Soya-saponins induce intestinal inflammation and barrier dysfunction in juvenile turbot (Scophthalmus maximus)

Fish and Shellfish Immunology 77 (2018) 264–272

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Full length article

Soya-saponins induce intestinal inflammation and barrier dysfunction in juvenile turbot (Scophthalmus maximus)

T

Min Gu, Qian Jia, Zhiyu Zhang, Nan Bai∗, Xiaojie Xu, Bingying Xu Marine College, Shandong University at Weihai, 180 Wenhua West Road, Weihai, 264209, PR China

A R T I C LE I N FO

A B S T R A C T

Keywords: Turbot Soya-saponins Intestinal inflammation Intestinal barrier Apoptosis Intestinal antioxidant defense

Soybean meal-induced enteritis (SBMIE) is a well-described condition in the distal intestine (DI) of several cultured fish species, but the exact cause is still unclear. The work on Atlantic salmon and zebrafish suggested soya-saponins, as heat-stable anti-nutritional factors in soybean meal, are the major causal agents. However, this conclusion was not supported by the research on some other fish, such as gilthead sea bream and European sea bass. Our previous work proved that soybean could induce SBMIE on turbot and the present work aimed to investigate whether soya-saponins alone could cause SBMIE and the effects of soya-saponins on the intestinal barrier function in juvenile turbot. Turbots with initial weight 11.4 ± 0.02 g were fed one of four fishmealbased diets containing graded levels of soya-saponins (0, 2.5, 7.5, 15 g kg−1) for 8 weeks. At the end of the trial, all fish were weighed and plasma was obtained for diamine oxidase (DAO) activity and D-lactate level analysis and DI was sampled for histological evaluation and quantification of antioxidant parameters and inflammatory marker genes. The activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and intestinal glutathione level were selected to evaluated intestinal antioxidant system. The distal intestinal epithelial cell (IEC) proliferation and apoptosis were investigated by proliferating cell nuclear antigen (PCNA) labelling and TdT-mediated dUTP nick end labeling (TUNEL), respectively. The results showed that soya-saponins caused significantly dose-dependent decrease in the growth performance and nutrient utilization (p < 0.05). Enteritis developed in DI of the fish fed diet containing soya-saponins. Significantly dose-dependent increases in severity of the inflammation concomitant with up-regulated expression of il-1β, il-8, and tnf-α, increased IEC proliferation and apoptosis, and decreases in selected antioxidant parameters were detected (p < 0.05). The epithelial permeability (evaluated by the plasma DAO activity and D-lactate level) was significantly increased with the increasing of dietary level of soya-saponins (p < 0.05), which was concomitant with the destroyed the intracellular junctions. In conclusion, the present work proved that soya-saponins induced enteritis and compromised the intestinal barrier functions. Based on the present work, strategies focus on regulation of cell apoptosis, epithelial permeability, intracellular junctions and redox homeostasis worth further investigating to develop new and efficient ways for SBMIE alleviation.

1. Introduction Fish meal is the primary protein source in feed of farmed fish, especially for carnivorous fish [1,2]. However, the production of fish meal has reached its maximum level over the last decade and price is increasing due to the increased demand [3–5]. The unbalance between the fish meal supply and demand has stimulated exploration of dietary alternative protein sources for the aquaculture industry [6,7]. Among the ingredients that are being investigated as alternatives to fish meal in fish diet, products derived from soybeans are some of the most promising because of the security of supply, price and protein/amino acid composition [8]. However, soybean meal (SBM) of standard quality can

∗

be used in carnivorous fish diets only at relatively low levels due to its negative effects on gut health in several fish species [9,10]. Specifically, SBM has been observed to cause proliferative or inflammatory conditions in the distal intestinal mucosa of cultured fish species such as Atlantic salmon, rainbow trout, common carp and zebrafish [11–16]. The histopathological changes, commonly referred to as soybean meal-induced enteritis (SBMIE), have been extensively studied [17]. SBMIE are characterized by a shortening of the mucosal folds, a swelling of the lamina propria and subepithelial mucosa, a strong infiltration of various inflammatory cells, and decreased numbers of absorptive vacuoles in the enterocytes [11,18–20]. Our previous study has shown that inclusion level of SBM in the range of

Corresponding author. E-mail address: [email protected] (N. Bai).

https://doi.org/10.1016/j.fsi.2018.04.004 Received 13 January 2018; Received in revised form 25 February 2018; Accepted 2 April 2018 Available online 04 April 2018 1050-4648/ © 2018 Elsevier Ltd. All rights reserved.

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260–540 g kg−1 caused the similar inflammatory changes in turbot as seen in other fish species [20]. The exact cause and mechanisms behind the inflammation and other negative effects are not fully understood. Work on the Atlantic salmon indicated that soya-saponins in soybean meal were the vital factors to induce SBMIE [21–26]. Saponins are heat-stable, triterpenoid or steroid, amphipathic glycosides [27]. The amphiphilic property provides saponins the ability to bind and form non-absorbable complexes with cholesterol [28,29]. In mammals, saponins apparently have the ability to bind to membrane cholesterol of intestinal epithelial cells (IEC) and thus form holes and alter membrane permeability, possibly facilitating the uptake of molecules, including antigens and potential toxins that normally are not absorbed by the enterocytes [30]. The previous study in Atlantic salmon clearly demonstrated that soya-saponins alone, supplemented at levels of 2–10 g kg−1, caused a dosedependent increase in the severity of inflammatory changes in the distal intestine tissue as described for SBMIE [25]. Soya-saponins were also proved to be responsible for the SBMIE in Zebrafish [16]. However, dietary soya-saponins at the level of 2–3 g kg−1 had no clear negative effects on channel fish [31], European sea bass [32,33] and gilthead sea bream [34,35] and rainbow trout [36]. The differences might be due to the differences in fish feeding habit, size and age, but also the basal diet, culture condition and experimental duration. Turbot Scophthalmus maximus has become the most important cultured flatfish in Europe and Asia because of its high quality flesh and rapid growth, with a global production of around 70 000 t per year [37]. The turbot faming is facing the problems of fish meal shortage. Our previous work proved soybean meal induced SBMIE on turbot [20]. However, the mechanisms behind SBMIE on turbot need to be further exploited. The purpose of this study was to investigate whether soyasaponins alone may induce enteritis and their effects on intestinal barrier function in turbot.

Table 1 Ingredients and compositions of experimental diets (dry-matter basis). Experimental dieta FM

SAP2.5

SAP7.5

SAP15

610 269 17 17 20 35 5 1 1 0.5 0.5 24 0

610 269 17 17 20 35 5 1 1 0.5 0.5 21.5 2.5

610 269 17 17 20 35 5 1 1 0.5 0.5 16.5 7.5

610 269 17 17 20 35 5 1 1 0.5 0.5 9 15

95.1 48.5 12.2 12.8 20.1

94.9 48.7 12.3 12.7 20.2

95.0 48.5 12.1 12.9 20.2

95.0 48.6 12.3 12.7 20.1

−1

Ingredients (g kg ) Fish mealb Wheat meal Fish oil Soybean oil Soybean lecithin Vitamin and mineral premixc Choline chloride Yttrium premix Calcium propionic acid Ethoxyquin Phaseomannite Cellulose Soya-saponinsd Proximate composition (%) Dry matter Crude protein Crude lipid Ash Gross energy (KJ/g) a

FM: a basal diet; SAP: soya-saponins included to the FM diet and 2.5, 7.5, and 15 indicate the levels of soya-saponins inclusion. b Fish meal: steam dried fish meal (COPENCA Group, Lima, Peru). c Vitamin premix supplied the diet with (mg kg−1 diet) the following compounds: retinyl acetate, 32; vitamin D3, 5; DL-α-tocopherol acetate, 240; vitamin K3, 10; thiamin, 25; riboflavin (80%), 45; pyridoxine hydrochloride, 20; vitaminB12 (1%), 10; Lascorbyl-2-monophosphate-Na (35%), 2000; calcium pantothenate, 60; nicotinic acid, 200; inositol, 800; biotin (2%), 60; folic acid, 20; cellulose, 11473. Mineral premix consisted of (mg kg−1 diet) the following ingredients: FeSO4·H2O, 80; ZnSO4·H2O, 50; CuSO4·5H2O, 10; MnSO4·H2O, 45; KI, 60; CoCl2·6H2O (1%), 50; Na2SeO3 (1%), 20; MgSO4·7H2O, 1200; calcium propionate, 1000; zoelite, 17485. d Soybean saponin (98% in purity) was obtained from Xi'an Chu-kang Biotechnology Co., Ltd (Shaanxi, China).

2. Materials and methods 2.1. Feed ingredients and diet formulation Based on our previous work [20], a fish meal based diet (FM diet) was formulated to contain 48% crude protein and 12% crude lipid with fish meal as the primary protein source, fish oil and soybean oil as lipid sources, and wheat flour as the carbohydrate source (Table 1). This diet was used as the control diet. Based on previous work on the soya-saponins on fish [25,38], another three isonitrogenous and isolipidic diets were formulated with the addition of 2.5, 7.5 and 15 g kg−1 soya-saponins (98% in purity, obtained from Xi'an Chukang Biotechnology Co., Ltd, Shaanxi, China) to FM diet, named as SAP2.5, SAP7.5 and SAP15 respectively. The level of soya-saponins in soybean meal ranges in 5–7 g kg−1 [39,40], the soya-saponin concentration of diet SAP2.5 was similar with that of the diet supplemented with 40% soybean meal, which was proved to induce SBMIE in turbot [20]. All four diet formulations were designed to meet the essential amino acid (EAA) requirements of juvenile turbot based on the whole body amino acid profile [41,42]. The diet preparation and storage were as described by our previous work [43]. Standard methods were used to analyze the experimental diets [44]. Moisture and ash content were determined gravimetrically to constant weight in an oven at 105 °C and 550 °C, respectively. Crude lipid was determined gravimetrically after extraction with ethylether (Extraction System B-811, BUCHI, Switzerland). Crude protein was determined by the Kjeldahl method with a FOSS Kjeltec System (2300, Sweden) using boric acid to trap released ammonia. Gross energy was determined by calorimetric bomb (Parr, Moline, IL, USA).

commercial farm in Haiyang, China and transferred to an indoor flowthrough water system in the Haiyang Yellow Sea Aquatic Product Co., Ltd. The fish were acclimated to the system and fed with the FM diet for 2 weeks. Next, turbot with initial body weight of about 11.4 g were randomly distributed into 12 tanks, 30 fish per tank (filled with 300 L seawater). The seawater was pumped from the adjacent coastal water, filtered through a sand filter, and distributed to each tank at approximately 2.0 L min−1. Each diet was fed to fish in three tanks. Fish were fed with the experimental diets to apparent satiation twice daily at 07:00 and 18:00 and the feed consumption was recorded. During the 8week feeding trial, the water temperature was 12–16 °C, pH 7.8–8.2, and the salinity was 28–30. 2.3. Sampling After 8 weeks of feeding, all experimental fish were anesthetized with eugenol (1: 10000, Shanghai Reagent Co., Shanghai, China) and the body weight was recorded before sampling. And then, eight fish from each tank were randomly selected and blood samples were collected from the caudal vein using heparinized syringes. Plasma samples were obtained by centrifugation (4000 × g for 10 min at 4 °C) and immediately stored at −80 °C until analysis. Subsequently, the fish were killed with a blow to the head and samples for body length were determined. The intestine was removed, cleared of any mesenteric, adipose tissue, and rinsed with ice-cold PBS to remove the eventual remaining gut contents. Only fish with food in the intestinal tract were sampled to ensure recent intestinal exposure to the diets. Four of the eight sampled fish were randomly selected and their distal intestines

2.2. Experimental procedures Apparent disease-free juvenile turbots were obtained from a 265

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2.7. Quantitative real time PCR (qPCR)

(DI) were removed individually and divided into three parts for histological and gene expression examination. For each fish, one part of the DI was placed in 4% phosphate-buffered formaldehyde solution for 24 h, and subsequently stored in 70% ethanol until further processing for light microscopy analysis, proliferating cell nuclear antigen (PCNA) labelling and TdT-mediated dUTP nick end labeling (TUNEL). The second part was fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.0) for transmission electron microscopy (TEM) analysis. The fixed samples were washed in buffer, dehydrated with graded ethanol, and embedded in Spurr's resin for ultrastructure observation. The third part for gene expression analysis was placed in RNAlater (Ambion), stored at 4 °C for 24 h, then at −20 °C temporarily and finally at −80 °C. In these three analyses, the DI from one fish was treated and stored individually and given a specific code. For enzyme activity assessment, the DI tissues from the other four fish per tank were frozen in liquid nitrogen and stored at −80 °C.

Total RNA was extracted and purified from DI tissue samples (approximately 50 mg) using the RNeasy Protect Mini Kit (Qiagen, 74126) according to manufacturer's instructions. RNA was quantified using NanoDrop® ND-1000 spectrophotometer (NanoDrop® Technologies Inc.) and its quality was checked by Agilent Bio-Analizer (Agilent Technologies, USA). The cDNA synthesis was performed with the QuantiTect Reverse Transcription Kit (Qiagen, 205311) using 1.0 μg of RNA and following the manufacturer indications. The expression profiles of interleukin-1 beta (il-1β), interleukin 8 (il8) and tumor necrosis factor α (tnf-α) were determined using real-time quantitative PCR (qPCR) using ribosomal protein S4 (rps4) as the housekeeping gene for sample normalization. The experimental was conducted followed our previous work [46,47]. 2.8. Plasma diamine oxidase activity and D-lactate level assay

2.4. Histology

According to the manufacturer's instructions, plasma diamine oxidase activity and D-lactate level were measured by Diamine oxidase (DAO) assay kit (A088-1, Nanjing Jiancheng Bioengineering Institute, China) and D-Lactic Acid ELISA kit (H263, Nanjing Jiancheng Bioengineering Institute, China), respectively.

Fixed DI tissue samples were processed according to standard histological techniques and stained with hematoxylin and eosin (H&E). Examination was conducted blindly with a light microscope using a continuous scoring scale from 0 to 10 as described by Penn et al. [45]. The following histological characteristics were evaluated: length and fusion of mucosal folds, cellular infiltration and width of the lamina propria and submucosa and enterocyte vacuolization. The DI samples from FM and SAP15 groups for TEM analysis were processed as follows: one ultrathin section for each intestinal sample (total of 12 intestine samples per dietary treatment) was cut and stained with 2% uranyl acetate, post-stained with 0.2% lead citrate and examined in a Jeol JEM-1200 TEM at 80 kV. All digital images were captured with Olymbus SIS software and analyzed using Image J version 1.36.

2.9. Antioxidant enzyme activity and glutathione level assay Intestinal samples were homogenized in 10 vol (w/v) of ice-cold physiological saline and centrifuged at 5000 × g for 20 min at 4 °C, then the supernatants were collected, equally divided into 10 pieces and stored at −80 °C until used for the antioxidant enzyme activity and glutathione level assay. The activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and intestinal glutathione (GSH) level were qualified using commercial kits according to the manufacturer's instructions (A001–1, A007–1, A005, A062, A006-1, respectively; Nanjing Jiancheng Bioengineering Institute, China).

2.5. Immunohistochemistry Detection of proliferating cell nuclear antigen (PCNA) in fixed sections of DI tissue was performed by immunohistochemistry [25]. Sections were deparaffinized using xylene and subsequently rehydrated. The PCNA antigen was retrieved by hot citrate buffer (pH = 6.0) for 20 min. Nonspecific antibody binding was reduced by incubating the sections for 20 min with blocking buffer (1 × PBS/5% normal goat plasma/0.3% Triton ™ X-100). This was followed by overnight incubation at 37 °C with the primary antibody (mouse monoclonal antiPCNA, ab29, Abcam) diluted 1:5000 in antibody dilution buffer (1 × PBS/1% BSA/0.3% Triton ™ X-100). Following rinsing with PBS, the slides were incubated with horseradish peroxidase-labeled goat anti-mouse secondary antibody for 20 min, according to the manufacturer's instructions for the PV-6002 2-step plus® Poly-HRP Anti-Mouse IgG Detection System kit (ZSGB-Bio, Beijing, China). The slides were then exposed to diaminobenzidine for visualization and hematoxylin for nuclear counterstaining. Staining was evaluated by measuring the PCNA staining height. The Pcna-positive proliferative compartment length (PCL) was measured and corrected for body length (PCLBL): PCLBL (μm/m) = [PCL (μm)/body length (cm)] × 100.

2.10. Calculations and statistical analysis Specific growth rate and feed efficiency ratio was applied for growth performance and feed utilization, respectively. The specific growth rate (SGR) was calculated using the tank means for initial body weight (IBW) and final body weight (FBW) and calculated as follows: SGR = [(ln FBW − ln IBW)/number of days] × 100 Feed efficiency ratio was calculated as: Feed efficiency ratio (FER) = (FBW − IBW) / total amount of the feed consumed. Effects of the inclusion levels of soya-saponins were evaluated using regression analysis. The results were fit to polynomial models of first, second, and third degrees. The model considered to fit the results best on the basis of visual examination and the observed R2 is reported. 3. Result

2.6. TUNEL analysis

3.1. Effect of soya-saponins on growth performance and nutrient utilization in turbot

The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was carried out with the In-Situ Cell Death Detection kit (ZK8005, ZSGB-Bio, Beijing, China) according to the manufacturer's instructions. The positive cells were countered in a blinded manner and corrected for 100 μm mucosal fold length. Four folds were detected in one sample.

During the feeding trial, no mortality was recorded. The effects of the experimental diets on turbot growth performance and nutrient utilization are shown in Table 2 and Fig. 1 (A, B). The regression analysis showed a significant inverse relationship between growth and soya-saponins level following a second-degree relationship and FER 266

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3.4. Effect of soya-saponins on electron microscopic structure of the distal intestine in turbot

Table 2 Results of regression analysis of effects of increasing doses of soya-saponins on growth performance, feed utilization, histology, cytokines gene expression, intestinal cell proliferation and apoptosis, intestinal permeability and antioxidant indices data from turbot.

Specific growth rate Feed efficiency ratio Mucosal folds height in DI Mucosal folds fusion in DI Lamina propria width in DI Lamina propria cellular infiltration in DI Submucosa width in DI Submucosa cellular infiltration in DI Enterocyte vaculization in DI Enterocyte nucleus position in DI il-1β expression in DI il-8 expression in DI tnf-α expression in DI PCL in DI PCLBL in DI Apoptotic epithelial cell in DI Serum diamine oxidase activity Serum D-lactate level Superoxide dismutase activity in DI Catalase activity in DI Glutathione peroxidase activity in DI Glutathione reductase activity in DI Glutathione level in DI

P (model)

R2

intercept

X

X2

< 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001

0.97 0.91 0.58 0.62 0.72 0.78

2.659 1.422 7.519 2.225 1.717 1.188

−0.5824 −0.1034 −0.1509 0.2333 0.1313 0.2424

0.1667 0.0194 −0.0001 −0.0067 0.0032 −0.0019

< 0.001 < 0.001

0.67 0.75

1.465 1.265

0.1716 0.2472

−0.0023 −0.0049

< 0.001

0.69

7.585

−0.2522

0.0046

< 0.001

0.56

1.837

0.1503

−0.0016

< 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001

0.87 0.95 0.86 0.93 0.96 0.83

1.128 0.457 1.103 113.290 9.472 0.045

0.2890 0.9463 0.1431 9.6450 1.2290 1.1686

0.0041 0.012 −0.0034 −0.255 −0.034 −0.038

< 0.001

0.97

8.131

11.3170

−0.3073

< 0.001 0.067

0.83 0.45

11.049 43.133

0.7862 −1.2135

−0.0124 0.036

0.021 < 0.001

0.78 0.94

3.881 25.369

−0.2451 −2.9468

0.0102 0.1025

0.002

0.76

4.621

0.1043

−0.0169

< 0.001

0.89

15.525

−0.8893

0.0152

All the 12 fish fed the SAP15 diet showed increased infiltration of leucocytes in the epithelium layer and intercellular space compared with fish fed the control diet by TEM analysis (Fig. 3). The intercellular junctions include apical tight junction (TJ), subjacent adherens junction (AJ) and desmosomes in all the 12 fish fed SAP15 diet were shorter and less developed than fish fed the control diet (Fig. 3 (C–D)). Cytolytic necrosis was observed in the IEC of fish fed the SAP15 diet (Fig. 4). The cytolytic necrotic cells were characterized by decreased cytoplasm with transparency increased, dissolved nucleus and organelles, and the cell separated from the surrounding cells. 3.5. Effect of soya-saponins on proliferation of distal IEC in turbot Results of the PCNA staining indicated that increases in soya-saponins levels significantly increased the height of the staining in the mucosal folds (Fig. 5A). The regression analyses showed that a seconddegree function fit the results best (Fig. 5B). 3.6. Effect of soya-saponins on apoptosis of distal IEC in turbot As can be seen from Fig. 6A, the numbers of apoptotic epithelial cell identified by TUNEL staining significantly increased with increasing level of soya-saponins inclusion. The regression analyses showed that a second-degree function fit the results best (Fig. 6B). 3.7. Effect of soya-saponins on intestinal epithelial permeability in turbot The regression analyses showed that the plasma DAO activity and Dlactate level increased with increasing soya-saponins inclusion in the diet, fitting a second-degree relationship (Table 2 and Fig. 1 (I, J)).

Abbreviations: DI, distal intestine; PCL, PCNA-positive compartment length; PCLBL, PCNA-positive proliferative compartment length corrected for body length. il-1β: interleukin-1 beta, il-8: interleukin 8, tnf-α: tumor necrosis factor α.

3.8. Effect of soya-saponins on intestinal antioxidant parameters in turbot The regression analyses showed that SOD, CAT, GPX and GR activities and GSH level significantly decreased with the increasing level of soya-saponins inclusion. All the indices followed a second-degree relationship best (Table 2 and Fig. 1 (K, L)).

showed the same picture.

4. Discussion

3.2. Effect of soya-saponins on light microscopic structure of the distal intestine in turbot

The present study showed negative effects of soya-saponins on growth performance and feed utilization in turbot, which was in agreement with the previous study in Japanese flounder with fish meal as the primary protein source [48]. In Atlantic salmon, supplementation of 2 g kg−1 soya-saponins to a diet containing high levels of pea protein concentrate resulted in significantly decreased growth performance [21]. However, dietary soya-saponins had no clear effects on neither growth nor feed utilization in channel fish at the inclusion level of 2.6 g kg−1 [31], European sea bass at the inclusion level of 2 g kg−1 [33] and gilthead sea bream at the inclusion level of 2 g kg−1 [34]. Even in Atlantic salmon, addition of soya-saponins (2, 4, 6 or 10 g kg−1) to two basal diet with fishmeal as the primary protein source or with a blend of fishmeal, wheat gluten and lupin meal did not affect the growth performance [25]. The diverging effects of soya-saponins seem to be related to fish species and dietary protein sources. In the present study, soya-saponins were added to a simple diet with protein provided only by fish meal and dose-dependently decreased the growth performance and feed utilization in turbot. The present study clearly demonstrated that soya-saponins, supplemented at levels of 2.5–15 g kg−1 of diet, caused a dose-dependent increase in the severity of inflammatory changes in DI tissue as described for SBMIE in turbot [20]. Our finding strengthened the conclusion of Krogdahl et al. that soya-saponins were the causal agent to

The soya-saponins inclusion level affected all the characteristics assessed and showed alterations typical for mucosal inflammation (Fig. 2). The severity increased with increasing level of soya-saponins inclusion. The soya-saponins inclusion decreased height and increased fusion of the mucosal folds, increased width and cellular (leucocyte) infiltration of the lamina propria and submucosa and reduced numbers of supra-nuclear absorptive vacuoles in enterocytes. All the characteristics followed a second-degree relationship best, with increasing effect with increasing soya-saponins level (Table 2 and Fig. 1 (C–E)).

3.3. Effect of soya-saponins on gene expression of distal intestinal cytokines in turbot As can be seen from Table 2 and Fig. 1 (F-H), the expressions of il1β, il-8 and tnf-α showed gradual and accelerating increases with increasing soya-saponins inclusion in the diet, fitting a second-degree relationship. Dietary soya-saponins at 2.5 g kg−1 dietary level elevated most of these parameters, indicating that even lower inclusion levels might affect the immune apparatus of the intestine.

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Fig. 1. Illustration of relationships between level of dietary soya-saponins supplementation (g kg−1) and (A) specific growth rate (SGR; % day−1), (B) feed efficiency ratio (g gain/g dry feed), (C) mucosal folds height in DI (score), (D) lamina propria cellular infiltration in DI (score), (E) submucosa cellular infiltration in DI (score), (F) mean normalized expression of il-1β in DI tissue, (G) mean normalized expression of il-8 in DI tissue, (H) mean normalized expression of tnf-α in DI tissue, (I) plasma diamine oxidase activity (nmol/ml), (J) plasma D-lactate level (U/l), (K) catalase activity in DI (U/mg protein) and (L) glutathione peroxidase activity (U/mg protein). The curves illustrate the regression that fits the results best according to the regressions shown in Table 2. DI: distal intestine, il-1β: interleukin-1 beta, il-8: interleukin 8, tnf-α: tumor necrosis factor α.

increased infiltration of leukocytes was also observed in epithelial monolayer. In mammals, pro-inflammatory cytokines, such as IL-1β, IL8 and TNF-α can lead to recruitment and activation of leukocytes. And these leukocytes emigrate from blood vessels, reach the sub-epithelial space, and migrate across the epithelial monolayer toward the intestinal lumen [49]. The results of the present study showed that turbot may have similar process of the leukocyte transepithelial migration when intestinal inflammation happens.

induce the SBMIE [25]. At the lowest dietary level, the effects were small, but trends were observed for changes of light microscope structure showing by increased width and cellular infiltration in lamina propria and submucosa and higher expression of pro-inflammatory cytokine genes including il-1β, il-8 and tnf-α. The inflammatory responses in SAP15 fed fish were strongest as shown by highest degree of cellular infiltration in both lamina propria and submucosa and expression of pro-inflammatory cytokines. The TEM data showed that 268

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Fig. 2. Representative histomorphological images from hematoxylin and eosin-stained sections of the distal intestine of turbot depicting the gradual increase in the severity of the inflammatory changes with increasing soya-saponins supplementation in turbot fed the FM (a, e, i), SAP2.5 (b, f, g), SAP7.5 (c, g, k) and SAP15 (d, h, l). (a–d) Representative images of decreased height and increased fusion of the mucosal folds with increasing soya-saponins level. (e–h) Representative images of increased width and cellular (leucocyte) infiltration of the lamina propria with increasing soya-saponins level and images of reduced numbers of supranuclear absorptive vacuoles in enterocytes. (i–l) Representative images of increased width and cellular (leucocyte) infiltration of the submucosa with increasing soya-saponins level. FM: a basal diet; SAP: soya-saponins included to the FM diet and 2.5, 7.5, and 15 indicate the levels of soyasaponins inclusion.

Fig. 3. Electron microscopic structure of the distal intestine epithelium of turbot fed with experimental diets. (A) and (C), FM diet, a basal diet; (B) and (D), 15 g kg−1 of the soya-saponins inclusion level to FM diet. The black arrow in (A) and (B) represents the infiltrated leucocytes into the epithelium layer. In (C) and (D), the black arrow, white arrow and white triangle represent apical tight junction (TJ), subjacent adherens junction (AJ) and desmosomes, respectively.

Fig. 4. Cytolytic necrosis in the fish fed the diet supplemented with 15 g kg−1 of the soya-saponins. The black arrows in (A), (B) and (C) represent cytolytic necrotic cells.

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Fig. 5. (A) Representative images of the localization and distribution of immunohistochemically labeled proliferating cell nuclear antigen (PCNA) protein in the epithelial cells of the distal intestine of turbot depicting the dose-dependent increase in the relative number of proliferating cells with increasing soya-saponin supplementation considered low for the FM diet, moderately elevated for the SAP2.5 and SAP7.5 diets, and highly elevated for the SAP15 diet. (B) Quantification of PCNA positive cells in the intestinal mucosa. PCL, PCNA-positive compartment length; PCLBL, PCNA-positive proliferative compartment length corrected for body length. FM: a basal diet; SAP: soya-saponins included to the FM diet and 2.5, 7.5, and 15 indicate the levels of soya-saponins inclusion.

compared with those fed with the diet based on fish meal [52]. However, the roles of other caspases members and their regulation mechanisms in the SBMIE are still unknown. The molecular pathway regulating caspases and apoptosis has been viewed as therapeutic tool in inflammatory bowel disease [53] and would probably be utilized as potential strategies for preventing SBMIE. Besides the apoptosis, the present work also proved that the dose-dependent manner of cell proliferation in turbot with the dietary level of soya-saponins, which was consistence with the Atlantic salmon [25]. The epithelium of the intestinal mucosa acts as a physical barrier that helps to block pathogens, while allowing dietary nutrients to enter the body. An intact intestinal mucosal barrier is therefore very important for intestinal function [54]. The DAO, an intracellular enzyme catalyzing the oxidation of diamines, exists in high concentrations in

In the present study, soya-saponins supplementation induced dosedependent decreasing in mucosal fold height, which can serve as a criterion reflecting intestinal damage condition induced by inflammation. The atrophy of mucosa was accompanied by the enhanced apoptosis, as investigated by TUNEL. In metazoans, apoptosis is central to the development and homeostasis and its dysregulation leads to a variety of pathologies [50]. The central components of the apoptotic response are caspases, which lead to the characteristic morphological and biochemical hallmarks of apoptosis, and ultimately lead to fragmentation of the DNA and the engulfment of dying cells or apoptotic bodies [51]. At least 14 distinct mammalian caspases have been identified, but the work on fish caspases is relatively scarce. Bakke-McKelle et al. proved that the reactivity of caspases-3 increased in the intestine of the Atlantic salmon fed the diet with SBM supplementation

Fig. 6. (A) Representative images of the TUNEL staining epithelial cells in the distal intestine of turbot depicting the dose-dependent increase in the relative number of apoptotic epithelial cells with increasing soya-saponin supplementation considered low for the FM diet, moderately elevated for the SAP2.5 and SAP7.5 diets, and highly elevated for the SAP15 diet. Apoptosis was evaluated with TUNEL (black arrow). Apoptotic cells in the ablation area can be visualized by brown-yellow nuclear staining. (B) Quantification of TUNEL-positive apoptotic cells corrected for 100 μm mucosal fold length. FM: a basal diet; SAP: soya-saponins included to the FM diet and 2.5, 7.5, and 15 indicate the levels of soya-saponins inclusion. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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ZR2017PC016), China Postdoctoral Science Foundation (Grant No. 2017M610428), The Key Laboratory of Mariculture (KLM), Ministry of Education, Ocean University of China (Grant No. KLM2018001) and Young Scholars Program of Shandong University, Weihai (Grant No. 2016WHWLJH04). All authors read and approved the final manuscript.

the intestinal mucosa, and the majority of DAO in the blood comes from the intestine [55]. The plasma activity of DAO is a reliable marker of intestinal mucosal integrity [56]. D-lactate is a metabolic product of bacteria, present in the intestinal lumen. The intact intestinal mucosa provides a barrier function to prevent DAO and D-lactate infiltrating the portal blood, which are indices of increase in permeability of the intestinal wall [57]. In the present study, the plasma DAO activity and Dlactate level increased with the increasing of soya-saponins inclusion, which indicated that soya-saponins increased the epithelial permeability in turbot. Similar results were also reported by Knudsen et al. on Atlantic salmon [26]. It has been proved in mammals that saponins could bind to membrane cholesterol of intestinal epithelial cells and thus form holes and alter membrane permeability [30] and our work added the information on the mechanisms of soya-saponins inducing epithelial permeability from the view of intercellular junctions and antioxidative system. Epithelial intercellular junctions are the key structures regulating paracellular trafficking of macromolecules and control the epithelial permeability [58]. As shown by the TEM data, the intercellular junctions were destroyed in fish fed the SAP-containing diet, and resulted in an increased intercellular space. The intercellular junctions include an apical tight junction (TJ), subjacent adherens junction (AJ), and desmosomes [59]. During the inflammation, epithelial intercellular junctions alter rapidly and regulated by pro-inflammatory cytokines, which induce endocytosis of TJ proteins in epithelial cells [60]. As in the present work, the intracellular junction destruction could be partly attributed to the higher expression of il-1β, il-8 and tnf-α, which are shown to impair the TJ in different experimental models [49]. Modulation of cytokine function have been used for therapy for chronic intestinal inflammation [61] and such strategies could be applied on fish for alleviating SBMIE. Oxidative stress in the inflammation is another reason for the disruption of epithelial and endothelial junctions and enhanced epithelial permeability [62]. Oxidative stress is defined as an imbalance between oxidants production and antioxidant defense system and involved in several symptoms of inflammation [63]. Reactive oxygen species, such as superoxide radical anion, are major oxidants and can promote disruption of both TJs and AJs and disruption of intercellular junctions [64]. Antioxidant defenses involve enzymes and non-enzymes. Superoxide radical anion undergoes dismutation by SOD and generates hydrogen peroxide, which can be transformed into water by CAT and by GPx with GSH converting to its oxidized form. The presence of GR is responsible for regeneration of GSH [65]. In the present work, dietary soya-saponins decreased above mentioned antioxidant enzymes activities and GSH level in dose-dependent manner. These results indicated that soya-saponins impaired the antioxidant systems of the turbot intestine, induced the imbalance in the redox homeostasis and eventually may impair the intracellular junction. Administration of antioxidants maybe beneficial in the treatment of human intestinal inflammation [66] and such substrates have the potential for alleviating SBMIE. In conclusion, soya-saponins induced dose-dependent increases in severity of inflammation in DI tissue as described for SBMIE in turbot. Dietary soya-saponins decreased mucosal folds height, induced intestinal cell proliferation and apoptosis and increased epithelial permeability with accompany of destroying the intracellular junctions and impairing the intestinal antioxidative system. The present work suggested that some strategies for inflammation therapy targeting cell apoptosis, epithelial permeability, intracellular junctions and redox homeostasis worth further investigating to evaluate their anti-SBMIE functions.

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Acknowledgments This study was jointly supported by the National Science Foundation of China (Grant No. 31502174 and 31702363), Shandong Provincial Natural Science Foundation (Grant No. 2014ZRCP003 and 271

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