Ellagic Acid Alleviates Oxidative Stress by Mediating Nrf2 Signaling Pathways and Protects against Paraquat-Induced Intestinal Injury in Piglets

The gastrointestinal tract is a key source of superoxide so as to be one of the most vulnerable to oxidative stress damage. Ellagic acid (EA), a polyphenol displays widely biological activities owing to its strong antioxidant properties. Here, we investigated the protective benefits of EA on oxidative stress and intestinal barrier injury in paraquet (PQ)-challenged piglets. A total of 40 weaned piglets were randomly divided into five groups: Control, PQ, 0.005% EA-PQ, 0.01% EA-PQ, and 0.02% EA-PQ. Piglets were intraperitoneally injected with 4 mg/kg (BW) PQ or saline on d-18, and sacrificed on d-21 of experiment. EA treatments eliminated growth-check induced by PQ and increased serum superoxide dismutase (SOD) activity but decreased serum malondialdehyde (MDA) level as compared to PQ group. EA supplementation promoted Nrf2 nuclear translocation and enhanced heme oxygenase-1 (HO-1) and quinone oxidoreductase 1 (NQO1) protein abundances of small intestinal mucosa. Additionally, EA improved PQ-induced crypt deepening, goblet cells loss, and villi morphological damage. Consistently, EA increased tight junction protein expression as was evident from the decreased serum diamine oxidase (DAO) levels. EA could ameliorate the PQ-induced oxidative stress and intestinal damage through mediating Nrf2 signaling pathway. Intake of EA-rich food might prevent oxidative stress-mediated gut diseases.


Introduction
Ellagic acid (EA) is mainly found in the form of free monomers, derivatives, and complex ellagic tannins in various plants such as pomegranates, berries, and walnuts [1]. EA-rich foods have shown a broad spectrum of bioactivities in pathological conditions, including antioxidative, anti-inflammatory, and antimicrobial properties, in animal models and human studies [2]. It has been reported that EA is able to counteract the detrimental reactive oxygen (ROS) and nitrogen species (RNS), and the mechanisms rely mainly on its capacity to accept electrons and to participate in antioxidation redox reactions [3]. Since EA could either prevent oxidation by acting as free-radical scavengers or retard the oxidation process by acting through indirect pathway, it has been classified as the multi-function 0.005% ellagic acid + 4 mg/kg paraquet; EM = 0.01% ellagic acid + 4 mg/kg paraquet; EH = 0 ellagic acid + 4 mg/kg paraquet. n = 8. Data are shown as mean ± SEM. * in (B) means the diffe was significant when compared to d-18. * in (C-E) means the difference was significant compared to the control group. ns means the difference was not significant when compared control group. a-c Values with different lowercase letters are significantly different among PQ EM, and EH groups (p < 0.05).   The BW and feed intake were measured weekly. Average daily gain (ADG) and average daily feed intake (ADFI) were calculated as follows: F/G = total feed intake/(final weight-initial weight)

Tissue Collection and Processing
At the end of experimental period, piglets were sacrificed for serum, jejunum, and ileum tissue collection. Blood samples of 10 mL were collected aseptically from the jugular vein and then centrifuged at 3000× g for 10 min at 4 • C after over fasting [27]. The jejunum and ileum were dissected and rinsed thoroughly with ice-cold isotonic saline. The middle segments of the jejunum (2 cm) and ileum (2 cm) were cut and fixed in 2.5% glutaraldehyde or 4% formaldehyde for morphological and immunohistochemical analysis. Samples of the jejunal and ileal mucosa were scraped, immediately snap frozen in liquid nitrogen, and stored at −80 • C for further analysis [28].

Determinations of Serum Diamine Oxidase and D-Lactate Concentrations
Diamine oxidase (DAO) and D-lactate (DLA) concentrations in serum were measured according to the manufacturing instructions contained in the porcine enzymelinked immunosorbent assay (ELISA) kits (Jiangsu Meimian Industrial Co., Ltd., Yancheng, Jiangsu, China).

Intestinal Histological Evaluation
The segments of the jejunum and ileum fixed in 4% formaldehyde were used to determine morphology using hematoxylin-eosin staining. After dehydration, embedding, sectioning and staining, the jejunum and ileum were observed with a microscope. The villus height, crypt depth, and the number of goblet cells were measured with Case-Viewer software [29,30].
As described by German [31] and Liu et al. [32], tissue segments fixed with cold 2.5% glutaraldehyde were used for scanning and transmission electron microscopy analysis. Briefly, segments of jejunum and ileum were fixed in 2.5% glutaraldehyde for 2 h, and washed 3 × 10 min in PBS at 4 • C. Then the tissues were postfixed in 1% osmium tetroxide for 12 h at 4 • C, washed 10 min in PBS, and the PBS washing process was repeated three times. After ethanol dehydration, tert-Butyl alcohol storage, quick-drying silver paint installation, and gold-palladium coating, the tissues were examined by scanning electron microscope [29]. Moreover, after being postfixed in 1% tetroxide, the segments were dehydrated, embedded, and cut into thin sections, then stained with uranyl acetate and lead citrate for 20 min before being observed using transmission electron microscopy [33].

Immunofluorescence Staining
The protein abundances of occludin and claudin-3 in jejunum and ileum were detected by immuno-histochemical analysis as described by previous study [34]. Briefly, slides were blocked with 5% bovine serum albumin (BSA), and then incubated with occludin antibody (1:500; Abcam; Cambridge, UK) and claudin-3 (1:150; Abcam; Cambridge, UK) overnight at 4 • C, washed three times for 5 min in phosphate buffer saline (PBS) (pH 7.4), and then incubated with secondary antibodies, horseradish peroxidase-conjugated goat anti-rabbit IgG (1:500; Wuhan service bio technology; Wuhan, China) for 50 min in the dark. Cell nucleus were stained with 40, 6-diamidino-2-phenylindole (DAPI) for 10 min and were washed with PBS (pH 7.4) three times for 5 min per wash, then treated with a self-fluorescence quenching agent for 5 min. After sealing, images were obtained under a fluorescence microscope (NIKON ECLIPSE C1; Nikon Corporation; Tokyo, Japan).

Quantitative Reverse Transcription-PCR Analyses
Total RNA was extracted from jejunal and ileal mucosa according to the instructions of Trizol kit of Biyuntian, complementary deoxyribonucleic acid (cDNA) was obtained on the basis of the instruments of reverse transcription kit, and the cDNA was diluted and used as reverse transcriptase-PCR (RT-PCR) template to evaluate gene expression. The reaction system was 20 µL. RT-PCR was carried out on a Roche 48 II Fluorescent quantitative PCR apparatus, and RT-PCR conditions were as follows: 95 • C for 30 s, 95 • C for 5 s, annealing for 15 s, 72 • C for 10 s, 35 cycles, melting curve. The expression levels of target genes were quantified by comparative threshold cycle (Ct) values method, and normalized by β-actin expression levels [35,36]. Primers used in the PCR assay are listed in Table 2.

Statistical Analysis
Data were analyzed by using SPSS 17.0 statistical software. The differences between Ctrl and PQ group were evaluated using independent t-test, and the differences among PQ, EL, EM, and EH were analyzed using one-way ANOVA. Tukey-Kramer multiple comparison procedure was used for post-hoc comparisons. The Kruskal-Wallis test was used when data were not normally distributed. Besides, data in Figure 1B were performed by paired t-test. The p values less than 0.05 were considered as statistically significant. Results are presented as means ± standard error of mean (SEM).

EA Alleviates the Oxidative Stress and Eliminates the Growth Arrest Induced by PQ in Piglets
Although there was no noticeable difference in final BW, ADG, ADFI, and F/G among five treatments (Table 3), PQ injection showed a negative effect on BW gain of piglets ( Figure 1). PQ challenge induced a growth check of piglets from d-18 to d-21 of study, whereas EL and EH treatments improved the growth check (p < 0.05) ( Figure 1B). Meanwhile, PQ induced a significant oxidative stress in piglets, which was exhibited by decreased serum SOD activities and increased serum MDA levels as compared to the control group (p < 0.05). Compared to the PQ group, EM remarkably enhanced serum SOD activities and decreased MDA levels (p < 0.05), and EH reduced serum MDA levels of piglets injected with PQ ( Figure 1C,E). PQ injection and EA treatments did not affect the activities of serum CAT in piglets ( Figure 1D).

EA Supplementation Stimulates the Nrf2-HO1/NQO1 Signaling Pathway in Small Intestine of Piglets
Nrf2 has been reported to involve in the redox-modulated cell signaling. In order to investigate whether EA eliminated the oxidative stress via regulating Nrf2 signaling pathway, we detected the mRNA and protein expressions of NQO1, HO-1, Nrf2, and Keap1 in small intestinal mucosa (Figure 2). Compared to the control group, PQ decreased the ileal mucosal Nrf2, Keap1, and HO-1 mRNA relative expressions but increased the jejunal mucosal HO-1 and NQO1 mRNA relative expressions (p < 0.05). EA treatments had no effect on the mRNA expression levels of Nrf2 signaling pathway in jejunal mucosa ( Figure S1A). However, compared to the PQ group, EM increased HO-1 mRNA levels, and EH up-regulated Nrf2, Keap1, and HO-1 mRNA levels in ileal mucosa (p < 0.05) ( Figure S1B). Nrf2 is blocked in the cytosol by its inhibitor Keap1 when cells stay at equilibrium redox state. It is released from Keap1 and translocated into the nucleus, where it binds  Nrf2 is blocked in the cytosol by its inhibitor Keap1 when cells stay at equilibrium redox state. It is released from Keap1 and translocated into the nucleus, where it binds with elements participated in transcription of genes coding antioxidant enzymes, when the redox state is out of balance [37]. In jejunal mucosa, EA stimulated the Nrf2-Keap1-HO1/NQO1 signaling pathway by enhancing Nrf2 protein levels in the EL and EM groups, and enhancing HO1 protein levels in the EH group. In ileal mucosa, EA stimulated Nrf2 signaling pathway mainly by inducing Keap1 protein expressions ( Figure S1C,D). We also detected the relative protein levels of Nrf2, Keap1, NQO1, and HO-1 in cytosol or nucleus of jejunal and ileal mucosa (Figure 2A,B). Compared with the control group, PQ increased Nrf2 but decreased Keap1 protein levels in cytoplasm of jejunal and ileal mucosa, as well as decreased Nrf2 protein levels in nucleus of jejunal and ileal mucosa. EL and EM treatments promoted the nuclear transportation of Nrf2 protein exhibited by increasing its levels in nucleus of jejunal and ileal mucosa. Meanwhile, PQ decreased HO-1 protein abundances in jejunal and ileal cytoplasm and nucleus, as well as decreased NQO1 protein abundances in ileal cytoplasm while increased that in jejunal cytoplasm. In jejunal and ileal mucosa, EL and EM treatments increased NQO1 protein expressions in nucleus, and EH increased HO-1 protein expressions in cytoplasm.

EA Supplementation Improves the Intestinal Morphology of PQ-Challenged Piglets
Oxidative stress induces gut epithelial barrier disruptions, which is associated with the intestinal morphological damage and the increased gut permeability [38]. As shown in Figure 3, PQ decreased the ratio of villus height to crypt depth (V/C) and the number of goblet cells in jejunum and ileum, while the above two indications increased in EA (p < 0.05). ( Figure 3A). Compared with the control group, PQ caused a decrease of villi density and villi folds in the jejunum and ileum, while supplementation of EA induced an increase in villi density and villi folds ( Figure 3B). These results indicated that EA might recover the intestinal morphological injury of PQ-challenged piglets.

EA Supplementation Maintains the Structure of Tight Junction and Decreases the Permeability of Intestinal Barrier in PQ Challenged Piglets
Because the tight junction plays a key role in maintaining the intestinal m barrier integrity and the impermeability [39], we also evaluated the structure and p abundances of tight junction ( Figure 4A,B). Piglets in PQ group were observed to shorter microvilli and a larger gap of enterocytes in jejunum and ileum as compa the control group. Conversely, EA treatments displayed longer microvilli and re intact tight junction protein structures in the jejunum and ileum ( Figure 4A). Consis PQ significantly decreased the protein abundances of claudin-3 and occludin in je and ileum as compared to control group (p < 0.05), whereas EA treatments enhanced protein expressions of in jejunum and ileum (p < 0.05) ( Figure 4B). Compared to the c group, PQ challenge increased DAO levels in serum (p < 0.05). Compared to the PQ EM and EH groups significantly reduced serum DAO levels (p < 0.05) ( Figure 4C). (B) Scanning electron microscopy analysis of jejunal and ileal mucosa (magnification 400×, 1000×). PQ = 4 mg/kg paraquet; EL = 0.005% ellagic acid + 4 mg/kg paraquet; EM = 0.01% ellagic acid + 4 mg/kg paraquet; EH = 0.02% ellagic acid + 4 mg/kg paraquet. n = 8. Data are shown as mean ± SEM. * means the difference was significant when compared to the control group. ns means the difference was not significant when compared to the control group. a-c Values with different lowercase letters are significantly different among PQ, EL, EM, and EH groups (p < 0.05).

EA Supplementation Maintains the Structure of Tight Junction and Decreases the Permeability of Intestinal Barrier in PQ Challenged Piglets
Because the tight junction plays a key role in maintaining the intestinal mucosal barrier integrity and the impermeability [39], we also evaluated the structure and protein abundances of tight junction ( Figure 4A,B). Piglets in PQ group were observed to have shorter microvilli and a larger gap of enterocytes in jejunum and ileum as compared to the control group. Conversely, EA treatments displayed longer microvilli and restored intact tight junction protein structures in the jejunum and ileum ( Figure 4A). Consistently, PQ significantly decreased the protein abundances of claudin-3 and occludin in jejunum and ileum as compared to control group (p < 0.05), whereas EA treatments enhanced these protein expressions of in jejunum and ileum (p < 0.05) ( Figure 4B). Compared to the control group, PQ challenge increased DAO levels in serum (p < 0.05). Compared to the PQ group, EM and EH groups significantly reduced serum DAO levels (p < 0.05) ( Figure 4C). These results implied that EA supplementations could maintain the structure of tight junction and decrease the permeability of intestinal barrier in PQ challenged piglets.

Discussion
Pigs are subjected to various adverse stimuli during the entire production cycle. These adverse stimuli eventually translate into imbalanced redox levels, which has limited the development of high-efficiency and high-quality swine industry. Numerous studies have reported the anti-oxidative benefits of EA or foods enrich with EA from pomegranates, barriers, and walnuts on various metabolic diseases (rodent model and in vitro model) [11,40,41], but few studies considered the direct effect of EA on oxidative damage of intestinal barrier in vivo. Our current study found that EA could alleviate the oxidative damage of intestinal barrier in piglets challenged by PQ, and these effects were associated with the key regulatory factor Nrf2 signaling pathway.
Piglets post weaning having immature intestine and immune systems are susceptible to oxidative stress attack, which has a direct negative impact on piglets growth performance [42]. Previous studies have shown that oxidative stress (e.g., piglets feeding with oxidized fish oil [43], peroxidized-lipids [44] or D-galactose containing diets [45], or received an injection of diquat [43,46]) decreases the feed intake and the final BW, and slows down the weight gain of piglets. Despite the fact that in our study PQ challenge did not affect the final BW, ADG, and ADFI during the whole experimental period, the PQ caused the growth-check after injection as compared to the control group. EA treatment, especially low dose and high dose EA supplementation, eliminated this growth-check induced by PQ, which might be as a result of EA antioxidative properties. Many studies have shown that EA, as a strong oxidant that can effectively scavenge free radicals, plays an important regulatory role in maintaining redox homeostasis and oxidative stress injury repair [47]. MDA, as biomarkers of cellular oxidative stress, is an end product of lipid peroxidation [48], and SOD and CAT provide the major antioxidant defense against reactive species [15]. EA supplementations in this study decreased the serum MDA levels but increased SOD activities of PQ-induced piglets, which is consistent with the previous studies [49][50][51][52]. The expression of defense genes coding antioxidant proteins is mediated by Nrf2-Keap1 pathway, which is switched on or off directly by ROS so as to be a signaling pathway committed to the oxidant elimination [53]. An increasing number of studies demonstrate that in oxidative stress induced diseases (such as diabetes induced tissue damage, memory impairment, and hepatic injury, but not gut diseases-related evidences), EA elevates the activities SOD and glutathione (GSH) and declines the MDA levels by increasing the nuclear translocation of Nrf2, thereby protecting cells from the free radical damage [54][55][56][57][58]. In the present study, EA treatments did not change the gene expression of nqo1 in the small intestine, but up-regulated ho-1, nrf2 and keap1 mRNA levels of ileal mucosa of piglets. Consistently, EA promoted Nrf2 nuclear translocation and enhanced the HO-1 and NQO1 protein abundances in both cytoplasm and nucleus of jejunal and ileal mucosa. Moreover, many researchers suggest that the bioactivities of EA at least partially depend on its intestinal microbial metabolites, urolithins. Besides, a previous study suggested that the antioxidant effect of EA on gastrointestinal tract is closely related to its metabolites, urolithins [59]. The microbial community loads and composition changes along the gastrointestinal tract, and this might cause the results in jejunum and ileum having slightly differences. Altogether, our results indicated EA could alleviate the oxidative stress of PQ-induced piglets, and this function might be associated with the activation of Nrf2-Keap1 signaling pathway.
In addition, Nrf2 plays a regulatory role in intestinal barrier integrity [60,61]. In the present study, we also observed that EA administrations alleviated microvilli shedding and crypt hyperplasia, as well as increased the number of goblet cells in piglets injected with PQ, which is consistent with previous studies [51]. Oxidative stress can cause the intestinal barrier injury mainly manifested as villi shorting, crypt hyperplasia, and goblet cells apoptosis, which contribute further to a reduced ability for nutritional absorption and presentation of pathogens [62,63]. Maintaining intestinal integrity is a prerequisite to ensure the intestinal barrier function [64]. Tight junction proteins, an important part of the intestinal barrier, have been shown to be enhanced by urolithin A via the Nrf2-Keap1 signaling pathway [17,65]. Here, our results showed that EA supplementations alleviated the occludin and claudin-3 proteins deficiency in small intestinal mucosa caused by PQ challenge. Occludin and claudin-3, as the tight junction components between intestinal epithelial cells, were responsible for the barrier tightness [66][67][68]. Similarly, transmission electron microscope results suggested that the intestinal tight junction structures in the EA group were more complete and clearer than these in PQ group. Serum DAO and DLA levels are two bio-markers of the intestinal barrier permeability [69]. EA treatments significantly reduced the serum level of DAO and tended to decrease the serum level of DLA, which is consistent with the decreased intestinal permeability. Data from this study supported that EA could restore the damaged intestinal morphology and tight junction structure, thereby reducing intestinal barrier permeability.

Conclusions
In summary, our results confirm that the protective effect of EA against PQ-induced oxidative damage in piglets and prove the protective mechanism may rely on two approaches: (1) Maintaining REDOX balance mediated by nuclear transport of Nrf2. (2) Improving intestinal mucosal barrier integrity by up-regulating tight junction protein expressions. Further researches are needed on the specific mechanism of Nrf2 regulating intestinal barrier. Notably, moderate intake of EA (0.001% EA) should have the best beneficial effect in weaned piglets injected with PQ. EA, as a powerful plant-based antioxidant, has the advantages of high safety and low cost, and our study indicated its application prospect in swine production, as well as provided an appropriate additive dose of EA in piglets. However, the application of EA in animal production are needed to be verified by more animal models and clinical trials.

Acknowledgments:
We are grateful to Hunan New Wellful Co., Ltd. for its support.

Conflicts of Interest:
There are no conflicts of interest to declare.