
==== Front
Poult Sci
Poult Sci
Poultry Science
0032-5791
1525-3171
Elsevier

S0032-5791(24)00783-1
10.1016/j.psj.2024.104204
104204
METABOLISM AND NUTRITION
Epigallocatechin gallate improves oleic acid-induced hepatic steatosis in laying hen hepatocytes via the MAPK pathway
Zhu Yifeng *
Zhao Xiyu †‡
Li Xinyan †‡
Hu Chengfang †‡
Zhang Yao †‡
Yin Huadong yinhuadong@sicau.edu.cn
†‡1
⁎ Institute of Animal Nutrition, Key Laboratory for Animal Disease-Resistance Nutrition of China, Ministry of Education, Sichuan Agricultural University, Chengdu 611130, Sichuan, China
† Key Laboratory of Livestock and Poultry Multi-omics, Ministry of Agriculture and Rural Affairs, College of Animal Science and Technology, Sichuan Agricultural University, Chengdu 611130, Sichuan, China
‡ Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu 611130, Sichuan, China
1 Corresponding author: yinhuadong@sicau.edu.cn
10 8 2024
11 2024
10 8 2024
103 11 10420422 5 2024
6 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Fatty liver disease in laying hens, characterized by excessive lipid accumulation in hepatocytes, poses significant challenges to poultry health and production efficiency. In this study, we investigated the therapeutic potential of epigallocatechin gallate (EGCG), a bioactive compound found in green tea, in mitigating oleic acid (OA)-induced hepatic steatosis in primary chicken hepatocytes. Treatment with EGCG effectively attenuated lipid deposition by downregulating lipid synthesis-related genes. Moreover, EGCG mitigated oxidative stress, inflammation, DNA damage, and apoptosis induced by OA, thereby preserving hepatocyte viability. Mechanistically, EGCG exerted its protective effects by modulating the p38 MAPK signaling pathway. Our findings suggest that EGCG holds promise as a therapeutic agent for managing fatty liver disease in poultry, offering insights into novel strategies for improving poultry health and production outcomes.

Key words

EGCG
OA
hepatic steatosis
lipid deposition
p38 MAPK pathway
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pmcINTRODUCTION

Hepatic lipidosis adversely affects liver function, laying performance, and egg quality, thereby impacting the economic sustainability of egg production (Cherian and Hayat, 2009; Van Elswyk, et al., 1994; Zhang, et al., 2008). The liver plays a crucial role in lipid metabolism, including synthesis, storage, and secretion of lipids (Rao, et al., 2023; Zhou, et al., 2023). When lipid metabolism is dysregulated, excessive accumulation of lipids occurs within hepatocytes, leading to hepatic lipidosis (Chu, et al., 2024). This condition not only compromises liver function but also predisposes laying hens to a range of health problems, including decreased egg production, poor egg quality, and increased susceptibility to metabolic disorders (Izuddin, et al., 2023).

OA, a predominant dietary fatty acid in poultry feed (Ali, et al., 2024), has been implicated in the development of hepatic lipidosis in laying hens. Excessive intake of OA disrupts lipid metabolism in the liver, leading to an imbalance between lipid synthesis and clearance. Moreover, OA can induce oxidative stress and inflammation in hepatocytes, further exacerbating lipid accumulation and hepatocellular damage (Song, et al., 2023).

Recent years have witnessed a surge in research exploring natural compounds as alternatives to traditional pharmacological interventions in animal husbandry (Gao, et al., 2023). EGCG, a bioactive polyphenol found abundantly in green tea, have emerged as potential therapeutic agents for mitigating hepatic lipidosis (Sahadevan, et al., 2023). Due to its antioxidative, anti-inflammatory, and lipid-reducing effects, EGCG stands out as a promising candidate for alleviating hepatic lipidosis in chickens (He, et al., 2023; Li, et al., 2024; Luo, et al., 2023). By targeting various signaling pathways involved in lipid synthesis, oxidation, and storage, EGCG may offer a multifaceted approach to counteract the deleterious effects of excessive lipid accumulation in hepatocytes (Wu, et al., 2023). For instance, the MAPK pathway, which is involved in regulating lipid metabolism and inflammatory responses, could be modulated by EGCG to reduce lipid accumulation and mitigate liver damage (Lawan and Bennett, 2017).

Studies in mammalian models have suggested that EGCG may hold promise in ameliorating hepatic lipidosis (Cahyani, et al., 2021; Meng, et al., 2023). However, the specific molecular mechanisms underlying its effects in laying hens remain inadequately explored. To address this gap in knowledge, we utilized an in vitro model in which primary liver cells isolated from laying hens were subjected to OA-induced lipid accumulation to mimic hepatic lipidosis. Subsequently, these cells were treated with EGCG to assess its effects on lipid accumulation and hepatic function. Understanding the molecular mechanisms underlying the therapeutic effects of EGCG is essential for developing targeted interventions to alleviate hepatic lipidosis in laying hens. By elucidating these mechanisms, we aim to provide valuable insights into the potential of EGCG for improving liver health which may have broader implications for treating liver conditions.

MATERIALS AND METHOD

Isolation and Culture of Primary Chicken Hepatocytes

We followed the method of Jing et al. (2023) to isolate primary chicken hepatocytes. In simple terms, Roman Chickens aged 22 to 25 wk were used. Subsequently, the abdominal cavity was opened and the whole liver was collected. The liver was first perfused for 15 min with 1,000 mL free-calcium HEPES buffer (pH 7.5), then washed with buffer containing calcium chloride for an additional 15 min. After perfusion, hepatocytes were isolated by type II collagenase (Biofroxx, Einhausen, Germany) and filtration. In collagen-coated dishes, cells were cultured in DMEM (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS, Gibco) and antibiotics (Solarbio, Beijing, China). Cultures were maintained at 37°C, 5% CO2, with medium changes every 2 d. Sichuan Agricultural University's Animal Welfare Committee approved the feeding and treatment of chickens (Approval number: 2023102023).

Cell Treatment

Primary chicken hepatocytes were seeded onto appropriate cell culture plates and cultured for 24 h. Afterward, the cells were treated with gradient concentrations (0, 0.2, 0.4, 0.6, 0.8, 1 mM) of OA (Sigma, St. Louis, MO) for 24 h. Following the OA treatment, gradient concentrations (0, 0.1, 0.5, 1, 1.5, 2 μM) of EGCG (Yuanye, Shanghai, China) were applied to the cells for an additional 24 h. Optimal concentrations of EGCG and OA for subsequent experiments were determined through screening. EGCG was dissolved in water, while OA was initially dissolved in dimethyl sulfoxide (DMSO, Solarbio) to prepare a 100 mM stock solution. This stock solution was then appropriately diluted in the cell culture medium to achieve the required treatment concentrations.

Cell Viability Assay

Primary chicken hepatocytes were seeded into 96-well cell culture plates. Cell viability was assessed using the Cell Counting Kit-8 (CCK-8, Meilunbio, Dalian, Liaoning, China). Following the treatment period, cells were incubated with CCK-8 solution (10% v/v in culture medium) for 2 h at 37°C. Absorbance was measured at 450 nm using a microplate reader (Thermo Fisher, Waltham, MA).

Quantitative Real-Time PCR Analysis

A commercial RNA extraction kit (FOREGENE, Chengdu, Sichuan, China) was used to extract total RNA from treated cells. The RNA concentration and purity were measured with a spectrophotometer (Thermo Fisher), and RNA integrity was verified by gel electrophoresis. The isolated RNA was converted into complementary DNA (cDNA) utilizing a reverse transcription kit (TaKaRa, Kusatsu, Shiga, Japan) according to the provided instructions. Gene expression analysis was carried out by Quantitative real-time PCR (qPCR) with gene-specific primers and SYBR Green Master Mix (TaKaRa). PCR amplification was conducted on a real-time PCR system (Biorad, Hercules, CA) with the following protocol: an initial denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 15 seconds, annealing at the specified temperature for 30 seconds, and extension at 72°C for 30 seconds. The relative gene expression levels were determined using the 2−ΔΔCt method with β-actin serving as the internal reference. The primers used are listed in Supplementary Table S1.

Western Blot Analysis

Total protein was extracted from hepatocytes using RIPA buffer (Solarbio) with added protease inhibitors. The cell lysates were centrifuged at 12,000 rpm for 15 min at 4°C, and the supernatant was collected. Protein concentration was measured using the BCA assay (Bestbio, Nanjing, Jiangsu, China). Proteins were then separated on a 12% SDS-polyacrylamide gel and transferred onto PVDF membranes (Beyotime, Shanghai, China). The membranes were blocked at room temperature for 1 h with blocking buffer (Beyotime) and then incubated overnight at 4°C with primary antibody. This was followed by a 1-h incubation at room temperature with an HRP-conjugated secondary antibody. Detailed antibody information is provided in Supplementary Table 2. Bands were visualized using enhanced chemiluminescence (ECL, Beyotime) and detected with the ChemiDoc system (Biorad). Densitometry analysis to quantify band intensity was performed using Image J software. A complete list of the antibodies used can be found in Supplementary Table S2.

Flow Cytometry Analysis

In this study, cells were plated in 6-well plates. After 24 h of treatment with EGCG or OA, cells were harvested via trypsin digestion. ROS levels were determined using 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA, Thermo Fisher). Post-treatment, cells were incubated with 10 μM DCF-DA for 30 min at 37°C, and fluorescence intensity was measured using CytExpert flow cytometry (Beckman, Brea, CA). Mitochondrial membrane potential (MMP) was evaluated with the JC-1 fluorescent probe (Invitrogen, Carlsbad, CA). Cells treated were incubated with 100 nM JC-1 for 15 min at 37°C, then analyzed with CytoFLEX flow cytometry (Beckman). Apoptosis detection was performed using Annexin V-FITC/propidium iodide (PI) staining. After treatment, cells were stained with Annexin V-FITC and PI according to the manufacturer's instructions and analyzed using CytoFLEX flow cytometry. Data analysis was conducted using Kaluza 2.1 software.

Oil Red O Staining

After the treatment period, cells were fixed in 4% paraformaldehyde for 30 min at room temperature. The fixed cells were rinsed with phosphate-buffered saline (PBS) and then stained with Oil Red O solution (Solarbio) for 30 min at room temperature. After staining, the cells were washed again with PBS to remove any excess dye. The stained cells were examined under a light microscope (Olympus, Tokyo, Japan), and images were taken to visualize the lipid droplets. Lipid droplet quantification was carried out using Image J software, by measuring the intensity of the stained cells in the images.

Enzyme-Linked Immunosorbent Assay

After the treatment period, cell levels of triglyceride (TG), total cholesterol (TC), malondialdehyde (MDA), catalase (CAT), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), interleukin 1 beta (IL-1β), and interleukin 6 (IL-6) were assessed using Enzyme-linked immunosorbent assay (ELISA) kits (R&D, Minneapolis, MN) following the manufacturer's guidelines. Cell culture supernatants were collected and centrifuged to eliminate debris. The supernatants were added to ELISA plates precoated with specific primary antibodies and incubated. After washing, HRP-conjugated secondary antibodies were added and incubated. Another wash was followed by the addition of the substrate solution, which was incubated for a designated time. The reaction was halted, and absorbance was read at a specific wavelength using a microplate reader (Biorad).

Immunofluorescence

Following treatment, cells were fixed with paraformaldehyde and permeabilized with Triton X-100 (Beyotime). After blocking with an appropriate solution (Beyotime), cells were exposed to the γ-H2AX antibody. Next, they were treated with a fluorescently labeled secondary antibody. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Immunofluorescently labeled cells were observed using a fluorescence microscope (Olympus). Images were captured at suitable magnifications to observe the subcellular localization and expression levels of γ-H2AX. Quantitative analysis of fluorescence intensity and colocalization studies were performed using Image pro plus software.

Transcriptome Sequencing Analysis

The RNA samples from NC group, EGCG group, OA group, and OA+EGCG group were submitted to Shanghai Majorbio Technology Co., LTD for library construction, sequencing and bioinformatics analysis in strict accordance with the company's standard operating procedures, which are available online (https://www.majorbio.com/). The raw sequencing reads were trimmed and subjected to quality control using SeqPrep and Sickle software. Subsequently, the processed clean reads were aligned to the Gallus gallus reference genome (version 5.0) using HISAT2. Transcript expression levels were quantified using the transcripts per million (TPM) method. Differential gene expression analysis was conducted with DESeq2 software, applying criteria of a fold change greater than 1.2 and a P-value less than 0.05 for significance.

Statistical Analysis

The results were presented as mean ± standard error (SEM) and visualized using GraphPad Prism software. Statistical analyses were conducted using SPSS 20 software. Student's t-test was employed for parametric data, and nonparametric data were analyzed using nonparametric tests for comparisons between 2 groups. One-way analysis of variance (ANOVA) followed by Tukey's test was utilized for comparisons among multiple groups. Significance levels were denoted as **P < 0.01, *P < 0.05, and lowercase letters (a, b, c) for P < 0.05.

RESULTS

Influence of Various Concentrations of EGCG on Lipid Synthesis and Transport in Chicken Hepatocytes

In this experiment, 5 gradient concentrations of EGCG were selected for treatment on primary chicken hepatocytes. The results indicated that different concentrations of EGCG can effectively reduce the mRNA expression of hepatic cell lipid synthesis-related genes, including ACACA, FASH, PPARγ and SREBP1, to varying degrees (Figures 1A-D, P < 0.05). Moreover, as the concentration of EGCG increases, the inhibitory effect on lipid synthesis-related gene expression first intensifies and then diminishes. Concurrently, the impact of EGCG on the mRNA expression levels of hepatic cell lipid transport-related genes, including AopA1, ApoVLDV-II, and CPT1, manifests similar trends (Figures 1E-G, P < 0.05). Overall, 1.0 μM EGCG demonstrated optimal efficacy in reducing the expression levels of genes related to lipid synthesis and transport in chicken hepatocytes. Additionally, through CCK-8 assays analyzing the influence of different EGCG concentrations on cell viability, the results suggested that lower concentrations of EGCG can enhance hepatocyte activity, whereas concentrations exceeding 1.0μM significantly reduce cell viability (Figure 1H, P < 0.05). In summary, 1.0 μM EGCG emerged as the optimal treatment concentration in this experiment. Hence, we had chosen this concentration for subsequent experiments.Figure 1 Influence of various concentrations of EGCG on Lipid synthesis and transport in chicken hepatocytes. (A-D) Relative mRNA expression levels of lipid synthesis-related genes ACACA, FASH, PPARγ and SREBP1. (E-G) Relative mRNA expression levels of lipid transport-related genes ApoA1, ApoVLDL-II and CPT1. (H) CCK-8 assay for assessing cell viability. Data represent the mean ± SEM (n = 3 independent samples). Bars having different superscript letters differed significantly (P < 0.05).

Figure 1

Construction of a Hepatic Steatosis Model in Chicken Hepatocytes Induced by OA

Treatment of primary chicken hepatocytes with 5 different concentrations of OA revealed significant upregulation in the mRNA expression of genes associated with lipid synthesis and transport (Figures 2A-G, P < 0.05). Concurrently, intracellular levels of triglycerides and cholesterol markedly increased following OA treatment. Moreover, the induction of hepatocyte steatosis by OA exhibited a biphasic response, with efficacy peaking at 0.6mM concentration. Additionally, CCK-8 analysis indicated a significant reduction in cell viability beyond 0.6mM OA concentration (Figure 2J, P < 0.05). Collectively, we elected to employ 0.6 mM OA for the establishment of a chicken hepatocyte steatosis model, poised for subsequent experimental investigations.Figure 2 Construction of a hepatic steatosis model in chicken hepatocytes induced by OA. (A–D) Relative mRNA expression levels of lipid synthesis-related genes ACACA, FASH, PPARγ and SREBP1. (E–G) Relative mRNA expression levels of lipid transport-related genes ApoA1, ApoVLDL-II and CPT1. (H-I) Cellular levels of TC and TG. (J) CCK-8 assay for assessing cell viability. Data represent the mean ± SEM (n = 3 independent samples). Bars having different superscript letters differed significantly (P < 0.05).

Figure 2

EGCG Alleviated OA-Induced Lipid Deposition in Chicken Hepatocytes

Observations from our investigation of TG and TC levels in chicken hepatocytes revealed that OA treatment significantly promoted lipid deposition in the cells (Figures 3A-B, P < 0.05). However, we found that EGCG markedly alleviates the elevation of TG and TC levels induced by OA (Figures 3A-B, P < 0.05). Additionally, Oil Red O staining results further demonstrated the efficacy of EGCG in effectively ameliorating the increased lipid droplet content induced by OA in hepatocytes (Figures 3C-D). Furthermore, EGCG facilitated the mRNA expression of fatty acid β-oxidation-related genes, including ACOX1, ACADS, and ACSL1 (Figures 3E-G, P < 0.05). Remarkably, EGCG treatment effectively counteracts the inhibition of fatty acid β-oxidation induced by OA treatment (Figures 3E-G, P < 0.05).Figure 3 EGCG alleviated OA-induced lipid deposition in chicken hepatocytes. (A–B) Cellular levels of TC and TG. (C) Oil red O staining of hepatic cells, with red representing lipid droplets. (D) The quantification results of the average gray level in the red region. (E–G) Expression levels of mRNA for fatty acid β-oxidation-related genes ACOX1, ACADS and ACSL1. Data represent the mean ± SEM (n = 3 independent samples). Bars having different superscript letters differed significantly (P < 0.05).

Figure 3

EGCG Mitigated Oxidative Stress, Inflammatory Response and DNA Damage Induced by OA in Chicken Hepatocytes

After treating chicken hepatocytes with OA, qPCR results revealed a significant decrease in the relative mRNA expression levels of antioxidant-related genes Trx, SOD1, GPX7, and GSR (Figures 4A-D, P < 0.05). ELISA experiments further demonstrated a notable downregulation in the cellular contents of antioxidant enzymes CAT, GSH-PX, and SOD, accompanied by a significant increase in MDA levels (Figures 4E-H, P < 0.05). Flow cytometry results indicated a significant elevation in the ROS-positive rate and MMP depolarization rate in hepatocytes following OA treatment (Figures 4I-L, P < 0.05). Subsequent treatment of OA-exposed cells with EGCG significantly ameliorated oxidative stress in hepatocytes (Figures 4I-L, P < 0.05). As depicted in Figures 5A-D, OA also induced the upregulation of inflammation-related genes, including TNF-α, IL-6, IL-8, and IFN-γ mRNA expression, as well as increased IL-6 protein expression in hepatocytes (Figure 5G-J, P < 0.05), thereby promoting the secretion of IL-6 and IL-1β (Figures 5E-F, P < 0.05). EGCG attenuated the exacerbated inflammatory response induced by OA in hepatocytes (Figures 5A-J, P < 0.05). Similarly, EGCG also reduced OA-induced DNA damage in hepatocytes, including the mRNA and protein expression levels of γ-H2AX (Figures 5K, T-W, P < 0.05), as well as the reduction in γ-H2AX-positive cell rates (Figures 5X-Y, P < 0.05). Moreover, EGCG can significantly upregulate the expression of DNA repair-related genes (Figures 5L-S, P < 0.05).Figure 4 EGCG mitigated oxidative stress induced by OA in chicken hepatocytes. (A–D) Relative mRNA expression levels of antioxidant-related genes Trx, SOD1, GPX7 and GSR. (E–H) Cellular contents of antioxidant enzymes CAT, GSH-PX, and SOD. (I–L) Flow cytometry analysis of cellular ROS activity and MMP depolarization level. Data represent the mean ± SEM (n = 3 independent samples). Bars having different superscript letters differed significantly (P < 0.05).

Figure 4

Figure 5 EGCG mitigated inflammatory response and DNA damage induced by OA in chicken hepatocytes. (A–D) Relative mRNA expression levels of inflammation-related genes IL-6, IL-8, IFN-γ and TNF-α. (E–F) Cellular contents of inflammatory factors IL-1β and IL-6. (G-J) Relative protein expression levels of IL-6. (K) Relative mRNA expression level of γ-H2AX. (L-S) Relative mRNA expression levels of DNA repair-related genes AAD51, DNA-Pk, LIG4, RAD23, MLH1, ER-β, OGG1 and MSH6. (T–W) Relative protein expression level of γ-H2AX. (X–Y) Immunofluorescence staining images of γ-H2AX, where blue represented cell nuclei and red represented γ-H2AX. Data represent the mean ± SEM (n = 3 independent samples). Bars having different superscript letters differed significantly (P < 0.05).

Figure 5

EGCG Attenuated the Elevated Levels of Autophagy and Apoptosis Induced by OA in Chicken Hepatocytes

The intervention of OA significantly increased the mRNA expression levels of Atg3, Atg8, and Beclin1 in hepatocytes, as observed in Figures 6A-C. Subsequent treatment with EGCG led to a partial restoration in the expression levels of autophagy-related genes (Figures 6A-C, P < 0.05). Consistently, Western blot experiments demonstrated a significant elevation in the protein expression levels of Beclin1 and LC3-II, along with a notable reduction in the expression level of the anti-autophagy protein p62, following OA induction (Figures 6D-I, P < 0.05). The addition of EGCG alleviated the OA-induced upregulation of autophagy in hepatocytes (Figures 6D-I, P < 0.05). Moreover, OA supplementation significantly promoted apoptosis in hepatocytes, as confirmed by qPCR, Western blot, and flow cytometry experiments (Figures 6G-S, P < 0.05). Following EGCG treatment, the mRNA and protein expression levels of pro-apoptotic genes significantly decreased, while the expression levels of anti-apoptotic genes markedly increased. Concurrently, the overall apoptosis rate of cells significantly decreased (Figures 6G-S, P < 0.05).Figure 6 EGCG attenuated the elevated levels of autophagy and apoptosis induced by OA in chicken hepatocytes. (A–C) Relative mRNA expression levels of autophagy-related genes Atg3, Atg8 and Beclin1. (D–I) Relative protein expression levels of Beclin1, p62 and LC3. (G–M) Relative mRNA expression levels of apoptosis-related genes Caspase-3, Caspase-8, Caspase-9 and Bcl-2. (N–Q) Relative protein expression levels of Caspase-3, Caspase-9 and Bcl-2. (R-S) Flow cytometry analysis of cell apoptosis levels. Data represent the mean ± SEM (n = 3 independent samples). Bars having different superscript letters differed significantly (P < 0.05).

Figure 6

EGCG Modulated Chicken Hepatocyte Functions Through the p38 MAPK Signaling Pathway

By conducting transcriptomic sequencing analysis on the NC group, EGCG group, OA group, and OA+EGCG group, we identified over 38,000 genes in each group (Figure 7A), with the majority of gene lengths exceeding 1800 bp (Figure 7B). Venn diagram analysis revealed 33,688 genes commonly expressed across all 4 groups (Figure 7C). Differential expression analysis demonstrated that 147 genes were differentially expressed between the NC and EGCG groups, with 75 upregulated and 72 downregulated (Figure 7D). Between the NC and OA groups, 212 genes were differentially expressed, with 130 upregulated and 82 downregulated (Figure 7E). In the OA and OA+EGCG groups, 164 genes were differentially expressed, with 70 upregulated and 94 downregulated (Figure 7F). Subsequent analysis of gene expression trends within the NC, OA, and OA+EGCG groups showed that genes in profile 2 exhibited decreased expression in the OA group, which was alleviated by EGCG treatment (Figure 7G). Conversely, genes in profile 5 showed increased expression in the OA group, which was reduced following EGCG treatment (Figure 7G). These findings suggested that the expression of these genes may be regulated by EGCG. KEGG enrichment analysis of genes in profiles 2 and 5 indicated significant enrichment in the MAPK pathway for both profiles (Figures 7H-I). Further investigation using western blotting to assess p38 MAPK protein phosphorylation levels revealed that OA treatment significantly increased p38 phosphorylation (Figures 7J-M). However, this increase was markedly attenuated with EGCG treatment (Figures 7J-M). Thus, EGCG appeared to modulate a range of chicken hepatocyte functions by inhibiting p38 phosphorylation.Figure 7 EGCG modulated chicken hepatocyte functions through the p38 MAPK signaling pathway. (A) Number of genes obtained from sequencing in each group. (B) Distribution plot of gene lengths. (C) Venn diagram analysis of commonly expressed genes in the 4 groups. (D–F) Volcano plots of differentially expressed genes among the groups. (G) Trend analysis of co-expressed genes in the NC group, OA group, and OA+EGCG group. (H–I) KEGG enrichment analysis of genes in profile 2 and profile 5 with trend analysis. (J-M) Western blot analysis of p38 MAPK phosphorylation levels. Data represent the mean ± SEM (n = 3 independent samples).

Figure 7

DISCUSSION

Energy imbalance is a key factor contributing to excessive fat deposition and fatty liver in both animals and humans (Sanyal, 2005). Excessive fat deposition in chickens not only reduces feed efficiency and increases the incidence of fatty liver disease but also directly impacts carcass quality, resulting in significant losses in the poultry industry (Liu, et al., 2018). Lipid metabolism encompasses the digestion and absorption of dietary fats exogenously, as well as the transport and breakdown of synthesized fats endogenously (Désert, et al., 2018; Wang, et al., 2017). This series of biochemical reactions involves the participation of various enzymes, with hepatic lipid metabolism being the most important and complex aspect in avian fat metabolism. Abnormalities in this process often lead to fatty liver syndrome (FLS) in laying hens (Tan, et al., 2020). Hepatic lipid metabolism primarily involves the synthesis of fatty acids (FA), the synthesis and transport of TG, and cholesterol metabolism (Alvarenga, et al., 2011). Key genes and transcription factors regulating this process include the FABP gene family, ELOVL gene family, FADS gene family, SREBP family, and PPAR family (Chen, et al., 2017a; Claire D'Andre, et al., 2013; Fu, et al., 2014; Huang, et al., 2015; Liu, et al., 2019; Payne et al., 2010 ). In this study, an OA-induced cellular steatosis model of chicken hepatocytes was established to investigate lipid accumulation. By examining the expression of genes related to hepatic lipid synthesis and transport, as well as the levels of TG, TC, and cell viability, the successful construction of the steatosis model was confirmed. Subsequent treatment with different concentrations of EGCG on the established cellular steatosis model revealed significant mitigation of hepatic lipid accumulation induced by OA. This is consistent with previous studies. Sugiura et al. demonstrated that EGCG can enhance lipid metabolism and inhibit hepatic inflammation in mice, suggesting that EGCG's impact on hepatic lipid metabolism may be mediated through the activation of certain signaling pathway (Sugiura, et al., 2012). Li et al. showed that AMPK is involved in EGCG-mediated regulation of genes related to lipid synthesis and degradation (Li, et al., 2018). Additionally, studies have indicated that EGCG can alleviate hepatic lipid metabolism disorders by reducing the absorption of bile acids and lipids in mice (Huang, et al., 2018).

Multiple studies have shown that dysregulated lipid metabolism is a key factor in inducing mitochondrial dysfunction, as it increases ROS production and decreases ATP generatio (Li, et al., 2020b). Moreover, it is widely recognized that hepatic steatosis is closely associated with mitochondrial dysfunction and oxidative stress (Ipsen, et al., 2018; Li, Yao, Zhao, Cao and Ma, 2020b). ROS accumulation is closely related to cellular damage, as excessive ROS can induce oxidative damage to proteins, enzymes, cell membrane lipids, and even directly affect nucleic acids, leading to DNA strand breaks and activating inflammatory pathways such as NF-κB, thereby exacerbating inflammatory response (Jaeschke, 2000; Schwabe and Brenner, 2006). Excessive ROS accumulation as a critical factor can trigger oxidative damage and apoptosis in cells, further contributing to the development of nonalcoholic fatty liver disease (NAFLD) (Simões et al., 2021). ROS accumulation in hepatocytes not only triggers metabolic disturbances but also serves as a signaling molecule to induce inflammatory reactions within the body (Li, et al., 2020a). Our study demonstrated that OA significantly increased ROS and MMP depolarization levels in chicken hepatocytes, thereby enhancing the inflammatory response. Studies have shown that EGCG can enhance antioxidant parameters in rat heart tissue to alleviate hepatic lipid peroxidation damage and reduce oxidative stress (Xu, et al., 2014). Moreover, EGCG exhibits potent anti-inflammatory and antioxidant activities in human corneal epithelial cells by inhibiting the phosphorylation levels of p38 and JNK MAPK (Cavet, et al., 2011). Consistently, our study found that EGCG significantly alleviated the levels of hepatic lipid peroxidation and inflammation induced by OA.

DNA damage response involves DNA repair, cell cycle regulation, and apoptosis, with autophagy also implicated (Roos and Kaina, 2013; Wang, et al., 2021). DNA damage activates autophagy in cells, and excessive autophagy leads to programmed cell death. Upon DNA damage, H2AX is phosphorylated to form γ-H2AX (Rahmanian, et al., 2021). Therefore, in this study, the extent of DNA damage in tissue and cells was determined by detecting γ-H2AX. Furthermore, OA-induced DNA damage in chicken hepatocytes was confirmed by qPCR and Western blotting, and EGCG was found to mitigate this process to some extent. It has been reported that OA can induce autophagy and apoptosis in cancer cells, and our experimental results similarly demonstrate that appropriate levels of OA promote apoptosis and autophagy in hepatocytes. Subsequent addition of EGCG led to a reduction in both autophagy and apoptosis levels. This is consistent with Chen et al.'s findings that EGCG reduces pathological autophagy flux and inhibits apoptosis (Chen, et al., 2017b).

Studies have indicated that ROS accumulation induced by hepatic steatosis is closely associated with MAPK signal transduction (Lawan and Bennett, 2017). Moreover, MAPK pathways play a crucial role in regulating NF-κB-induced inflammation (Wu, et al., 2020). The MAPK signaling pathway is an essential component of eukaryotic signal transduction networks, transmitting signals from the cell surface to the nucleus. Various kinases in this pathway interact to regulate cell proliferation, differentiation, apoptosis, and stress responses to external environments and pathological conditions (Aghazadeh and Yazdanparast, 2010). Transcriptome sequencing in our study revealed enrichment of the MAPK signaling pathway, and Western blotting experiments confirmed that EGCG alleviated the increased phosphorylation of p38 MAPK induced by OA in chicken hepatocytes. This may represent a crucial mechanism by which EGCG reduces hepatic lipid accumulation, inhibits hepatocyte autophagy and apoptosis, and other cellular activities. Consistently, MAPK pathways have been shown to play a vital role in regulating the expression of genes related to hepatic lipid metabolism. Li et al. demonstrated that EGCG isomer GCG inhibits adipocyte differentiation and inflammation levels through MAPK signaling. Liu et al. found that EGCG can inhibit endotoxin-induced inflammation and oxidative stress in human hepatocytes by suppressing NF-κB and MAPK pathways (Liu, et al., 2014).

CONCLUSIONS

EGCG exhibits multifaceted effects on chicken hepatocytes, particularly in mitigating OA-induced hepatic steatosis. It effectively regulates lipid metabolism, reduces oxidative stress and inflammation, diminishes DNA damage, and modulates autophagy and apoptosis. EGCG shows significant potential in counteracting the adverse effects induced by OA, underscoring its promise as a therapeutic agent for managing lipid-related disorders. Mechanistically, EGCG achieves these beneficial effects through the modulation of the p38 MAPK signaling pathway. These findings highlight EGCG's value as an intervention for improving hepatocyte health and addressing hepatic steatosis in poultry farming practices.

DISCLOSURES

The authors declare that they have no known competing financial interests.

Appendix Supplementary materials

Image, application 1

ACKNOWLEDGMENTS

This research was funded by China Agriculture Research System of MOF and MARA, grant number CARS-40; Sichuan Science and Technology Program, grant number 2021YFYZ0007 , 2021YFYZ0031 and 2022YFYZ0005 .

Supplementary material associated with this article can be found in the online version at doi:10.1016/j.psj.2024.104204.
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