The state of equilibrium of lipids content and composition is controlled by lipid metabolic pathways in the body (Seebacher et al., 2020). Lipid metabolism is complex and involved in a variety of metabolic processes, usually including lipid transport, absorption, synthesis, oxidation, and catabolism (Fernández-Ruiz, 2020). Lipid metabolism is composed of the metabolism of fatty acids (FA), triglycerides (TG), cholesterol, and other lipids (Anand, 2020; Mutlu et al., 2021). FA metabolism is fundamental to other lipid metabolisms, which can be converted into other lipids through anabolism. Other lipids can also be converted into FA through catabolism, which will be further oxidized and decomposed into smaller FA chains through β-oxidation and generate energy (Poitelon et al., 2020). However, the metabolism of TG and cholesterol is related to the lipid transfer protein, including high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C). Both HDL-C and LDL-C can combine with lipids, which were transported to different parts of the body for metabolism (Joseph et al., 2014). Maintaining normal metabolism and energy homeostasis is strictly dependent on lipid metabolism. However, the cases of dyslipidemia increased in recent decades. Dyslipidemia is part of metabolic syndrome in the body, resulting in excessive or insufficient lipids in various tissues (Bahiru et al., 2021). Intracellular dyslipidemia includes abnormal lipid regulation, accumulation of TG, and free fatty acids (FFA). Extracellular dyslipidemia includes abnormal lipid deposition and abnormal secretion of adipokines (Fu et al., 2019). Dyslipidemia can lead to obesity, type 2 diabetes mellitus, atherosclerosis (AS), and other diseases (Vekic et al., 2019; Wang et al., 2020; Valanti et al., 2021). Moreover, lipid peroxidation is a common oxidation reaction of polyunsaturated FA in living organisms. Excessive oxidation products are harmful to proteins, lipids, and nucleic acids, leading to the damage to cellular structure and function (Cháfer-Pericás, 2021). Thereby, reactive oxygen species-induced lipid peroxidation could contribute to chronic diseases, such as cardiovascular disease, diabetes, cancer, and senescence (Clemente et al., 2020; Gianazza et al., 2020; Kuzgun et al., 2020). Due to the side effects of drug treatment on dyslipidemia and lipid peroxidation, finding bioactive compounds from natural products has attracted more and more attention.

Ellagic acid (C₁₄H₆O₈, EA), an active natural polyphenol ingredient, is extensively found in a variety of fruits and nuts (such as pomegranate, gallnut, blueberry, and grape; Zuccari et al., 2020). The molecular structure of EA contains multiple phenolic hydroxyl groups with strong antioxidant functions (Dávalos et al., 2019). In addition, a large number of studies have confirmed that EA has antibacterial, anti-inflammatory, liver protection, prevention and treatment of obesity, and anti-tumor effects (Chen et al., 2018). Furthermore, dietary supplementation with EA substantially decreased de novo synthesis of FA in mature adipocytes and hepatic TG levels by downregulating adipogenic markers, such as CCAAT/enhancer-binding protein alpha and peroxisome proliferator-activated receptor gamma (Okla et al., 2015; Woo et al., 2015).

As mentioned above, several investigations have expounded that EA can modulate lipid metabolism and enhance antioxidant activities. However, to our knowledge, the underlying molecular mechanisms of EA on lipid metabolism and oxidative damage in mice have not been fully elucidated. Thus, the present study aims to evaluate the possible hepatoprotective effects of EA and explore the underlying mechanisms in vivo.

The experimental procedures in the study were approved by the Ethics Review Committee for Animal Experimentation of Sichuan Academy of Animal Science (Chengdu, China; P202002028). All experimental protocols were performed following the practical animal protection law and the Guide for the Care and Use of Laboratory Animals formulated by the National Research Council (China).

The imbalance of fat deposition and consumption caused by the dysfunction of lipid metabolism often leads to excessive fat deposition, which is detrimental to the health of animals and humans. Dyslipidemia is often manifested as a variation of hyperlipidemia indicators, such as serum TC, TG, abnormal elevation of LDL-C, or abnormal reduction of HDL-C (Tietge, 2014). Hyperlipidemia is not only the causative factor for many cardiovascular diseases, such as atherosclerosis, coronary artery disease, and myocardial infarction but also closely related to obesity, fatty liver, hypertension, and hyperglycemia (Alloubani et al., 2020; Yang et al., 2020). Hence, maintaining the balance of lipid metabolism is vital to the health of animals and humans. Previous studies have demonstrated that EA might serve as a potential agent in the therapy of hepatic steatosis in patients with NAFLD by activation of AKT/mTORC1 signaling in the liver (Zhang et al., 2019). Our results demonstrated that dietary supplementation with EA significantly increased the concentration of HDL-C and decreased the concentrations of T-CHO and LDL-C, which, to some extent, supported the notion that EA administration ameliorated the dysfunction of lipid metabolism. The molecular mechanisms, including lipogenesis, lipolysis, and fatty acid oxidation (FAO), are complex and critical for lipid metabolism. Therefore, we further explored whether EA supplementation affects the process of lipid metabolism through these key steps in the liver of mice. ACC, a biotin-dependent allosteric carboxylase, is the rate-limiting enzyme of the process of de novo fatty acid synthesis and catalyzes the first reaction in the fatty acid synthesis pathway (Hunkeler et al., 2018). As a neutral lipase, HSL mainly catalyzes the hydrolysis of TG, diacylglycerol (DG), and monoacylglycerol (MAG) in animal adipose tissue, releasing FFA to participate in the body oxidation energy supply (Recazens et al., 2020). Besides, CPT1 is the rate-limiting step in the process of FA transportation into mitochondria. Overexpression of CPT1B improved insulin sensitivity and FAO to lower lipid contents (Khan and Alhomida, 2013). Meanwhile, PPARα can regulate the activity of CPT1B. Our data showed a dramatic increase in the hepatic protein abundances of p-HSL, CPT1B, and PPARα and a marked decrease in the hepatic protein abundance of ACC, indicating that EA promoted the lipid metabolism by improving hepatic lipolysis and FAO and suppressing hepatic lipogenesis. However, dietary supplementation with 0.1% EA showed little effect on the lipid metabolism comparing with 0.3% EA, which possibly supported the notion that a higher concentration of EA possessed a better function in regulating the lipid metabolism of the liver.

Previous research has demonstrated that chronic oxidative stress is considered one of the critical factors responsible for the liver injury (Rezzani and Franco, 2021). Lipid peroxidation resulting from oxidative stress produces a large amount of MDA, which can also contribute to hepatocyte damage, thereby aggravating the lesions (Tsikas, 2017). By contrast, SOD and CAT are important antioxidant enzymes, responsible for scavenging free radicals and inhibiting lipid peroxidation, thus protecting the hepatocytes against toxicity (Mansuroğlu et al., 2015; Nowicki and Kashian, 2018). A kinetic DFT study found that EA was more reactive toward HO•, but less reactive toward CCl₃OO• with good rate constants for the reactions (Tošović and Bren, 2020). Moreover, EA could restore the antioxidant defense system by scavenging free radicals, leading to the abatement of oxidative damage in the livers and brains of D-gal-induced aging (Chen et al., 2018). Therefore, the antioxidant capacity of EA was assessed in this study. Dietary supplementation with EA dose-dependently increased the contents of CAT and SOD and decreased the content of MDA, implying EA exerts a protection effect by elevating the hepatic antioxidant capacity. It is reported that the elevation of antioxidant activity was regulated by several critical antioxidant genes. Nrf2 is the most critical transcription factor for intracellular ROS clearance and the most important defense system against oxidative stress caused by pathogen infection (Tian et al., 2020). Nrf2 regulates the expression of the antioxidant gene mainly by nuclear translocation and interacting with the antioxidant response element (ARE) at the 5′ end of the gene (Johnson et al., 2008). After the ARE is activated, a series of antioxidant proteins/enzymes, such as NQO1 and HO-1, are induced to maintain the redox balance (Liang et al., 2013). Our study suggested that dietary supplementation with EA increased the hepatic protein abundances of p-Nrf2, NQO1, and HO-1. These results implied that dietary EA supplementation could enhance the antioxidant capacity by elevating the Nrf2-ARE signaling pathway.

To summarize, our study demonstrated that dietary supplementation with 0.1 and 0.3% EA has beneficial effects on lipid metabolism by regulating the proteins expressions related to lipogenesis, lipolysis, and FAO. Besides, the enhanced antioxidant capacity caused by EA is relevant to the activation of the Nrf2-ARE signaling pathway. Our results provided a scientific basis for the potential contribution of EA to maintain liver health.

The original contributions presented in the study are included in the article/supplementary material, and further inquiries can be directed to the corresponding author.

The animal study was reviewed and approved by the Ethics Review Committee for Animal Experimentation of Sichuan Academy of Animal Science (Chengdu, China).

HD: conceptualization, methodology, validation, resources, writing—review and editing, supervision, and funding acquisition. QX: formal analysis, investigation, and writing—original draft preparation. SL, WT, and JY: data curation. XW and MZ: project administration. All authors have read and agreed to the published version of the manuscript.

This work was supported by the Sichuan Science and Technology Program (2021NZZJ0019).

Author SL was employed by company Sichuan Animtech Biology Development Co., Ltd, and authors WT, JY, XW, and MZ were employed by company Sichuan Animtche Feed Co. Ltd.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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