Resveratrol alleviates lipid deposition, endoplasmic reticulum stress, inflammation and apoptosis in the livers of large yellow croaker (Larimichthys crocea) fed with a soybean oil-based diet

Abstract

Resveratrol has been shown to alleviate endoplasmic reticulum stress (ERS), lipid deposition and inflammation induced by high-fat diets in fish and mammals. However, whether dietary resveratrol supplementation in vegetable oil-based diets of fish can exert these beneficial effects remains unclear. A 56-d feeding trial was conducted to explore the impacts of resveratrol supplementation in a soybean oil-based diet (SO) on the growth and liver lipid deposition, ERS, inflammation and apoptosis in large yellow croaker (Larimichthys crocea). The fish (35.56 ± 0.26 g) were randomly distributed into 18 cages (50 fish/cage) corresponding to triplicate cages of the six diets, including a fish oil-based diet (FO), SO, and SO supplemented with 0.05% (SR0.05), 0.10% (SR0.1), 0.20% (SR0.2), and 0.40% (SR0.4) resveratrol, respectively. The results showed that the total replacement of fish oil by soybean oil significantly reduced final body weight (FBW) and percent weight gain (PWG) (P < 0.05), and significantly increased feed conversion ratio (FCR) and feed intake (FI) (P < 0.05). The SR0.1 group exhibited significantly higher PWG and lower FCR than the SO group (P < 0.05) and similar FBW and FI to the FO group (P > 0.05). The FO and SR0.1 groups shared similar liver crude lipid and triglyceride contents (P > 0.05), which were significantly lower than those of the SO groups (P < 0.05). Compared to the FO treatment, the livers of the SO groups showed severe vacuolation, increased size and number of lipid droplets, and swollen endoplasmic reticulum lumen. These changes were markedly alleviated in the SR0.1 group. Dietary soybean oil significantly increased the relative mRNA expression levels of ERS-related genes (grp78, xbp1, atf6α, atf4, and chop), pro-inflammatory cytokines (tnfα and il-1β) and pro-apoptotic genes (bax, caspase 3, and caspase 9), as well as the protein expression levels of ERS-related proteins (GRP78, XBP1, and ATF6) and phosphorylation levels of PERK, NF-κB p65 and JNK (P < 0.05). Meanwhile, dietary soybean oil decreased the relative mRNA expression levels of sirt1, anti-inflammatory cytokines (arg1 and il-10), anti-apoptotic gene (bcl-2), and the protein expression level of SIRT1 in the liver (P < 0.05). Resveratrol supplementation in the SO restored the expression of the above genes and proteins to some extent. In conclusion, supplementation with 0.10% resveratrol in the SO improved PWG and feed utilization, promoted SIRT1 expression and alleviated lipid deposition, ERS, inflammation and apoptosis in the liver of large yellow croaker.

1. Introduction

Fish oil, with ample long-chain polyunsaturated fatty acids (LC-PUFA), has been a high-quality lipid source in aquafeed. Nevertheless, with the rapid development of aquaculture, the limited fish oil resources can no longer meet the growing demands of the aquafeed industry. Vegetable oils have the advantage of plentiful unsaturated fatty acids, low cost, and wide source, thus the application of vegetable oils as excellent alternative lipid sources for fish oil in aquafeeds is becoming more extensive. However, there is increasing evidence suggesting that high proportions of dietary vegetable oils not only inhibit growth and feed utilization, but also induce abnormal lipid deposition in the liver of fish (Mu et al., 2020b; Qin et al., 2020; Shen et al., 2022). Excessive accumulation of lipids could further trigger the inflammatory response, apoptosis and tissue damage, which ultimately weakens anti-stress capability and disease resistance of fish (Caballero et al., 2002; Sargent et al., 2003; Shen et al., 2023). Therefore, it is essential to explore key targets and nutritional strategies to alleviate hepatic lipid accumulation in fish caused by the replacement of dietary fish oil with high proportions of vegetable oils.

Vegetable oil and fish oil possess distinct fatty acid compositions. The variations in dietary fatty acid profile induced by the substitution of dietary fish oil with vegetable oil usually alter the fatty acid profile of the biomembrane in fish, which further affects the membrane fluidity and cellular function (such as signal transduction) (Almaida-Pagán et al., 2015; Arts and Kohler, 2009). The endoplasmic reticulum is an organelle that plays a vital role in cellular signal transduction and homeostasis maintenance (Fu et al., 2012). Characterized by the intricate membranous network structure, the endoplasmic reticulum is responsible for regulating intracellular lipid synthesis and secretion, protein folding and processing, as well as the maintenance of Ca²⁺ homeostasis in cells (Han and Kaufman, 2016). Exposure to adverse stimuli (e.g., nutrient deficiency or excess, or viral infection) could trigger endoplasmic reticulum stress (ERS) in organisms and lead to the accumulation of misfolded or unfolded proteins in the endoplasmic reticulum, thereby activating the unfolded protein response (UPR) to restore endoplasmic reticulum homeostasis (Almanza et al., 2019). Three key UPR mediators involved in the UPR activation are inositol-requiring enzyme 1 (IRE1), activating transcription factor 6 (ATF6) and protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) (Cnop et al., 2012). Under normal cellular conditions, the domains located in the endoplasmic reticulum lumen of these three endoplasmic reticulum transmembrane proteins bind to the glucose-regulated protein 78 (GRP78). However, when ERS occurs, GRP78 dissociates from these three proteins, triggering a series of cascade reactions (Cnop et al., 2012). Prolonged activation of UPR can induce lipid deposition, the inflammatory response and apoptosis. For example, activated PERK stimulated the expression of genes related to lipogenesis to promote lipid deposition via activating sterol-regulatory element-binding protein-1 (SREBP-1) (Lauressergues et al.,2012). When ERS persists, the UPR can arouse the transcription of pro-inflammatory cytokines to induce inflammation by promoting the nuclear translocation of nuclear factor-κB (NF-κB) (Jiang et al., 2003). Fatty acids could change the fatty acid composition and fluidity of the endoplasmic reticulum membrane through selectively incorporating into its lipid bilayer, which influences the activities of a series of proteins on the endoplasmic reticulum membrane (Volmer and Ron, 2015). It is shown that saturated fatty acids (SFA) trigger ERS and activate UPR, which can be reversed by n-3 unsaturated fatty acids (Nivala et al., 2013; Zhang et al., 2011b). Compared with fish oil, soybean oil with rich linoleic acid content has been found to result in ERS, lipid droplet accumulation and hepatocyte damage in rabbit (Zhu et al., 2016). A study in black seabream (Acanthopagrus schlegelii) concluded that totally replacing fish oil by soybean oil or palm oil rich in palmitic acid significantly upregulated the expression of ERS-related genes, and downregulated the expression of genes related to fatty acid oxidation in liver, accompanied by hepatic lipid deposition, inflammation and apoptosis (Shen et al., 2022).

Plentiful studies have revealed that silent information regulator 1 (SIRT1) alleviates ERS-induced lipid accumulation, inflammatory response and apoptosis by deacetylation modification of ERS-sensor proteins (Chou et al., 2019; Prola et al., 2017). As a natural agonist of SIRT1, resveratrol (3,5,4′-trihydroxystilbene) is widely used in the prevention and treatment of metabolic diseases such as obesity, hyperlipidemia and fatty liver because of its ability to improve lipid metabolism and inhibit inflammation. This natural polyphenol compound of the stilbene group is a white to light yellow odorless powder, insoluble in water, but readily soluble in organic solvents. It is present in various kinds of plants and can be obtained through natural extraction or synthetic production (Căpruciu, 2025). Resveratrol has been demonstrated to ameliorate ERS and lipid droplet accumulation in mouse liver induced by a high-fat diet through activating SIRT1 to promote the deacetylation of ATF6 (Zhou et al., 2018). Similarly, it was recently reported that resveratrol activated SIRT1 to deacetylate IRE1, thereby attenuating high-fat diet-evoked hepatic ERS, lipid deposition, inflammation, and apoptosis in black seabream (Jin et al., 2024). Meanwhile, resveratrol clearly mitigated the abnormal lipid accumulation and inflammatory response caused by poor nutritional conditions (such as high-fat and low-fishmeal diets) in common carp (Cyprinus carpio) (Wu et al., 2022) and turbot (Scophthalmus maximus) (Tan et al., 2019). However, previous studies on the application of resveratrol in low fish oil diets only focused on its effects on growth and tissue fatty acid composition of fish (Torno et al., 2017, 2018). It remains unknown whether the activation of SIRT1 by dietary resveratrol supplementation can mitigate hepatic lipid deposition and inflammatory response induced by high proportions of dietary vegetable oils through regulating ERS in fish.

Large yellow croaker (Larimichthys crocea), with an output of 292,615 tons in 2024 (Bureau of Fisheries of the Ministry of Agriculture and Rural Affairs of China, 2025), has been a commercially important mariculture fish species in China. However, this species is highly sensitive to dietary lipid sources. Consuming diets with high levels of vegetable oils is easy to trigger abnormal lipid accumulation and liver injury of large yellow croaker (Li et al., 2019a; Mu et al., 2020a; Tan et al., 2023). Soybean oil has become widespread in aquafeed due to its high yield, low cost, and richness in n-6 polyunsaturated fatty acids (PUFA) (Shen et al., 2022; Zhao et al., 2023). A previous study showed that substituting dietary fish oil with a high level of soybean oil not only inhibited growth, but also induced an imbalance in the n-3/n-6 PUFA ratio, a significant increase in crude lipid content, and severe inflammation in the liver of large yellow croaker (Mu et al., 2018). Therefore, the current study aims to explore whether resveratrol supplementation could ameliorate the impacts of a high proportion of soybean oil used as a replacer for dietary fish oil on the growth and liver damage of large yellow croaker by activating SIRT1 and modulating ERS. The findings will provide data support for the development and utilization of resveratrol in low fish oil diets and contribute to offering a key target and nutritional strategy to alleviate liver injury of fish caused by the substitution of dietary fish oil with vegetable oils.

2. Materials and methods

2.1. Animal ethics statement

All animal experiments in this study were performed under the guidelines of the Animal Care and Use Committee of Jiangsu Ocean University (approval No. 2020–37).

2.2. Diet preparation and animal feeding experiment

As shown in Table 1, six diets containing 45% crude protein and 11% crude lipid were prepared according to previous approach (Chen et al., 2025). The 6.50% fish oil was used as the main lipid source to formulate the fish oil-based diet (FO) by referring to a previous study (Mu et al., 2018). The soybean oil-based diet (SO) was designed via totally replacing fish oil in the FO with soybean oil. Based on previous descriptions (Torno et al., 2018; Wilson et al., 2015; Wu et al., 2022), the SO was supplemented with 0.05%, 0.10%, 0.20%, and 0.40% resveratrol, respectively to obtain the resveratrol-supplemented diets designated as SR0.05, SR0.1, SR0.2, and SR0.4, respectively. Resveratrol (purity 98%) was provided by the Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Ingredients were ground to fine powder and mixed fully, followed by blending with fish oil or soybean oil and water to produce pellets using a pellet-making machine (F-26 [II], Guangzhou Huagong Optoelectromechanical Technology Co., Ltd., Guangzhou, Guangdong, China). After drying, the experimental diets were kept at −20 °C for feeding use.

Table 1.

Ingredients and nutrient levels of the experimental diets (dry matter basis,%)¹.

Items	FO	SO	SR0.05	SR0.1	SR0.2	SR0.4
Ingredients
Fish meal	40.00	40.00	40.00	40.00	40.00	40.00
Soybean meal	24.00	24.00	24.00	24.00	24.00	24.00
Wheat meal	22.00	22.00	22.00	22.00	22.00	22.00
Fish oil	6.50	0.00	0.00	0.00	0.00	0.00
Soybean oil	0.00	6.50	6.50	6.50	6.50	6.50
Resveratrol	0.00	0.00	0.05	0.10	0.20	0.40
Ca(H2PO4)2	0.50	0.50	0.50	0.50	0.50	0.50
Chloride choline	0.30	0.30	0.30	0.30	0.30	0.30
Soybean lecithin	2.00	2.00	2.00	2.00	2.00	2.00
Vitamin and mineral premix2	1.00	1.00	1.00	1.00	1.00	1.00
Attractant3	0.50	0.50	0.50	0.50	0.50	0.50
Mold inhibitor4	0.10	0.10	0.10	0.10	0.10	0.10
Tertiary butylhydroquinone	0.05	0.05	0.05	0.05	0.05	0.05
Microcrystalline cellulose	3.05	3.05	3.00	2.95	2.85	2.65
Total	100.00	100.00	100.00	100.00	100.00	100.00
Nutrient levels5
Moisture	8.03	7.09	7.76	7.40	7.24	7.75
Crude protein	45.38	44.73	45.31	44.75	45.23	44.99
Crude lipid	10.92	11.10	11.86	10.96	10.99	11.13
Organic matter	82.58	83.49	82.68	83.10	83.26	82.79
Gross energy, MJ/kg	20.21	20.96	20.35	20.28	20.74	20.44

¹FO, fish oil-based diet; SO, soybean oil-based diet; SR0.05, SO supplemented with 0.05% resveratrol; SR0.1, SO supplemented with 0.10% resveratrol; SR0.2, SO supplemented with 0.20% resveratrol; SR0.4, SO supplemented with 0.40% resveratrol.

²Provides the following per kg of premix: vitamin A 450,000 IU; vitamin D₃ 100,000 IU; vitamin E 5000 mg; vitamin K 500 mg; vitamin B₁ 500 mg; vitamin B₂ 700 mg; nicotinic acid 3500 mg; calcium pantothenate 2000 mg; vitamin B₆ 600 mg; vitamin B₇ 6 mg; folic acid 150 mg; vitamin B₁₂ 2 mg; inositol 8000 mg; vitamin C 10,000 mg; cobalt 80 mg; copper 1500 mg; iodine 100 mg; iron 2000 mg; magnesium 20,000 mg; manganese 2000 mg; selenium 10 mg; zinc 7500 mg.

³The attractant comprises 25% of glycine (Shijiazhuang Donghua Jinlong Chemical Co., Ltd., Shijiazhuang, Hebei, China) and 75% of betaine (Shijiazhuang Donghua Jinlong Chemical Co., Ltd., Shijiazhuang, Hebei, China).

⁴The mold inhibitor comprises 50% of fumaric acid (Shijiazhuang Donghua Jinlong Chemical Co., Ltd., Shijiazhuang, Hebei, China) and 50% of calcium propionate (Shijiazhuang Donghua Jinlong Chemical Co., Ltd., Shijiazhuang, Hebei, China).

⁵Moisture, crude protein, crude lipid, and gross energy were measured values, and organic matter was calculated value.

A 56-d feeding trial was performed on large yellow croaker obtained from Ningde Fufa Fisheries Co., Ltd. (Ningde, Fujian, China). After 14 d of acclimation, nine hundred fish (average initial body weight: 35.56 ± 0.26 g) were randomly distributed into 18 outdoors sea cages (1.0 m × 1.0 m × 1.5 m, 50 fish/cage). Each experimental diet was casually assigned to cages in triplicate. Fish were hand-fed the experimental diets to the satiation level twice daily (05:30 and 17:00). During the feeding period, the water temperature (21–30 °C), salinity (31–37) and dissolved oxygen content (above 6.0 mg/L) were determined utilizing a mercury thermometer, salinometer (DLX-ARH100, DELIXI Group Instruments Co., Ltd., Wenzhou, Zhejiang, China) and portable dissolved oxygen analyzer (AR8406, Dongguan Wanchuang Electronic Products Co., Ltd., Dongguan, Guangdong, China), respectively.

2.3. Sample collection

After being fasted for 24 h, fish in each cage were anesthetized with MS-222 (1:10,000; Sigma–Aldrich, Merck Life Science A/S, Søborg, Denmark) and then batch-weighed and counted to compute the parameters correlated with growth performance. Five fish per cage were randomly collected to record the body weight, body length, and liver and viscera weight for the calculation of condition factor (CF), hepatosomatic index (HSI), and viscerosomatic index (VSI). The liver tissues from these five fish were stored at −80 °C for the analysis of crude lipid and triglyceride (TAG) contents. Another five fish per cage were obtained to collect blood samples from the caudal vasculature. After standing at 4 °C, serum samples were isolated by centrifugation (3000 × g, 10 min, 4 °C) and then frozen at −80 °C for biochemical analysis. After sampling the blood, the liver tissues of these five fish were dissected, flash frozen in liquid nitrogen and stored at −80 °C for the determination of gene expression and Western blot analysis. Liver tissues were sampled from six fish in each cage and soaked in 4% paraformaldehyde for hematoxylin and eosin (H&E) staining, Oil Red O staining, immunohistochemical staining, immunofluorescence staining, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. Further liver tissues dissected from three fish in each cage were fixed in 2.5% glutaraldehyde solution for transmission electron microscope (TEM) analysis.

2.4. Proximate composition and fatty acid analysis

According to the standard methods (AOAC, 1995), moisture content of diets was determined by drying to constant weight at 105 °C (method 950.46). Crude protein and crude ash contents in diets were determined by the Kjeldahl method (method 968.06) and the incineration method (method 923.03), respectively. Crude lipid content in diets and liver tissues was determined using the Soxhlet extraction method (method 954.02). The organic matter level in diets was calculated as 100 – moisture – crude ash content. An oxygen bomb calorimeter (ZDHW-3, Hebi Longhua Instrument Co., Ltd., Hebi, Henan, China) was used to determine the gross energy in diets, following the method GB/T 45104-2024 (China National Standard, 2024).

The separation and identification of fatty acids in fish oil, soybean oil and experimental diets were performed according to the methods reported in detail previously (Chen et al., 2024; Liu et al., 2022). In brief, total lipid in lipid sources and diets was extracted using the chloroform-methanol method (Liu et al., 2022). Then, fatty acid methyl esters (FAMEs) were separated and identified by the gas chromatography-mass spectrometry (GCMS-QP2010, Shimadzu Corporation, Kyoto, Japan) equipped with a DB-23 column (60 m × 0.15 mm, 0.25 μm) (Chen et al., 2024). The contents of fatty acids were presented as percentages of total identified fatty acids (Table 2).

Table 2.

Fatty acid profiles of lipid sources and experimental diets (% of total identified fatty acids).

Items	Lipid sources		Dietary treatments1
	Fish oil	Soybean oil	FO	SO	SR0.05	SR0.1	SR0.2	SR0.4
C12:0	0.07	N.D.	0.08	0.03	0.04	0.02	0.03	0.04
C14:0	3.82	N.D.	4.81	1.69	1.67	1.69	1.62	1.73
C16:0	14.53	10.73	18.94	14.03	13.84	14.29	13.86	14.09
C17:0	0.65	N.D.	0.51	0.10	0.08	0.09	0.12	0.11
C18:0	3.81	3.78	4.24	3.91	4.01	3.89	3.95	3.92
C20:0	0.19	0.34	0.13	0.33	0.31	0.34	0.36	0.31
C21:0	N.D.	N.D.	0.03	0.05	0.06	0.04	0.04	0.05
C22:0	0.17	N.D.	0.14	0.03	0.04	0.03	0.03	0.02
C23:0	0.08	N.D.	0.06	0.14	0.15	0.13	0.12	0.16
C24:0	0.13	N.D.	0.08	N.D.	N.D.	N.D.	N.D.	N.D.
∑SFA	23.46	14.85	29.03	20.31	20.20	20.52	20.13	20.43
C14:1	0.50	N.D.	0.54	0.14	0.15	0.14	0.17	0.12
C16:1	5.68	N.D.	5.18	1.65	1.71	1.61	1.65	1.62
C17:1	0.35	N.D.	0.45	0.20	0.21	0.18	0.20	0.23
C18:1n-9t2	0.11	N.D.	0.11	N.D.	N.D.	N.D.	N.D.	N.D.
C18:1n-9c2	23.39	24.80	18.87	20.63	20.68	20.61	20.68	20.69
C20:1n-9	0.28	N.D.	0.23	0.04	0.04	0.03	0.06	0.04
C22:1n-9	0.13	N.D.	0.10	N.D.	N.D.	N.D.	N.D.	N.D.
C24:1n-9	0.35	N.D.	0.30	0.09	0.07	0.09	0.08	0.07
∑MUFA	30.78	30.66	25.78	22.75	22.86	22.66	22.84	22.77
C18:2n-6t3	0.15	0.21	0.15	0.08	0.07	0.07	0.09	0.09
C18:2n-6c3	18.57	53.33	20.00	44.09	43.41	44.23	44.26	43.52
C18:3n-6	2.61	0.61	1.66	0.51	0.49	0.48	0.49	0.51
C20:3n-6	1.89	N.D.	1.07	0.02	0.02	0.02	0.02	0.02
C20:4n-6	0.90	N.D.	0.85	0.27	0.28	0.26	0.28	0.24
∑n-6 PUFA	24.12	54.15	23.72	44.98	44.27	45.06	45.14	44.38
C18:3n-3	3.16	5.86	3.01	5.17	5.78	5.16	5.07	5.28
C20:3n-3	0.11	N.D.	0.04	0.04	0.03	0.04	0.05	0.04
C20:5n-3	6.34	N.D.	7.47	3.20	3.21	3.13	3.22	3.26
C22:5n-3	1.06	N.D.	1.01	0.34	0.36	0.32	0.34	0.35
C22:6n-3	10.59	N.D.	9.50	2.79	2.86	2.61	2.78	2.96
∑n-3 PUFA	21.27	5.86	21.03	11.53	12.24	11.26	11.46	11.89
n-3/n-6 PUFA	0.88	0.11	0.89	0.26	0.28	0.25	0.25	0.27
∑n-3 LC-PUFA	18.10	N.D.	17.98	6.33	6.43	6.06	6.34	6.57
C20:2	0.29	0.34	0.30	0.32	0.32	0.31	0.31	0.35
C22:2	0.08	N.D.	0.15	0.16	0.17	0.17	0.14	0.16

N.D. =- not detected; ∑SFA = total saturated fatty acids; ∑MUFA = total monounsaturated fatty acids; ∑n-6 PUFA = total n-6 polyunsaturated fatty acids; ∑n-3 PUFA = total n-3 polyunsaturated fatty acids; ∑n-3 LC-PUFA = total n-3 long-chain polyunsaturated fatty acids.

¹FO, fish oil-based diet; SO, soybean oil-based diet; SR0.05, SO supplemented with 0.05% resveratrol; SR0.1, SO supplemented with 0.10% resveratrol; SR0.2, SO supplemented with 0.20% resveratrol; SR0.4, SO supplemented with 0.40% resveratrol.

²C18:1n-9t and C18:1n-9c are trans-9-octadecaenoic acid (elaidic acid) and cis-9-octadecaenoic acid (oleic acid), respectively.

³C18:2n-6t and C18:2n-6c are linoelaidic acid and cis-9, cis-12-octadecadienoic acid (linoleic acid), respectively.

2.5. Determination of serum biochemical parameters and liver TAG level

The amounts of TAG (A110-1-1), non-esterified fatty acid (NEFA; A042-1-1), total cholesterol (TC; A111-1-1), high density lipoprotein cholesterol (HDL-C; A112-1-1) and low density lipoprotein cholesterol (LDL-C; A113-1-1) in serum, as well as liver TAG level (A110-1-1) were evaluated by commercial assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China).

2.6. Histological and ultrastructural observation of liver tissue

The methods for H&E staining and Oil Red O staining of liver tissue were as described previously (Tan et al., 2023). For H&E staining, after fixation in 4% paraformaldehyde, gradient dehydration, paraffin embedding, sectioning, staining with H&E and microscopic examination of liver tissues were carried out. For Oil Red O staining, liver tissues fixed in 4% paraformaldehyde were embedded in optimal cutting temperature (OCT) compound, followed by freezing and cutting into 10 μm thick sections. The slices were then stained with Oil Red O to visualize lipid droplets. For ultrastructural observation, liver tissues fixed in a 2.5% glutaraldehyde solution were further fixed with 1% osmium tetroxide again. After gradient dehydration in ethanol solutions followed by 100% acetone, embedding and polymerization, ultrathin sections of 60 and 80 nm in thickness were obtained and stained with uranyl acetate and lead citrate. The TEM (Hitachi HT7800, Hitachi High-Tech Corporation, Tokyo, Japan) was then used to observe the ultrastructures and capture images.

2.7. Gene expression analysis

Based on the protocols detailed previously (Mu et al., 2023), total RNA was extracted from the liver samples using a total RNA isolation kit (Sangon Biotech Co., Ltd., Shanghai, China), followed by integrity check and concentration detection of RNA, as well as reverse transcriptase reaction with a reverse transcription kit (Vazyme Biotech Co., Ltd., Nanjing, Jiangsu, China). The amplification in a 20 μL reaction volume and procedure of quantitative real-time PCR (qRT-PCR) were conducted as reported previously (Mu et al., 2023). The β-actin with stable expression served as the housekeeping gene. After melting curve analysis and amplification efficiency verification, the comparative Ct approach was adopted to estimate the mRNA levels of candidate genes (Livak and Schmittgen, 2001). The specific primers designed using Primer 5.0 software and their amplification efficiencies are detailed in Table S1. The full names of the abbreviations of all genes can be found in supplementary file Abbreviations and Full Names of Genes and Proteins.

2.8. Western blot analysis

Based on the methods mentioned previously (Chen et al., 2025), total proteins were extracted from liver tissues and protein concentrations were then measured, followed by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, transfer of separated proteins to polyvinylidene fluoride membranes, as well as incubation with primary antibodies and secondary antibody. Subsequently, the membrane was developed with a DAB horseradish peroxidase color development kit (P0203, Beyotime Biotechnology Co., Ltd., Shanghai, China). The ImageJ 1.54k software (National Institutes of Health, Bethesda, MD, USA) was employed to quantify the band intensity. The following primary antibodies were used: GAPDH from Hangzhou Goodhere Biotechnology Co., Ltd. (Hangzhou, Zhejiang, China); and SIRT1, GRP78, XBP1, ATF6, PERK, p-PERK, NF-κB p65, p–NF–κB p65, JNK, and p-JNK from Wanlei Biotechnology Co., Ltd. (Shenyang, Liaoning, China). Horseradish peroxidase-labeled goat anti-rabbit IgG (Beyotime Biotechnology Co., Ltd., Shanghai, China) was used as the secondary antibody. Details of antibodies are provided in Table S2. The full names of the abbreviations of all proteins can be found in supplementary file Abbreviations and Full Names of Genes and Proteins.

2.9. Immunohistochemical staining, immunofluorescence analysis, and TUNEL assay

The method for immunohistochemical staining of liver samples was as reported previously (Chen et al., 2025), including dewaxing of paraffin sections, antigen repair, blocking of endogenous peroxidase, serum closure, incubation with primary antibody anti-SIRT1 (WL02995, Wanlei Biotechnology Co., Ltd., Shenyang, Liaoning, China), secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit IgG) (GB23303, Wuhan Servicebio Technology Co., Ltd., Wuhan, Hubei, China) and DAB color developing solution (G1212, Wuhan Servicebio Technology Co., Ltd., Wuhan, Hubei, China). After counterstain of cell nucleus with hematoxylin, images were captured using a light microscope (Eclipse E100, Nikon Corporation, Tokyo, Japan).

Immunofluorescence analysis of liver samples was carried out by standard procedures. Paraffin sections were dewaxed with environmentally friendly dewaxing solution (G1128, Wuhan Servicebio Technology Co., Ltd., Wuhan, Hubei, China) before hydration to distilled water by graded ethanol, followed by the antigen repair, serum closure, incubation with primary antibodies, secondary antibodies, 4′, 6-diamidino-2-phenylindole (DAPI) solution (G1012, Wuhan Servicebio Technology Co., Ltd., Wuhan, Hubei, China) and autofluorescence quencher solution (G1221, Wuhan Servicebio Technology Co., Ltd., Wuhan, Hubei, China). Images were obtained adopting a fluorescent microscope (Nikon Eclipse C1, Nikon Corporation, Tokyo, Japan) after mount with anti-fade mounting medium (G1401, Wuhan Servicebio Technology Co., Ltd., Wuhan, Hubei, China). The primary antibodies applied were GRP78 (WL03157, Wanlei Biotechnology Co., Ltd., Shenyang, Liaoning, China), ATF6 (WL02407, Wanlei Biotechnology Co., Ltd., Shenyang, Liaoning, China), p-PERK (Thr 982) (137056, Absin Bioscience Inc., Shanghai, China) and NF-κB p65 (WL01980, Wanlei Biotechnology Co., Ltd., Shenyang, Liaoning, China). The secondary antibody used was Cy3-conjugated goat anti-rabbit IgG (GB21303, Wuhan Servicebio Technology Co., Ltd., Wuhan, Hubei, China).

The TUNEL assay of liver tissues was based on the protocol mentioned in detail previously (Wang et al., 2020). In brief, after dewaxing of paraffin sections, repair and rupture, the sections were then incubated with reagents from the TUNEL apoptosis assay kit (G1504, Wuhan Servicebio Technology Co., Ltd., Wuhan, Hubei, China) and DAPI solution (G1012, Wuhan Servicebio Technology Co., Ltd., Wuhan, Hubei, China). The slices were sealed with anti-fade mounting medium (G1401, Wuhan Servicebio Technology Co., Ltd., Wuhan, Hubei, China) and photographed by a fluorescent microscope (Nikon Eclipse C1, Nikon Corporation, Tokyo, Japan).

2.10. Calculations and statistical analysis

The following formulas were applied to compute the growth and feed performance parameters, as well as morphological indices:

Survival rate (SR,%) = 100 × N₁/N₀;

Percent weight gain (PWG,%) = 100 × (W₁ – W₀)/W₀;

Specific growth rate (SGR,%/d) = 100 × (Ln W₁ − Ln W₀)/Days;

Feed conversion ratio (FCR) = Consumed feed weight/Weight gain;

Feed intake (FI,%/d) = 100 × Consumed feed weight/[(W₀ + W₁)/2]/Days;

CF (g/cm³) = 100 × W/L³;

HSI (%) = 100 × (W₂/W);

VSI (%) = 100 × (W₃/W),

where N₀ and N₁ are the initial number and final number of fish, respectively; W₀ and W₁ are the initial and final average weight of fish (g), respectively; W and L are the fish body weight (g) and body length (cm), respectively; W₂ and W₃ are the liver weight (g) and viscera weight (g), respectively.

SPSS 17.0 (SPSS Inc., Chicago, IL, USA) was used to analyze the significant differences at P < 0.05 by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. The statistical model for one-way ANOVA is the following:

Y_ij = μ + α_i + ϵ_ij,

where Y_ij denotes the dependent variable; μ is the overall mean of all observations;α_i denotes the fixed treatment effect; and ϵ_ij represents the random error. The results are given as the means and standard error of the mean (SEM) in all tables. The impacts of different resveratrol levels in the SO were evaluated linearly and quadratically by applying regression analysis.

3. Results

3.1. Growth and feed performance, and morphological indices

As shown in Table 3, the final body weight (FBW) of the SO, SR0.05, and SR0.4 groups was significantly decreased compared with the FO group (P = 0.004). Compared with the other five groups, the fish fed FO exhibited significantly higher PWG and SGR (P < 0.05), while significantly lower FCR (P < 0.001). The PWG of SR0.1 group was significantly higher than that of the SO, SR0.05, and SR0.4 treatments (P < 0.001). The FCR (P < 0.001) and FI (P = 0.001) of the SR0.05, SR0.1, and SR0.2 groups were significantly lower than those of the SO treatment. However, the FI displayed a significantly higher value in the SO and SR0.4 groups than in the FO treatment (P = 0.001). Meanwhile, a quadratic relationship was observed between FCR (P < 0.001) or FI (P = 0.005) and dietary resveratrol supplementation. The HSI in the SO group was significantly higher than that in the FO and SR0.1 groups (P = 0.002). Additionally, HSI in the SR0.1 group was significantly lower than that in the SO group, with linear (P = 0.012), quadratic (P = 0.011) and ANOVA (P = 0.002) effects.

Table 3.

Effects of dietary soybean oil and resveratrol levels on growth and feed performance, and morphological indices of large yellow croaker.

Items	Dietary treatments1						SEM	P-value2
	FO	SO	SR0.05	SR0.1	SR0.2	SR0.4		ANOVA	Linear	Quadratic
SR, %	88.00	80.67	83.33	85.33	84.67	82.67	0.832	0.162	0.363	0.122
IBW, g	35.00	36.00	35.33	36.00	36.00	35.00	0.258	0.740	0.532	0.722
FBW, g	75.37a	64.50b	62.88b	69.87ab	66.48ab	61.62b	1.327	0.004	0.792	0.181
PWG, %	115.28a	79.11c	77.88c	94.16b	84.45bc	76.12c	3.475	<0.001	0.970	0.068
SGR, %/d	1.37a	1.04bc	1.03c	1.18b	1.09bc	1.01c	0.032	<0.001	0.983	0.065
FCR	1.46c	2.38a	2.06b	1.86b	1.96b	2.30a	0.076	<0.001	0.536	<0.001
FI, %/d	1.90c	2.41a	2.06bc	2.12bc	2.08bc	2.27ab	0.044	0.001	0.422	0.005
CF, g/cm3	1.40	1.46	1.50	1.43	1.49	1.45	0.013	0.212	0.905	0.968
HSI, %	1.69c	2.10a	2.04ab	1.81bc	1.91abc	1.88abc	0.037	0.002	0.012	0.011
VSI, %	4.74	4.63	5.03	4.84	4.86	4.78	0.075	0.765	0.838	0.554

SR = survival rate; IBW = initial body weight; FBW = final body weight; PWG = percent weight gain; SGR = specific growth rate; FCR = feed conversion ratio; FI = feed intake; CF = condition factor; HSI = hepatosomatic index; VSI = viscerosomatic index; SEM = standard error of the mean.

Means within the same row without a common superscript significantly differ at P < 0.05 (n = 3).

¹FO, fish oil-based diet; SO, soybean oil-based diet; SR0.05, SO supplemented with 0.05% resveratrol; SR0.1, SO supplemented with 0.10% resveratrol; SR0.2, SO supplemented with 0.20% resveratrol; SR0.4, SO supplemented with 0.40% resveratrol.

²The P-value of ANOVA denotes the difference among all the treatments. The P-values of linear and quadratic denote the differences among the SO and resveratrol-supplemented groups.

3.2. Serum biochemical parameters

As shown in Table 4, serum TAG content in the SO and SR0.05 groups was significantly higher than that in the other four groups (P < 0.001). Additionally, dietary resveratrol levels had linear and quadratic effects on the serum TAG amount (P < 0.05), with the SR0.1 group showing the lowest value when compared to the SO treatment. Except for the SR0.2 group, the remaining four groups showed significantly lower serum NEFA levels compared with the SO group (P = 0.001). Fish fed the FO diet had significantly higher serum HDL-C levels than all other groups (P = 0.003).

Table 4.

Effects of dietary soybean oil and resveratrol levels on serum biochemical parameters, and contents of crude lipid and triglyceride in liver of large yellow croaker.

Items	Dietary treatments1						SEM	P-value2
	FO	SO	SR0.05	SR0.1	SR0.2	SR0.4		ANOVA	Linear	Quadratic
Serum
TAG, mmol/L	2.29c	6.60a	5.61a	2.99bc	4.14b	3.23bc	0.378	<0.001	<0.001	<0.001
NEFA, μmol/L	31.49c	68.65a	48.64bc	47.52bc	54.26ab	49.41b	2.946	0.001	0.072	0.028
TC, mmol/L	3.30	3.08	2.75	2.81	3.36	3.19	0.089	0.263	0.257	0.344
HDL-C, mmol/L	1.75a	1.49b	1.43b	1.46b	1.46b	1.44b	0.031	0.003	0.567	0.823
LDL-C, mmol/L	0.47	0.56	0.58	0.55	0.47	0.60	0.017	0.053	0.848	0.475
Liver
Crude lipid, % of wet basis	21.62b	28.80a	22.74b	21.49b	22.51b	22.05b	0.672	<0.001	0.007	<0.001
TAG, mmol/g prot	0.59d	1.34a	1.07ab	0.69cd	0.87bcd	0.97bc	0.064	<0.001	0.032	<0.001

TAG = triglyceride; NEFA = non-esterified fatty acid; TC = total cholesterol; HDL-C = high density lipoprotein cholesterol; LDL-C = low density lipoprotein cholesterol; SEM = standard error of the mean.

Means within the same row without a common superscript significantly differ at P < 0.05 (n = 3).

¹FO, fish oil-based diet; SO, soybean oil-based diet; SR0.05, SO supplemented with 0.05% resveratrol; SR0.1, SO supplemented with 0.10% resveratrol; SR0.2, SO supplemented with 0.20% resveratrol; SR0.4, SO supplemented with 0.40% resveratrol.

²The P-value of ANOVA denotes the difference among all the treatments. The P-values of linear and quadratic denote the differences among the SO and resveratrol-supplemented groups.

3.3. Lipid deposition in liver

As shown in Table 4, the crude lipid content in the liver of the SO group was significantly higher than that of the other treatments (P < 0.001). Meanwhile, the FO, SR0.1 and SR0.2 treatments displayed significantly lower hepatic TAG level than the SO group (P < 0.001). Moreover, a quadratic relationship was observed between liver crude lipid or TAG content and dietary resveratrol supplementation (P < 0.05).

As shown in Fig. 1A, the results of H&E staining and Oil Red O staining indicated that compared with the FO group, the SO group presented hepatic steatosis, characterized by severe vacuolation, a decreased number of nuclei, nuclei pushed to one side of the cell, and accumulation of numerous lipid droplets. Ultrastructural observation of liver tissue further showed that both the number and size of lipid droplets in the SO group were obviously increased. However, the SR0.1 group exhibited marked improvement in hepatocellular vacuolation, with partial restoration of nuclear location and number, as well as reductions in both the number and size of lipid droplets.

Fig. 1.

Impacts of dietary soybean oil and resveratrol levels on liver lipid deposition in large yellow croaker. (A) Histological and ultrastructural observation of liver tissue (hematoxylin and eosin [H&E] staining: scale bar = 50 μm, magnification 400 ×; Oil Red O staining: scale bar = 100 μm, magnification 200 ×; transmission electron microscopy [TEM] analysis: scale bar = 20 μm, magnification 500 ×; L = lipid droplet; N = nucleus). (B) Relative mRNA expression levels of lipogenesis-related genes in liver. (C) Relative mRNA expression levels of lipolysis-related genes in liver. A, P-value of one-way ANOVA denoting the difference among all the treatments; L, P-value of linear regression denoting the difference among the SO and resveratrol-supplemented groups; Q, P-value of quadratic regression denoting the difference among the SO and resveratrol-supplemented groups; FO, fish oil-based diet; SO, soybean oil-based diet; SR0.05, SO supplemented with 0.05% resveratrol; SR0.1, SO supplemented with 0.10% resveratrol; SR0.2, SO supplemented with 0.20% resveratrol; SR0.4, SO supplemented with 0.40% resveratrol. Results were presented as means with standard error of the mean (n = 3). Data columns with different letters indicate statistical differences (P < 0.05).

The transcription levels of genes related to lipogenesis in fish liver are presented in Fig. 1B. The relative mRNA expression levels of srebp-1 and fas in the SR0.1, SR0.2 and SR0.4 groups were significantly lower than those in the SO and SR0.05 groups, but significantly higher than those in the FO group (P < 0.05). The SO and SR0.05 groups showed significantly higher acc1 relative mRNA expression than the other groups (P < 0.001). The relative mRNA expression level of acc2 was significantly lower in the FO and SR0.2 groups than in the other four groups (P < 0.001). The relative mRNA expression level of dgat2 was significantly lower in the SR0.2 group than in the FO, SO, and SR0.05 groups (P < 0.001). Dietary resveratrol levels had linear and quadratic effects on the relative mRNA expression of all the aforementioned genes (P < 0.05). As shown in Fig. 1C, regarding the expression of lipolysis-related genes in fish liver, the relative mRNA expression levels of atgl and cpt1 in the FO group were significantly higher than those in the other groups (P < 0.05). The SR0.1 and SR0.2 groups exhibited significantly higher relative mRNA expression of atgl compared with the SO and SR0.05 groups (P < 0.001). All resveratrol-supplemented groups showed significantly higher relative mRNA expression of cpt1 than the SO group (P < 0.001), with the SR0.1 group showing the highest value. Except for the SR0.05 group, the remaining four groups exhibited significantly higher relative mRNA expression of pparα compared with the SO group (P = 0.001). The relative mRNA expression levels of the above three genes all displayed a significantly quadratic relationship with dietary resveratrol supplementation (P < 0.05).

3.4. The SIRT1 expression and ERS of liver

As shown in Fig. 2A, except for the SR0.05 group, the remaining four groups showed significantly higher hepatic sirt1 relative mRNA expression level than the SO group (P < 0.001). The relative mRNA expression levels of grp78 and xbp1 in the liver of SR0.1 and SR0.2 groups were significantly lower than those in the SO group, but significantly higher than those in the FO group (P < 0.001). The relative mRNA expression levels of atf6α and atf4 were significantly higher in the SO and SR0.05 groups than in the other four groups (P < 0.05). Fish fed the SO diet exhibited significantly higher chop relative mRNA expression level than the other groups (P < 0.001). Among all the aforesaid genes, xbp1 relative mRNA expression level exhibited a significantly quadratic relationship with dietary resveratrol levels, while the relative mRNA expression levels of other genes were all affected linearly and quadratically by dietary resveratrol supplementation (P < 0.05).

Fig. 2.

Impacts of dietary soybean oil and resveratrol levels on the expression of silent information regulator 1 (SIRT1) and endoplasmic reticulum stress (ERS)-related genes, and ultrastructure of liver tissue in large yellow croaker. (A) Relative mRNA expression levels of sirt1 and ERS-related genes in liver. (B) Transmission electron microscopy images of liver tissue (scale bar = 5 or 1 μm, magnification 2000 × or 8000 × ; L = lipid droplet; N = nucleus; RER = endoplasmic reticulum). A, P-value of one-way ANOVA denoting the difference among all the treatments; L, P-value of linear regression denoting the difference among the SO and resveratrol-supplemented groups; Q, P-value of quadratic regression denoting the difference among the SO and resveratrol-supplemented groups; FO, fish oil-based diet; SO, soybean oil-based diet; SR0.05, SO supplemented with 0.05% resveratrol; SR0.1, SO supplemented with 0.10% resveratrol; SR0.2, SO supplemented with 0.20% resveratrol; SR0.4, SO supplemented with 0.40% resveratrol. Results were presented as means with standard error of the mean (n = 3). Data columns with different letters indicate statistical differences (P < 0.05).

Ultrastructural observation of liver tissue in large yellow croaker from the FO, SO, and SR0.1 groups (Fig. 2B) showed that totally replacing dietary fish oil with soybean oil induced abnormal lipid droplet accumulation in hepatocytes, which compressed the nucleus and organelles, leading to swelling of the endoplasmic reticulum lumen. However, the morphologies of nuclei and endoplasmic reticulum in the SR0.1 group were partially restored.

As shown in Fig. 3A, the SO group presented the lowest liver SIRT1 protein expression level, closely followed by the FO group, and the SR0.1 group had the highest level (P < 0.001). The protein expression levels of liver GRP78 (P < 0.001), XBP1 (P < 0.001), ATF6 (P = 0.007), and p-PERK (P < 0.001) in the SO group were significantly higher than those in the FO and SR0.1 groups. Compared with the FO group, the SR0.1 group displayed a significantly higher protein expression level of XBP1 and a lower protein expression level of p-PERK (P < 0.05). The p-PERK/PERK ratio in the liver of the SO group was significantly higher than that in the SR0.1 group (P = 0.006). The observations of immunohistochemical staining and immunofluorescence analysis (Fig. 3B–E) indicated that compared with the FO and SR0.1 groups, the SO group gave an obviously decreased SIRT1 protein expression level, and increased protein expression of GRP78, ATF6, and p-PERK in the liver.

Fig. 3.

Impacts of dietary soybean oil and resveratrol levels on the expression of SIRT1 and endoplasmic reticulum stress (ERS)-related proteins in liver of large yellow croaker. (A) Expression levels of SIRT1 and ERS-related proteins in liver determined by Western blot. (B) Images of immunohistochemical staining for SIRT1 protein in liver (scale bar = 50 μm, magnification 400 ×; brown-yellow areas indicated by the black arrows represented the positive signals). (C) Images of immunofluorescence staining for GRP78 protein in liver (scale bar = 20 μm, magnification 400 ×). (D) Images of immunofluorescence staining for ATF6 protein in liver (scale bar = 50 μm, magnification 400 ×). (E) Images of immunofluorescence staining for p-PERK protein in liver (scale bar = 20 μm, magnification 400 ×). A, P-value of one-way ANOVA denoting the difference among all the treatments; L, P-value of linear regression denoting the difference among the SO and resveratrol-supplemented groups; Q, P-value of quadratic regression denoting the difference among the SO and resveratrol-supplemented groups; FO, fish oil-based diet; SO, soybean oil-based diet; SR0.1, SO supplemented with 0.10% resveratrol. Results were presented as means with standard error of the mean (n = 3). Data columns with different letters indicate statistical differences (P < 0.05). DAPI = 4′, 6-diamidino-2-phenylindole.

3.5. Inflammatory response of liver

The transcriptions of inflammatory cytokines in fish liver are shown in Fig. 4A. The relative mRNA expression levels of tnfα and il-1β in the SR0.1 group were significantly lower than those of the SO group, while these values were significantly higher than those in the FO group (P < 0.05). The FO and SR0.1 groups showed significantly higher relative mRNA expression levels of arg1 and il-10 compared with the SO group (P < 0.05). The relative mRNA expression level of tgfβ was significantly higher in the FO group than in the other groups (P = 0.003). Excluding tgfβ, the relative mRNA expression levels of other genes all showed a significantly quadratic trend responding to the increasing resveratrol level in the SO (P < 0.05).

Fig. 4.

Impacts of dietary soybean oil and resveratrol levels on liver inflammatory response of large yellow croaker. (A) Relative mRNA expression levels of inflammatory cytokine in liver. (B) Protein expression levels of NF-κB p65 and p–NF–κB p65 in liver. (C) Images of immunofluorescence staining for NF-κB p65 protein in liver (scale bar = 20 μm, magnification 400 ×). A, P-value of one-way ANOVA denoting the difference among all the treatments; L, P-value of linear regression denoting the difference among the SO and resveratrol-supplemented groups; Q, P-value of quadratic regression denoting the difference among the SO and resveratrol-supplemented groups; FO, fish oil-based diet; SO, soybean oil-based diet; SR0.05, SO supplemented with 0.05% resveratrol; SR0.1, SO supplemented with 0.10% resveratrol; SR0.2, SO supplemented with 0.20% resveratrol; SR0.4, SO supplemented with 0.40% resveratrol. Results were presented as means with standard error of the mean (n = 3). Data columns with different letters indicate statistical differences (P < 0.05). DAPI = 4′, 6-diamidino-2-phenylindole.

As presented in Fig. 4B, the SO group had significantly higher p–NF–κB p65 expression level in liver compared with the FO and SR0.1 groups (P = 0.006). Meanwhile, the p–NF–κB p65/NF-κB p65 ratio in the SO group was also significantly higher than that in the FO group (P = 0.016). The immunofluorescence staining result of NF-κB p65 (Fig. 4C) showed that compared with the FO and SR0.1 groups, the protein expression of NF-κB p65 in the liver of the SO group was markedly increased, with fluorescence signals mostly overlapping with the nuclei, indicating that nuclear translocation of NF-κB p65 was enhanced.

3.6. Apoptosis of liver tissue

The expression levels of apoptosis-related genes in fish liver are given in Fig. 5A. The SO and SR0.05 groups showed significantly higher relative mRNA expression levels of bax and caspase 9 compared with the other groups (P < 0.05). The FO group showed significantly lower caspase 3 relative mRNA expression level than the other five groups (P < 0.001). The bcl-2 relative mRNA expression level in the SR0.1, SR0.2, and SR0.4 groups was significantly higher than that in the SO treatment, while it was significantly lower than that of the FO group (P < 0.001). With the increase of resveratrol supplementation in the SO, the relative mRNA expression levels of the above genes all exhibited significantly linear and quadratic trends (P < 0.05).

Fig. 5.

Impacts of dietary soybean oil and resveratrol levels on apoptosis of liver tissue in large yellow croaker. (A) Relative mRNA expression levels of apoptosis-related genes in liver. (B) Protein expression levels of JNK and p-JNK in liver. (C) Images of TUNEL assay of liver tissue (scale bar = 20 μm, magnification 400 ×). A, P-value of one-way ANOVA denoting the difference among all the treatments; L, P-value of linear regression denoting the difference among the SO and resveratrol-supplemented groups; Q, P-value of quadratic regression denoting the difference among the SO and resveratrol-supplemented groups; FO, fish oil-based diet; SO, soybean oil-based diet; SR0.05, SO supplemented with 0.05% resveratrol; SR0.1, SO supplemented with 0.10% resveratrol; SR0.2, SO supplemented with 0.20% resveratrol; SR0.4, SO supplemented with 0.40% resveratrol. Results were presented as means with standard error of the mean (n = 3). Data columns with different letters indicate statistical differences (P < 0.05). DAPI = 4′, 6-diamidino-2-phenylindole; TUNEL = terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling.

As shown in Fig. 5B, the FO group presented the lowest protein expression of p-JNK (P = 0.001) and p-JNK/JNK ratio (P < 0.001), followed closely by the SR0.1 group, while the SO group had the highest values. An in situ apoptosis analysis of liver tissue (Fig. 5C) indicated that the TUNEL signals in the SO group were obviously stronger than those of the FO treatment, while these strong signals were attenuated in the SR0.1 group.

4. Discussion

To date, a large number of studies have been dedicated to exploring the influences of the substitution of dietary fish oil with vegetable oil on farmed fish, with growth and feed performance being essential evaluation parameters (Caballero et al., 2002; Chen et al., 2024; Liu et al., 2022). In this study, totally replacing dietary fish oil by soybean oil significantly reduced the FBW, PWG, and SGR of large yellow croaker, while increasing the FCR and FI. Similar results were also found in previous studies on this fish species (Du et al., 2017; Wang et al., 2023). The impaired growth and feed utilization observed in the SO group may be related to the changed linoleic acid content (increased by 120.45%), n-3/n-6 PUFA ratio (decreased by 70.79%) and n-3 LC-PUFA level (decreased by 64.79%) in the diet caused by the substitution of fish oil with soybean oil in the present study. High consumption of n-6 PUFA (e.g., linoleic acid) could result in lipid metabolic disorders and inflammation (Du et al., 2020; Li et al., 2016), while a proper level of dietary n-3 LC-PUFA has been demonstrated to improve growth by strengthening anti-oxidative ability and immunity, and relieving lipid deposition and inflammation of fish (An et al., 2023; Jin et al., 2017). Meanwhile, a study in spotted seabass (Lateolabrax maculatus) indicated that a suitable n-3/n-6 PUFA ratio promoted growth and nutrient utilization via facilitating body protein retention and improving liver health (Dong et al., 2023). Interestingly, the SR0.1 group showed a significantly higher PWG than the SO group and a similar FBW and FI to the FO group. Meanwhile, the FCR in the SR0.05, SR0.1, and SR0.2 groups was significantly lower than that in the SO group, though it remained significantly higher than that in the FO group. These revealed that the suitable inclusion content of resveratrol in the SO tended to improve the growth and feed performance of large yellow croaker, which may be ascribed to its ability to improve antioxidant capacity, immune response and lipid metabolism, and inhibit inflammation (Wu et al., 2022). It was also demonstrated that dietary resveratrol supplementation could stimulate growth of southern flounder (Paralichthys lethostigma) through exerting antioxidant protective effects to reduce protein and lipid oxidative damage (Wilson et al., 2015). However, resveratrol inclusion in low fish oil diets showed no significant effects on the growth and feed efficiency of gilthead sea bream (Sparus aurata) (Torno et al., 2018). The supplemental level of 0.3% resveratrol even markedly inhibited the growth of rainbow trout (Oncorhynchus mykiss) fed low fish oil diets, which may be connected with a reduction in intake induced by a high dosage of resveratrol (Torno et al., 2017). Therefore, these varying results may be imputed to species differences, as well as variations in resveratrol inclusion levels and feed formulations (e.g., the substitution level of fish oil by vegetable oil).

Abnormal lipid accumulation in the liver which is a vital metabolic organ, can severely affect the growth, development, and health status of fish (Caballero et al., 2002; Sargent et al., 2003; Shen et al., 2023). In this study, a high proportion of dietary soybean oil markedly increased the HSI and contents of liver crude lipid and TAG in the current study, indicating that total substitution of dietary fish oil with soybean oil may trigger liver lipid accumulation in large yellow croaker. A similar phenomenon was also observed in previous studies on related fish species (Du et al., 2017; Mu et al., 2018), sharpsnout seabream (Diplodus puntazzo) (Piedecausa et al., 2007) and groupers (Epinephelus coioides) (He et al., 2021). Serum lipid levels are key parameters reflecting lipid metabolism in organisms, and abnormal lipid accumulation in the liver is often accompanied by the development of dyslipidemia (Cohen and Fisher, 2013; Zhao et al., 2023). The present data revealed that replacing 100% fish oil with soybean oil markedly elevated the TAG and NEFA levels while decreasing HDL-C content in serum, which is consistent with previous findings in large yellow croaker (Gu et al., 2019) and black seabream (Shen et al., 2022). Research on gilthead sea bream showed that the substitution of fish oil with vegetable oils markedly promoted the lipolysis rate in adipose tissue (Cruz-Garcia et al., 2011). The NEFA generated from the degradation of TAG in adipose tissue can enter the liver via the blood circulation and serve as the primary substrate for liver TAG synthesis (Donnelly et al., 2005; Postic and Girard, 2008). Therefore, the heightened lipolysis rate in adipose tissue may be the reason why high proportions of dietary vegetable oils raised serum NEFA level and liver lipid content. However, as the resveratrol inclusion level in the SO gradually increased, the HSI, hepatic crude lipid and TAG levels, and serum TAG and NEFA contents of large yellow croaker exhibited significantly quadratic trends. These five parameters reached their lowest values in the SR0.1 group, being significantly lower than those in the SO group but not significantly different from those in the FO group. These results denoted that 0.10% of dietary resveratrol supplementation may alleviate the hepatic lipid deposition induced by the complete substitution of fish oil with soybean oil in large yellow croaker. To further validate this hypothesis, the FO, SO, and SR0.1 groups were selected for the histological and ultrastructural observation of liver tissue. The H&E staining and Oil Red O staining revealed that a high proportion of dietary soybean oil triggered hepatic steatosis, characterized by severe vacuolation, a decline in the nuclei number, nuclei extruding to one side of the cell, and abundant lipid droplet accumulation. The TEM analysis further confirmed that both the number and size of lipid droplets in the liver of the SO group increased obviously. However, supplementing 0.10% resveratrol in the SO obviously improved hepatocyte vacuolation, increased nuclei number, and diminished both the quantity and diameter of lipid droplets, which corresponded with the results of crude lipid and TAG contents in liver. Likewise, resveratrol inclusion in a high-fat diet significantly reduced the contents of crude lipid and TAG, and mitigated lipid droplet accumulation in liver of black seabream (Jin et al., 2024). Meanwhile, dietary resveratrol also significantly decreased TAG level in serum of rainbow trout (Afzali-Kordmahalleh and Meshkini, 2023), and in plasma of blunt snout bream (Megalobrama amblycephala) (Zhang et al., 2018) and zebrafish (Danio rerio) (Ran et al., 2017). In addition, resveratrol has been proven effective in alleviating the elevation of serum NEFA and TAG amounts induced by a high-fat diet in rats (Huang et al., 2019). It has been concluded that dietary resveratrol downregulated the transcriptions of lipogenesis-related genes and/or upregulated the transcriptions of lipolysis-related genes to attenuate the high-fat diet-evoked hepatic lipid accumulation in black seabream (Jin et al., 2024), blunt snout bream (Zhang et al., 2018), and red tilapia (Oreochromis niloticus) (Zheng et al., 2022). In this study, dietary inclusion with an appropriate content of resveratrol significantly suppressed the upregulation of genes related to lipogenesis (srebp-1, fas, acc1, and acc2) and the downregulation of genes connected with lipolysis (atgl, pparα, and cpt1) in liver caused by the high level of soybean oil. Notably, the SR0.1 and SR0.2 groups exhibited similar relative mRNA expression levels of acc1 and pparα to the FO group. These results suggested that 0.10% resveratrol supplementation may alleviate hepatic lipid deposition induced by the substitution of fish oil with soybean oil through inhibiting lipogenesis and promoting lipolysis in large yellow croaker.

The endoplasmic reticulum is an organelle with a complex membranous network structure and its function is critically dependent on the fatty acid composition and fluidity of the membrane (Volmer and Ron, 2015). The endoplasmic reticulum is highly sensitive to nutrients, and both dietary lipid levels and fatty acid composition may influence its function (Jin et al., 2024; Nivala et al., 2013; Zhang et al., 2011b). In the current study, a high proportion of dietary soybean oil significantly heightened the relative mRNA expression levels of ERS-related genes (grp78, xbp1, atf6α, atf4, and chop) and the expression levels of ERS-related proteins (GRP78, XBP1, ATF6, and p-PERK) in fish liver. Meanwhile, the accumulation of substantial lipid droplets in hepatocytes compressed the nuclei and organelles in the SO group, which triggered swelling of endoplasmic reticulum lumen. These findings collectively demonstrated that complete substitution of dietary fish oil with soybean oil may evoke liver ERS in large yellow croaker. A previous research on related fish species also revealed that replacing 100% fish oil with soybean oil significantly promoted the relative mRNA expression levels of ERS-related genes (grp78, xbp1, atf6, and chop) and GRP78 protein expression level in head kidney, and that linoleic acid incubation observably elevated the expression of these genes and proteins in macrophages as well (Du et al., 2022). Meanwhile, the ERS marker genes and proteins were stimulated significantly in muscle by a high level of dietary palm oil or in myocytes by palmitic acid incubation (Zhao et al., 2024). Furthermore, complete replacement of dietary fish oil with either soybean oil or palm oil significantly upregulated the relative mRNA expression levels of ERS-related genes (grp78, ire1, and atf6α) in the liver of black seabream, accompanied by the lipid droplet accumulation and liver lesions (Shen et al., 2022). In addition, it has been reported that compared to fish oil, soybean oil markedly promoted the expression of ERS marker proteins in rabbit hepatocytes, triggering ERS and lipid droplet accumulation (Zhu et al., 2016). The ERS is strongly linked to lipid metabolism. For instance, IRE1 and PERK could promote lipid deposition by activating peroxisome proliferator-activated receptor (PPARγ) and SREBP-1, respectively to regulate the expression of genes related to lipid metabolism (Lauressergues et al., 2012; Zhang et al., 2011a). Tunicamycin-induced ERS has been found to result in excessive lipid accumulation via disturbing lipid metabolism in the intestine of large yellow croaker (Fang et al., 2021). Taken as a whole, the ERS may play a pivotal role in liver lipid deposition of farmed fish triggered by the substitution of fish oil with high proportions of vegetable oils.

There is building evidence that SIRT1 activation by resveratrol alleviates ERS and lipid deposition via deacetylation modification of ERS-sensor proteins (Jin et al., 2024; Zhou et al., 2018). In this study, totally replacing dietary fish oil by soybean oil markedly impaired the sirt1 relative mRNA expression level and the SIRT1 protein expression level in fish liver. However, supplementation with 0.10%-0.40% resveratrol significantly elevated hepatic sirt1 expression to a level similar to that observed in the FO group. Moreover, the SR0.1 group showed significantly higher hepatic SIRT1 protein expression than both the FO and SO groups. These results manifested that dietary supplementation with 0.10% resveratrol could effectively suppress the soybean oil-induced decline in liver SIRT1 expression of large yellow croaker. A previous study on this fish species demonstrated that palmitic acid incubation of hepatocytes not only significantly increased TAG content, but also markedly downregulated sirt1 transcription (Wang et al., 2017). However, subsequent incubation with eicosapentaenoic acid and docosahexaenoic acid significantly enhanced sirt1 relative mRNA expression level and SIRT1 deacetylase activity. Similarly, resveratrol incubation also markedly raised SIRT1 deacetylase activity (Wang et al., 2017). Correspondingly, the present study found that dietary inclusion with suitable amounts of resveratrol significantly reduced the relative mRNA expression levels of ERS-related genes (grp78, xbp1, atf6α, atf4, and chop) in liver. Notably, compared to the FO treatment, the SR0.1 group exhibited similar relative mRNA expression levels of atf6α, atf4, and chop, comparable GRP78 and ATF6 protein levels, and lower p-PERK expression. Furthermore, the morphologies of nuclei and endoplasmic reticulum were partially restored in the SR0.1 group. The above results collectively revealed that 0.10% resveratrol inclusion could alleviate liver ERS evoked by the complete substitution of fish oil with soybean oil in large yellow croaker. It was recently reported that resveratrol ameliorated high-fat diet-evoked hepatic ERS and abnormal lipid accumulation by activating SIRT1 to promote IRE1 deacetylation in black seabream (Jin et al., 2024). Concurrently, resveratrol has been shown to mitigate ovarian ERS in Nothobranchius guentheri (Zhu et al., 2023) and abnormal hepatic lipid deposition in largemouth bass (Micropterus salmoides) (Huang et al., 2021) through SIRT1 activation. Moreover, resveratrol significantly depressed protein expression of p-PERK and ATF4 in mice liver, thereby attenuating high-fat diet-aroused hepatic ERS and lipid deposition (Zhao et al., 2019). Taken together, the favorable impacts produced by dietary resveratrol on soybean oil-induced hepatic lipid deposition may be partially ascribed to the SIRT1 activation and alleviated ERS in liver of large yellow croaker, which remains to be further studied.

The substitution of dietary fish oil with high levels of vegetable oils usually leads to the concurrence of the inflammatory response and lipid deposition in fish liver. The NF-κB is an important transcription factor that regulates inflammation. External stimuli can trigger the nuclear translocation of cytoplasmic NF-κB, thus initiating the transcriptions of inflammatory cytokines (such as tnfα and il-1β) (Hayden and Ghosh, 2008). The present data showed that a high proportion of dietary soybean oil significantly promoted the transcriptions of pro-inflammatory cytokines (tnfα and il-1β), while inhibiting the transcriptions of anti-inflammatory cytokines (arg1, il-10, and tgfβ), and increasing the NF-κB p65 protein phosphorylation and its nuclear translocation in fish liver. These findings indicated that replacing 100% fish oil with soybean oil evoked liver inflammatory response by inducing NF-κB nuclear translocation in large yellow croaker. Similar observations were also verified in adipose tissue of large yellow croaker (Xu et al., 2022). Meanwhile, complete substitution of dietary fish oil by vegetable oils also led to a significant increment in pro-inflammatory cytokines expression and a marked decline in anti-inflammatory cytokines expression in head kidney and liver of large yellow croaker (Du et al., 2020; Li et al., 2019a,b), as well as in liver of black seabream (Shen et al., 2022). Additionally, linoleic acid incubation also triggered inflammatory response in hepatocytes of large yellow croaker (Du et al., 2020). It has been well-documented that when ERS persists, three key UPR mediators (IRE1, ATF6, and PERK) can arouse the transcriptions of inflammatory cytokines (such as tnfα and il-1β) via promoting the nuclear translocation of NF-κB (Jiang et al., 2003). In both intestinal tissues and cells of large yellow croaker, tunicamycin-induced ERS has been observed to exacerbate inflammatory response (Fang et al., 2021). Therefore, the hepatic inflammatory response found in the SO group of this study may be imputed to the liver ERS caused by the complete substitution of dietary fish oil with soybean oil. Research on black seabream revealed that a high-fat diet or oleic acid incubation triggered inflammation in liver or hepatocyte by activating IRE1 (Shen et al., 2023). However, SIRT1 activation by resveratrol not only alleviated liver/hepatocyte ERS evoked by a high-fat diet/oleic acid incubation but also weakened the inflammatory response through decreasing NF-κB p65 protein phosphorylation in black seabream (Jin et al., 2024). In the current study, as the dietary resveratrol inclusion level gradually increased, the expression of pro-inflammatory cytokines (tnfα and il-1β) in liver quadratically decreased first and then elevated, while the expression of anti-inflammatory cytokines (arg1 and il-10) quadratically enhanced first and then reduced. Specifically, the fish fed SR0.1 presented markedly diminished transcriptions of tnfα and il-1β, while markedly raised relative mRNA expression levels of arg1 and il-10 when compared to the SO group. Furthermore, similar arg1 relative mRNA expression level was observed in the SR0.1 and FO groups. Concurrently, the SR0.1 group displayed obviously reduced nuclear translocation of NF-κB p65 in liver, with NF-κB p65 protein phosphorylation being markedly lower than that of the SO group but showing no significant difference compared with the FO group. In common carp and gibel carp (Carassius gibelio), resveratrol suppressed the elevated expression of hepatic pro-inflammatory cytokines and NF-κB induced by a high-fat diet or acute ammonia exposure, while upregulated anti-inflammatory cytokine expression (Wu et al., 2022, 2023). Meanwhile, SIRT1 expression in the ovarian of Nothobranchius guentheri was significantly promoted by dietary resveratrol, accompanied by the decline in expression of NF-κB, pro-inflammatory cytokines and ERS marker gene (grp78) (Zhu et al., 2023). Additionally, resveratrol has been reported to alleviate emamectin benzoate-induced inflammatory response in hepatocytes of grass carp (Ctenopharyngodon idellus) via mitigating ERS (Bi et al., 2023). Overall, dietary inclusion with 0.10% resveratrol could relieve soybean oil-induced liver inflammation by suppressing NF-κB nuclear translocation in large yellow croaker, which corresponded with the alleviation role of resveratrol on liver ERS aroused by the total substitution of fish oil with soybean oil in this study. However, it still remains unclear whether SIRT1 directly depressed liver inflammatory response by regulating NF-κB or indirectly inhibited inflammation by influencing ERS and thereby modulating NF-κB, which warrants further investigation.

When ERS persists, the UPR can induce apoptosis through the IRE1/JNK, ATF6/CHOP, and PERK/ATF4/CHOP pathways (Wang and Kaufman, 2012). Given the effects of different dietary treatments on liver ERS in large yellow croaker, this study further analyzed apoptosis-related parameters in fish liver. The results showed that a high proportion of dietary soybean oil heightened the transcriptions of pro-apoptotic genes (bax, caspase 3, and caspase 9), the JNK protein phosphorylation and TUNEL signals, while impaired the transcription of anti-apoptotic gene bcl-2 in liver. Similarly, totally replacing dietary fish oil by soybean oil not only induced liver ERS but also significantly upregulated the transcriptions of jnk and its downstream pro-apoptotic genes (bax, caspase 7, and caspase 9), while significantly downregulated bcl-2 expression in liver of black seabream (Shen et al., 2022). Concurrently, the protein phosphorylation level of JNK in liver of large yellow croaker was markedly promoted by the complete substitution of fish oil with olive oil (Li et al., 2019a). The activation of IRE1 under ERS can activate JNK to allow it to enter the nucleus to regulate the transcriptions of apoptosis-related genes, thus triggering apoptosis (Urano et al., 2000). It was concluded that a high-fat diet/oleic acid incubation promoted the JNK protein phosphorylation by activating IRE1, ultimately arousing apoptosis in liver/hepatocytes of black seabream (Shen et al., 2023). Intriguingly, SIRT1 activation by resveratrol not only alleviated liver/hepatocyte ERS evoked by a high-fat diet/oleic acid incubation, but also attenuated apoptosis of black seabream via depressing JNK phosphorylation (Jin et al., 2024). In this study, supplementation with appropriate levels of resveratrol in the SO markedly diminished the transcriptions of bax and caspase 9, while markedly enhanced bcl-2 expression in fish liver. Notably, the SR0.1 and FO groups shared similar relative mRNA expression levels of bax and caspase 9. Although the JNK phosphorylation level in the SR0.1 group remained markedly higher than in the FO group, it was observably lower than that of the SO group. Moreover, the liver TUNEL signal intensity in the SR0.1 group was weakened compared to that in the SO group. A study in grass carp have revealed that resveratrol could relieve emamectin benzoate-induced pyroptosis in hepatocytes through mitigating ERS (Bi et al., 2023). In addition, resveratrol has been shown to ameliorate ERS-evoked cell death after traumatic brain injury in mice (Cao et al., 2024), and to attenuate cadmium-induced ERS and pyroptosis via activating SIRT1 to promote XBP1 deacetylation in human renal tubular epithelial cells (Chou et al., 2019). Taken together, the current study demonstrated that dietary supplementation with 0.10% resveratrol could alleviate soybean oil-induced liver apoptosis in large yellow croaker. However, whether this protective effect of dietary resveratrol is mediated through its alleviation role on ERS or direct regulation of the JNK pathway merits further exploration.

5. Conclusion

In summary, total substitution of dietary fish oil by soybean oil significantly suppressed the growth performance, feed utilization and hepatic SIRT1 expression, as well as triggered abnormal lipid deposition, ERS, inflammation and apoptosis in the liver of large yellow croaker. Supplementation with 0.10% resveratrol in the SO improved PWG and feed utilization of fish to some extent, significantly promoted hepatic SIRT1 expression and alleviated lipid deposition, ERS, inflammatory response, and apoptosis in the liver induced by the substitution of dietary fish oil with soybean oil. The above-mentioned findings revealed that resveratrol could be applied as a nutritional additive in a low fish oil diet of large yellow croaker to improve growth and alleviate liver injury. The present data also demonstrated that SIRT1 may be a key target for fish liver damage triggered by high levels of dietary vegetable oils.

Credit Author Statement

Hua Mu: Writing – original draft, Visualization, Project administration, Funding acquisition, Conceptualization. Zhinan Zhang: Investigation, Formal analysis, Data curation. Ningning Liu: Investigation. Chenbin Yang: Methodology. Jiashun Wu: Investigation. Na Li: Data curation. Shengdi Chen: Investigation. Binlun Yan: Supervision. Huan Gao: Writing – review & editing. Chaoqing Wei: Writing – review & editing, Validation, Project administration, Methodology, Funding acquisition. Lu Zhang: Writing – review & editing, Supervision, Conceptualization.

Declaration of competing interest

We declare that we have no financial or personal relationships with other people or organizations that could inappropriately influence our work. There are no professional or other personal interests of any nature in any product, service or company that could be construed as influencing the content of this paper, except for the following potential competing interest: Lu Zhang is currently employed by Tongwei Agricultural Development Co., Ltd.

Appendix A. Supplementary data

The following is the Supplementary data to this article.