Resveratrol Attenuates Oxidative Stress-Induced Intestinal Barrier Injury through PI3K/Akt-Mediated Nrf2 Signaling Pathway

Oxidative stress is implicated in a wide range of intestinal disorders and closely associated with their pathological processes. Resveratrol (RSV), a plant extract, plays a vital role in protecting various organs in vitro and in vivo. However, the benefits of RSV are controversial, and underlying mechanisms for its antioxidant effects on intestinal epithelial cells remain unclear. In this study, we evaluated the effects of RSV on oxidative stress induced by H2O2 in IPEC-J2 cells. We found that pretreatment with RSV significantly increased cell viability; increased expression levels of tight junction (TJ) proteins (claudin-1, occludin, and ZO-1); improved activities of superoxide dismutase-1 (SOD-1), catalase (CAT), and glutathione peroxidase (GSH-Px); and decreased intracellular reactive oxygen species (ROS) levels and apoptosis induced by H2O2 (P < 0.05). In addition, RSV upregulated Akt phosphorylation, Nrf2 phosphorylation, and expression levels of antioxidant genes HO-1, SOD-1, and CAT in a dose-dependent manner (P < 0.05) under oxidative stress. Knockdown of Nrf2 by short-hairpin RNA (shRNA) abrogated RSV-mediated protection against H2O2-induced apoptosis, RSV-induced increase of TJ protein levels, and antioxidant gene expression (SOD-1, CAT, and GSH-Px) (P < 0.05). Consistent with Nrf2 knockdown, the PI3K/Akt inhibitor LY294002 significantly suppressed RSV-induced Nrf2 phosphorylation and RSV-induced increase of TJ protein levels and antioxidant gene expression under H2O2 treatment (P < 0.05). Collectively, these results demonstrate that RSV can directly protect IPEC-J2 cells against oxidative stress through the PI3K/Akt-mediated Nrf2 signaling pathway, suggesting that RSV may be an effective feed additive against intestinal damage in livestock production.


Introduction
The intestine not only is a major digestive and absorptive organ for nutrients but also functions as a selective barrier against toxins, pathogens, and antigens from the luminal environment [1]. The intestinal barrier primarily consists of tight junction proteins (TJs), enterocyte membrane, antibac-terial peptides, and the mucous layer and immune system. When the intestinal barrier is disrupted, the luminal toxins and antigens will penetrate subepithelial tissues through the barrier, causing a mucosal oxidative stress and systemic inflammatory response [1]. Meanwhile, overproduction of reactive oxygen species (ROS) and proinflammatory cytokines disrupts the intestinal barrier and dysfunction.
However, due to the complex physiological and/or chemical environment of the intestine, it is extremely susceptible to oxidative stress.
Oxidative stress, defined as the imbalance between the antioxidant systems and oxidative system causing overdose of ROS, can disrupt cellular signaling and function. It is believed that oxidative stress is involved in the development of intestinal diseases such as inflammatory bowel disease (IBD), irritable bowel syndrome, and colon cancer [2][3][4][5]. Under the physiological condition, ROS is maintained at certain levels and excessive free radicals are usually scavenged by antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPH-Px). However, under the imbalance between the antioxidant and the oxidative system, overdose of ROS can disturb epithelial cell integrity and intestinal barrier by decreasing tight junctions and cell quantity [6]. Recent studies have shown that ROS or other free radicals can disturb cell functions by influencing transcription factors and the redox-sensitive signaling pathway. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that has an important regulative effect on oxidative statues through induction of the expression of the antioxidant and phase 2 detoxifying enzymes and related proteins [7,8]. In terms of the possible importance of ROS in intestinal injury, it is essential for cells to effectively upregulate antioxidants, decrease ROS production, and scavenge free radicals, which may contribute to increased intestinal permeability and epithelial apoptosis.
Plant extracts are considered as a potential source of antioxidant and anti-inflammatory molecules which have been identified and proposed as therapeutic agents to counteract oxidative stress-related disease [9]. Resveratrol (RSV) is a plant-derived stilbene (polyphenol) associated with a wide range of health benefits [10][11][12][13]. Many studies have suggested that RSV acts on multiple cellular targets such as Nrf2, NF-Kβ, Sirt1, and AMPK to control processes and signaling pathways related to reduction of oxidative stress, apoptosis, and anticancer effects [1,[14][15][16]. However, scientific research on healthy effects of dietary RSV has been controversial because of low bioavailability of RSV in vivo [17]. The doses of RSV in target tissues are extremely low and hardly reach the level of pharmacological concentration in in vitro studies [18]. Even though the function of RSV is still controversial, we hypothesize that RSV can directly protect intestines from oxidative stress through its rapid metabolism in intestinal cells. Therefore, we used an in vitro oxidative stress model induced by H 2 O 2 to investigate whether RSV can prevent intestinal impairment. Our results provide insights for the future application of RSV as feed additives against intestinal damage in livestock production.
2.3. Establishment of Nrf2-Knockdown IPEC-J2 Cell Line. Short-hairpin RNAs (shRNAs) were designed using the online shRNA design tool (https://portals.broadinstitute .org/gpp/public/) and cloned into the lentiviral vector pLKO.01 via Age I/EcoR I. The plasmids pLKO-shNrf2 or pLKO-scramble, pCMV-DR8.9, and pCMV-VSVG were transfected into 293T cells to produce lentiviruses. At 48 hours posttransfection, the lentiviruses were harvested and used to infect IPEC-J2 cells in the presence of polybrene (8 μg/mL). At 48 hours postinfection, the infected cells were selected and maintained with puromycin (2 μg/mL). The sequences of shRNAs are shown in Table 1      samples were incubated at room temperature (25°C) in dark conditions for 10 min. Finally, flow cytometric analysis was performed according to the manufacturer's instructions (Beckman, Fullerton, California, USA).

Quantitative Real-Time PCR (qRT-PCR).
After the extraction of total RNA in treated cells, reverse transcription and qRT-PCR were performed as described previously. Primers were designed with the online primer design tool Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primerblast/index.cgi?LINK_LOC=BlastHome) according to the gene sequences of the pig, and the sequences of the primers are shown in Table 2. β-Actin was used to normalize the levels of target gene transcripts, and the relative abundance of gene transcripts was normalized to the values of the control treatment.
2.9. Western Blot Analysis. Cells were treated with the indicated agents according to the experimental design and lysed with RIPA buffer (Beyotime) to extract total cellular proteins. The concentration of proteins was quantified using a BCA protein assay kit (Beyotime, China) according to the instructions. Western blot analysis was performed as described previously. The indicated proteins were normalized to β-actin and analyzed using ImageJ (National Institutes of Health, Bethesda, MD, USA).

Statistical Analyses
Results from a representative of three independent experiments were expressed as means ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) followed by the LSD test was used for multiple comparisons, and a value of P < 0:05 was accepted as statistically significant. The statistical analyses were performed by GraphPad Prism 7. The columns with the same superscript capital letters and with different superscript capital letters mean no significant difference (P > 0:05) and significant difference (P < 0:05), respectively.

Resveratrol Elevates Antioxidant Ability and the
Expression of Antioxidant Genes in IPEC-J2 Cells. The activities and expression of antioxidant enzymes are the main response for relieving oxidative stress damage in organs or cells. As shown in Figure 4, no significant ROS difference was found between cells treated with RSV alone, whereas RSV significantly decreased levels of ROS in the H 2 O 2treated cells in a dose-dependent manner (Figure 4(a)). It was shown that RSV significantly elevated the activities of T-SOD, CAT, and GSH-Px in the cells treated with or without H 2 O 2 (Figures 4(b)-4(d)). qRT-qPCR results revealed   (Figure 6(a)). Western blot analysis showed that RSV enhanced the phosphorylation of Nrf2 and Akt in a dose-dependent manner (P < 0:05), whereas no significant changes were found in the protein levels of Nrf2, Keap1, and Akt ( Figure 6(b)).   (Figure 8(a)), antioxidant genes (Figure 8(b)), and TJ protein levels (Figure 8(c)) and H 2 O 2 -induced apoptosis (Figure 8(d)). In addition, LY294002 treatment decreased the protein levels of Akt, Nrf2, p-Akt, and p-Nrf2 compared with the control (Figure 8(e)).

Discussion
The intestine is a major digestive and absorptive organ for nutrients and provides a selective barrier against endogenous and exogenous antigens. Numerous studies have revealed that intestinal health, especially the integrity of the intestinal barrier, plays a crucial role in maintaining an organism's healthy state. Oxidative stress is a critical factor involved in the intestinal barrier disruption in intestinal diseases. Therefore, identification of appropriate plant extracts as therapeutic agents to counteract oxidative stressrelated diseases is a research hotspot at present. In this study, IPEC-J2 cells were exposed to H 2 O 2 to induce oxidative stress. We found that H 2 O 2 treatment resulted in the reduction of cell viability and TJ protein expression, overproduction of ROS, disturbance of oxidation and antioxidation, and, finally, induced apoptosis. Moreover, the results also showed that RSV significantly attenuated H 2 O 2 -induced cell injury through increasing cell viability and upregulation of the expression of antioxidant genes. As a dietary supplement, RSV is a polyphenolic compound contained in various fruits and herbs. Ever since RSV was identified to have multiple biological functions (antioxidant, anti-inflammatory, anticancer, etc.), it has been paid great attention for practical applications [10,11,[20][21][22]. In the present study, our results showed that resveratrol potently protected IPEC-J2 cells against oxidative stressinduced intestinal injury using lower toxicity. The small intestinal has been found to be the major site of RSV absorption and metabolism; thus, we hypothesized that the intestinal epithelium may be a potential target for the beneficial effects of RSV though the first-pass effect by the intestine and microbe. Meanwhile, the anti-inflammatory and antioxidative effects of RSV were studied in many cases, such as models of ischemia/reperfusion injury and LPS induction [23,24]. However, according to modern medical perspectives, oxidative stress plays a crucial role in the progression of many diseases such as the intestinal inflammatory bowel. Intestinal integrity is the basic guarantee for intestinal function. Intestinal integrity relies on TJs, which are the principal determinants of intestinal barrier function that seal the paracellular space between neighboring epithelial cells [25,26]. Occludin, claudins, and ZO-1 are major intestinal barrier proteins [27]. In the present study, the expression of ZO-1, occludin, and claudin-1 showed a significant reduction after H 2 O 2 treatment, whereas RSV attenuated dysfunction of the intestinal barrier by upregulating the expression of TJ proteins under H 2 O 2 -induced oxidative stress. Moreover, the results showed that RSV significantly attenuated H 2 O 2 -induced cell damage through increasing cell viability and decreasing apoptotic rate in IPEC-J2 cells. It is easy to speculate that RSV attenuates intestinal injury and especially barrier disruption mainly by upregulating the oxidative status.
Oxidative stress is due to the imbalance between pro-and antioxidant factors. Organisms or cells have antioxidant systems, which involve SOD, catalase, and GSH peroxidase, to scavenge overproduced ROS. This study showed that RSV could upregulate the activities of catalase (CAT), SOD, and GSH peroxidase and maintain intracellular ROS homeostasis under H 2 O 2 -induced oxidative stress or normal condition. On the one hand, RSV can directly clear free radicals by  upregulating antioxidant enzyme activity in vitro and in vivo [21,28]. Wang and his colleagues showed that RSV can protect the Caco-2 cell against H 2 O 2 -induced oxidative damage by reducing the malondialdehyde level and intracellular ROS accumulation. RSV has been reported to improve mitochondrial biogenesis and redox status by upregulating SOD and GSH-Px activity in suckling piglets [29]. On the other hand, RSV can also upregulate the expression of antioxidant genes in many studies, which is consistent with our results. In the present study, RSV increased the expression of SODs, GSXs, and HO-1. In the intracellular antioxidant system, HO-1, SODs, CAT, and GPXs are phase 2 genes which are known as target genes mediated by the Nrf2/Keap1 signaling pathway. Meanwhile, many studies have revealed that RSV attenuates oxidative stress-induced intestinal dysfunction by upregulating HO-1 expression [30]. Upregulation of antioxidant genes is cellular adaptation to oxidative stress and is mainly or partly regulated by the Nrf2 pathway [31]. Nrf2, a pivotal sensor of oxidative stress in cells, is a transcription factor that plays a central role in the regulation of antioxidant and phase 2 detoxifying enzymes and related proteins [32]. Keap1 binds to Nrf2 in the cytoplasm and facilitates Nrf2 ubiquitination which can prevent Nrf2 translocation into the nucleus under physiological conditions [33]. When the levels of intracellular ROS sharply increase, overproduced ROS modifies the reactive cysteine residues on Keap1, and then Keap1 dissociates from Nrf2, leading to the translocation of Nrf2 into the nucleus and the expression of its target genes [33,34]. Recent studies have revealed that the phosphorylation of Nrf2 facilitates Keap1/Nrf2 dissociation and Nrf2 nuclear translocation. In the present study, RSV upregulated the levels of p-Nrf2 and p-Akt in a dose-dependent manner, suggesting that RSV attenuates oxidative status likely by activating the Nrf2 signaling pathway. Many studies have revealed that RSV treatment dramatically upregulates the expression of Nrf2 and HO-1 in vivo and in vitro [35,36]. Furthermore, RSV-induced upregulation of antioxidant genes and the intestinal epithelial cell barrier was inhibited by Nrf2 knockdown, suggesting that Nrf2 may be a major target of RSV in relieving oxidative stress. However, the phosphorylation levels of the Akt protein were increased upon RSV treatment in our study. The PI3K/Akt signaling pathway is commonly involved in the Nrf2-dependent transcription in diverse cell types of responding ROS insults [37,38]. Han's study showed that chlorogenic acid protects MC3T3-E3 cells against oxidative stress through the PI3K/Akt-mediated Nrf2 signaling pathway to induce HO-1 expression, suggesting that Nrf2 may be a downstream signal target of PI3K/Akt [19]. Therefore, Western blot analysis showed that RSV-induced increase of the Nrf2 phosphorylation level was inhibited by the specific PI3K/Akt inhibitor LY290042. In addition, RSV-induced changes of cell viability, apoptosis, antioxidant genes, and TJ expression were also inhibited by the inhibitor. These results were similar to effects of Nrf2 knockdown on IPEC-J2 cells. Moreover, the phosphorylation level of Akt was not affected when Nrf2 was knocked down. It indicates that the PI3K/Akt pathway plays a pivotal role in the cytoprotective effects of RSV-induced Nrf2 activation against oxidative stress.
Collectively, the present findings provide important evidence of potential cytoprotective effects of RSV against H 2 O 2 -induced dysfunction in the intestinal barrier. Pretreatment with RSV protects IPEC-J2 cells against oxidative stress by promoting antioxidant systems' activities, decreasing The columns with the same superscript capital letters and with different superscript capital letters mean no significant difference (P > 0:05) and significant difference (P < 0:05), respectively. intracellular ROS and the apoptotic rate, and upregulating the intestinal barrier. These cytoprotective effects of RSV at least partly depend on the PI3K/Akt-mediated Nrf2 signaling pathway.

Data Availability
The data used to support the findings of this study are available from the corresponding authors upon request.

Conflicts of Interest
The authors declare no conflict of interest.