Dietary Sodium Butyrate Supplementation Alleviates High-Fat Diet-Induced Liver Injury by Activating Nrf2 in Common Carp (Cyprinus carpio)

Aquafeed costs are a major consideration in aquaculture, generally accounting for 40%-50% of the total cost of production (Craig and Helfrich, 2002). Protein is considered as the most expensive part in aquafeed (Craig and Helfrich, 2002). Given this, many strategies have been implemented in aquaculture to decrease the protein content with other nutrients (Sargent et al., 2003), such as lipids. The protein-sparing effects of dietary lipids have been proved in many fish species, such as Atlantic cod (Gadus morhua) (Morais et al., 2001), blunt snout bream (Megalobrama amblycephala) (Li et al., 2012), and hybrid fish tambatinga (female Colossoma macropomum × male Piaractus brachypomus) (Welengane et al., 2019).

Common carp (Cyprinus carpio) is a worldwide-distributed species (cultured in over 100 countries) and accounts for up to 10% (over 4 million metric tons in 2018) of annual freshwater aquaculture production in the world (FAO, 2020). In China, the aquaculture production of common carp has reached 2.89 million metric tons (China Fishery Stastical Yearbook, 2020). For the sake of maximum culture profit, inclusion of large amounts of non-protein energy (especially high fat) into diets of common carp has been a common phenomenon in China (Abasubong et al., 2018). Intake of high-fat diets reduced the growth performance and disrupted the lipid metabolism in common carp (Abasubong et al., 2018; Ze et al., 2015). Fish liver is a multifunctional organ acting in detoxification, metabolism of carbohydrates and fat, and scavenging foreign substances (Dalmo et al., 1997; Moeller et al., 2014). Therefore, it is becoming increasing urgent to explore dietary strategies for counteracting the adverse effects of high-fat diets on the liver health in common carp.

Sodium butyrate (NaBT) is a salt of butyric acid and a commonly used additive to improve fish gut health and growth performance (Abdel-Latif et al., 2020; Tran et al., 2018). Recently, numerous studies on mice have suggested that dietary supplementation with NaBT could reduce the negative effects of high-fat diets on liver health (Fang et al., 2019; Matheus et al., 2017; Zhai et al., 2019). In addition, ingestion of 300 mg/kg NaBT via gavage in rats fed high-fat diets reduced the oxidative stress (Sun et al., 2019), fat accumulation and inflammation in the liver (Sun et al., 2018). However, much less research has investitgated the effects of NaBT on the liver health of aquatic animals fed high-fat diets. Given this, we hypothesized that dietary supplementation with NaBT could improve the liver health of common carp fed high-fat diets.

To that end, the present study investigated the effect of dietary NaBT supplementation on the growth performance, lipid deposition, oxidative stress, and inflammation in liver of common carp fed high-fat diets.

Three isonitrogenous (31% crude protein) diets with different fat content were formulated. The control diet (Control) contained medium fat (5.8%); the high fat diet (HF) contained high crude lipid (10.8%); the NaBT diet (NaBT) supplemented 0.1% NaBT in HF diet (Table I).

All ingredients were mixed thoroughly. Then the mixture were pelleted to pellets (2 mm) using a feed machine (Laifu Tk-12B, Guangdong, China). Pellets were dried to a moisture content of 8-10% and kept at -20 °C until used.

Common carp juveniles were purchased from a local fish pond (Luoyang, China) and were acclimated to laboratory conditions for 2 weeks. After starvation for 24 h, 216 experimental fish were randomly divided into 9 tanks (100 L). Each diet was assigned to triplicate tanks. Experimental fish were fed twice daily (8:00 and 17:00). Feed consumption of each tank was adjusted based on fish body weight which were weighted every two weeks. The experiment lasted for 8 weeks, during which water temperature was 25.7±1.4 °C, dissolved oxygen was above 6 mg/L, ammonia-nitrogen and nitrite were both below 0.1 mg/L.

1Sodium butyrate was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., Shanghai, China.

At the end of the feeding trial, all fish were starved for 24 h before sampling. All fish in each tank were anesthetized with benzocaine (50 mg/L), counted and weighed. Then the body weight and body length of 3 fish in each tank were recorded for the determination of condition factor (CF) and their blood were drawn from caudal vein to detect the activity levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Then liver samples of another 9 fish in each tank were sampled and divided to three portions. The first portion was collected and preserved at 4% paraformaldehyde for Oil Red O staining, and the second and third portions were stored at -80 °C for enzyme activity assay and quantitative real-time polymerase chain reaction (PCR).

The study protocol and all experimental procedures were approved by Experimental Animal Ethics Committee of Henan University of Science and Technology.

Proximate composition of experimental diets was tested according to the procedures described by AOAC (1995). Moisture content was determined through drying samples to a constant weight at 105°C. Crude protein content was measured with the Kjeldahl method. Crude lipid content was determined in a Soxtec system. Ash content was determined with a muffle furnace for 2 h at 600°C.

Fixed liver samples were first dehydrated in a graded series of ethanol with concentration increasing from 75% to 100% and were embedded in parafflin. Then liver samples were sliced into sections (4 μm). Liver sections were stained with Oil Red O. The area stained with Oil Red O solution was analyzed by Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA).

Activity levels of ALT and AST in plasma were measured according to 2, 4-dinitrophenyl hydrazine (DNPH) method (Reitman and Frankel, 1957). Triglyceride (TG) content was measured with a peroxidase-coupled method (McGowan et al., 1983) and TCHO content were tested following the method described by Allain et al. (1974).

MDA content was determined with thiobarbituric acid (TBA) following the method of Esterbauer and Cheeseman (1990). Commercial reagent kits (Nanjing Jiancheng Bioengineering Institute, China) were used to detect the activity levels of catalase (CAT) and glutathione peroxidase (GPx), and contents of glutathione (GSH). Activity level of total superoxide dismutase (T-SOD) was assayed with xanthine/xanthine oxidase method (McCord and Fridovich, 1969). Protein content in liver samples was tested with coomassie brilliant blue method following Bradford (1976).

Total RNA in liver was isolated by acid guanidinium thiocyanate-phenol-chloroform extraction. After determination of RNA concentration and quality, total RNA (1 μg) was used to synthesize first-strand cDNA for RT-PCR with a commercial reagent kits (TransGen Biotech Co., Ltd.)

Primers were synthesized commercially from Tsingke Biotechnology (Wuhan, China) (Table II). Real time PCRs were performed on a Light Cycler 96 (Roche Diagnostics, Meylan, France). The total reaction volume was 15 μL, containing 7.5 μL Light Cycler 480 SYBR Green I Master mix (Roche Diagnostics), 2.0 μL cDNA template, 1.5 μL of primer (2.5 μM), and 4.0 μL PCR-grade water. Each sample was analyzed in duplicate with the following thermal cycling conditions: 95 °C for 10 min; followed by 40 cycles of 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 20 s. Relative quantification of target gene transcripts were calculated using the 2-ΔΔCt method (Pfaffl, 2001).

The growth performance parameters were calculated according to following formulae:

SR (%) = 100 × (1 - dead fish number/initial fish number)

WGR (%) = 100 × (final body weight - initial body weight)/initial body weight

SGR (% d-1) = 100 × [ln (final body weight) - ln (initial body weight)]/ days

Feed efficiency (FE, %) = 100 × (fresh body weight gain)/dry feed intake

CF (g/cm3) = 100 × body weight/body length3

Nrf2, nuclear factor erythroid 2-related factor 2; HO-1, heme oxygenase-1; TNF-α, tumor necrosis factor α; IL-1β, interleukin-1β; IL-6, interleukin-6.

Prior to analysis, the Shapiro-Wilk and Levene tests were used to examine data normality and homogeneity of variance, respectively. Then Data that met the requirements were analyzed with one-way ANOVAs in SPSS v.20.0. P <0.05 was considered as statistically significant. All data were expressed as mean ± standard error of means (SEM).

There was no difference among groups in final body weight (FBW), WGR, SGR, FE, SR, and CF (Table III).

IBW, initial body weight; FBW, final body weight; WGR, weight gain rate; SGR, specific growth rate; FE, feed efficiency; SR, survival rate; CF, condition factor.

Experimental diets did not affect plasma ALT activity level. Diet HF increased AST activity level compared with the control. However, diet NaBT significantly decreased ALT activity level compared to diet HF (P<0.05) (Fig. 1).

Common carp fed diet HF significantly increased liver TG and TCHO contents compared with that fed control diet. However, fish fed diet NaBT had a lower TG (P>0.05) and TCHO (P<0.05) contents in liver compared to that fed diet HF (Fig. 2). More lipid droplets in fish fed diet HF were observed than that in fish fed diets Control and NaBT (P<0.05) (Fig. 3).

Compared with diet control, diet HF significantly increased MDA content, and simultaneously decreased activity levels of T-SOD, CAT and GPx, as well as contents of GSH. However, diet NaBT significantly decreased MDA contents, and significantly increased T-SOD and CAT activity levels (P<0.05), compared with diet HF (Fig. 4).

Compared to control, common carp at the HF group had a lower expression level of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) (P<0.05). However, common carp fed diet NaBT showed a significantly higher expression levels of Nrf2 and HO-1 compared with that fed the HF diet and exhibited a significantly lower expression levels of Nrf2 and HO-1 relative to that fed diet control (P<0.05) (Fig. 5).

Compared with the control, diet HF significantly upregulated the mRNA expression of tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and IL-6 in liver (P<0.05), whereas diet NaBT significantly reduced the mRNA expression of TNF-α, IL-1β, and IL-6 (P<0.05) compared to fish fed diet HF. There were no significant difference in these genes in liver of fish fed diets control and NaBT (P>0.05) (Fig. 6).

In the current study, high-fat diets did not affect fish growth and feed utilization, similar phenomenon were also found in previous studies on common carp (Abasubong et al., 2018) and blunt snout bream (Chen et al., 2016). Moreover, dietary supplementation with 0.1% NaBT has no influence on fish growth, in agreement with results of common carp fed diets containing 300 mg/kg microencapsulated sodium butyrate (MSB) (Liu et al., 2014). However, in other studies, enhancement of growth performance was observed in fish fed diets contacting NaBT, such as grass carp fed diets added with 0.1% powdery sodium butyrate or 0.05%-0.2% MSB (Tian et al., 2017), and rice field eel (Monopterus albus) fed with high soybean meal diets containing 0.025%-0.1% MSB (Zhang et al., 2020), and turbot (Scophthalmus maximus) fed 0.2% NaBT-containing diets (Liu et al., 2019). This discrepancy may be partly explained by the difference in fish species, dose and form of NaBT, feed composition, and rearing environment (Biagi et al., 2007; Liu et al., 2014, 2019).

In line with previous studies (Dai et al., 2019; Du et al., 2006), we found that high-fat diets significantly increased the deposition of TG and TCHO in the liver. However, dietary 0.1% NaBT supplementation significantly decreased hepatic TCHO content and lipid droplets. At present, few researches have examined the effect of dietary NaBT supplementation on the liver TCHO and lipid droplets in aquaculture. However, reports on rats and pigs suggested that NaBT could reduce lipid deposition through peroxisome proliferator-activated receptor α-mediated activation of β oxidation (Sun et al., 2018). In addition, studies on pigs suggested that dietary 0.1% NaBT supplementation could reduce triglyceride content in liver by reducing lipogenesis and enhance lipolysis via regulating related hormones and genes, such as down-regulating the expression of fatty acid synthase (Jiao et al., 2020). Thus NaBT could decrease TCHO contents and lipid droplets by enhancing lipolysis and reducing lipogenesis, further study is needed to reveal the underlying mechanism.

The increased activity level of AST in plasma usually indicates liver damage or dysfunction (Ashouri et al., 2015; Wang et al., 2006). In the current study, dietary NaBT supplementation in high-fat diets significantly decreased AST activity levels, indicating that NaBT decreased the impairment of liver function induced by high fat. At present, reports on the effect of NaBT supplementation on the blood AST activity levels are scarce. However, accumulating data in terrestrial animals suggested that dietary NaBT supplementation could significantly reduce AST activity levels in blood of mice or rats fed high-fat diets (Mattace-Raso et al., 2013; Zhou et al., 2017, 2018). The reduction of AST activity in plasma of common carp fed NaBT diets may be due to antioxidant effect of NaBT. NaBT could reduce the hepatic oxidative stress, thereby stabilizing the membrane permeability and reducing the leakage of AST into the blood (Nasr, 2014).

High-fat diets often leads to oxidative stress in fish (Jia et al., 2020; Lu et al., 2017; Zhou et al., 2020). These oxidative stress includes increasing MDA contents and decreasing activity levels of antioxidant enzymes (Jia et al., 2017; Zhong et al., 2020). In this study, high-fat diets significantly increased MDA content and decreased the activity levels of antioxidant enzymes (e.g, SOD and CAT), indicating that oxidative stress occurred in fish fed on high-fat diets. However, dietary 0.1% NaBT supplementation significantly decreased MDA content and increased the activity levels of SOD and CAT, suggesting that NaBT alleviated the high-fat diet-induced oxidative stress.

Decrease in hepatic oxidative stress by NaBT could be due to the activation of Nrf2/HO-1 pathway. Nrf2/HO-1 pathway plays an important role in defending oxidative stress (Loboda et al., 2016). Nrf2 controls the expression of many antioxidant response element dependent genes and has been reported to upregulate the mRNA expression of CAT and SOD (Ma, 2013). Nrf2 could activate the transcription of HO-1 which degrades heme and generates the antioxidant molecules (Loboda et al., 2016). In the present study, lower and higher expression levels of Nrf2 and HO-1 were found in fish fed high-fat diets and NaBT diet, respectively, indicating NaBT increased the activation of Nrf2/HO-1 pathway. NaBT is known as an activator of Nrf2 (Dong et al., 2017; Wu et al., 2018; Yaku et al., 2013). In mammals, it has been proved that NaBT increased the expression of Nrf2 by inhibiting histone deacetylase (Dong et al., 2017; Wang et al., 2012). Whether NaBT activated Nrf2 expression through inhibiting HDAC in fish is an interesting question which was worthy of further investigation.

Liver inflammation is a common phenomenon in aquatic animals (Cao et al., 2020; Dai et al., 2019). TNF-α, IL-1β, and IL-6 are commonly proinflammatory cytokines (Rauta et al., 2012) and have been identified as markers of inflammation in fish (Dai et al., 2019; Urán et al., 2008). In this study, high-fat diets significantly upregulated the mRNA expression levels of these proinflammatory cytokines in the liver, implying the occurrence of the liver inflammation in fish fed high-fat diets. However, dietary addition with NaBT in the high-fat diets significantly decreased the mRNA expression of these proinflammatory cytokines. Oxidative stress may partly account for the phenomenon. Oxidative stress and inflammation are closed linked; continued oxidative stress is known for leading to chronic inflammation through activating a variety transcription factors (Reuter et al., 2010). In this study, NaBT reduced the oxidative stress, thus alleviating the liver inflammation.

In the present study, dietary 0.1% NaBT supplementation significantly reduced hepatic fat deposition and improved liver function of common carp fed high-fat diets. In addition, dietary supplementation with 0.1% NaBT in high-fat diets reduced hepatic oxidative stress and inflammation in common carp by activating Nrf2/HO-1 pathway.

The research is supported by the Doctoral Scientific Research Foundation of Henan University of Science and Technology (13480088), the Student Research Training Program (SRTP) in Henan University of Science and Technology (2021383, 2021373) and Henan Province (S202110464058), and the Key Research and Development and Promotion of Special (Science and Technology) Project of Henan Province (212102110368).

The authors have declared no conflict of interest.