The protective role of daidzein in intestinal health of turbot (Scophthalmus maximus L.) fed soybean meal-based diets

Soybean meal-induced enteropathy (SBMIE) is prevalent in aquaculture. The aim of this study is to evaluate the role of daidzein on SBMIE of juvenile turbot (Scophthalmus maximus L.) by feeding with fish meal diet (FM), soybean meal diet (SBM, 40% fish meal protein in FM replaced by soybean meal protein) and daidzein diet (DAID, 40 mg/kg daidzein supplemented to SBM) for 12 weeks. We found that daidzein supplementation elevated the gene expression of anti-inflammatory cytokine TGF-β, decreased gene expression of pro-inflammatory cytokines TNF-α and signal molecules p38, JNK and NF-κB. SBM up-regulated the genes expression related to oxidative stress and apoptosis, but dietary daidzein restored it to the similar level with that in FM group. Moreover, dietary daidzein up-regulated gene expression of tight junction protein, and modified the intestinal microbial profiles with boosted relative abundance of phylum Proteobacteria and Deinococcus–Thermus, genera Sphingomonas and Thermus, species Lactococcus lactis, and decreased abundance of some potential pathogenic bacteria. In conclusion, dietary daidzein could ameliorate SBM-induced intestinal inflammatory response, oxidative stress, mucosal barrier injury and microbiota community disorder of turbot. Moreover, p38, JNK and NF-κB signaling might be involved in the anti-inflammatory process of daidzein, and daidzein itself might act as an antioxidant to resist SBM-induced oxidative damage.

. Effects of daidzein on gene expression of cytokines and signaling molecules of turbot fed with soybean meal. FM fish meal diet, SBM soybean meal diet, DAID 40 mg/kg daidzein included into SBM diet, TNF-α tumor necrosis factor-α, p38 p38 mitogen-activated protein kinase, TGF-β transforming growth factor-β, JNK c-Jun N-terminal kinase, ERK extracellular regulated kinase; NF-κB nuclear transcription factor-kappa B; Values are mean ± SEM, n = 3 and values shared different letters are significantly different (P < 0.05).  Table S1). The β-diversity analysis, such as Non-Metric Multi-Dimensional Scaling (NMDS) (Fig. 7A), principal co-ordinates analysis (PCoA) (Fig. 7B) as well as Unweighted Pair-group Method with Arithmetic Mean (UPGMA) analysis (Fig. 7C) based on weighted unifrac distances, indicates that the clusters and intestinal bacteria composition of DAID were more similar to FM group, and distinctly separated from SBM. MetaStat analysis on genus and species level showed that daidzein significantly boosted the abundance of unidentified_Desulfobacteraceae, unidentified_Xanthomonadales, bacterium_enrich-ment_culture_clone_AOM-SR-B34, Steroidobacter_sp._WWH78 and Lactococcus lactis (Table 1). Besides, according to analysis of linear discriminant analysis (LDA) Effect Size (LEfSe), at phylum level, the supplementation of daidzein strikingly increased the abundance of Proteobacteria and Deinococcus-Thermus, and decreased    www.nature.com/scientificreports/ the abundance of Bacteroidetes (Fig. 8). Moreover, daidzein highly decreased the relative abundance of family Bacteroidales S24-7 from 53.00% (SBM group) to 0.25% (DAID group) (Supplementary Table S2). At genus and species levels, daidzein significantly increased the relative abundance of Sphingomonas and Thermus, and significantly decreased the relative abundance of Alistipes, Lachnospiraceae NK4A136, Bacteroides and Bacteroides acidifaciens compared with the SBM group (Fig. 8).

Discussion
Our previous study showed that dietary daidzein improved SBM induced typical inflammatory symptoms such as profound infiltration of eosinophil, disarranged microvilli, wizened and shorter intestinal folds 28 . However, the involved mechanisms were still unclear. The release of inflammatory cytokines plays an important role in mediating SBMIE. In present study, dietary daidzein significantly down-regulated the gene expression of proinflammatory cytokine TNF-α and up-regulated the expression of anti-inflammatory cytokine TGF-β. These results were consistent with the study on DSS-induced colitis mouse model, which showed oral administration of daidzein ameliorated the ulcerative colitis and suppressed the expression of TNF-α, interleukin-1β (IL-1β), and IL-6 22 . The decreased gene expression of pro-inflammatory cytokines responded to daidzein was also found in intestine of turbot fed with fish meal-based diet 27 . It has been reported that many signaling pathways are involved in the anti-inflammatory effects of daidzein, of which MAPK and NF-κB are the most deeply studied 21,22,29 . MAPKs, a family of serine/threonine protein kinases, are potential molecular targets for anti-inflammatory therapeutics 30 , the classical MAPKs are consisted of ERK1/2, p38, JNK and ERK5 31 . NF-κB is a central mediator involved in inflammation development and progression 32 . Tomar et al. 21 found pretreatment with 100 mg/kg daidzein diminished the activation of ERK1/2, JNK, and p38 phosphorylation on cisplatin-induced rat-kidney injury with ameliorated oxidative stress, apoptosis, and inflammation. Moreover, daidzein modulated NF-κB, p38, MAPK and TGF-β1 pathway to counteract angiotensin II-induced inflammation on mice 29 . In line with previous findings, the present results showed that supplementation with daidzein inhibited the overexpression of p38, JNK, ERK and NF-κB induced by SBM, which suggested that daidzein might attenuate the inflammation via modulating MAPKs and NF-κB signaling.
To resist the assault of reactive oxygen species (ROS), an antioxidant system, including enzymatic and nonenzymatic antioxidant of fish have developed to either prevent or repair the oxidative damage 33,34 , which is tight regulated by Nrf2-keap1 signaling 35 . In the condition of oxidative stress, Nrf2 translocates to the nucleus by detaching from Keap1, and bounds to antioxidant response element to induce the expression of antioxidant genes such as superoxide dismutase (SOD), NQO-1 and HO-1 35 . In the current study, the mRNA levels of HO-1, NQO, prdx-6, as well as signal molecule Nrf2, were significantly up-regulated by SBM treatment, suggesting Indeed, previous studies revealed that daidzein could elevate antioxidant enzymes activities to inhibit oxidative stress 36 . Besides, daidzein is also a direct free radical scavenger, and possesses high antioxidant power to scavenge free radical such as superoxide anion radical, hydroxyl radical and hydrogen peroxide 37 . Thus, a possible www.nature.com/scientificreports/ explanation for the seemingly opposite results may be that daidzein acts as a free radical scavenger, resulting in alleviative oxidative stress of turbot caused by SBM. Accordingly, the antioxidative effects and daidzein on fish and the mechanisms involved merits further study. The enhanced intestinal epithelial cells turnover and apoptosis usually emerged with SBMIE 11,38 . Proteins of the B-cell lymphoma-2 (BCL-2) family composed of anti-apoptotic proteins (Bcl-2, BCL-XL , ect.) and proapoptotic (Bax, Bid, BAX, etc.) control the intrinsic apoptosis pathway 39 . Once apoptosis initiates, caspases, Table 1. The MetaStat analysis of intestinal microbiota of turbot at genus and species levels (× 10 −5 ). Values shared different letters in the same row are significantly different (q-value (corrected p value) < 0.05), n = 3. FM fish meal diet, SBM soybean meal diet, DAID 40 mg/kg daidzein included into SBM diet. www.nature.com/scientificreports/ a family of endoproteases playing an essential role in apoptosis, will be activated; caspases-8 and -9, initiator caspases, are activated by upstream signaling, which subsequently activated executioner caspases-3, -6 and -7 to induce efficient cell death 40 . In the current study, the up-regulated gene expression of Bax, Bid, and caspase-9 in SBM group was significantly inhibited by dietary daidzein, indicating a suppression of apoptosis. The results were in line with a previous study on 7,12-dimethylbenz[a]-anthracene (DMBA) treated mice, which showed that daidzein restored the increased Bax and caspase-3 caused by DMBA to normal values 41 . Aras et al. 42 also provided evidence that administration of daidzein decreased caspase-3 and caspase-9 immunoreactivity in the brain of rat suffered middle cerebral artery occlusion. TJs protein are the apical-most adhesive junctional complexes in epithelial cells, which constitute a major competent of intestinal physical barrier together with intestinal epithelial cells 43,44 . In this study, the decreased expression of barrier-forming TJs claudin-4, JAM-1 and ZO-1 transcript variant 1 observed in SBM group were in accord with previous studies on turbot 3,4,13,14 , indicating a disrupted TJs barrier. As expected, dietary daidzein reversed the changes of those gene expression. The result was in agreement with Ou's 27 finding which showed that dietary daidzein (fish meal-based diet) enhanced the intestinal mucosal barrier function of turbot via significantly up-regulating tricellulin, ZO-1, claudin-like and occludin expression. These findings suggest that daidzein could inhibit apoptosis of epithelial cells and modulate TJs to maintain the intestinal structural integrity. The intestinal bacterial community was also included in present study, similar with previous studies which showed that dietary daidzein could modulate intestinal bacterial community 27 . The results showed that dietary SBM significantly increased the abundance of Bacteroidetes, and decreased the abundance of Proteobacteria compared with FM group. The increased abundance of Bacteroidetes was consistent with previous studies carried out on turbot fed with soybean meal 3,10,14 . Bacteria of Bacteroidetes phylum possess a lot of genes encoding for carbohydrate-degrading enzymes, and are considered primary degraders of polysaccharides 45 . Bacteroidales S24-7, a family of Bacteroidetes, accounting for 53.00% of intestinal bacterial community in SBM group in present study, have been proved owing the ability to degrade complex carbohydrate 46 , which might help to explain the rich abundance of Bacteroidetes in SBM treatment. However, most carnivorous fish species are considered to be "glucose intolerant" 47,48 , the improved the usability of carbohydrate might increase the glucose load of fish, which eventually disturbed glucose homeostasis, even induced inflammation and oxidative stress 49 . While, administration of daidzein significantly reversed the situation with boosted Proteobacteria and Deinococcus-Thermus instead of Bacteroidetes compared with SBM group and made the microbial composition close to the FM group in terms of beta diversity analysis. Although the increased abundance of Proteobacteria is usually found to be associated with many human disease 50,51 , some marine Proteobacteria can produce bioactive compounds with anti-cancer and antibiotic activity 52 . Proteobacteria are the prominent gut microbiota in intestine of fish 53 , and its relative abundance is even up to 90.64% in turbot 54 . Sphingomonas, the most predominant genus in daidzein group, mainly contributed to the increment of Proteobacteria in the current study. Previously, Sphingomonas was found to be abundant in the intestine of healthy Atlantic salmon compared with unhealthy fish 55 . Sphingomonas is metabolically versatile, which can utilize many kinds of compounds and some environmental contaminants such as hydrophobic polycyclic aromatic hydrocarbons 56,57 , and was considered as a potential probiotic in aquaculture due to the removal of ammonia nitrogen and inhibition of Vibrio spp 58 . Bacteria from the phylum Deinococcus-Thermus are highly resistant to extreme stresses such as radiation, oxidation, desiccation and high temperature. Some studies revealed that Deinococcus-Thermus bacteria such as genus Thermus, can synthesize carotenoids 59-62 which can reduce oxidative stress by interrupting the propagation of free radicals. The prevalence of them in daidzein treatment might contribute the antioxidant ability of daidzein. Moreover, the relative abundance of a lactic acid-producing bacteria Lactococcus lactis, was significant increased by dietary daidzein. The increased abundance of lactic acid bacteria was also observed in the intestine of turbot fed fishmeal-based diets containing 400 mg/kg daidzein 27 . As a probiotic, Lactococcus lactis is widely studied in aquaculture, Adel et al. 63 found that diets containing Lactococcus lactis subsp. Lactis improved the growth performance, disease resistance, digestive enzyme activity and intestinal microbiota of white shrimps (Litopenaeus vannamei). Compared with SBM group, dietary daidzein significantly decreased the relative abundance of potential pathogenic bacterium Alistipes and Lachnospiraceae NK4A136 group. Alistipes, a genus of Bacteroidetes, one of the most abundant genera, existed in human colorectal adenoma 64 . Previous studies demonstrated that Alistipes could produce lipopolysaccharide (LPS) 65 and promote inflammation and tumorigenesis 66 . A recent review done by Parker et al. 67 indicated that Alistipes are highly related to dysbiosis and disease. The abundance of Lachnospiraceae NK4A136 group is positively correlated with gut dysbiosis 68 . In study of Cui et al. 69 , the abundance of Lachnospiraceae NK4A136 group was lower in ameliorated gut dysbiosis. However, dietary daidzein also down-regulated the abundance of Bacteroides and Bacteroides acidifaciens. By a meta-analysis, Zhou and Zhi 70 revealed lower levels of Bacteroides in IBD patients. Bacteroides acidifaciens was beneficial for treating insulin resistance and preventing obesity in mice 71 , while its function on fish was unclear. All in all, these results suggested daidzein had a deep influence on intestinal microbiota of turbot, and the influence of daidzein on intestinal microbiota in fish merits further investigation.
In conclusion, the present study suggested that 40 mg/kg daidzein in the diet could remarkably mitigate the SBMIE of turbot. The dietary supplementation of daidzein displayed strong inhibition of key genes involved in inflammatory response, antioxidant enzyme, and apoptosis, improved intestinal integrity, and had a positive influence on intestinal microbiota profiles. Besides, daidzein might modulate p38, JNK and NF-κB signaling pathway to counteract SBM-induced inflammation. The present results indicate that daidzein is a promising feed additive for fish feed. Healthy juvenile turbot was purchased from a local company in Weihai City (Shandong Province, China). After acclimating to the experimental conditions for 2 weeks, turbot with even size (9.55 ± 0.04 g), high vitality and normal appearance were randomly assigned to 9 tanks (300 L, 30 fish per tank). Fish were fed twice daily to apparent satiation (8:00 a.m. and 6:00 p.m.) for 12 weeks. During the feeding period, the water temperature was 19-25 °C, pH 7-8, salinity 23-26, dissolved oxygen > 7.0 mg/L, and NH 4 + -N < 0.3 mg/L. The protocols of animal care and treatment performed in this study has been approved by the Institutional Animal Care and Use Committee of Ocean University of China. Moreover, this study was carried out in compliance with the ARRIVE guidelines. All methods involved in present study were strictly conducted following the Guide for the Use of Experimental Animals of Ocean University of China.
Sampling. Fish were anesthetized with eugenol (1:10,000) (purity 99%, Shanghai Reagent Corp, Shanghai, China) before sampling. Distal intestine for real-time quantitative polymerase chain reaction (qRT-PCR) was collected from three fish per tank. The samples of intestinal microbiota were obtained under alcohol burner, and the surface of three fish per tank was sterilized by 75% alcohol. All samples were frozen in liquid nitrogen immediately, then stored at − 80 °C.
qRT-PCR. The RNA of intestine samples was extracted by using RNAiso Plus (9108; Takara Biotech., Dalian, China). Then, the quality of RNA was determined by electrophoresis and NanoDrop ND-1000 (Nano-Drop Technologies, Wilmington, DE, USA), evaluating the integrity and concentration, respectively. The RNA was reversed transcribed to cDNA with PrimeScript RT reagent Kit (Perfect Real Time, Takara, Japan).
The specific primers presented in present study were synthesized by TSINGKE (Beijing, China) and presented in Table 3. β-actin was used as reference gene. The program of the qRT-PCR was described in our previous papers 72   Bacterial DNA extraction, sequencing and bioinformatic analysis. Genomic DNA of intestinal bacteria was extracted using QIAamp DNA Stool Mini Kit (Qiagen, Germany) following the method described by Liu et al. 14 . The high-quality of DNA determined by integrity and quality via electrophoresis on 1.2% denatured agarose gel and NanoDrop ND-1000 (Nano-Drop Technologies, USA) was amplified with 515F (GTG CCA GCMGCC GCG GTAA) /806R (GGA CTA CHVGGG TWT CTAAT) primers for amplifying the V4 region of 16S rDNA of intestinal bacteria. The specific amplification program was presented in a previous work 14 .

Target genes
Forward primer (5′-3′) Reverse primer (5′-3′) GenBank number GGA CAG GGC TGG TAC AAC AC  TTC AAT TAG TGC CAC GAC AAA GAG AJ276709.1   TGF-β  CTG CAG GAC TGG CTC AAA GG  CAT GGT CAG GAT GTA TGG TGGT  KU238187.1   p38  GAA CGC CCC CAA CAT CTC TA  CTC GGC TGC TGT TAT TCG   Statistical analysis. All data except microbiota data underwent the Bartlett test and the Shapiro-Wilk W goodness of fit test for homogeneity and normality of variance. Then, one-way analysis of variance (ANOVA) of the data was performed via SPSS 22.0. Tukey's test was applied for comparing the means among treatments, and the data were presented as means ± standard error. P < 0.05 was regarded as statistically significant.

Data availability
Raw reads of 16s rDNA sequencing were deposited to the National Center for Biotechnology