Water extract of Artemisia capillaris alleviates fatty liver hemorrhagic syndrome in laying hens by modulating the gut microbiota–bile acid axis

Abstract

Fatty liver hemorrhagic syndrome (FLHS) is a major metabolic disorder that compromises the health and productivity of laying hens. This study aimed to evaluate the protective effects of the water extract of Artemisia capillaris (ACTE) against FLHS induced by a high-energy, low-protein (HELP) diet in laying hens. Ninety 50-wk-old Hy-Line Brown laying hens were randomly assigned to 5 groups: CON, HELP, HELP-L, HELP-M, and HELP-H, each with 6 replicates of 3 hens. ACTE supplementation alleviated metabolic disturbances induced by the HELP diet, improving production performance, reducing hepatic and abdominal lipid accumulation, and ameliorating liver dysfunction, as evidenced by reduced serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, with the high-dose treatment showing the most pronounced protective effects. ACTE also enhanced antioxidant capacity and suppressed inflammatory responses. Gene expression analysis revealed downregulation of lipogenic genes and upregulation of genes involved in fatty acid oxidation and bile acid signaling. Ileal 16S rRNA gene sequencing showed that the HELP diet decreased microbial α-diversity, reduced the abundance of Lactobacillus, and enriched dysbiosis-associated genera, including Romboutsia and Clostridium sensu stricto 1. ACTE supplementation partially restored microbial community structure and increased Lactobacillus abundance. Targeted bile acid metabolomics further demonstrated increased primary bile acids, including cholic acid (CA) and chenodeoxycholic acid (CDCA), and decreased secondary and conjugated bile acids, including taurodeoxycholic acid (TDCA) and lithocholic acid (LCA). These findings suggest that ACTE supplementation mitigates FLHS associated with HELP diet feeding in laying hens by modulating lipid metabolism, oxidative stress, inflammation, gut microbiota composition, and bile acid homeostasis.

Introduction

Fatty liver hemorrhagic syndrome (FLHS) is a major metabolic disorder affecting cage-reared laying hens (You et al., 2023). This condition is typically associated with abnormal lipid deposition in the liver, marked hepatomegaly, increased tissue fragility with a propensity for rupture, and the presence of focal or petechial hemorrhagic and necrotic lesions (Wolford and Polin, 1972). In addition to impairing laying performance and egg quality, FLHS can lead to sudden mortality in affected flocks, thereby causing substantial economic losses to the poultry industry (Anene et al., 2023; Khodaei et al., 2025a). At present, caged laying hens continue to dominate commercial egg production systems. The widespread use of high-producing laying hen strains, intensive rearing practices, and energy-dense diets may increase the risk of FLHS. Therefore, its prevention and control have become important concerns in poultry nutrition and health research (Li et al., 2024; Imtiaz et al., 2025).

The pathogenesis of FLHS involves multiple factors and signaling pathways, and the disease is not confined to the liver. Rather, it reflects a systemic metabolic disorder characterized by multi-organ dysregulation along the gut–liver–adipose axis (Fleishman and Kumar, 2024). Accumulating evidence suggests that gut microbial imbalance and altered bile acid metabolism may contribute to the development of FLHS (Wang et al., 2023; Zhang et al., 2026). Therefore, the gut–liver axis is increasingly recognized for its critical role in the regulation of hepatic metabolic diseases, with particular emphasis on the bidirectional interactions between intestinal microbiota and bile acids that influence hepatic metabolism and inflammatory responses (Zhao et al., 2024a; Li et al., 2025a). Recent research has shown that dietary supplementation with bile acids, such as chenodeoxycholic acid (CDCA) and hyodeoxycholic acid (HDCA), alleviates hepatic lipid accumulation and reduces inflammatory mediator levels in a high-fat diet–induced FLHS model in laying hens, highlighting bile acids as signaling molecules capable of directly modulating hepatic lipid metabolism (Wang et al., 2024b). Accordingly, interventions targeting intestinal microbial communities and bile acid composition may provide a promising strategy for alleviating FLHS in laying hens (Wang et al., 2024a; Zhang et al., 2025).

Artemisia capillaris Thunb. (A. capillaris) is a traditional medicinal herb that has long been used for the treatment of hepatic disorders. Extensive evidence has demonstrated that it confers hepatoprotective and choleretic benefits, while also exerting anti-inflammatory, antioxidant, lipid-regulatory, and anti-fibrotic actions, and is commonly administered in the form of water extracts for traditional applications, including heat-clearing, detoxification, and liver protection (Jang et al., 2015; Hsueh et al., 2021). The water extract of Artemisia capillaris (ACTE) contains diverse bioactive constituents, including coumarins, flavonoids, phenolic acids, and polyacetylenes. Representative monomeric compounds identified in ACTE include 6,7-dimethoxycoumarin (scoparone), 6‑methoxy-7-hydroxycoumarin (scopoletin), and chlorogenic acid, among others (Gao et al., 2024; Yuan et al., 2025). A recent study demonstrated that A. capillaris–derived components attenuated cholestatic liver injury in mice, partly through reshaping the gut microbial community and alleviating hepatic oxidative and inflammatory stress (Cai et al., 2024). In another study, a combined extract of A. capillaris and bamboo shoots was administered to mice with dextran sulfate sodium (DSS)–induced colitis. The treatment attenuated colitis, suppressed expression of proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β), reduced reactive oxygen species (ROS) generation, and preserved intestinal epithelial barrier integrity (Kim et al., 2022). In studies of fatty liver disorders, ACTE has been reported to significantly alleviate nonalcoholic fatty liver disease (NAFLD) induced by high-fat diets in murine models. Mechanistic investigations further revealed that ACTE activated the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway and promoted the phosphorylation of adenosine monophosphate–activated protein kinase (AMPK). These changes were accompanied by reduced expression of sterol regulatory element–binding protein 1c (SREBP-1c) and coordinated downregulation of multiple lipogenic genes, including fatty acid synthase (FASN), acetyl-CoA carboxylase (ACC), stearoyl-CoA desaturase 1 (SCD1), and diacylglycerol acyltransferase 2 (DGAT2) (Liang et al., 2022, 2025). Recent evidence indicates that regulation of bile acid homeostasis and the gut–liver axis represents an important component of ACTE-mediated hepatoprotection, particularly through farnesoid X receptor (FXR)–fibroblast growth factor 15/19 (FGF15/19) signaling (Stofan and Guo, 2020; Li et al., 2025b).

To date, research on the effects of ACTE in fatty liver disease has focused predominantly on NAFLD, with most studies centered on hepatic metabolism. In contrast, integrative investigations that simultaneously consider the gut, bile acids, and hepatic metabolic pathways remain limited, and mechanistic evidence regarding the role of ACTE in FLHS is particularly scarce. Although studies on A. capillaris and its extracts in livestock and poultry remain limited, their well-documented hepatoprotective, choleretic, and lipid-regulatory properties suggest that they may serve as promising candidates for improving bile acid metabolic disturbances and gut–liver metabolic imbalance.

Accordingly, the present study aimed to investigate whether ACTE could alleviate FLHS in cage-reared laying hens by modulating ileal microbial community structure and bile acid profiles. We hypothesized that ACTE would alleviate FLHS by coordinating the regulation of lipid metabolism, gut microbiota composition, and bile acid homeostasis. By integrating the traditional hepatoprotective use of A. capillaris with the modern framework of the gut–liver axis and bile acid metabolism, this study sought to elucidate the potential mechanisms underlying ACTE-mediated improvements in hepatic lipid metabolism and to provide a scientific basis for the development of safe and sustainable functional feed additives for the prevention and control of FLHS (Khodaei et al., 2025b).

Materials and methods

Ethics statement

All animal procedures were conducted in accordance with the ethical standards established by the Institutional Animal Care and Use Committee of Northeast Agricultural University (Harbin, China) and were approved by the Animal Ethics Committee of Northeast Agricultural University (approval No. NEAUEC2024-03-86).

Experimental design and animal management

A total of 90 healthy Hy-Line Brown laying hens at 50 wk of age were selected for the present experiment. After a 1-wk acclimation period, hens were randomly assigned to 5 groups (18 hens per group), with 6 replicate cages per treatment and 3 hens per cage; each cage was considered one replicate. The hens were assigned to the following treatments: a control group (CON) fed a basal diet; a model group (HELP) fed a HELP diet; and three ACTE-treated groups (HELP-L, HELP-M, and HELP-H) fed the HELP diet and received ACTE by oral gavage at doses equivalent to 0.5, 1.0, and 2.0 g crude A. capillaris/kg BW, respectively. ACTE was provided as a freeze-dried powder, dissolved in distilled water (0.5 g/mL), and administered once daily by oral gavage based on body weight. Hens in the CON and HELP groups received an equal volume of distilled water daily. Based on the extraction yield of ACTE (15.6%), the doses corresponded to 78, 156, and 312 mg ACTE/kg BW, with gavage volumes of 0.156, 0.312, and 0.624 mL/kg, respectively. The ingredient composition and nutrient levels of the basal and HELP diets are shown in Table 1. Hens were housed under controlled environmental conditions (22 ± 2°C) with a 16 h light:8 h dark cycle and ad libitum access to feed and water. The experiment lasted 15 wk (51 to 65 wk of age). The doses and duration were determined based on preliminary trials and previous studies.

Table 1.

Composition and nutrient levels of the experimental diets.

Items	Normal diet	HELP diet
Ingredient (%)
Corn	63.7	65.2
Soybean meal	23.6	14.3
Lard	0	7.8
Limestone	7.4	7.4
Salt	0.3	0.3
Premix 1	5	5
Total	100	100
Analyzed nutrient levels 2
ME, (MJ/kg)	11.1	12.7
Crude protein (%)	18.2	13.7
Crude fat (%)	2.8	10.1
Ca (%)	3.25	3.25
Available P (%)	0.38	0.38
Lysine (%)	0.94	0.67
Methionine (%)	0.32	0.27

¹Vitamin–mineral premix supplied the following per kilogram of diet: vitamin A, 12,500 IU; vitamin B1, 1.5 mg; vitamin B2, 6 mg; vitamin B12, 0.025 mg; vitamin D3, 2,500 IU; vitamin E, 10 mg; vitamin K3, 2 mg; niacin, 40 mg; biotin, 0.3 mg; folic acid, 1.25 mg; calcium pantothenate, 12 mg; Fe, 80 mg; Cu, 8 mg; Mn, 80 mg; Zn, 75 mg; I, 1.2 mg; Se, 0.25 mg.

²Nutrient levels were determined by chemical analysis.

Preparation of ACTE

A. capillaris was purchased from Harbin Songshantang Pharmaceutical Co., Ltd. (Harbin, China). Following removal of impurities, the dried plant material was coarsely ground and subjected to aqueous extraction using distilled water at a solid-to-liquid ratio of 1:10 (w/v). After a 30-min soaking period, the suspension was refluxed at 100°C for 2 h and subsequently filtered. The residue was extracted with 8 volumes of distilled water (1:8, w/v) under the same conditions for an additional 1 h. The two aqueous extracts were combined and lyophilized at −50°C for 48 h to obtain a powder. The average extraction yield of A. capillaris was 15.6%. The lyophilized extract was stored at −20°C until use.

Identification of chemical constituents in ACTE

The chemical constituents of ACTE were profiled using untargeted LC–MS/MS analysis. Briefly, 20 mg of freeze-dried ACTE was extracted by ultrasonication with 80% methanol, followed by centrifugation at 12,000 × g for 10 min at 4°C. The supernatant was filtered through a 0.22 µm membrane prior to analysis. LC–MS/MS analysis was performed using a Thermo Vanquish UHPLC system interfaced with an Orbitrap Exploris 120 mass spectrometer. Separation was achieved on an ACQUITY UPLC® HSS T3 column (100 × 2.1 mm, 1.8 μm; Waters, Milford, MA, USA) at a column temperature of 40°C, with a flow rate of 0.3 mL/min and an injection volume of 2 μL. The mobile phases consisted of 0.1% formic acid in water (A) and acetonitrile (B) for positive mode, and 5 mM ammonium formate in water (A) and acetonitrile (B) for negative mode, using the following gradient program: 0–1 min, 10% B; 1–5 min, 10–98% B; 5–6.5 min, 98% B; 6.5–6.6 min, 98–10% B; and 6.6–8 min, 10% B. Mass spectra were acquired using electrospray ionization in both positive and negative ion modes. The spray voltage was set at 3.5 kV (positive) and −2.5 kV (negative). Full-scan detection was performed over an m/z range of 100–1000. The major compounds were annotated based on accurate mass measurements, MS/MS fragmentation profiles, and database comparisons.

Sample collection

At the end of the 15-wk feeding trial, hens were fasted for 12 h with free access to water. One hen from each replicate was randomly selected for sample collection (n = 6 per group). Blood was drawn from the brachial vein and centrifuged at 3,000 × g for 15 min at 4°C. Serum was then separated, aliquoted into microcentrifuge tubes, and stored at −80°C for subsequent analysis. Body weight was recorded before euthanasia. Birds were euthanized by cervical dislocation and then subjected to exsanguination. The liver and abdominal fat were immediately excised and weighed. Liver tissue, intestinal segments, and intestinal contents were collected, snap-frozen in liquid nitrogen, and stored at −80°C until analysis.

Production performance

Production performance of the hens was evaluated at wk 15 of the experimental period. Laying rate was calculated as the percentage of eggs produced daily relative to the number of hens housed. Egg weight was measured daily and averaged within each replicate. ADFI was calculated based on total feed consumption and the number of hens per replicate. Feed conversion ratio (FCR) was calculated as feed intake (g) divided by egg mass (g) per replicate. Broken and soft-shelled eggs were excluded from the calculations of laying rate and egg weight.

Measurement of serum and hepatic biochemical parameters, antioxidant capacity, and inflammatory cytokines

Serum samples were analyzed to determine lipid metabolism–related biochemical indices, antioxidant capacity, and inflammatory markers. Serum concentrations of triglycerides (TG), total cholesterol (TC), alanine aminotransferase (ALT), aspartate aminotransferase (AST), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were measured using commercial assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Antioxidant status was evaluated by measuring glutathione peroxidase (GSH-Px), total superoxide dismutase (T-SOD), catalase (CAT), total antioxidant capacity (T-AOC), and malondialdehyde (MDA). Serum concentrations of inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6), were determined using ELISA kits (Beijing Chenglin Biotechnology Co., Ltd., Beijing, China).

For liver analysis, approximately 0.1 g of tissue was homogenized in ice-cold saline (1:9, w/v). The homogenate was then centrifuged at 3,000 × g for 15 min at 4°C, and the supernatant was collected. The hepatic biochemical, antioxidant, and inflammatory parameters were determined, with results normalized to total protein content measured using a BCA protein assay kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China).

Histological analysis

Liver samples were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 5 µm for hematoxylin–eosin (H&E) staining. For Oil Red O staining, fresh liver samples were embedded in OCT compound, cryosectioned (8–10 µm), and stained to evaluate lipid accumulation. Representative histological images were obtained using a light microscope (Olympus, Japan).

Reverse transcription-quantitative PCR (RT-qPCR)

Total RNA was isolated from liver and ileal tissues using TRIzol reagent (Guangzhou Meiji Biotechnology Co., Ltd., Guangzhou, China) according to the manufacturer’s instructions. cDNA was synthesized using a BioRT High-Sensitivity Reverse Transcription Kit (Hangzhou Bioer Technology Co., Ltd., Hangzhou, China). Quantitative real-time PCR was performed using ChamQ qPCR Mix (Vazyme, China) on a LightCycler® 480 system (Roche, Germany). β-actin was used as the reference gene. All reactions were performed in triplicate, and relative expression levels were calculated using the 2−ΔΔCt method. Primers were designed and synthesized by Sangon Biotech Co., Ltd. (Shanghai, China), and the primer sequences are listed in Table S1.

16S rRNA gene sequencing and analysis

Given that the HELP-H group exhibited the most pronounced phenotypic improvement, the CON, HELP, and HELP-H groups were selected for 16S rRNA gene sequencing. Total genomic DNA was extracted from ileal contents using a commercial kit (Dalian Meilun Biotechnology Co., Ltd., China). The V3–V4 region of the bacterial 16S rRNA gene was amplified using primers 338F and 806R and sequenced on an Illumina NovaSeq 6000 platform. Raw reads were quality-filtered using Trimmomatic and Cutadapt, and high-quality non-chimeric sequences were obtained using the DADA2 pipeline implemented in QIIME2 (v2020.6). Taxonomic assignment was performed using a naïve Bayesian classifier against the SILVA 138 database (Callahan et al., 2016). Alpha diversity indices (Ace, Chao1, Shannon, and Simpson) and beta diversity metrics were calculated. Differences in microbial community structure were further assessed using principal coordinates analysis (PCoA), nonmetric multidimensional scaling (NMDS), and permutational multivariate analysis of variance (PERMANOVA). Differential taxa were identified using LEfSe (LDA > 4) and Metastats (P < 0.05) (Segata et al., 2011).

LC–MS/MS–based bile acid metabolomics analysis

For bile acid metabolomics analysis, the CON, HELP, and HELP-H groups were selected. Ileal contents (20 mg) were extracted with methanol containing an internal standard (10 µg/mL), vortex-mixed, precipitated at −20°C, and centrifuged (12,000 × g, 10 min, 4°C). The resulting supernatants were analyzed by LC–MS/MS using an ExionLC™ AD UHPLC system coupled to a QTRAP® 6500+ mass spectrometer. The instrument was operated with electrospray ionization in negative ion mode and multiple reaction monitoring (MRM). Separation was achieved on an ACQUITY UPLC HSS T3 C18 column using a water–acetonitrile mobile phase containing acetic acid and ammonium acetate. Bile acids were identified based on retention times and ion transitions matched to reference standards. Multivariate statistical analyses, including principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA), were conducted after unit variance scaling. Differential metabolites were screened using the criteria of variable importance in projection (VIP) > 1 and |log2FC| ≥ 1.

Statistical analysis

Data are expressed as mean ± SEM. Statistical analyses were performed using SPSS (version 26.0) and GraphPad Prism (version 8.0). Prior to statistical analysis, data were examined for normality and homogeneity of variance. Group differences were analyzed by one-way ANOVA, followed by Tukey’s multiple-comparison test. Statistical significance was set at P < 0.05.

Results

LC–MS/MS analysis of ACTE

Untargeted LC–MS/MS analysis was conducted to characterize the chemical composition of ACTE. Total ion chromatograms (TICs) recorded in both positive and negative ion modes are presented in Fig. 1a–b, revealing multiple well-resolved chromatographic peaks distributed across the retention time range, indicating the chemical complexity of ACTE.

Fig. 1.

LC–MS/MS analysis of the chemical constituents of ACTE. A, Positive ion mode. B, Negative ion mode.

A total of 43 compounds were tentatively characterized in ACTE through integrated analysis of accurate mass measurements, MS/MS fragmentation profiles, and spectral database comparisons (Table S2). These constituents mainly included organic acids, flavonoids, coumarins, terpenoids, chromones, and isocoumarins. Among them, organic acids accounted for the largest proportion (39.53%), followed by flavonoids (34.89%) and coumarins (16.28%), while chromones (4.65%), terpenoids (2.33%), and isocoumarins (2.33%) were present in smaller proportions. These findings suggest that the diverse phytochemical constituents of ACTE may contribute to its potential hepatoprotective and lipid-regulatory activities.

Production performance of laying hens

The HELP diet impaired production performance in laying hens. As shown in Table 2, hens fed the HELP diet exhibited a reduced laying rate and an increased FCR compared with the CON group (P < 0.05), indicating reduced laying efficiency and poorer feed utilization. ACTE supplementation attenuated these adverse effects, with the HELP-H group having a higher laying rate and lower FCR than the HELP group (P < 0.05). Neither ADFI nor egg weight differed among groups, suggesting that ACTE primarily improved feed utilization efficiency rather than increasing feed intake.

Table 2.

Effects of ACTE on production performance of laying hens.

Items	CON	HELP	HELP-L	HELP-M	HELP-H	SEM	P-value
Laying rate, %	83.65 ᵃ	58.41ᵈ	62.45ᶜᵈ	66.24 ᶜ	73.54 ᵇ	1.742	<0.001
Egg weight, g	63.35	60.67	61.68	62.57	63.20	0.498	0.418
ADFI, g	118.34	120.59	120.72	117.93	117.15	0.724	0.426
FCR	2.24ᶜ	3.42ᵃ	3.15ᵃᵇ	2.85ᵇ	2.53ᶜ	0.084	<0.001

Abbreviations: FCR: Feed conversion ratio, denotes the ratio of feed intake to egg weight. CON, control group fed a basal diet; HELP, model group fed a high-energy low-protein diet; HELP-L, HELP diet + 78 mg/kg ACTE; HELP-M, HELP diet + 156 mg/kg ACTE; HELP-H, HELP diet + 312 mg/kg ACTE (administered by oral gavage).

Data are presented as mean ± SEM (n = 6). Mean values within a row with different superscript letters indicate statistically significant differences (P < 0.05).

Analysis of liver morphology, lipid accumulation, and biochemical parameters in laying hens

Representative liver gross morphology and histological images are shown in Fig. 2. Compared with the CON group, hens fed the HELP diet exhibited typical fatty liver characteristics, including enlarged pale-yellow livers with apparent lipid deposition (Fig. 2a). ACTE supplementation attenuated these changes, with the HELP-H group showing the most pronounced improvement. Histological examination further confirmed these observations. H&E staining revealed severe hepatocellular vacuolation in the HELP group, indicating pronounced hepatic steatosis. In contrast, these pathological changes were reduced in ACTE-treated groups, particularly in the HELP-H group, where hepatocyte architecture appeared better preserved and lipid vacuolation was reduced (Fig. 2b). Consistently, Oil Red O staining showed extensive neutral lipid deposition in hepatocytes of the HELP group, whereas ACTE treatment reduced hepatic lipid accumulation (Fig. 2c).

Fig. 2.

Effects of ACTE on liver morphology and histopathology in laying hens. A, Gross liver morphology. B, Hematoxylin–eosin (H&E) staining. C, Oil Red O staining. Scale bars: 200 µm (upper panels) and 50 µm (lower panels).

Quantitative analysis further supported these findings (Table 3). Compared with the CON group, the HELP diet increased liver weight, liver index, abdominal fat weight, and abdominal fat index (P < 0.05). Hepatic TG and TC concentrations were also increased (P < 0.05). In addition, serum TG, TC, and LDL-C concentrations and AST and ALT activities were increased (P < 0.05), whereas HDL-C concentration was decreased (P < 0.05). Compared with the HELP group, ACTE supplementation attenuated these changes. Among the ACTE-treated groups, the improvement was generally dose-related, with the HELP-H group showing the most pronounced effects.

Table 3.

Effects of ACTE on hepatic lipid accumulation and serum biochemical indices in laying hens.

Items	CON	HELP	HELP-L	HELP-M	HELP-H	SEM	P-value
Body weight, kg	1.79d	2.22a	2.10ab	1.96bc	1.86cd	0.035	<0.001
Liver weight, g	33.94c	55.00a	47.80b	39.42c	35.85c	1.723	<0.001
Liver index, %	1.89b	2.48a	2.31a	2.01b	1.99b	0.059	0.002
Abdominal fat weight, g	40.05d	164.55a	107.75b	78.25c	64.21c	8.441	<0.001
Abdominal fat index, %	2.20d	7.27a	5.10b	3.98c	3.39c	0.340	0.022
Hepatic TG, mmol/g protein	0.15c	0.29a	0.27a	0.22b	0.16c	0.011	<0.001
Hepatic TC, mmol/g protein	0.45b	0.64a	0.58a	0.52ab	0.52ab	0.021	0.036
Serum TG, mmol/L	0.33c	0.64a	0.64a	0.52b	0.43b	0.026	<0.001
Serum TC, mmol/L	2.20d	5.57a	4.77b	4.44b	2.95c	0.244	<0.001
Serum LDL-C, mmol/L	0.96c	1.44a	1.32ab	1.12bc	1.12bc	0.048	0.006
Serum HDL-C, mmol/L	2.50d	1.59c	1.86bc	2.01c	2.16d	0.082	0.003
Serum AST, U/L	40.32bc	87.05a	78.42b	58.76ab	39.38c	3.761	<0.001
Serum ALT, U/L	2.31c	5.99a	4.91b	4.53b	2.75c	0.271	<0.001

Abbreviations: TG, triglycerides; TC, total cholesterol; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; AST, aspartate aminotransferase; ALT, alanine aminotransferase. CON, control group fed a basal diet; HELP, model group fed a high-energy low-protein diet; HELP-L, HELP diet supplemented with 78 mg/kg ACTE; HELP-M, HELP diet supplemented with 156 mg/kg ACTE; HELP-H, HELP diet supplemented with 312 mg/kg ACTE (administered by oral gavage).

Data are presented as mean ± SEM (n = 6). Mean values within the same row with different superscript letters indicate significant differences (P < 0.05).

Antioxidant capacity and inflammatory cytokines in laying hens

The HELP diet reduced antioxidant capacity and exacerbated oxidative stress and inflammatory responses in laying hens (Fig. 3). Relative to the CON group, hens fed the HELP diet showed decreased antioxidant indices in both serum and liver, increased levels of lipid peroxidation, and elevated concentrations of inflammatory cytokines (P < 0.05).

Fig. 3.

Effects of ACTE on oxidative stress– and inflammation-related parameters in the serum and liver of HELP-fed laying hens. A, Serum SOD activity. B, Serum T-AOC activity. C, Serum GSH-Px activity. D, Serum CAT activity. E, Serum MDA content. F, Serum TNF-α content. G, Serum IL-1β content. H, Serum IL-6 content. I, Hepatic SOD activity. J, Hepatic T-AOC activity. K, Hepatic CAT activity. L, Hepatic GSH-Px activity. M, Hepatic MDA content. N, Hepatic TNF-α content. O, Hepatic IL-1β content. P, Hepatic IL-6 content. Values are presented as mean ± SEM (n = 6). Data were analyzed by one-way ANOVA followed by Tukey's test. *P < 0.05, **P < 0.01, ***P < 0.001.

Compared with the HELP group, ACTE supplementation increased antioxidant enzyme activities and reduced lipid peroxidation in laying hens. In serum, ACTE supplementation increased the activities of SOD, T-AOC, GSH-Px, and CAT and reduced MDA concentrations (P < 0.05, Fig. 3a–e). Among the ACTE supplementation groups, a dose-dependent trend was observed, with the HELP-H group showing the highest antioxidant enzyme activities and the lowest MDA concentrations (P < 0.01). With respect to inflammatory parameters, ACTE supplementation reduced serum IL-1β concentration (P < 0.05), whereas serum IL-6 and TNF-α levels did not differ among groups (Fig. 3f–h). Consistent with the serum results, compared with the HELP group, ACTE supplementation increased hepatic SOD, T-AOC, CAT, and GSH-Px activities and reduced hepatic MDA concentrations (P < 0.05, Fig. 3i–m). In addition, hepatic TNF-α, IL-1β, and IL-6 concentrations were lower in the HELP-H group than in the HELP group following ACTE supplementation (P < 0.05, Fig. 3n–p).

Expression of lipid and bile acid metabolism–related genes in laying hens

Relative to the CON group, hens fed the HELP diet showed elevated hepatic expression of genes involved in lipogenesis, including ACC, SCD1, FASN, SREBP-1c, and liver X receptor alpha (LXRα) (P < 0.05, Fig. 4a). ACTE supplementation reduced the expression of these lipogenic genes (P < 0.05), with a dose-responsive effect observed among ACTE supplementation groups; the HELP-H group showed the lowest expression levels (Fig. 4a).

Fig. 4.

Effects of ACTE on the expression of genes related to hepatic lipogenesis, fatty acid oxidation, and gut–liver bile acid metabolism in laying hens with FLHS. A, Hepatic expression of genes associated with de novo lipogenesis. B, Hepatic expression of genes involved in fatty acid oxidation and lipid transport. C, Expression of bile acid metabolism–related genes in the ileum and liver. Relative gene expression levels were normalized to β-actin. Values are presented as mean ± SEM (n = 6). Data were analyzed by one-way ANOVA followed by Tukey's multiple-comparison test. *P < 0.05, **P < 0.01, ***P < 0.001.

Among genes associated with fatty acid oxidation and transport, the HELP diet reduced the expression of peroxisome proliferator-activated receptor α (PPARα) and acyl-CoA oxidase 1 (ACOX) (P < 0.05), whereas the expression of FXR and peroxisome proliferator-activated receptor γ (PPARγ) increased (P < 0.05, Fig. 4b). Following ACTE supplementation, the expression levels of PPARα increased (P < 0.05), whereas the HELP diet–induced upregulation of hepatic FXR was reduced (P < 0.05), with expression levels partially returning toward those observed in the CON group. Among the ACTE supplementation groups, these effects showed a graded response.

With respect to bile acid metabolism–related genes, the HELP diet increased the expression of ileal FXR, FGF19, and ileal bile acid-binding protein (IBABP), whereas it reduced hepatic cholesterol 7α-hydroxylase (CYP7A1) expression (P < 0.05, Fig. 4c). In contrast, the expression of apical sodium-dependent bile acid transporter (ASBT) did not differ among groups. Among the ACTE supplementation groups, the effects progressively improved with increasing dose, with the most evident changes observed in the HELP-H group, in which ileal FXR and FGF19 expression decreased and hepatic CYP7A1 expression increased toward levels observed in the CON group (P < 0.05).

Gut microbial diversity and community structure in laying hens

The HELP diet reduced ileal microbial α-diversity indices, with a decrease in the richness index (P = 0.039). Although the Chao1, Simpson, and Shannon indices did not differ among groups (P > 0.05), lower values were detected in the HELP group (Fig. 5a–d). Following ACTE supplementation, microbial richness was higher in the HELP-H group (Fig. 5b). β-diversity analysis revealed separation in microbial community structure among the three groups (Fig. 5e–f). The HELP diet altered the ileal microbial community composition, whereas ACTE supplementation shifted the microbial structure toward that of the CON group. At the phylum level, Firmicutes constituted the predominant phylum in the ileal microbiota of all three groups. Relative to the CON group, the HELP group showed broadly similar phylum-level profiles, whereas the HELP-H group showed a slight enrichment of several less abundant phyla (Fig. 5i). Genus-level analysis further revealed distinct differences among groups (Fig. 5h). In the CON group, Lactobacillus was the dominant genus, whereas Romboutsia accounted for a relatively small proportion of the microbial community. In the HELP group, the relative abundance of Lactobacillus decreased, whereas the abundances of Romboutsia, Turicibacter, Clostridium sensu stricto 1, and Gallibacterium increased, indicating pronounced alterations in the intestinal microbial community. Following ACTE supplementation, the microbial community exhibited partial recovery, with the overall distribution pattern positioned between those of the CON and HELP groups. LEfSe analysis (LDA > 4) further identified differentially abundant genera among the groups (Fig. 5g). The CON group was characterized by enrichment of lactobacilli-related taxa, including Lactobacillus and its higher taxonomic lineages. In contrast, the HELP group showed enrichment of several taxa associated with microbial dysbiosis, such as Clostridium sensu stricto 1, Turicibacter, and Clostridium. Notably, the HELP-H group was enriched in Bacteroides and taxa affiliated with the Lachnospiraceae family.

Fig. 5.

Effects of ACTE on ileal microbial diversity and community structure in laying hens. A-D, Alpha diversity indices of the ileal microbiota, including Chao1, Richness, Shannon, and Simpson, with statistical significance assessed using the Kruskal–Wallis test. E and F, Beta diversity analysis based on Bray–Curtis and Jaccard distance matrices, visualized using principal coordinates analysis (PCoA). G, Linear discriminant analysis effect size (LEfSe) analysis showing differentially enriched taxa among groups (LDA score > 4). H, Relative abundance of dominant bacterial genera. I, Relative abundance of dominant bacterial phyla.

Ileal bile acid profiles in laying hens

Bile acid metabolomic profiling indicated that the HELP diet modified the composition of the ileal bile acid pool, whereas ACTE supplementation partially normalized the bile acid profile. Relative abundance analysis showed that bile acid profiles in the CON and HELP-H groups were compositionally similar, mainly consisting of taurochenodeoxycholic acid (TCDCA), CDCA, cholic acid (CA), and taurocholic acid (TCA), with primary and conjugated bile acids predominating (Fig. 6a). In contrast, the HELP diet altered bile acid composition, characterized by an increase in TCDCA and lower relative abundances of several primary bile acids, including CA, CDCA, and TCA. Multivariate analysis further confirmed these alterations. PCA showed clear separation among the three groups, with the HELP group deviating markedly from the CON group, whereas the HELP-H group clustered closer to the CON group (Fig. 6b). Similar discrimination was observed in the OPLS-DA analysis (Fig. 6c), indicating systematic differences in bile acid metabolic profiles. Differential metabolite analysis revealed that the HELP diet increased several secondary and conjugated bile acids, including deoxycholic acid (DCA), taurodeoxycholic acid (TDCA), and tauroursodeoxycholic acid (TUDCA), whereas it reduced certain microbiota-related bile acids such as ILCA and THCA. ACTE supplementation shifted these bile acid profiles by increasing specific bile acids, including isolithocholic acid (ILCA) and isoallolithocholic acid (IALCA), and reducing several secondary bile acids. Hierarchical clustering analysis further revealed that the bile acid profile of the HELP-H group was positioned between those of the CON and HELP groups (Fig. 6f).

Fig. 6.

Effects of ACTE on ileal bile acid metabolomic profiles in laying hens. A, Relative abundance of major bile acid classes among the three groups. B, Principal component analysis (PCA) score plot showing the distribution of bile acid metabolic profiles among groups. C, Orthogonal partial least squares discriminant analysis (OPLS-DA) score plot illustrating the separation among the CON, HELP, and HELP-H groups. Model parameters: R²X = 0.604, R²Y = 0.976, Q² = 0.674 (Q², p = 0.04). D, Volcano plot showing differentially abundant bile acids between the HELP and CON groups. E, Volcano plot showing differentially abundant bile acids between the HELP-H and HELP groups. F, Hierarchical clustering heatmap of representative bile acids, with colors indicating relative metabolite abundance. Each point in the PCA and OPLS-DA plots represents one biological replicate. In the heatmap, red indicates higher abundance and green indicates lower abundance.

Correlation analysis between bile acids and gut microbiota

To investigate the relationship between bile acids and the gut microbiota, redundancy analysis (RDA) was performed using bile acids as explanatory variables and major bacterial genera as response variables. The results indicated that bile acid composition accounted for part of the variation in microbial community structure (Fig. 7a). Among the bile acids, glycochenodeoxycholic acid (GCDCA), cholic acid-3-sulfate (CA-3S), ursodeoxycholic acid (UDCA), and lithocholic acid (LCA) exhibited relatively long vectors, indicating relatively strong associations with microbial distribution patterns. At the microbial level, Lactobacillus and Romboutsia were positively correlated with bile acids such as UDCA and CA-3S, whereas several dysbiosis-related taxa were correlated with secondary and conjugated bile acids including LCA and TDCA.

Fig. 7.

Integrated analysis of bile acids and gut microbiota. A, Redundancy analysis (RDA) showing the relationships between bile acid composition and the gut microbial community. Arrows represent bile acid variables, and their directions indicate correlations with major bacterial genera. B, Spearman correlation–based hierarchical clustering heatmap illustrating the associations between differentially abundant bile acids and dominant bacterial genera. Red indicates positive correlations, whereas blue indicates negative correlations. Asterisks indicate significant correlations (*P < 0.05, **P < 0.01). The columns represent differentially abundant bile acids, and the rows represent differential bacterial genera.

Spearman correlation analysis further supported these patterns (Fig. 7b). Secondary and conjugated bile acids, including TDCA, 12-dehydrocholic acid (12-DHCA), and LCA, were positively correlated with genera such as Negativibacillus and Clostridium sensu stricto 1, but negatively correlated with Lactobacillus. In contrast, primary bile acids including CA, CDCA, and UDCA were positively correlated with beneficial genera such as Lactobacillus and Leuconostoc. These results indicate close associations between bile acid composition and intestinal microbial community structure.

Discussion

This study systematically evaluated the protective effects of ACTE against HELP diet–induced FLHS in laying hens through multiple levels of evidence, including production performance, hepatic phenotypes, biochemical parameters, and gut–liver–related metabolic pathways. The HELP diet effectively induced characteristic manifestations of FLHS in laying hens, including excessive hepatic lipid deposition, compromised liver function, reduced antioxidant capacity, and disruption of the gut–bile acid axis. These findings are consistent with recent reports in laying hen FLHS models and diet-induced fatty liver models (Lv et al., 2024; Sun et al., 2025b). Accumulating evidence indicates that energy–protein imbalance promotes hepatic lipogenesis, suppresses fatty acid oxidation, and disrupts bile acid metabolism. This imbalance is therefore considered a critical driver in the development of FLHS (Chu et al., 2024; Sun et al., 2025a). On the basis of this stable and reproducible model, this study aimed to clarify the therapeutic efficacy and potential mechanisms of ACTE, providing experimental support for its potential application as a sustainable nutritional strategy for the prevention and mitigation of FLHS.

Our results demonstrated that ACTE markedly reduced HELP diet–induced hepatic TG and TC accumulation, restored serum lipid profiles, and improved hepatic histological architecture. These findings are consistent with previous studies reporting the protective effects of A. capillaris or its bioactive constituents against NAFLD and hepatic steatosis (Hong et al., 2004; Xu et al., 2024). The improvements in metabolic status and liver phenotypes were accompanied by improved production performance. Compared with the HELP group, ACTE-treated hens exhibited a higher laying rate and a lower FCR, with the greatest improvements observed in the HELP-H group. Egg weight did not differ among groups, indicating that ACTE improved production performance primarily by enhancing laying efficiency and feed utilization rather than altering individual egg weight. This observation may be associated with hepatic metabolic regulation. Given the central role of the liver in yolk precursor synthesis, improvements in hepatic lipid metabolism may facilitate the production and secretion of key precursors such as very low-density lipoprotein (VLDL) and vitellogenin (VTG), thereby supporting a higher laying rate. These findings support the practical value of ACTE supplementation and are consistent with its effects on attenuating hepatic lipid deposition while improving serum lipid parameters and liver function indices (You et al., 2023). Collectively, these observations suggest that improved hepatic lipid homeostasis is an important metabolic basis for the observed improvements in production performance (Rozenboim et al., 2016).

The present findings further indicate that ACTE reduced the expression of lipogenic genes, including SREBP-1c, FASN, ACC, and SCD1, while increasing the expression of genes involved in fatty acid oxidation, such as PPARα and ACOX. These regulatory patterns are consistent with previously reported mechanisms by which chlorogenic acid and related constituents modulate lipid metabolism through the AMPK and PI3K–AKT signaling pathways (Jang et al., 2014; Liang et al., 2022). Nevertheless, the specific upstream signaling events and molecular targets involved in ACTE-mediated regulation of hepatic lipid metabolism remain to be elucidated and warrant further investigation. In addition, the HELP diet led to downregulation of CYP7A1 expression, whereas the restorative effect of ACTE suggests improved bile acid synthetic capacity and enhanced cholesterol conversion into the bile acid biosynthetic pathway. Previous studies indicate that plant-derived constituents can regulate bile acid and lipid metabolic homeostasis through pathways involving FXR, LXR, and AMPK (Cai et al., 2018; Zhao et al., 2024b), which is consistent with the improved hepatic metabolic status observed in the present study. When considered together with the alterations in the ileal FXR–FGF19 axis and the ileal bile acid profile identified herein, these findings suggest that ACTE may participate in metabolic remodeling by modulating the classical “bile acid–ileal FXR–FGF15/19–hepatic CYP7A1” gut–liver bile acid negative feedback loop. This mechanism may contribute to the regulation of hepatic bile acid synthesis and lipid homeostasis (Kim et al., 2007; Modica et al., 2012). Collectively, the hepatoprotective effects of ACTE can be attributed to the coordinated regulation of lipogenesis, fatty acid oxidation, and bile acid synthesis through multiple interconnected pathways.

Previous studies have shown that a HELP diet induces oxidative stress and inflammatory responses in laying hens, thereby promoting the progression of fatty liver hemorrhagic syndrome (Zhao et al., 2024a; Guo et al., 2025). In the present study, hens in the HELP group exhibited reduced SOD, GSH-Px, and CAT activities and lower T-AOC, accompanied by elevated MDA concentrations, indicating impaired endogenous antioxidant defenses and enhanced lipid peroxidation (Tauil et al., 2024; Niu et al., 2025). ACTE increased antioxidant capacity while lowering malondialdehyde concentrations, in agreement with earlier findings showing that plant-derived extracts attenuate inflammatory and oxidative damage (Jang et al., 2014; Xue et al., 2024; Guo et al., 2026). In addition, the HELP diet increased inflammatory cytokine concentrations, whereas ACTE supplementation partly suppressed these inflammatory responses, particularly in the liver. Growing evidence suggests that A. capillaris mitigates hepatic inflammation by suppressing major inflammatory signaling pathways, including NF-κB, STAT3, and the NLRP3 inflammasome (Han et al., 2023), which further supports the combined anti-inflammatory and antioxidant effects observed in this study. Notably, these effects of ACTE may help attenuate the pathological cascade of lipid accumulation, inflammation, and oxidative damage, thereby contributing to the alleviation of FLHS progression. Imbalance of the gut microbiota has been considered a key contributor to FLHS development and is closely associated with alterations in bile acid metabolism (Albillos et al., 2020; He et al., 2026). Based on the dose-gradient design, three representative groups with distinct phenotypes were selected for further analyses: CON as the physiological baseline, HELP as the disease model, and HELP-H as the high-dose ACTE supplementation group. These groups were used for 16S rRNA sequencing and bile acid metabolomics. This targeted grouping strategy allowed focused assessment of key alterations in the gut microbiota and bile acid profiles during the transition from disease to recovery, while also improving the ability to detect biologically meaningful associations. Our results showed that the HELP group exhibited a reduction in gut microbial α-diversity, accompanied by a decreased relative abundance of Lactobacillus and increased abundances of Romboutsia and Clostridium sensu stricto 1. These changes indicate that the ileal microbiota shifted from a homeostasis-associated community toward a dysbiosis-related microbial profile. Such alterations have been frequently reported in models of metabolic liver disease or energy surplus and are often associated with elevated inflammatory status and disruption of metabolic homeostasis (Dai et al., 2022). In the HELP-H group, ACTE supplementation partially restored the gut microbiota, characterized by an increased abundance of homeostasis-associated genera such as Lactobacillus and a concomitant suppression of several taxa enriched by the HELP diet. Importantly, Lactobacillus species commonly exhibit bile salt hydrolase (BSH) activity, which allows them to deconjugate bile acids and consequently influence the intestinal bile acid composition (Zhou et al., 2022; Xue et al., 2024). Bile acid metabolomic analysis further demonstrated that the HELP diet resulted in reduced concentrations of primary bile acids, including CA and CDCA, and increased concentrations of secondary and conjugated bile acids, including TDCA, 12-DHCA, and LCA. This bile acid disturbance pattern is consistent with those reported in previous studies on poultry FLHS and mammalian fatty liver disease models (He et al., 2026). ACTE supplementation increased the levels of certain primary or specific bile acids while reducing multiple secondary bile acids, particularly those associated with inflammatory and cytotoxic risks, such as LCA and TDCA (Calzadilla et al., 2022; Camilleri, 2022). Collectively, the present data indicate that ACTE facilitates the re-establishment of gut–liver bile acid homeostasis through modulation of the bile acid profile (Gillard and Leclercq, 2023). Earlier studies indicate that primary bile acids modulate hepatic lipid metabolism via the FXR–FGF15/19–CYP7A1 signaling axis, whereas excessive accumulation of secondary bile acids compromises intestinal barrier integrity and amplifies inflammatory responses (Tang et al., 2024; Wang et al., 2024c), which is consistent with the patterns observed in the present study.

The findings of this study indicate that ACTE exerts protective effects against HELP diet–induced FLHS, likely involving restoration of the gut microbiota–bile acid–liver axis. ACTE increased the abundance of Lactobacillus, a genus commonly possessing bile salt hydrolase activity, while reducing taxa enriched under HELP feeding, including Clostridium sensu stricto 1 and Negativibacillus. These microbial shifts suggest that ACTE may improve the intestinal microenvironment for bile acid biotransformation (Xu et al., 2023; Sun et al., 2024). Partial restoration of the microbial community was accompanied by a shift in the bile acid pool from a HELP diet–induced pattern enriched in secondary and conjugated bile acids toward a profile closer to the physiological baseline. In the HELP-H group, concentrations of TDCA, LCA, and 12-DHCA were reduced, whereas CA, CDCA, and several less hydrophobic bile acids, such as UDCA and CA-3S, showed recovery toward physiological levels (Reichardt et al., 2025; Yan et al., 2025). This coordinated remodeling of the gut microbiota and bile acid composition further supports a role of ACTE in re-establishing gut–liver bile acid homeostasis during FLHS. Rebalancing of the bile acid profile may alleviate bile acid–associated epithelial stress and promote the restoration of beneficial genera such as Lactobacillus (Stenman et al., 2013; Foley et al., 2023). ACTE-induced alterations in bile acid composition were accompanied by partial restoration of the ileal FXR–FGF19 axis and hepatic CYP7A1 expression, suggesting the involvement of the gut–liver bile acid negative feedback loop. This mechanism may subsequently influence hepatic lipid metabolism and inflammatory status (Hsu and Schnabl, 2023). Spearman correlation and RDA analyses revealed that primary bile acids enriched by ACTE were positively correlated with genera such as Lactobacillus, whereas secondary and conjugated bile acids accumulated under HELP feeding were mainly associated with Clostridium sensu stricto 1, Negativibacillus, and other model-associated taxa. These findings suggest that ACTE is associated with metabolic coupling between intestinal microbes and bile acid metabolism, which represents a key mechanistic component underlying its protective effects against FLHS (Larabi et al., 2023; Wahlström et al., 2024).

Overall, ACTE does not appear to act independently on either the gut microbiota or bile acids. Instead, it appears to coordinately regulate multiple components, including microbial community structure, bile acid pool composition, the ileal FXR–FGF19 axis, and hepatic lipid metabolism. The pathological process induced by the HELP diet is characterized by gut microbiota dysbiosis and bile acid disturbances, along with the accumulation of potentially deleterious secondary bile acids. These alterations may impair the gut–liver bile acid feedback loop (FXR–FGF19–CYP7A1), thereby contributing to hepatic lipid metabolic dysregulation and increased inflammatory and oxidative stress responses. This multi-node regulatory model is consistent with emerging evidence showing that plant-derived extracts can ameliorate metabolic liver diseases by remodeling the gut microbiota–bile acid–liver axis (Tang et al., 2023; Asar et al., 2024). Importantly, the present study extends this concept to a laying hen FLHS model and provides evidence that ACTE exerts protective effects by partially restoring the microbiota–bile acid–liver network. These findings provide new insights and practical directions for developing nutritional strategies and functional feed additives for the prevention and mitigation of FLHS.

Conclusions

The present study demonstrates that ACTE mitigates FLHS caused by the HELP diet in laying hens. ACTE supplementation at doses equivalent to 0.5–2.0 g crude A. capillaris/kg body weight for 15 wk improved laying performance, alleviated hepatic lipid deposition and liver injury, and reduced oxidative stress and inflammatory responses. These effects were accompanied by decreased expression of lipogenic genes, enhanced fatty acid oxidation, and improved gut–liver bile acid homeostasis. In addition, ACTE modulated the ileal microbial community and improved bile acid profiles, suggesting involvement of the FXR–FGF19–CYP7A1 signaling axis. Collectively, these findings indicate that ACTE may serve as a promising nutritional strategy for preventing and mitigating FLHS in laying hens (Fig. 8).

Fig. 8.

Schematic illustration of the mechanisms by which ACTE alleviates HELP diet–induced FLHS in laying hens through modulation of the gut microbiota–bile acid–liver axis.

CRediT authorship contribution statement

Miao Chen: Writing – original draft, Visualization, Investigation, Data curation. Shuhui Liu: Visualization, Investigation. Chen Xu: Data curation. Binglin Wang: Data curation. Shuqi Zhang: Supervision, Conceptualization. Wenkai Xie: Methodology. Wenhui Yu: Writing – review & editing. Xiaowen Jiang: Writing – review & editing, Methodology.

Disclosures

The authors have no conflicts of interest to declare.