A novel traditional Chinese medicine compound alleviates broiler ascites syndrome by modulating the IL-6/STAT3/FOXO3a signaling pathway

Abstract

Broiler ascites syndrome (BAS), a non-infectious group disease caused by relative hypoxia in broilers, is one of the three most serious nutritional metabolic diseases that jeopardize the chicken industry. At present, traditional Chinese medicine (TCM) is widely used to treat BAS, however, its mechanism of action remains unclear. Therefore, this study aimed to screen a new traditional Chinese medicine compound (NCMC) for the treatment of BAS and to elucidate its therapeutic mechanism through an integrated strategy of data mining, network pharmacology, molecular docking, and animal experiments. To this end, a novel NCMC comprising Poria, Pericarpium Citri Reticulatae, Radix Scutellariae, Radix Astragali, Semen Plantaginis, and Rhizoma Zingiberis Recens was successfully formulated based on data mining analysis. Utilizing network pharmacology, STAT3, SRC, and EGFR were identified as core therapeutic targets for BAS. Gene pathway analysis further suggested that the NCMC might exert its effects by modulating hypoxia response, oxidative stress, and the FOXO signaling pathway. These in silico predictions were subsequently validated by in vivo experiments, which demonstrated that NCMC treatment significantly alleviated systemic inflammation, reduced the ascites heart index, and mitigated lung tissue damage in broilers. Concurrently, it markedly enhanced the activities of the antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) while decreasing the content of malondialdehyde (MDA) in lung tissue. Ultimately, our integrated research approach revealed that the therapeutic effect of NCMC against BAS is primarily achieved through inhibition of the IL-6/STAT3/FOXO3a signaling pathway activation. This study not only provides a promising candidate drug for the treatment of BAS, but also offers a feasible systems pharmacology paradigm for modern research on traditional Chinese medicine compounds.

Introduction

Under modern poultry breeding conditions with advanced epidemic prevention technologies, nutritional and metabolic diseases such as Broiler Ascites Syndrome (BAS) are becoming increasingly detrimental to the fast-growing broilers (Li et al., 2022). BAS induces relative hypoxia in chickens, leading to a range of pathophysiological alterations including pulmonary arterial hypertension and ascites fluid accumulation (Cheng et al., 2021, Peng et al., 2024). In severe cases, it results in large-scale mortality within broiler flocks, causing substantial economic losses to the poultry industry. Current clinical treatments primarily rely on western medical approaches such as diuretics, antibiotics, hormone drugs, yet their efficacy remains inconsistent (Fathi et al., 2022, Obaid Saleh et al., 2022). Since 2020, China has fully implemented policies aimed at reducing or restricting antibiotic use (Yang et al., 2024). In response to national initiatives and to ensure food safety, there is an urgent need to identify efficient therapeutic agents that can accurately and rapidly treat BAS.

In recent decades, Traditional Chinese Medicine (TCM) has attracted worldwide attention due to its recognized efficacy and favorable safety profile. Several studies have indicated that TCM and its active constituents exhibit significant therapeutic effects against BAS. For example, Scutellaria baicalensis has been shown to enhance the activities of antioxidant enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GPX), and ameliorate pulmonary vascular remodeling by regulating oxidative stress (Wang et al., 2021). Quercetin exhibits significant antioxidant activity by scavenging free radicals and elevating GSH levels. Under hypoxic conditions, it directly eliminates ROS and hydroxyl radicals, thereby restoring endogenous redox homeostasis and demonstrating therapeutic efficacy against oxidative liver injury (Qi et al., 2022, Katsaros et al., 2024). Additionally, quercetin exhibits broad-spectrum antibacterial properties, effectively inhibiting the growth of pathogenic bacteria such as Salmonella, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus (Nguyen and Bhattacharya, 2022). Tanshinone IIA, as the core active ingredient of the TCM Salvia miltiorrhiza, has been fully proven to possess anti-inflammatory effects and the ability to improve microcirculation (Hu et al., 2017). Baicalin-copper complex alleviates intestinal damage in avian pathogenic Escherichia coli-infected chicks by targeting the AKT/NF-κB pathway (Cao et al., 2025). The scientifically formulated combination of various herbal medicines can leverage the unique strengths of each component, reduce potential toxicity and side effects, and yield synergistic therapeutic outcomes (Luan et al., 2020). However, the complexity of TCM compositions, including multi-component and multi-target characteristics, along with insufficiently elucidated mechanisms, has complicated the study of compatibility principles and optimal compound formulation.

Data mining and network pharmacology, as emerging interdisciplinary technologies integrating computer science and bioinformatics, are increasingly applied in medical and drug development fields due to their systematic and holistic research advantages (Li et al., 2021, Wu et al., 2021). While the two approaches differ in their research focus, both provide innovative methodological support for the modernization of TCM and veterinary TCM. The application of data mining techniques in TCM research dates back to the 1990s. After decades of development, its scope now spans nearly all TCM research domains, including herbal resource evaluation, drug quality identification, formula combination pattern analysis, and clinical efficacy assessment (Subrahmanya et al., 2022). Among these, drug quality control and formula combination mechanism exploration represent current research hotspots (Wu et al., 2021, Zhang et al., 2023). Compared to its mature application in TCM, data mining in the field of traditional Chinese veterinary medicine remains in its infancy, with relatively limited overall application scenarios.

Network pharmacology is an original discipline in drug systems research born in the era of artificial intelligence and big data. Its core strength lies in integrating multidimensional data to construct complex “component-target-pathway” interaction networks. (Noor et al., 2023, Li et al., 2023). This technology aligns perfectly with the holistic action characteristics of traditional Chinese medicine compounds. It overcomes the limitations of TCM research, which has been constrained by a “single-component, single-target” approach. It enables systematic elucidation of the mechanisms of action and the scientific principles of formula combinations at the molecular level (Zhang et al., 2023). This approach is widely applied in research directions such as screening active components in formulas, predicting action targets, analyzing formula combination patterns, and validating potential mechanisms (Nguyen and Bhattacharya, 2022, Zhao et al., 2023). However, few studies have applied these computational approaches to the treatment of BAS.

This study identified a New Chinese Medicine Compound (NCMC) as a candidate drug for BAS through data mining. Network pharmacology was employed to predict NCMC's primary active components, key target molecules, and signaling pathways for treating BAS. Molecular docking technology and animal experiments were used to jointly verify the screening results and explore the signaling pathway of NCMC in treating BAS. This provides reliable data and technical support for screening traditional Chinese medicine formulas for the treatment of BAS.

Materials and methods

Data mining

A comprehensive literature search was performed using multiple electronic databases, including China National Knowledge Infrastructure, PubMed, and the National Center for Biotechnology Information. The search strategy employed the combined keywords “broiler ascites syndrome” and “traditional Chinese medicine” to identify relevant publications. The identified traditional Chinese medicines were standardized according to the 2020 edition of the Veterinary Pharmacopoeia of the People's Republic of China and the Chinese Materia Medica. Their medicinal properties, including nature, flavor, meridian tropism, and efficacy, were systematically documented. Association rule analysis and cluster analysis were conducted using IBM SPSS Modeler, with a minimum support threshold of 10% and a confidence level of 80%.

Network pharmacology analysis

The chemical constituents of NCMC were identified using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform and the Encyclopedia of Traditional Chinese Medicine. Screening criteria included oral bioavailability (OB) ≥ 30%, drug-likeness (DL) ≥ 0.18, or a drug-likeness rating of moderate to good. Targets of these components were predicted using the SwissTargetPrediction database. BAS-related targets were retrieved from the human gene databases GeneCards and Online Mendelian Inheritance in Manby searching with the keywords “Ascites Syndrome” and “Broiler Ascites Syndrome”. Common targets between drug and disease were identified as potential targets using Venny 2.1 and subsequently imported into Cytoscape software (version 3.9.1) to construct a drug–active ingredient–potential target network. Key active ingredients were identified based on centrality analysis performed with the CytoHubba plugin. A protein–protein interaction (PPI) network was generated by submitting the potential targets to the STRING database, with the organism limited to Gallus gallus and a minimum interaction confidence score set to 0.4.

Gene pathway analysis

To elucidate the biological significance of the potential targets, functional enrichment analysis was performed using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database. The DAVID bioinformatics resource was utilized for systematic functional annotation and pathway analysis. Significance thresholds were set at a p-value of < 0.01 and a false discovery rate of < 0.05 to ensure robust identification of enriched biological processes and signaling pathways.

Molecular docking

Molecular docking analysis was performed to validate the predicted interactions between the key active compounds and the core targets identified through network pharmacology. The three-dimensional structures of the ligands were obtained from the PubChem database, while the crystal structures of the core targets were obtained from the Protein Data Bank. Molecular docking simulations were carried out using AutoDock to assess the binding affinities and modes of interaction between the ligands and their respective protein targets.

Preparation of animals

Sixty 1-day-old male Ross broiler chicks underwent a 14-day adaptation period before being randomly assigned to six experimental groups using a completely randomized design(Table 1): Control group, Model group, L-arginine (L-Arg, 10 g/kg, equivalent to a 1% supplementation rate (Wideman et al., 1995, Ahmadipour et al., 2018)), NCMC (Low: 1.25 g/kg, Medium: 2.5 g/kg, Hight: 5 g/kg) group. The control group received only the basal diet with standard drinking water at a room temperature of 25 °C. The BAS model was established with a low temperature (9–11 °C) from the age of 8 days, with the basic diet was supplemented with 3% lard and 4% fish meal, combined with high-salt drinking water (0.12% NaCl) (Kang et al., 2025). The drug dosage was determined based on the “Veterinary Pharmacopoeia of the People's Republic of China” (Commission, 2020) and and the results of preliminary experiments (Supplementary materials). Broilers were monitored daily for health status, and body weight was measured every 7 days. The nutritional composition of the basal diet is shown in Table 2. All the procedures and animal care mentioned in this study were approved by the Institutional Animal Care and Use Committee of Shandong Agricultural University (SDAUA-2021-007).

Table 1.

Composition of the basal diet.

Ingredients	Content (༅)
Corn	55
Soybean meal	30
Extruded soybean	8
Soybean oil	1.5
Limestone	1.3
Dicalcium phosphate	1.2
Sodium bicarbonate	0.15
Choline chloride (50༅)	0.1
Sodium chloride	0.2
L-Lysine. HCl (98.5༅)	0.4
DL-Methionine (99༅)	0.3
Premix11	1
Total	100
Nutrient levels
Metabolizable energy (MJ/kg)	12.3
Crude protein	21.5
Calcium	0.95
Available phosphorus	0.48
Lysine	1.35
Methionine	0.55
Methionine + cysteine	0.95

¹Premix provided per kilogram of diet: VA 5400 IU, VD3 2000 IU, VE 31 mg, VK3 16 mg, VB1 1.7 mg, VB2 7.5 mg, VB6 3.5 mg, VB12 0.015 mg, pantothenate 14 mg, nicotinamide 15 mg, biotin 0.05 mg, folic acid 1.5 mg; Cu 9.5 mg, Fe 70 mg, Mn 121 mg, Zn 60 mg, I 1.40 mg, Se 0.45 mg.

Table 2.

Animal grouping and handling.

	Control	Model	L-Arg	Low	Medium	Hight
Drink	basal diet	High-salt1	High-salt	High-salt	High-salt	High-salt
Feed	basal diet	high-energy diet2	high-energy diet	high-energy diet	high-energy diet	high-energy diet
Medication	——	——	L-Arg 10 g/kg	NCMC3 1.25 g/kg	NCMC 2.5 g/kg	NCMC 5 g/kg

¹High-salt drinking (0.12 % Na+)

²Mixed 3 % Lard+4 % Fishmeal

³New traditional Chinese medicine compound

Preparation of NCMC

Based on the pre-test results, it was determined that the NCMC had a therapeutic effect on BAS, then a uniform design experiment was carried out for different drug ratios. After adaptive rearing of 30 one-day-old Ross broiler chickens up to 14 days of age (male), the broilers were randomly divided into six groups of A-F (n = 5 in each group), different dosing ratios for each group (Table S1). According to the single factor screening Poria, Pericarpium Citri Reticulatae, Radix Scutellariae, Radix Astragali, Semen Plantaginis, Rhizoma Zingiberis Recens six TCM the participating factors of the uniform design, each factor was set at the six-factors, six-levels U66 uniform design table was used to conduct the experiment design (Table S2). The results of the homogeneous design experiment and the AHI of broiler counted during the experiment were obtained (Table S1 and S3). With its AHI as the objective function (Y), the data were analyzed by quadratic polynomial stepwise regression with DPS to get the regression equation and tested it, if the regression equation is highly significant, it means that the equation had a higher degree of confidence (Table S4).

The equation has the interaction of X2 (Pericarpium Citri Reticulatae) with X4 (Radix Astragali), X2 (Pericarpium Citri Reticulatae) with X5 (Semen Plantaginis), X4 (Radix Astragali) with X5 (Semen Plantaginis), and the p-value of the squared term of the 2-factor of X2 (Pericarpium Citri Reticulatae) were less than 0.01, which indicated that they have a significant effect on the AHI index at the highly significant level. The observed values, fitted values and fitting errors for the samples are shown in the following table (Table S5). The results shown that NCMC with a ratio of 15:10:5: 30:15:9 had the best therapeutic effect. NCMC is soaked in distilled water, decocted, and collected. The final concentration of the drug solution is 1 g/mL, and stored at 4°C for future use.

Sample collection

On days 35 and 42, five broilers were randomly selected from each group and blood samples were collected from the brachial vein using sodium heparin-coated vacuum tubes. The blood was centrifuged at 3000 × g for 15 min at 4 °C to obtain serum, which was subsequently stored at −80 °C. Subsequently, the above broilers were slaughtered for segregating visceral organs. Lung tissues were dissected and divided into two portions: one was fixed in 4% paraformaldehyde for histopathological examination, and the other was snap-frozen in liquid nitrogen and stored at −80 °C for subsequent molecular analyses. Hearts were excised and the atria were removed along the coronary groove. After clearing residual fat and clots, the total ventricle (TV) was weighed. The right ventricle (RV) was then separated along the interventricular groove and weighed. The ascites cardiac index (AHI) was calculated as RV/TV and used as a criterion for BAS diagnosis.

Indicators of oxidative stress

Quantification of MDA in serum and GPX, CAT, and SOD levels in lung tissue using commercial detection kits. Specifically, MDA levels were measured using the Lipid Oxidation (MDA) Detection Kit (S0131S) according to the manufacturer's protocol. GPX levels were determined using the Glutathione Peroxidase Detection Kit (S0056). CAT levels were assessed using the Catalase Assay Kit (S0051), while SOD levels were evaluated with the Total SOD Activity Assay Kit (S0109). All kits were purchased from Jiangsu Biyuntian Company, Jiangsu, Republic of China, and all assays were strictly performed in accordance with the manufacturers’instructions.

Detection of nitric oxide in broiler chickens

Serum samples were stored at 4 °C and thawed gradually before measurement. Nitric oxide (NO) concentration in serum was determined following the instructions provided with the NO assay kit (S0021S, Jiangsu Biyuntian Company, Jiangsu, China.).

Inflammatory cytokine protein expression detection

In order to detect the effect of NCMC on the expression of IL-6 protein in broiler lungs. Tissue samples were assayed according to the instructions of the ELISA kit (JB233-Ch, Shanghai Jinma Biotechnology Co., Ltd., Shanghai, China).

Western blot

Total protein was extracted from lung tissues using RIPA lysis buffer (WB3100, New Saimai Biologicals Co., Ltd., Suzhou,China) according to the manufacturer's instructions and quantified with a bicinchoninic acid assay kit (CW0014S, Kangwei Century Biotechnology Co., Ltd., China). Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane. The membrane was blocked with 5% skim milk or 5% bovine serum albumin for 1.5–2 h at room temperature, followed by incubation with the appropriate primary antibody at 4 °C overnight. Subsequently, the membrane was incubated with a horseradish peroxidase -conjugated secondary antibody for 1 h at room temperature. Protein band signals were detected using an enhanced chemiluminescence system, and grayscale values were quantified with ImageJ software.

Data analysis

All data are expressed as mean ± SEM. SPSS 23.0 statistical analysis software was used for one-way analysis of variance to compare differences between multiple groups. Different letters indicate significant differences between groups (P < 0.05, n = 5).

Results

Data mining identifies core traditional Chinese medicines

After screening, filtering, and removal of duplicates, 128 prescriptions for the treatment of BAS were identified, comprising 164 herbal medicines and 28 flavor attributes with a frequency of use exceeding 10 (Table S6). Herbs with cold nature and sweet flavor were the most prevalent (Fig. 1A, B). In terms of meridian tropism, the majority of herbs were associated with the liver and spleen meridians (Fig. 1C). Association rule analysis of the top 20 most frequently used herbs yielded 34 commonly employed herbal combination patterns (Table S7-S8). These 20 high-frequency herbs were categorized into six distinct groups through systematic cluster analysis (Fig. 1D). Based on the above results, a NCMC was formulated according to the “monarch, minister, assistant and guide” compatibility principle (Fig. 1E).

Fig. 1.

Data mining to screen NCMC for the treatment of BAS. (A) Medicinal nature distribution of NCMC. (B) Flavors distribution of NCMC. (C) Distribution of meridians entry of NCMC. (D) Clustering diagram of drug variables of NCMC. (E) Explanation of NCMC.

Active ingredients and related targets

After removal of duplicate entries, a total of 89 active ingredients, 951 drug-related targets, 759 disease-associated targets, and 166 potential therapeutic targets were identified (Table S9, Fig. 2A). The Drug–Active Ingredient–Potential Target network revealed that the most highly connected compounds included Rivularin, 5-Hydroxy-7,8-dimethoxyflavone, 5-Hydroxy-3,7,8,2′-tetramethoxyflavone, Paradol, Baicalein, and Epiberberine, suggesting that these may represent the core bioactive constituents of NCMC responsible for its therapeutic effects (Fig. 2B). PPI analysis identified 20 core targets, among which the top 10 ranked by relevance were STAT3, SRC, BCL2, GAPDH, CCND1, MMP9, EGFR, IL-6, ESR1, and CXCR4 (Fig. 2C).

Fig. 2.

Network pharmacology predicts the mechanism of action of NCMC in the treatment of BAS. (A) Venny diagram of NCMC active ingredients versus disease targets. (B) The "drug-active ingredient-target" network of NCMC for the treatment of BAS. Note: The purple part is the TCM monomer, the round part is the active ingredient of the TCM monomer, and the rectangle is the target point for treating BAS. (C) Key targets of NCMC for the treatment of BAS. Note: The redder the color, the higher the target ranking and the more important the target. (D) GO function analysis. (E) KEGG enrichment analysis.

Gene pathway analysis

Enrichment analysis was conducted using the DAVID database (Fig. 2D). The results suggested that NCMC may exert its therapeutic effects on BAS by modulating biological processes including cellular response to hypoxia, oxidative stress, protein phosphorylation, and the regulation of cell proliferation.

KEGG pathway enrichment analysis revealed that the most significantly enriched pathways associated with BAS included the MAPK signaling pathway, FOXO signaling pathway, cellular senescence, apoptosis, and the calcium signaling pathway (Fig. 2E).

Molecular docking validation

To validate the predictions from network pharmacology, molecular docking was performed between the core targets and the key bioactive components (Fig. 3A). A stronger binding affinity is generally indicated by a redder color in the heatmap, representing lower docking scores. The results demonstrated that hydrogen bonding was the primary interaction between the major active components and core targets, with binding energies below –2.0 kcal·mol⁻¹ for most complexes. The exception was the pair of 5-Hydroxy-3,7,8,2′-tetramethoxyflavone and FOXO3a. These findings suggest strong binding activity between the components and targets (Fig. 3B).

Fig. 3.

Molecular docking. (A) Docking diagram of the active ingredient with the core target. Note: Small molecule ligands are shown in red and large molecule receptor residues are shown in purple. (B) Molecular docking binding energy. Note: The redder the color, the greater the binding energy and the stronger the docking activity.

The effect of NCMC on growth performance and incidence rate

To further evaluate the therapeutic efficacy of NCMC on BAS, in vivo validation was performed using an animal model. Broilers in the model group exhibited characteristic clinical symptoms, including feather soiling, lethargy, abdominal distension with palpable fluctuation, and a significant reduction in body weight (P < 0.05). Postmortem examination revealed ascites with yellow transparent fluid, pericardial effusion, cardiac hypertrophy, pulmonary congestion, and hepatosplenomegaly, showing significant differences compared with the control group.

In contrast, broilers treated with L-Arg and NCMC displayed improved general conditions, characterized by clean feathers, alertness, normal appetite, and attenuated weight loss. Lung color and morphology also appeared normal in these groups. Pericardial effusion was still observed in some individuals in both the low- and high-dose NCMC groups (Fig. S1), and pulmonary hemorrhaging was noted in a subset of broilers receiving the high dose of NCMC (Fig. 4A, C).

Fig. 4.

Therapeutic effect of NCMC on BAS. (A) Morphological changes of broiler lungs. (B) Effect of NCMC on cardiac index of broiler ascites. (C) Effect of NCMC on the body weight of broilers. Low: 1.25 g/kg, Medium: 2.5 g/kg, high: 5 g/kg. Different letters indicate significant differences between groups, (n = 5), P<0.05.

The ascites heart index (AHI), a key indicator of BAS severity, was significantly elevated in the model group at both 35 and 42 days compared to the control group (P < 0.05), with all values exceeding the clinical threshold of 0.25. In comparison, AHI values were significantly reduced in both the L-Arg and NCMC treatment groups (P < 0.05; Fig. 4B). These results demonstrate that NCMC confers a protective effect against BAS in broilers.

Effect of the NCMC on lung lesions in broiler chickens

Histopathological examination of lung tissue sections revealed structurally normal architecture in the control group. In contrast, the model group exhibited severe pathological alterations, including diffuse tissue edema, hemorrhage, extensive accumulation of edematous fluid in alveolar spaces, dilation of respiratory capillaries, marked thickening of vascular walls, and inflammatory cell infiltration. Compared with the model group, both the L-Arg and NCMC treatment groups showed varying degrees of improvement in these pathological features. The low-dose NCMC group displayed interstitial erythrocyte extravasation and mild inflammatory infiltration. The medium-dose NCMC group presented with mild vascular wall thickening. The high-dose NCMC group was characterized by significant vascular wall thickening and persistent inflammatory cell infiltration (Fig. 5A). Semi-quantitative analysis of lung tissue sections revealed that the inflammatory infiltration area in the model group was significantly larger than that in the control group, while the inflammatory infiltration area in the L-Arg group and all dose groups of the NCMC was significantly lower than that in the model group (P<0.05, Fig. 5B). Furthermore, analysis of vascular wall thickness revealed that the model group exhibited significantly greater vascular wall thickness than the control group, whereas the L-Arg group and both low- and medium-dose NCMC groups demonstrated significantly reduced vascular wall thickness compared to the model group (P<0.05, Fig. 5C).

Fig. 5.

Effect of NCMC on lung lesions in broiler chickens. (A) HE staining pathological section. Red arrows indicate respiratory capillaries, black arrows indicate vessel walls, blue arrows indicate edema fluid, green arrows indicate erythrocytes, yellow arrows indicate inflammatory cell infiltration. Degree of inflammatory cell infiltration and vascular wall thickness. (B) Area of inflammatory cell infiltration, (n = 5), (P<0.05). (C) Degree of inflammatory cell infiltration, (n = 5), (P<0.05).

Detection of oxidative stress indicators

In the model group, the serum MDA level of broilers was significantly higher than that in the control group (P < 0.05), whereas the activities of SOD, GPX, and CAT were significantly lower than those in the control group (P < 0.05). Compared with the model group, serum MDA levels in broilers of both the L-Arg group and the NCMC treatment group were significantly decreased (P < 0.05), while the activities of SOD, GPX, and CAT were significantly increased (P < 0.05) (Fig. 6).

Fig. 6.

Effect of NCMC on oxidative stress levels in broiler chickens. (A) MDA content. (B) SOD content. (C) GPX content. (D) CAT content. Different letters denote significant differences between groups, (n = 5), P<0.05.

Nitric oxide content testing

NO is a core molecule regulating the pathological process of BAS. Results showed that serum NO levels in the model group were significantly elevated compared to the control group (P < 0.05). In comparison with the model group, both the L-Arg and NCMC treatment groups exhibited a significant reduction in serum NO (P < 0.05), with the medium-dose NCMC group showing the most pronounced decrease. These results suggest that systemic hypoxia induced increased NO release, and that NCMC administration effectively attenuated this response (Fig. 7A).

Fig. 7.

Effect of NCMC on NO levels and protein expression levels in broiler chickens. (A) NO content. (B) IL6 expression level. (C) STAT expression level. (D) FOXO3a expression level. Different letters indicate significant differences between groups, (n = 5), P<0.05.

Based on these findings, the medium-dose NCMC group (2.5 g/kg) demonstrated the most significant therapeutic effect against BAS and was therefore selected for subsequent experiments.

Expression of key pathway factor proteins

To elucidate the mechanism of action of NCMC, we assessed the protein expression levels of IL-6, phosphorylated STAT3 (P-STAT3) and FOXO3a in lung tissues. As shown in Fig. 7, compared with the control group, broilers in the model group exhibited significantly elevated levels of IL-6 and P-STAT3 proteins (P < 0.05), along with a significant reduction in FOXO3a protein expression (P < 0.05). Administration of the medium-dose NCMC significantly decreased IL-6 protein levels (P < 0.05; Fig. 7B). Furthermore, at 42 days, the NCMC medium-dose group significantly reduced P-STAT3 expression and increased FOXO3a expression (P < 0.05), while a similar but non-significant trend was observed at 35 days (Fig. 7, Fig. 7). These results suggest that IL-6 contributes to the pathogenesis of BAS, and that medium-dose NCMC effectively attenuates its expression. Additionally, NCMC treatment counteracted the alterations in P-STAT3 and FOXO3a protein levels observed in BAS broilers, indicating a modulatory role in the associated signaling pathways.

Discussion

With the growing prominence of BAS in poultry farming, developing safe and effective natural drug alternatives has become a research hotspot in veterinary medicine. However, current research on TCM formulas for treating BAS remains limited. This study aimed to explore the therapeutic efficacy and underlying molecular mechanism of a NCMC on BAS.

We formulated NCMC through data mining results, adhering to the “Monarch, Minister, Assistant and Guide” formula principle of TCM (Liu et al., 2021). The Monarch herbs Poria and Pericarpium Citri Reticulatae directly target the core pathogenesis of BAS, which is characterized by internal retention of water-dampness and blood stasis. Poria (Lu et al., 2024) induces eliminate dampness and promote diuresis, invigorates the spleen and tranquilize the mind; Pericarpium Citri Reticulatae (Li et al., 2021) regulates qi and invigorates the spleen. The Minister herbs Radix Scutellariae and Radix Astragali enhance the dampness elimination and diuresis promotion efficacy of the Monarch herbs. Radix Scutellariae (Hu et al., 2021) also can clear heat and detoxify; Radix Astragali (Zhang et al., 2024) tonifying qi and promoting diuresis. The Assistant herb Semen Plantaginis (Meng et al., 2023) clears heat and induces diuresis, eliminates residual damp-heat, and modulates the inherent properties of the Monarch and Minister herbs. The Guide herb Rhizoma Zingiberis Recens (Zhang et al., 2023) relieves exterior syndrome and dispels cold, mediates the effects of all herbs, neutralizes the cold nature of other herbs, and directs the therapeutic efficacy to the target meridians. This formula integrates etiological treatment with systemic protection, aligning with the multi-target synergistic therapeutic characteristics of traditional Chinese herbal formulas. Further application of network pharmacology analysis predicted that the primary active components of NCMC are Rivularin, Paradol, and Baicalein. Both Rivularin and Baicalein belong to the flavonoid class of compounds. Research has demonstrated that natural flavonoids can improve pulmonary arterial hypertension through multi-target synergistic actions, including antioxidant effects, anti-inflammatory properties, ion channel regulation, and intervention in proliferation-related signaling pathways (Yeh et al., 2015, Zhang et al., 2024). Paradol, as a phenolic compound, exhibits distinct anti-inflammatory, antioxidant, and immunomodulatory effects (Ayustaningwarno et al., 2024). The synergistic effects of these active ingredients provide the material basis for NCMC treatment of BAS.

The reliability of animal experiments relies on stable disease models. We employed a combined induction approach using low temperature, high-energy diet adnd high-salt drinking water to establish a BAS model (Guo et al., 2023). Cold stress increases energy expenditure for thermoregulation, while high-energy + high-salt conditions impose additional metabolic and cardiovascular load. These factors redirect nutrient partitioning from growth to stress response and homeostasis (Mohebodini et al., 2025, Zhang et al., 2025). Broilers in the model group exhibited weight loss, worsened pulmonary histological damage, and elevated AHI, consistent with the typical BAS phenotype observed in previous studies (Chen et al., 2025). The NCMC treatment group significantly alleviated these symptoms, increasing broiler weight, improving pulmonary histopathology, and lowering AHI values, confirming its clear therapeutic effect on BAS.

Oxidative stress is a key pathway in BAS pathogenesis. BAS-affected chickens exhibit reduced oxygen radical scavenging capacity, with ROS accumulation triggering lipid peroxidation. This leads to elevated MDA levels and decreased antioxidant enzyme activities, including SOD and GPX (Rawat et al., 2022, He et al., 2025). Previous studies have demonstrated that dietary supplementation with vitamin E, vitamin C, or α-lipoic acid can effectively mitigate this oxidative stress damage (Villar-Patiño et al., 2002, Díaz-Cruz et al., 2003). In this study, the model group broilers exhibited typical oxidative stress characteristics, whereas the NCMC-treated group significantly increased SOD, GPX, and catalase (CAT) activity while reducing MDA levels. This suggests NCMC exerts protective effects by regulating antioxidant system balance and mitigating oxidative stress damage, which function is consistent with Qiling Jiaogulan Powder (Yu et al., 2023).

NO, as a key regulator of vascular endothelial function, shows a metabolic imbalance closely associated with BAS pathogenesis, though the imbalance pattern varies with the modeling approach (Chen et al., 2025, Khajali et al., 2011). In arginine-deficient BAS, insufficient substrate for endothelial nitric oxide synthase (eNOS) leads to reduced NO synthesis; supplementation with L-Arg restores physiological NO levels (Shirzadi et al., 2024); whereas the cold stress combined with a high-energy + high-salt diet employed in this study induces hypoxia-stress-mediated overexpression of inducible nitric oxide synthase, leading to excessive NO production. excessive NO combines with superoxide anion to form highly toxic peroxynitrite, exacerbating pulmonary vascular endothelial injury and vasoconstriction (Hannemann and Böger, 2022). Simultaneously, hypoxia stress in this model also triggers enhanced oxidative stress, leading to dysfunction of eNOS. This causes eNOS, which is normally responsible for synthesizing NO, to instead produce superoxide radicals and/or hydrogen peroxide. This not only reduces NO bioavailability and weakens its physiological protective effects on blood vessels, but also further converts limited intracellular NO into highly toxic peroxynitrite. This synergizes with iNOS-mediated toxic damage, ultimately exacerbating endothelial cell injury and endothelial dysfunction (Chen et al., 2025, Wu et al., 2021). In this study, serum NO levels were significantly elevated in the model group. The positive control L-Arg achieved balanced regulation by feedback inhibition of iNOS activity and promotion of NO metabolic clearance (Lee et al., 2003, Sayed et al., 2023). In contrast, the medium-dose group of NCMC precisely regulated NO within physiological ranges, preserving its vasoprotective and vasodilatory functions while avoiding toxic damage from excessive NO. This superior regulatory specificity may be attributed to the synergistic effects of NCMC's multiple components.

KEGG pathway enrichment analysis of the NCMC for treating BAS indicates that the FOXO signaling pathway serves as its core regulatory pathway. Combined with PPI network analysis and GO functional enrichment results, the core targets molecules of NCMC include IL-6, and STAT3. These targets primarily participate in biological processes such as hypoxia response, oxidative stress, inflammatory response, protein phosphorylation, and cell proliferation regulation, aligning closely with the core pathogeneis of BAS. Hypoxia and inflammatory responses are important common triggers of pulmonary diseases. Both can synergistically induce macrophages to polarize toward a pro-inflammatory phenotype (M1 type), leading to massive secretion of pro-inflammatory factors such as IL-6 and TNF-α (Kishimoto et al., 2015, Liang et al., 2021). In this study, IL-6 protein levels in lung tissues of model group broilers were significantly higher than those in the control group at all detection time points. Conversely, IL-6 protein levels in lung tissues of NCMC-treated broilers at the dose group were lower than those in the model group at all time points. Previous studies have confirmed that IL-6 is a key pro-inflammatory factor in the pathological process of BAS, and IL-6-deficient mice exhibit significant resistance to hypoxic pulmonary hypertension (Xu et al., 2023). Furthermore, as a core upstream signal for STAT3 activation, the overexpression of IL-6 can trigger a cascade of downstream pathway activations (Costa-Pereira, 2014, Zhang et al., 2025). Under the synergistic effects of IL-6 overload and hypoxia stress, the expression levels of phosphorylated STAT3 (P-STAT3) protein in lung tissues of broiler chickens in the model group were higher than those in the control group at all detection time points. This finding is consistent with previous conclusions that hypoxia stress, as a core trigger for BAS onset, can significantly activate STAT3 phosphorylation. In contrast, P-STAT3 protein expression in the NCMC medium-dose group was lower than that in the model group, suggesting that NCMC may block abnormal signaling by inhibiting STAT3 phosphorylation. Previous studies have confirmed that FOXO family members are core molecules regulating host stress responses, participating in stress reactions such as hypoxia and DNA damage, their downstream signaling pathways regulate key processes including oxidative stress, apoptosis, and cell cycle arrest (Li et al., 2024, Rodriguez-Colman et al., 2024). As a core member of the FOXO family, FOXO3a plays a vital role in multiple biological processes including development, proliferation, apoptosis, metabolism, and differentiation, and its functional activity and localization are strictly regulated by phosphorylation modifications (Link, 2019, Link and Ferreira, 2025, Orea-Soufi et al., 2022). Research indicates that p-STAT3 directly mediates the phosphorylation of FOXO3a, once phosphorylated, FOXO3a remains confined to the cytoplasm, unable to enter the nucleus to bind target gene promoters and exert transcriptional regulatory functions (Xu et al., 2023). Concurrently, this study found that total FOXO3a protein expression levels in lung tissues of model group broilers were significantly lower than those in the control group at all detection time points. The combined effects of these dual factors markedly weakened FOXO3a's inhibitory action on abnormal proliferation of vascular smooth muscle cells, thereby exacerbating pulmonary circulation disorders (Grobs et al., 2021). In contrast, FOXO3a protein expression in lung tissue from NCMC-treated broilers was significantly higher than that in the model group. This suggested that NCMC may exert its therapeutic effect on BAS by dual mechanisms. Inhibiting the abnormal activation of STAT3 reduces the phosphorylation modification of FOXO3a, and restores its nuclear localization and transcriptional function, while simultaneously increasing total FOXO3a protein expression. This dual mechanism restores FOXO3a's functional activity, ultimately exerting therapeutic effects against BAS by suppressing abnormal vascular smooth muscle cells proliferation and improving pulmonary circulation dysfunction.

Conclusion

This study identified a NCMC for the treatment of BAS. Using network pharmacology and molecular docking approaches, STAT3, SRC, and EGFR were predicted as core therapeutic targets. Mechanistic analyses suggested that NCMC may alleviate BAS through modulation of pathways involved in hypoxia response, oxidative stress, and FOXO signaling. The therapeutic potential of NCMC was further validated in a successfully established BAS model, which demonstrated that NCMC reduces systemic inflammation and enhances antioxidant capacity in broiler lungs via the IL-6/STAT3/FOXO3a signaling pathway. These findings provide valuable insights for the development of traditional Chinese medicine-based therapies and clinical management of BAS.

CRediT authorship contribution statement

Yifan Gao: Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Conceptualization. Shijing Shan: Writing – review & editing, Investigation, Formal analysis, Data curation. Yang Liu: Writing – review & editing, Investigation. Shuo Cao: Writing – review & editing. Linjue Li: Writing – review & editing. Yue Yu: Writing – review & editing. Jianzhu Liu: Project administration, Methodology, Funding acquisition. Xiaona Zhao: Project administration, Methodology, Funding acquisition. Pimiao Zheng: Supervision, Project administration, Funding acquisition, Conceptualization.

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.