Effects of Bacillus subtilis on hepatic lipid metabolism and oxidative stress response in grass carp (Ctenopharyngodon idellus) fed a high-fat diet

Grass carp is one of the most important freshwater species in China that can consume artifcial diets and water plants (Du et al. 2006; Kong et al. 2017b). Feeding carp a high-fat diet may lead to fatty liver disease in this species and adversely infuence aquaculture production of grass carp (Huang et al. 2018). The literature clearly shows that dietary lipids can bring protein sparing efect and enhance growth. However, high levels of dietary lipids may have a negative efect on fsh growth. A study by Du et al. (2005) showed a positive efect only when dietary lipids were below 4% and a negative efect was observed when dietary lipids were higher than 6% in the growth of grass carp. Furthermore, consumption of a high-fat diet may lead to unwanted hepatic fat deposition (Li et al. 2016; Wang et al. 2015), dyslipidemia (Sabzi et al. 2017; Zhang et al. 2018), afect the antioxidative system (Chen et al. 2016; Huang et al. 2018; Ma et al. 2018), and afect normal gut microbiota leading to dysbiosis (Al-Muzafar and Amin 2017; Falcinelli et al. 2017; Tian et al. 2016). The above-mentioned efects of consuming a high-fat diet may ultimately impact health and reduce fsh harvest yields. Given this, it is of vital importance to fnd solutions to alleviate the adverse efects of consuming a high-fat diet.

Published studies have shown that probiotics can modify gut microbiota and signifcantly reduce the risk for fatty liver disease in mammals, improve liver function, and have signifcant therapeutic efects on fatty liver disease induced by high-fat diet (Al-Muzafar and Amin 2017; Awaisheh et al. 2013; Xu et al. 2012). Recently, two published studies showed that using the probiotic Lactobacillus rhamnosus as a feed additive could improve intestinal epithelium structures and induce transcriptional decrease of genes related to triglyceride and cholesterol metabolism, concomitantly decreasing the body triglyceride and cholesterol levels in zebrafsh induced by a high-fat diet (Falcinelli et al. 2015, 2017). B. subtilis, a gram-positive, sporeforming, non-pathogenic bacterium, is widely used in aquaculture as a probiotic (Ren et al. 2017). B. subtilis has been shown to have a positive impact on the size and structure of the microbial community in the intestinal chyme, on the activity of some digestive enzymes, and on the digestibility and weight gain of carp (Zuenko et al. 2017). It has also been shown to improve growth performance and disease resistance in shrimp by enhancing the immune response (Zokaeifar et al. 2012). Our studies also show that B. subtilis has a protective efect, specifcally on the intestines in grass carp. This protective efect is based on the following knowledge about B. subtilis. (1) B. subtilis can modulate intestinal microfora and improve digestive enzyme activity and growth performance of grass carp (Wu et al. 2012). (2) B. subtilis has a protective efect on the intestinal mucosal structure, which can reduce damage to the intestinal mucosal barrier, and decrease infammation in grass carp (Huang et al. 2017). (3) B. subtilis efectively protects fsh against oxidative stress damage (Tang et al. 2018). However, few studies examine the efect of B. subtilis on liver lipid metabolism at present.

Based on the above rationale and results of our previous studies, we aimed to determine the impact of B. subtilis on liver lipid metabolism and oxidative stress of grass carp by evaluating growth performance and morphology, hepatic lipid accumulation and histochemistry, serum biochemical indicators, expression of genes involved in lipid metabolism, and oxidative stress indicators. Our fndings can inform solutions for reducing the risk of fatty liver disease in grass carp as well as provide a theoretical basis for the application of B. subtilis in aquaculture practice.

Data on the growth performance and morphological parameters of grass carp are presented in Table 1. After 8 weeks, the SGR and WGR of grass carp in the two groups fed the high-fat diet signifcantly increased compared to those in the control group (P < 0.05). The grass carp in the HF+B. subtilis group showed the highest WGR (P < 0.05). Furthermore, the HF group and HF+B. subtilis group had a higher FI and lower FCR compared to the control group (P < 0.05). Lastly, no signifcant diference was observed in SR, CF, HSI, and VSI among all the experimental groups (P > 0.05).

Results of oil-red O staining are presented in Fig. 1a. Lipids are red colored and nuclei are blue colored after staining with oil-red O. The images show that the amount and volume of lipid droplets stained by oil-red O were higher in the HF group than those in the Con group, but lower than the HF + B. subtilis group. The relative areas of lipid droplets stained with oil-red O (Fig. 1b) were consistent with the results of hepatic lipid accumulation. As shown in Fig. 1c, the hepatic lipid content of the HF + B. subtilis group was between the control group and HF group, and was significantly different from the control group and HF group (P < 0.05).

Figure 1.  Efect of B. subtilis on hepatic lipid accumulation in grass carp: (a) efect of the B. subtilis diet and high-fat diet on histochemistry (oil-red O staining) (original magnifcation×400, bars 50 μm); (b) the relative areas of the lipid droplets in oil-red O stained grass carp were analyzed by Image-Pro Plus 6.0; (c) efect of the B. subtilis diet and high-fat diet on the lipid content in the liver of grass carp. Values are the mean± SE (n=6). Diferent letters indicate signifcant diferences among groups (P < 0.05)

As shown in Table 2, analysis of key indicators for lipid metabolism showed that the serum CHO and LDL-C levels in the HF group significantly increased compared to the control group (P < 0.05). However, in the HF + B. subtilis group, the serum CHO and LDL-C levels decreased to nearly the same level as the control group (P > 0.05). Moreover, fish fed the high-fat diet with B. subtilis had lower serum AST than fish fed the high-fat diet (P < 0.05). There was no significant difference in the serum TG, HDL-C content, ALT, TP, and Alb among the control group, HF group, and HF + B. subtilis group (P > 0.05).

As shown in Fig. 2, the groups displayed diferences in relative mRNA expression of lipid metabolism genes and transcription factors. The L-FABP was signifcantly up-regulated in the HF group (P < 0.05) with no signifcant diference in the HF + B. subtilis group compared to the control group and HF group (P > 0.05), respectively. The FAS, ACCα, SCD, LPL, CPTIα1a, SREBP-1c, PPARγ and PPARα were signifcantly down-regulated after fsh were fed the highfat diet for 8 weeks (P < 0.05). Moreover, the expression of FAS was signifcantly down-regulated and the expression of CPTIα1a was signifcantly up-regulated in the HF + B. subtilis group compared with the HF group (P < 0.05). The expression of LPL and PPARα in the HF + B. subtilis group was regulated to nearly the same level as the control group (P > 0.05).

Figure 2.  Efect of B. subtilis on lipid metabolism gene and transcription factor mRNA expression in liver of grass carp. Values are the mean±SE (n=6). Diferent letters indicate signifcant diferences among groups (P < 0.05)

As shown in Fig. 3, in the HF + B. subtilis group, GSH showed a signifcant increase compared to the HF group (P < 0.05), and the T-AOC higher than the control group (P < 0.05). H₂O₂ and MDA levels in HF + B. subtilis group were lower than in the HF group (P < 0.05) with no signifcant diference in the control group.

Figure 3.  Efect of B. subtilis on MDA and H₂O₂ content and activities of antioxidant enzymes in liver of grass carp. Values are the mean ±SE (n=6). Diferent letters indicate signifcant diferences among groups (P < 0.05)

Our results show that feeding a high-fat diet to grass carp for 8 weeks signifcantly increased SGR, WGR, and FI. This fnding is similar to fndings from a study conducted in zebrafsh fed a high-fat diet (Shimada et al. 2015; Zang et al. 2015) but difers from the results of the study in Atlantic salmon, Japanese seabass, and Jade Perch (Hillestad et al.1998; Xu et al. 2011). The inconsistency across studies may be related to the diferent feed formula, FI, aquaculture environment, and the species of fsh. Many study authors have reported that some probiotics, such as commercial mixed-species probiotics (Standen et al. 2016), Lactobacillus acidophilus (Chelladurai et al. 2012), Saccharomyces cerevisiae (Abass et al. 2018), B. subtilis, and Lactobacillus rhamnosus (Munirasu et al. 2017), are conducive to increasing the growth performance of fsh. In the present study, we observed that administration of B. subtilis was also benefcial to body weight gain as evidenced by a signifcant increase on WGR with no signifcant diference on FI compared to the HF group. In addition, no signifcant diferences were noted for CF, HSI, and VSI among grass carp fed two dietary lipid levels, which is similar to the study results in meagre Argyrosomus regius with increased dietary lipid (Chatzifotis et al. 2010).

Lipid content and histochemical analyses of the liver demonstrated that a high-fat diet caused hepatic lipid accumulation and dietary supplementation probiotic B. subtilis decreased the hepatic lipid content with one study showing that Lactobacillus rhamnosus could decrease the lipid content of zebrafsh (Falcinelli et al. 2015). Similar results were also observed in juvenile Senegalese sole (Solea senegalensis, Kaup 1858) receiving a diet containing probiotic Shewanella spp. strains Pdp11 and Pdp13 signifcantly decreased numbers of lipid droplets in the liver and modulated the sole intestinal microbiota. This regulation of intestinal fora was mainly manifested as the increased presence of Shewanella spp. members and the decreased presence of Vibrio spp. members in the microbiota (Banda et al. 2010; Tapia-Paniagua et al. 2014). In our experiments, we only measured the hepatic lipid content of grass carp supplemented with B. subtilis. The change of intestinal fora after B. subtilis treatment will be a measurement in our follow-up study. Additionally, this observation was also similar to results of treating mammals with fatty liver disease induced by high-fat diet with probiotics (Lee et al. 2006; Ma et al. 2008; Xu et al. 2012).

Once fatty acids enter the liver, they are esterifed into TG and then can be stored into the hepatocytes in lipid droplets or enter the blood through TG-rich lipoproteins (Yuan et al. 2016), which can lead to a signifcant change in CHO and TG in blood (Jung et al. 2013; Rincon-Cervera et al. 2016). Therefore, serum lipid metabolism-related indicators, such as CHO, TG, HDL-C and LDL-C, were evaluated. Our data showed that the content of CHO in the HF group was higher than those of the other two groups. In addition, compared with the HF group, adding B. subtilis to the high-fat diet resulted in a decreased LDL-C content. Similar studies confrmed that intestinal probiotics are benefcial for lowering CHO levels in rats (Awaisheh et al. 2013) and treatment with Bifdobacterium spp. decreased LDL-C in serum in high-fat diet-induced obese rats (An et al. 2011). This suggests that B. subtilis supplementation alleviates dyslipidaemia, which corresponds to results that B. subtilis could reduce liver lipid contents in our study. AST and ALT are the best clinical biomarkers of hepatic functions and are mainly distributed in the mitochondria and cytoplasmic water-soluble phase, respectively. Once the liver cells are damaged, a large amount of AST and ALT will enter into the extracellular space and ultimately into circulation (Ozer et al. 2008). Hassaan et al. (2018) observed a signifcant decrease in AST and ALT in Nile tilapia (Oreochromis nilotcus) fed a diet containing B. subtilis. We found that the AST in serum decreased in the HF + B. subtilis group, similar to results observed with specifc probiotic strains in fatty liver disease of mice and rats induced by a high-fat diet (Al-Muzafar and Amin 2017; Xin et al. 2014).

To explore the molecular mechanisms involved in B. subtilis regulation of lipid deposition, the expression of several key genes in the liver was analyzed. We frst examined the mRNA expression of L-FABP, which is related to fatty acid transport. L-FABP is a type of fatty acid binding protein that transports lipids to lipid droplets for storage and to mitochondria for β-oxidation (Schroeder et al. 1998; Venkatachalam et al. 2013). Briskey (2015) observed that probiotics restored expression of L-FABP gene to a level similar to that in mice fed with a chow diet, which was similar to our results. The main lipogenic enzymes, such as FAS, ACC, and SCD1 (Chen et al. 2013; Song et al. 2016), and important lipogenic factors, such as SREBP-1c and PPARγ, can regulate hepatic fatty acid synthesis, which in turn, drives hepatic triglyceride synthesis (Eberle et al. 2004; Rosen et al. 1999; Tontonoz et al. 1994). PPARα regulates lipid catabolism by inducing the expression of CPT (Ji et al. 2011; Kerner and Hoppel 2000; Morash et al. 2008). Our results showed that the high-fat diet caused downregulation of both the enzymes involved in fatty acid synthesis (FAS, ACCα, SCD) and transcription factors (PPARγ and SREBP-1c). The down-regulation of these mRNA expressions is a form of adaption of grass carp to a high-fat diet to achieve a dynamic balance between endogenous fatty acids and exogenous fatty acids (Li et al. 2016). Down-regulated CPTIα1a mRNA expression suggests that the high-fat diet caused the synthesis and β-oxidation of fatty acids to be reduced. B. subtilis down-regulated FAS mRNA expression and up-regulated CPTIα1a mRNA expression, indicating that adding B. subtilis to the diet may decrease the hepatic lipid content by inhibiting the synthesis and promoting the β-oxidation of fatty acids, which is consistent with the result of serum CHO and LDL-C content. Studies by Briskey (2015) and Yoo et al. (2013) showed results similar to ours.

Oxidative stress and altered redox balance play an important role in the pathogenesis of steatosis (Roberto et al. 2011). SOD, CAT, and GSH are important antioxidant enzymes that play an important role in protecting cells from oxidative stress and preventing or repairing oxidative damage. For example, the dismutation of two superoxide radicals to H₂O₂ was shown to be catalyzed by SOD, whereas H₂O₂ was degraded by CAT (Wang et al. 2009). MDA is a product of lipid oxidative damage and it is used to evaluate lipid peroxidation (Tang et al. 2018; Yang et al. 2010). Our experimental results showed that after feeding grass carp the diet containing B. subtilis, their antioxidant capacity was signifcantly improved and the oxidative stress response was also reduced, which was mainly refected in the signifcant improvement of liver GSH, the lower content of MDA and H₂O₂ compared with HF group and the higher T-AOC compared with control group. The supplementation of B. subtilis in the diet after 40 days could signifcantly increase the activity of T-AOC and SOD and signifcantly decrease the MDA levels in the serum of Litopenaeus vannamei (Shen et al. 2010). After feeding the basic diet containing B. subtilis for 4 weeks, the activity of T-AOC, SOD, CAT and GSH was signifcantly increased and the content of MDA was signifcantly decreased in the liver of grass carp (Li et al. 2012). These results are similar to ours. Consequently, B. subtilis efectively enhanced the antioxidant capacity and improved oxidative damage of grass crap fed a high-fat diet.

In conclusion, the present study demonstrated that a high-fat diet intake induced hepatic lipid accumulation, but dietary supplementation of probiotic B. subtilis may inhibit the synthesis and promote the β-oxidation of fatty acids in the liver to signifcantly reduce hepatic lipid accumulation, alleviate dyslipidaemia and liver oxidative damage in grass carp, which contribute to the amelioration of diet-induced fatty liver disease progress. Additionally, B. subtilis promotes the growth performance of grass carp, which has a certain guiding signifcance for aquaculture production.

The bacteria used in this study were isolated from the gut of grass carp. The isolated bacterial strain was identifed as B. subtilis Ch9 and stored in the Laboratory of Aquatic Animal Medicine, Fisheries College of Huazhong Agricultural University. B. subtilis was inoculated onto a Luria–Bertani (LB) agar plate, then incubated for 24 h at 37 ℃. A single clone was selected and inoculated into LB broth and cultured in a shaker at 37 ℃ for 3 days. Bacterial cells were harvested by centrifugation at 3500 rpm for 15 min. The supernatant was discarded. The pellet was re-suspended in sterile phosphate-bufered saline (PBS) to remove metabolic waste from the bacterial solution. It was then diluted.

Three diets were prepared and used in this study control (basal) diet (4% lipids of the dry matter), high-fat diet (8% lipids of the dry matter), and high-fat diet supplemented with B. subtilis (8% lipids of the dry matter). The composition and chemical analyses of the three diets are presented in Table 3. All ingredients were purchased from Hubei Haida Feed Co., Ltd. (Wuhan, China). After ingredients were thoroughly mixed (including B. subtilis solution), pellets with a diameter of 2 mm were produced by a granulator in 30 min. After the pellets were air dried, they were stored in a freezer at - 20 ℃ until use. Before use, the survival of B. subtilis was 1 × 10⁷ CFU g^-1 as determined by the plate counting method.

Grass carp were obtained from a commercial freshwater fsh farm (Bai Rong Aquaculture Co., Ltd., Hubei Province) and reared in circular polyester tanks. After 2 weeks, 135 healthy grass carp (50.24 ± 1.38) g were randomly divided into one of the three groups: control (fed a basal diet), HF (fed a high-fat diet), and HF + B. subtilis (fed a high-fat diet supplemented with B. subtilis), with each group in three replicate tanks with a capacity of 300 L (15 fsh per tank). All grass carp were fed to apparent satiation twice daily (08:30 and 16:30) for 8 weeks. During the entire experimental period, one-third of the water was replaced daily. The recirculating water temperature was maintained at (25 ± 1) ℃, pH at 7.5 ± 0.3, dissolved oxygen at (7 ± 0.45) mg·L^-1, ammonia at (0.015 ± 0.002) mg·L^-1 and nitrate at (0.05 ± 0.008) mg·L^-1.

At the end of the 8-week feeding trial, approximately 24 h after the last feeding, the survival rate and body weight gain of fsh in every tank were calculated. A total of 24 fsh per group were then randomly selected and euthanized (MS-222, 10 mg·L^-1). Six fsh were measured for their individual body length and body weight to calculate the condition factor (CF). Blood samples were collected from the caudal vein before dissection to measure serum biochemical indicators. The fsh were then dissected on ice. The liver weight and visceral mass weight were measured to calculate the hepatosomatic index (HSI) and viscera index (VSI). Six fsh were immediately removed using sterile forceps, frozen in liquid nitrogen and stored at - 80 ℃ (not longer than 2 weeks) for total RNA extraction and antioxidant activities, six fsh for determination of liver lipid content, and another six fsh for histochemical observation.

Live samples were freeze dried for 24 h at - 50 ℃ to obtain the dried liver sample. The crude lipid content of individual samples was determined using Soxhlet extraction.

Histochemical analysis was performed as described by Song et al. (2016) with slight modifcations. Briefy, histochemical observations were carried out by oil-red O staining. First, the liver samples were fxed with paraformaldehyde, dehydrated with sucrose, embedded in optimal cutting temperature compound (OCT), sliced (thickness 8 μm) using a cryostat microtome, and stained with oil-red O. Photos were then taken under a microscope (400 ×). Ten felds of view were randomly selected from each sample to calculate the relative area of lipid droplets in oil-red-O stained liver tissue.

The CHO, TG, HDL-C, LDL-C, Alb, TP, AST, and ALT contents were determined using an automatic biochemical analyser (Selectra-xl, the Netherlands) at the Fisheries College of Huazhong Agricultural University (Wuhan, Hubei, China). All kits were purchased from Biosino Bio-Technology and Science, Inc.

TriPure Reagent Kit (Aidlab, RN0102) was used according to its instructions to extract RNA from the liver tissue of grass carp. The quality and quantity of RNA were then assessed via agarose gel (1%) electrophoresis and spectrophotometric (A260:280 nm ratio) analysis, respectively. RNA was reverse transcribed into cDNA using a TRUE script 1st Strand cDNA Synthesis Kit with gDNA Eraser (Aidlab, PC5402). β-Actin is a commonly used reference gene to normalize cDNA loading in grass carp in our laboratory (Kong et al. 2017a, b; Tang et al. 2018) and was used in this study. Specifc primers were designed according to the published sequences of grass carp and are presented in Table 4. Real-time quantitative PCR was performed on a Roche LightCycler 480 real-time PCR instrument. The total volume of the reaction was 20 μL and included 2 μL cDNA, 0.5 μL each primer, 7 μL aqueous diethylpyrocarbonate (DEPC), and 10 μL 2 × SYBR Green qPCR Mix (Aidlab, PC5902). The relative mRNA expression levels were calculated using the 2^-ΔΔCt method.

The total antioxidant capacity (T-AOC), the malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) concentration, the superoxide dismutase (SOD) and catalase (CAT) activity, and glutathione (GSH) levels were determined using commercial kits provided by Nanjing Jiancheng Bioengineering Institute (China). Kits were used according to manufacturer instructions.

One-way analysis of variance was used to analyze the data. All results are expressed as the mean ± SE (standard error of the mean). Multiple comparisons were performed using the Duncan multiple range test among the groups. Statistical signifcance was P < 0.05. All statistical analyses were performed using SPSS 22.0.

This work was supported by the National Natural Science Foundation of China (Grant nos. 31472310 and 31672683) and the Technical Innovation Project of Hubei Province (Grant No. 2018ABA103).

Conceptualization, HZ and YL; methodology, HZ, YL, YZ, XC, HW, DG and ZW; validation, HZ and YL; writingoriginal draft preparation, HZ and YL; writing-review and editing, XC and ZW; supervision, ZW; project administration, ZW.

The authors declare that there are no conficts of interest.

All applicable international, national, and institutional guidelines for the care and use of animals were followed by the authors.