Two experiments were performed in vivo and in vitro. All procedures were approved by the Northwest A&F University Institutional Animal Care and Use Committee.
2.1. Experiment 1: in vivo study
2.1.1. Experimental diets
Three isonitrogenous and isoenergetic purified diets were formulated by adding different concentrations of DHA (Net content of 50%，Xunda Marine Biological Products Co., Ltd., Jiangsu, China). The fish were divided into three groups based on the concentrations of DHA, including Control (0% DHA), and 0.5% and 1% DHA groups. The formulation and proximate composition of the experimental diets are demonstrated in Table 1. Soybean oil (Kerry Oils & Grains Co., Shenzhen, China) and linseed oil (Hoval Seasons Bio-Sci Co., Changchun, China) were added to supplement 1% linoleic acid (LA) and 1% alpha-linolenic acid (LNA) essential fatty acid requirements, respectively. The experimental diets were prepared according to previously described methods .
2.1.2. Experimental procedure
Fish culture and management procedures used in this study were described by Shi et al. . A total of 108 juvenile grass carp (initial weight: 29.76 ± 2.34 g) were distributed into 9 tanks (225-L) with 12 fish per tank. Diets were randomly assigned to three replicate tanks, and the feeding experiment lasted for 8 weeks.
At the end of the experiment, the fish were euthanized via immersion in tricaine methanesulfonate (MS-222; 100 mg/L) (Sigma, St. Louis, MO, USA). The final body weight (FBW, g), body length (BL, cm), relative intestine length (RIL, %), hepatosomatic index (HSI, %), condition factor (CF, g/cm3), survival rate (SR, %), viscerosomatic index (VSI, %), kidney index (KI, %) and spleen index (SI, %) of each fish were determined. Subsequently, triglyceride (TG) content, hematoxylin & eosin (H&E) staining, fatty acid composition, antioxidant enzymatic activities and relative gene expression levels of lipid synthesis, inflammation and ER stress were analyzed in the liver tissues.
An automatic biochemical analyzer (Hitachi 7180, Tokyo, Japan) was used to examine the serum biochemical indices, including alanine aminotransferase (ALT, U/mL), aspartate aminotransferase (AST, U/mL), total protein (TP, g/L), albumin (ALB, g/L), globulin (GLB, g/L), albumin and globulin (A/G) ratio, high-density lipoprotein cholesterol (HDL-c, mmol/L) and low-density lipoprotein cholesterol (LDL-c, mmol/L).
2.2. Experiment 2: in vitro study
2.2.1. Cell culture and treatment
We cultured the grass carp hepatocyte L8824 cell line as described previously . The palmitic acid (PA; article no.: P5585) and DHA (article no.: D2534) were obtained from Sigma Aldrich (St. Louis, Missouri, USA). PA and DHA were dissolved in ethanol (Zhiyuan Chemical Reagent Co., Ltd., Tianjin, China) to prepare 100 mM stock solution and then diluted with a medium containing 2% free fatty acid (FFA)-free bovine serum albumin (BSA) to obtain the designed concentrations before use. PA (200 μM) with or without DHA (50 or 100 μM) was used to treat hepatocytes for 24 h. We added 10 μM Compound C (CC; an AMPK inhibitor, HY-13418; Medchem Express Inc., Monmouth Junction, NJ, USA) in water, and the AMPK inhibitor was added 2 h before fatty acid treatment. Briefly, the experiment was divided into four experimental groups: Control, 200 μM PA, 200 μM PA +DHA (50 or 100 μM) and 200 μM PA +100 μM DHA+10 μM CC (pretreatment for 2 h).
2.3. Sample analysis
2.3.1. Fatty acid composition analysis in experimental diets and liver samples
We used gas chromatography (GC) to detect fatty acid composition as described previously . The fatty acid composition of the experimental diets is observed in Table 3.
2.3.2. TG content, malondialdehyde (MDA) content and enzymatic activity assays on the liver and hepatocytes
The TG content was measured using a tissue triglyceride assay kit (Applygen Technologies Inc., Beijing, China). Then, antioxidant enzymatic activities, such as superoxide dismutase (SOD) and catalase (CAT), were measured . The end products of lipid peroxidation MDA were measured using assay kits (Jian Cheng Biochemical Co. Nanjing, China).
2.3.3. Liver histological processing and morphological evaluation, cell viability assays and Nile red staining
We prepared liver sample sections and stained them with H&E, as described previously . These histological samples were observed and photographed under upright microscopy (Leica Biosystems, Germany), and relative cell viability was determined following standard instructions of the Cell Counting Kit-8 (K009-100T, Zeta Life Inc., USA) . Briefly, hepatocytes were plated in a 96-well plate (1 × 104 cells/well) and cultured until these reached 85-90% confluence. Then, hepatocytes were treated with different concentrations of PA (100 or 200 μM), DHA (50 or 100 μM) and 200 μM PA +100 μM DHA for 24 h. Then, we added 10 μl CCK-8 solution to each well (total reaction volume of 110 µL) and incubated for another 3 h. The absorbance was detected at the wavelength of 450 nm. For observing the effect on lipid deposition, Nile red staining was used to view the lipid droplets in hepatocytes .
2.3.4. Investigation of the concentrations of pro-inflammatory cytokines in serum by ELISA assay
Pro-inflammatory cytokine concentrations, including TNFα and NFκB, were examined using Fish ELISA Assay kits (FANKEL Industrial Co., Ltd, Shanghai, China).
2.3.5. Transcriptomic analysis of the liver samples
We detected the transcriptome of DHA-fed grass carp liver as described by Jing et al. . Briefly, total liver RNA (2 fish/tank) was extracted, and its concentration, purity and integrity were determined. Subsequently, the total RNA from fish in each tank was mixed in equivalent amounts (3 replicates per treatment). RNA was enriched, fragmented and reverse transcribed into complementary DNA (cDNA), and the second cDNA strand was synthesized. Consequently, the cDNA fragments were purified using magnetic bead precipitation and terminally repaired. poly (A) was added and attached to Illumina sequencing adapters. Finally, ligation products were size-selected through agarose gel electrophoresis, amplified using polymerase chain reaction (PCR) and sequenced using the Illumina novaseq 6000 platform (Sangon Biotech (Shanghai) Co., Ltd., China) following the manufacturer instructions.
2.3.6. Quantitative real-time PCR (qPCR) and western blot analysis using liver samples and hepatocytes
We investigated gene transcription levels based on our previous study . The various primers used in this study are given in Table 2. The comparative Ct method (2−ΔΔCt) was used to calculate the target gene expression values . We obtained protein translation levels from our previous study . The primary antibodies against FAS (WL03376, 1:1000, Wanleibio) and β-Actin (3779, 1:1000, Prosci) were applied. Finally, the secondary antibody, goat anti-rabbit horseradish peroxidase conjugate (A02010, 1:2000, Abbkine), was added.
2.4. Statistical analysis
All statistical analyses were performed with SPSS 25.0 software (SPSS, Chicago, IL, USA). Percentage data, such as the fatty acid composition of the liver, were arcsine-transformed before analysis . The Shapiro–Wilk test was done to analyze the normality of the data distribution, and Levene’s test was performed to examine the homogeneity of the variances. The differences among the treatments were analyzed by one-way analysis of variance (ANOVA) and compared by Duncan’s test. The student’s t-test was used for 2-group comparisons in vivo and in vitro. All data were then expressed as mean ± standard deviation (SD), and P < 0.05 was considered significant.