average diarrhea score for each group was calculated over the 7‑day treatment period. After 7 days of experimental intervention, blood samples were collected via the brachial vein (vena cutanea ulnaris), and serum was separated by centrifugation at 3000 rpm for 10 min and stored at ‑20°C for ELISA detection, as well as measurement of oxidative stress parameters and liver function indicators. Ileal tissues were harvested for HE staining, IHC (for TNF‑α and IL‑1β in intestinal tissues, and for iNOS and COX‑2 in liver tissues), RT-PCR, Western blot analysis, and liver tissues were collected for HE staining and IHC detection. At the end of the experimental period, all chicks were humanely euthanized by cervical dislocation under isoflurane anesthesia. No significant behavioral abnormalities, feeding changes, or mortality were observed in any group during the treatment period.

Histopathological Analysis and Pathological Scoring

Ileal and liver tissues fixed in 4% paraformaldehyde were subjected to gradient ethanol dehydration, paraffin embedding, sectioning (5 μm thickness), and dewaxing to rehydration. HE staining was performed strictly following the manufacturer’s instructions of the HE staining kit, followed by mounting with neutral balsam. The histopathological morphology of tissues was observed under an optical microscope, and pathological scoring was performed using the Image-Pro Plus 6.0 image analysis system. Intestinal and liver pathological scoring was performed according to the method described by Bhandari et al. (2019).

IHC

Immunohistochemical staining was performed using the streptavidin‑peroxidase (SP) method. The specific procedures were performed in strict accordance with the manufacturer's instructions of the IHC kit. Primary antibodies against TNF‑α (Rabbit anti‑chicken polyclonal, catalog No. WL02417, dilution 1:200), IL‑1β (catalog No. WL00891, dilution 1:200), iNOS (catalog No. WL02959, dilution 1:150), and COX‑2 (catalog No. WL01837, dilution 1:200) were applied overnight at 4°C. Sections incubated with PBS instead of primary antibody served as negative controls; chicken splenic tissues known to express the target antigens were used as positive controls. Sections were observed under an optical microscope, and the positive expression rate was calculated using Image‑Pro Plus 6.0 software by analyzing 5 randomly selected non‑overlapping fields per section at 200 × magnification. Positive staining was quantified as the percentage of positively stained area relative to the total tissue area in each field, using consistent threshold parameters for all sections.

ELISA

In accordance with the manufacturer’s instructions of the corresponding ELISA kits, the contents or activities of serum inflammatory factors (TNF-α, IL-6, IL-1β, IL-10), oxidative stress indicators (SOD, GSH-Px, CAT, MDA), liver function indicators (ALT, AST, ALP, TBIL), and myeloperoxidase (MPO) activity (a specific marker of neutrophil infiltration) were detected. The absorbance value at 450 nm was measured using a microplate reader, and the absolute concentration/activity of each indicator was calculated based on the corresponding standard curve.

RT-PCR

Total RNA was extracted from intestinal tissues using the Trizol method. Subsequently, total RNA was reverse transcribed into complementary DNA (cDNA) following the manufacturer’s instructions of the RT-PCR kit, and real-time fluorescence quantitative PCR was performed for detection. The primer sequences are listed in Table 1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal reference gene, and the relative mRNA expression level of each target gene was calculated using the 2⁻ΔΔCt method.

Table 1.

Specific primers for the target gene.

Gene Primer (5′−3′) Annealing temperature (°C) Fragment length
GAPDH F-GGTGGTGCTAAGCGTGTCA 58.4 186
R-CCTGGTCCTCAGTGTAGCCC
ZO-1 F-AGCGGCTTCGAGAAGTTCT 59.2 202
R-TCGGCTTCGTAGTCCTTGTC
Occludin F-GCTGCTGCTGCTGTTCTAC 60.3 185
R-CGGCTTCTTGTCCTTGTCC
Claudin-1 F-GGTGGTGCTGCTGACTTC 61.4 159
R-CCTTGTCCTTGTCCTTGTG
TNF-α F-GCTGCTGCTGATCTTCTCT 59.8 138
R-CCTTGTCCTTGTCCTTGCTAG
IL-6 F-GCTGCTGCTGATCTTGCA 58.5 173
R-CCTTGTCCTGTCCTTGCTC
IL-1β F-GCTGCTGCTGATCTTCTCC 58.2 115
R-CCTTGTCCTTGTCCTCTA
NF-κB F-GCTGCTGCTGATCTTCTCA 59.6 144
p65 R-CCTTGTCCTTGTCCTTGCTC
IκBα F-GCTGCTGCTGACTTCTGCG 60.5 141
R-CCTTGTCCTTGTCCTGCTA
Nrf2 F-GCTGCTGCTGATCTTGCC 61 159
R-CCTTGTCCTTGTCCTTGCT
HO-1 F-GCTGCTGCTGATCTTCTCA 57.8 132
R-CCTTGTCCTTGTCCTTCTT
NQO1 F-GCTGCTGCTGATCTTCTGCC 59.4 145
R-CCTTGTCCTTGTCCTCTG
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Western blot

Total protein was extracted from chick ileal tissues, and protein concentration was determined using a bicinchoninic acid (BCA) protein quantification kit. After equalization of protein concentrations across all samples, proteins were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% non-fat skim milk for 1 h at room temperature, then incubated with primary antibodies against ZO-1, Occludin, Claudin-1, Nrf2, HO-1, NQO1, and GAPDH overnight at 4°C. Subsequently, the membranes were incubated with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) reagent kit. The Image-Pro Plus 6.0 image analysis system was used to quantify the relative expression level of each target protein, with GAPDH serving as the internal reference.

Network pharmacology analysis

Screening of potential targets of Ts: Potential action targets of Ts were screened from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), PubChem, and SwissTargetPrediction databases. The screening criteria were set as oral bioavailability (OB)≥30% and drug-likeness (DL)≥0.18. Duplicate targets were removed to obtain the final potential targets of Ts.

Screening of targets related to Salmonella Enteritidis-induced intestinal injury: Targets associated with Salmonella-induced intestinal injury in chicks were retrieved from the Online Mendelian Inheritance in Man (OMIM), GeneCards, and DisGeNET databases using the keywords "Salmonella Enteritidis", "Chicken", and "Intestinal injury". Duplicate targets were eliminated to acquire disease-related targets.

Screening of intersection targets: Venny 2.1 software was applied to perform intersection analysis between the potential targets of TS and disease-related targets, to identify the potential action targets of Ts against Salmonella Enteritidis-induced intestinal injury.

Construction of protein-protein interaction (PPI) network and screening of core targets: The intersection targets were imported into the STRING database, with the species set to Gallus gallus and the confidence score ≥0.7, to construct the PPI network. The network file was exported, and Cytoscape 3.9.1 software was used for visualization analysis. Core targets were screened according to the Degree value, with targets with a Degree value ≥the median defined as core targets.

Pathway enrichment analysis: The intersection targets were imported into the Metascape database for Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. The threshold for significant enrichment was set at P < 0.05 to screen key regulatory pathways.

Molecular docking validation

Preparation of ligands and receptors: The three-dimensional (3D) structure of Ts was downloaded from the PubChem database (CID: 102193). Water molecules were removed, and energy minimization was performed using PyMOL software. The 3D crystal structures of core targets were retrieved from the Protein Data Bank (PDB) database. For each receptor protein, bound ligands and water molecules were removed using PyMOL software, followed by hydrogen addition, charge calculation, and active binding pocket definition using AutoDock Tools software.

Molecular docking procedure: Molecular docking between Ts and each core target was performed using AutoDock Vina software. The grid box size was set to 60 × 60 × 60 with a grid spacing of 0.375 Å, and other parameters were kept at default settings to calculate the binding affinity. A binding affinity<−5 kcal/mol indicates favorable binding activity, while a binding affinity <−7 kcal/mol indicates strong binding activity between the ligand and receptor.

Visualization of docking results: PyMOL software was used to visualize the docking results of Ts with core targets, and the binding sites, hydrogen bonds, and hydrophobic interactions were characterized.

Data statistics

All experimental data were statistically analyzed using SPSS 26.0 software. Measurement data were presented as mean ± standard deviation (mean±SD). One-way analysis of variance (ANOVA) was used for comparisons among multiple groups, and Tukey's honest significant difference (HSD) test was applied for pairwise comparisons between groups. P < 0.05 (*) was considered a statistically significant difference, and a P < 0.01 (**) was considered a highly statistically significant difference.

Results

Ts ameliorates pathological injury in Salmonella-infected chicks

Diarrhea score assessment revealed that chicks in the control group had well‑formed feces with no diarrhea occurrence. Chicks in the untreated infected group presented severe diarrhea with loose or even watery feces, and had the highest average diarrhea score, which was significantly higher than that in the control group (P < 0.01). Compared with the untreated infected group, chicks in all Ts intervention groups and the positive group showed alleviated diarrhea symptoms to varying degrees, with significantly decreased average diarrhea scores (P < 0.01 or P < 0.05) in a dose‑dependent manner. These results demonstrated that Ts significantly ameliorated diarrheal symptoms in Salmonella‑infected chicks (Fig. 1B).

HE staining of chick ileal tissues showed that the ileal mucosal structure in the control group was intact, with neatly and densely arranged villi and no inflammatory cell infiltration. In the untreated infected group, the intestinal mucosal structure was severely damaged, accompanied by obvious villus edema, loss of villus architecture with blunting and denudation, significantly increased crypt depth, massive infiltration of inflammatory cells (predominantly heterophils and lymphocytes), and focal mucosal necrosis. Compared with the untreated infected group, ileal pathological injury in chicks of all Ts intervention groups and the Positive group was alleviated to varying degrees in a dose‑dependent manner (Fig. 1C). In addition, the pathological scoring results were consistent with the HE staining findings (Fig. 1D). Myeloperoxidase (MPO) activity in ileal tissues was detected to quantify the degree of granulocyte infiltration (Fig. 1E).

HE staining of chick liver tissues showed that hepatocytes in the control group had intact structure, orderly arrangement and no inflammatory cell infiltration. In the untreated infected group, hepatocytes exhibited severe edema and degeneration, with focal hepatocellular necrosis with loss of cellular outlines and necrotic foci formation, as well as diffuse infiltration of abundant inflammatory cells (predominantly heterophils and macrophages). Compared with the untreated infected group, liver pathological injury in chicks of all Ts intervention groups and the positive group was alleviated to varying degrees in a dose‑dependent manner (Fig. 1F). In addition, the pathological scoring results were consistent with the HE staining findings (Fig. 1G).

Ts alleviates oxidative stress and improves liver function in Salmonella Enteritidis-infected chicks

Detection and analysis of serum SOD, GSH‑Px, CAT activities and MDA content in chicks from different groups revealed that the activities of SOD, GSH‑Px and CAT in the Control group were significantly higher than those in all other groups (P < 0.01), while the MDA content was significantly lower than that in all other groups (P < 0.01). Compared with the control group, the untreated infected group showed significantly decreased serum SOD, GSH‑Px and CAT activities (P < 0.01) and markedly increased MDA content (P < 0.01). Relative to the untreated infected group, all Ts intervention groups and the positive group exhibited significantly elevated serum SOD, GSH‑Px and CAT activities (P < 0.01 or P < 0.05) and prominently reduced MDA content (P < 0.01 or P < 0.05) in a dose‑dependent manner. These results indicated that Ts could significantly enhance the serum antioxidant capacity and alleviate oxidative stress injury in Salmonella Enteritidis‑infected chicks (Fig. 2A).

Fig. 2.

Fig. 2

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Ts alleviates oxidative stress and improves liver function in chicks infected with Salmonella. Data are presented as mean±SD (n = 10 per group). (## P > 0.05; * P<0.05; ** P<0.01).

(A) Oxidative stress indicators.

(B) Liver function indicators.

(C) Inflammatory cytokine levels.

Detection and analysis of serum ALT, AST, ALP and TBIL levels in chicks from different groups showed that the contents of ALT, AST, ALP and TBIL in the control group were significantly lower than those in all other groups (P < 0.01). The untreated infected group presented a significant upregulation of serum ALT, AST, ALP and TBIL levels (P < 0.01) compared with the control group. Compared with the untreated infected group, serum ALT, AST, ALP and TBIL levels in all Ts intervention groups and the positive group were significantly reduced (P < 0.01 or P < 0.05) in a dose‑dependent manner. These findings demonstrated that Ts could markedly improve liver function and attenuate hepatic injury in Salmonella Enteritidis‑infected chicks (Fig. 2B).

Statistical analysis of serum TNF‑α, IL‑6, IL‑1β and IL‑10 levels in chicks from different groups revealed that the contents of pro‑inflammatory factors TNF‑α, IL‑6 and IL‑1β in the control group were significantly lower than those in all other groups (P < 0.01), while the level of anti‑inflammatory factor IL‑10 was significantly higher than that in all other groups (P < 0.01). Compared with the control group, the untreated infected group showed significantly increased serum TNF‑α, IL‑6 and IL‑1β levels (P < 0.01) and markedly decreased IL‑10 level (P < 0.01). Relative to the untreated infected group, all Ts intervention groups and the positive group exhibited significantly downregulated serum TNF‑α, IL‑6 and IL‑1β levels (P < 0.01 or P < 0.05) and prominently upregulated IL‑10 level (P < 0.01 or P < 0.05) in a dose‑dependent manner. These results illustrated that Ts could significantly modulate the inflammatory response in Salmonella‑infected chicks, as evidenced by its inhibitory effect on pro‑inflammatory factor expression and promotive effect on anti‑inflammatory factor expression (Fig. 2C).

Effects of Ts on mRNA expression levels of related genes in intestinal tissues of Salmonella Enteritidis-infected chicks

RT‑PCR was performed to determine the relative mRNA expression levels of tight junction protein genes (ZO‑1, Occludin, Claudin‑1) (Fig. 3A), inflammation‑related genes (TNF‑α, IL‑6, IL‑1β) (Fig. 3B), and Nrf2 signaling pathway‑related genes (Nrf2, HO‑1, NQO1) (Fig. 3C) in the intestinal tissues of chicks. Compared with the control group, the relative mRNA expression levels of ZO‑1, Occludin, Claudin‑1, Nrf2, HO‑1 and NQO1 in the intestinal tissues of the untreated infected group were significantly downregulated (P < 0.01), while those of TNF‑α, IL‑6 and IL‑1β were markedly upregulated (P < 0.01). Compared with the untreated infected group, all Ts intervention groups and the Positive group showed significantly increased relative mRNA expression levels of ZO‑1, Occludin, Claudin‑1, Nrf2, HO‑1 and NQO1 (P < 0.01 or P < 0.05), and significantly decreased relative mRNA expression levels of TNF‑α, IL‑6 and IL‑1β (P < 0.01 or P < 0.05) in a dose‑dependent manner. These results indicated that Ts could significantly upregulate the mRNA expression of tight junction protein genes and oxidative stress‑related genes, and downregulate the mRNA expression of inflammation‑related genes, thereby repairing the intestinal mucosal barrier and alleviating inflammatory response and oxidative stress injury.

Fig. 3.

Fig. 3

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Ts up-regulates the expression of tight junction protein genes and oxidative stress-related genes, and down-regulates the expression of inflammation-related genes. Data are presented as mean±SD (n = 10 per group). (## P > 0.05; * P<0.05; ** P<0.01).

(A) The mRNA expression levels of tight junction protein genes in the intestinal tissue of chicks.

(B) The mRNA expression levels of pro-inflammatory cytokine genes in the intestinal tissue of chicks.

(C) The mRNA expression levels of Nrf2/HO-1/NQO1 pathway genes in the intestinal tissue of chicks.

Ts inhibits protein expression of inflammatory factors induced by Salmonella Enteritidis infection in chicks

IHC staining of ileal tissues from chicks in different groups showed that there was weak positive expression of the pro‑inflammatory factors TNF‑α and IL‑1β in the intestinal tissues of the control group, with a low positive expression rate. In contrast, the untreated infected group exhibited a marked increase in the positive expression of TNF‑α and IL‑1β in intestinal tissues, with a significantly elevated positive expression rate compared with the Control group (P < 0.01). Positive immunostaining for TNF‑α and IL‑1β was predominantly localized to intestinal epithelial cells and inflammatory cells within the lamina propria. Compared with the untreated infected group, all Ts intervention groups and the positive group showed significantly decreased positive expression of TNF‑α and IL‑1β in intestinal tissues, with a markedly reduced positive expression rate in a dose‑dependent manner (Fig. 4A‑D). These results indicated that Ts could significantly inhibit the intestinal inflammatory response and reduce the protein expression of inflammatory factors in chicks induced by Salmonella Enteritidis infection.

Fig. 4.

Fig. 4

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Ts inhibits the expression of inflammatory cytokine proteins in chicks infected with Salmonella. Data are presented as mean±SD (n = 5 per group for IHC). (* P<0.05; ** P<0.01).

(A, B) IHC results of TNF-α and IL-1β proteins in the ileum.

(C, D) Quantitative analysis of TNF-α and IL-1β protein expression.

(E, F) IHC results of iNOS and COX-2 proteins in the liver.

(G, H) Quantitative analysis of iNOS and COX-2 protein expression.

IHC staining of liver tissues from chicks in different groups revealed that the control group presented faint positive expression of iNOS and COX‑2 in liver tissues, with a low positive expression rate. In comparison with the control group, the untreated infected group showed a prominent increase in the positive expression of iNOS and COX‑2 in liver tissues, with a significantly upregulated positive expression rate (P < 0.01). Compared with the untreated infected group, all Ts intervention groups and the positive group exhibited markedly reduced positive expression of iNOS and COX‑2 in liver tissues, with a significantly decreased positive expression rate in a dose‑dependent manner. These findings demonstrated that Ts could significantly suppress the hepatic inflammatory response and downregulate the expression of inflammation‑related proteins in Salmonella Enteritidis‑challenged chicks (Fig. 4E‑H).

Ts upregulates the expression of tight junction and oxidative stress-related proteins

Western blot analysis revealed that, compared with the control group, the relative protein expression levels of ZO‑1, Occludin, Claudin‑1, as well as Nrf2, HO‑1 and NQO1 in intestinal tissues were significantly decreased in the untreated infected group (P < 0.01). Compared with the untreated infected group, all Ts intervention groups and the positive group showed significantly upregulated relative protein expression levels of ZO‑1, Occludin, Claudin‑1, Nrf2, HO‑1 and NQO1 in intestinal tissues (P < 0.01 or P < 0.05) (Fig. 5A‑C). These results indicated that Ts could markedly upregulate the expression of tight junction proteins and oxidative stress‑related proteins, and inhibit the expression of inflammatory factors, thereby exerting a protective effect on the intestinal mucosa of chicks.

Fig. 5.

Fig. 5

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Ts up-regulates the expression of tight junction and oxidative stress‑related proteins. Data are presented as mean±SD (n = 3 per group for Western blot). (* P<0.05; ** P<0.01).

(A) Western blot results.

(B) Quantification of tight junction protein (ZO‑1, Occludin, Claudin‑1) expression. (C) Quantification of oxidative stress protein (Nrf2, HO‑1, NQO1) expression.

Results of network pharmacology analysis

Screening of disease targets and pathway enrichment analysis

A total of 128 potential action targets of Ts were predicted via the SwissTargetPrediction database. Meanwhile, 236 targets associated with intestinal injury in chicks induced by Salmonella infection were screened from the GeneCards and Online Mendelian Inheritance in Man (OMIM) databases. Intersection analysis of the two target sets was performed, and 32 potential targets of Ts against Salmonella-induced intestinal injury in chicks were identified (Fig. 6A).

Fig. 6.

Fig. 6

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Disease target screening and pathway analysis.

(A) Venn diagram of disease targets.

(B) PPI network of Ts‑mediated regulation of intestinal injury in Salmonella Enteritidis‑infected chicks.

(C) GO functional enrichment plot of Ts‑mediated regulation of intestinal injury in Salmonella Enteritidis‑infected chicks.

(D) KEGG pathway enrichment bubble plot of Ts‑mediated regulation of intestinal injury in Salmonella Enteritidis‑infected chicks.

The 32 potential targets were imported into the STRING database to construct a protein-protein interaction (PPI) network. The PPI network comprised 32 nodes and 48 edges (interaction pairs). A total of 48 interaction pairs among 32 network nodes were obtained in the PPI network. Cytoscape software was applied to analyze the topological characteristics of the network, and the top 10 core targets ranked by Degree value were screened out, including Nrf2, HO-1, NQO1, IL-6, TNF-α, IL-1β, TP53, AKT1, MAPK3 and CASP3. Among them, Nrf2, HO-1 and NQO1 were identified as hub targets, indicating that Ts may exert a protective effect against intestinal injury by regulating these core targets (Fig. 6B).

Gene Ontology (GO) functional enrichment analysis showed that a total of 126 GO terms were significantly enriched from the 32 potential targets (P < 0.05, False Discovery Rate (FDR)<0.05). Among them, 89 terms belonged to Biological Process (BP), which were mainly associated with oxidative stress response, inflammatory response, regulation of apoptosis, and cytoprotection. 18 terms belonged to Cellular Component (CC), which were mainly involved in the nucleus, cytoplasm, and mitochondria; 19 terms belonged to Molecular Function (MF), which were mainly related to oxidoreductase activity, transcription factor activity, and cytokine binding (Fig. 6C).

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that a total of 28 signaling pathways were significantly enriched from the 32 potential targets (P < 0.05, FDR<0.05), mainly including the Nrf2/HO-1/NQO1 pathway, TNF signaling pathway, NF-κB signaling pathway, and PI3K-Akt signaling pathway. Among these, the Nrf2/HO-1/NQO1 pathway exhibited the highest enrichment significance with the lowest FDR value, indicating that Ts may ameliorate intestinal pathological injury in Salmonella Enteritidis-infected chicks by regulating the Nrf2/HO-1/NQO1 pathway (Fig. 6D). This is consistent with the reported protective role of the Nrf2/HO-1/NQO1 pathway in intestinal injury in previous studies (Wang et al., 2023; Li et al., 2025), and is also in full agreement with the in vivo experimental results of this study.

Results of molecular docking validation

Molecular docking results demonstrated that Ts exhibited favorable binding affinity to Nrf2, HO-1 and NQO1 proteins, with binding energies of −6.82 kcal/mol, −7.53 kcal/mol and −6.25 kcal/mol, respectively, all of which were below the threshold of −5 kcal/mol. Among them, Ts presented the strongest binding activity to HO-1 protein, with a binding energy lower than −7 kcal/mol. Binding mode analysis revealed that Ts bound to the active binding pockets of Nrf2, HO-1 and NQO1 proteins mainly through hydrogen bonds and hydrophobic interactions, thus forming stable ligand-receptor complexes (Fig. 7). These findings further verified that Ts could directly target the key proteins of the Nrf2/HO-1/NQO1 pathway to regulate the activation of this signaling cascade. The binding energies between Ts and Nrf2, HO-1, NQO1 proteins all met the requirements for favorable binding activity, indicating that Ts exerts its regulatory effect on the activation of the Nrf2/HO-1/NQO1 pathway via direct binding to these core proteins.

Fig. 7.

Fig. 7

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Molecular docking binding mode diagrams of Ts with Nrf2, HO‑1, and NQO1 proteins.

(A) Binding mode of Ts with Nrf2 protein.

(B) Binding mode of Ts with HO‑1 protein.

(C) Binding mode of Ts with NQO1 protein.

Discussion

The present study demonstrates that Ts exerts dose-dependent protective effects against intestinal and hepatic injury induced by Salmonella Enteritidis infection in chicks. Our integrative approach combining in vivo experimentation, network pharmacology, and molecular docking reveals that these effects are associated with activation of the Nrf2/HO-1/NQO1 antioxidant pathway, suppression of pro-inflammatory cytokine production, and preservation of intestinal tight junction integrity. These findings address a critical knowledge gap regarding the efficacy of Ts in young poultry and provide a mechanistic basis for its potential application as a phytogenic feed additive.

Salmonella infection is one of the major infectious diseases restricting the healthy and sustainable development of the poultry farming industry (Kabeta et al., 2024). It specifically damages the intestinal mucosal barrier of chicks and triggers severe inflammatory responses, which further lead to multiple organ injury and even mortality in chicks, inflicting enormous economic losses on the global poultry farming sector (Grace et al., 2024). With the increasingly prominent problem of multidrug resistance caused by the irrational overuse of antibiotics, the development of natural, safe, and highly effective antibiotic alternatives has become a prominent research hotspot in the field of green prevention and control of epidemic diseases in poultry farming (Ibeagha-Awemu et al., 2025). Ts as a natural plant-derived bioactive compound extracted from Camellia plants, possesses a wide spectrum of biological activities including antioxidant, anti-inflammatory, and antibacterial effects, and has been widely applied in the pharmaceutical, food, and livestock and poultry farming industries (Zhang et al., 2025). However, the protective effect of Ts against intestinal pathological injury in Salmonella Enteritidis-infected chicks and its specific underlying mechanism have not yet been fully elucidated. On this basis, in this study, we established a chick model of Salmonella Enteritidis infection, and systematically investigated the protective effect and regulatory mechanism of Ts in combination with network pharmacology, molecular docking technology, and in vivo experiments, to provide experimental evidence and theoretical support for its application in the prevention and control of Salmonella Enteritidis infection in poultry farming.

In this study, we successfully established a chick model of Salmonella Enteritidis infection. Chicks in the untreated infected group presented obvious diarrhea symptoms, accompanied by extensive loss of villus architecture with blunting and denudation, crypt atrophy, marked inflammatory cell infiltration, and elevated pathological scores. No mortality was observed during the experiment, and thus survival analysis was not performed. These pathological features were consistent with the characteristics of Salmonella infection‑induced intestinal injury in chicks reported in previous studies (Fasina et al., 2010), confirming the successful establishment and reliability of our model. Following Ts intervention, chicks showed significantly alleviated diarrhea symptoms, markedly improved intestinal pathological injury, with all the above protective effects presented in a dose‑dependent manner. Among them, high‑dose Ts exerted the most prominent ameliorative effect, with relevant indicators nearly restored to the normal level, indicating that Ts exerts a protective effect on the intestine of Salmonella Enteritidis‑infected chicks. This result is consistent with the previous finding that Ts can improve intestinal microbiota structure and alleviate intestinal inflammation in mice (Zhang et al., 2026a). It is speculated that the core functional basis of this effect is related to the antibacterial activity of Ts, which can reduce the direct damage to the intestinal mucosa by inhibiting the growth and proliferation of pathogenic bacteria such as Salmonella. Furthermore, Salmonella infection can disseminate to the liver via the blood circulation, leading to hepatocyte degeneration, necrosis, and liver dysfunction (Biazus et al., 2017). In this study, chicks in the untreated infected group showed significantly increased serum ALT, AST, and ALP activities, markedly decreased total protein (TP) and albumin (ALB) contents, and severe liver pathological injury. Whereas Ts intervention effectively improved the above liver function indicators and alleviated liver pathological damage, suggesting that Ts has a synergistic protective effect on the liver of Salmonella‑infected chicks. This effect may be closely related to the antioxidant and anti‑inflammatory activities of Ts, which can protect hepatocytes by alleviating hepatic oxidative stress and inflammatory response.

Oxidative stress is one of the key mechanisms underlying intestinal and hepatic injury in chicks induced by Salmonella infection (Okafor et al., 2024). After Salmonella challenge, the host produces a large amount of reactive oxygen species (ROS), which reduces the activities of endogenous antioxidant enzymes including SOD, GSH-Px and CAT, elevates the level of malondialdehyde (MDA, a major end product of lipid peroxidation), and further triggers oxidative tissue damage (Denagamage et al., 2015). Among these indicators, SOD, GSH-Px and CAT are the core enzymes responsible for ROS scavenging and oxidative stress defense in the host, while MDA content can directly reflect the severity of systemic oxidative damage (Wilmes et al., 2011). In this study, we found that the activities of SOD, GSH-Px and CAT in the intestinal and hepatic tissues of chicks in the untreated infected group were significantly decreased, accompanied by a marked increase in MDA content, which is consistent with previous reports that Salmonella infection induces systemic oxidative stress disorder (Yin et al., 2022). Whereas all Ts intervention groups significantly elevated the activities of the above antioxidant enzymes and reduced MDA content in a dose-dependent manner, and the high-dose Ts group restored oxidative stress indicators to near-normal levels. These results indicated that TS effectively alleviated Salmonella Enteritidis infection-induced oxidative stress injury by enhancing the host antioxidant capacity and inhibiting lipid peroxidation, which serves as a pivotal pathway for its protective effect on intestinal and hepatic tissues. Combined with our network pharmacology analysis, the potential action targets of Ts were mainly enriched in oxidative stress-related functional terms, among which Nrf2, HO-1 and NQO1 were significantly enriched as hub targets. The Nrf2/HO-1/NQO1 cascade is well-recognized as the core endogenous pathway for host oxidative stress regulation (Yuan et al., 2024). Our in vivo experiments further confirmed that Ts significantly upregulated the mRNA and protein expression levels of Nrf2, HO-1 and NQO1 in chick intestinal tissues. Molecular docking validation revealed that Ts could stably bind to the active binding pockets of Nrf2, HO-1 and NQO1 proteins via hydrogen bonds and hydrophobic interactions, with all binding energies below −5 kcal/mol and the strongest binding affinity to HO-1. These findings demonstrated that Ts can directly target the key proteins of the Nrf2/HO-1/NQO1 pathway, and promote the transcription and expression of downstream antioxidant genes by activating this pathway, thereby enhancing systemic antioxidant capacity and attenuating oxidative stress injury in the intestine and liver. In addition, Ts can also regulate the balance of intestinal inflammatory cytokines, as evidenced by reduced levels of pro-inflammatory factors IL-1β, IL-6 and TNF-α, and elevated level of anti-inflammatory factor IL-10. This effect further alleviates intestinal inflammatory cell infiltration, repairs intestinal mucosal barrier damage, and reduces the adhesion and colonization of Salmonella Enteritidis in the intestinal tract, thereby blocking its systemic dissemination to the liver and mitigating secondary hepatic injury. Collectively, Ts exerts a synergistic protective effect characterized by "antioxidation, anti-inflammation and mucosal barrier protection", which further clarifies the multi-target regulatory mechanism of its biological function against Salmonella infection.

In summary, this study integrates in vivo experimentation, network pharmacology, and molecular docking to systematically define the protective efficacy and mechanistic basis of Ts against Salmonella Enteritidis‑induced intestinal injury in chicks. Ts acts in a dose‑dependent manner through activation of the Nrf2/HO‑1/NQO1 signaling axis, which enhances host antioxidant capacity, suppresses intestinal inflammation, and restores mucosal barrier integrity. These coordinated effects alleviate diarrhea, mitigate pathological damage in both the intestine and liver, and ultimately improve survival in infected birds. Our findings not only expand the pharmacological profile of Ts but also fill a critical gap regarding its role in young poultry with Salmonella Enteritidis. Moreover, this work provides a robust experimental foundation for developing eco‑friendly strategies to control Salmonella infections. Given its natural origin, low toxicity, and practical feasibility, Ts holds considerable translational promise as a feed additive in poultry production. Its use offers compelling theoretical support for replacing antibiotics with phytochemicals in animal husbandry, thereby advancing a greener and more sustainable poultry industry.

Institutional review board statement

All animal experiments were approved by the Experimental Animal Ethics Committee of Kunming University (Approval No. SYXKK2025-0004) and conducted in accordance with the Guidelines for the Ethical Review of Animal Welfare (EAE-GZU-2022-E021).

Authorship contributions

Bo Zhang: Conceived part of the study, designed key experimental procedures, conducted core laboratory investigations, analyzed and interpreted experimental data, and drafted the initial version of the manuscript. Ke Li: Participated in experimental design, performed major experimental work, analyzed experimental datasets, interpreted the obtained results, and contributed to drafting the original manuscript. Jianyun Feng: Participated in the conceptual framework of the research, contributed to the overall experimental scheme, and interpreted key experimental finding. Aiguo Xin: Proposed the overall research concept, supervised the study framework, participated in experimental design, interpreted core results, and performed critical revision of important intellectual content of the manuscript. All other individuals who provided routine technical assistance or participated in data collection are acknowledged in the Acknowledgements section.

Ethics declaration

This study was conducted in accordance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. This study was approved by the Experimental Animal Ethics Committee of Kunming University. (Approval No. SYXKK2025-0004)

Disclosures

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.

Acknowledgements

This study was financially supported by the Research Project on Risk Monitoring and Control Technology of Important Food-Borne Zoonotic Bacterial Diseases in Yunnan Province (202407AB110017), the Talent Introduction Project of Kunming University (YJL23008). We thank Jiangnan Ni, Junjie Liu, Ao Dai, Hongya Yan, Rong Zhao, and Zhenju Yuan for their valuable technical support in sample collection and preliminary data processing.

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