The Role of Nrf2 Signaling Pathway in Eucommia ulmoides Flavones Regulating Oxidative Stress in the Intestine of Piglets

Hindawi Oxidative Medicine and Cellular Longevity Volume 2019, Article ID 9719618, 9 pages https://doi.org/10.1155/2019/9719618 Research Article The Role of Nrf2 Signaling Pathway in Eucommia ulmoides Flavones Regulating Oxidative Stress in the Intestine of Piglets Dingfu Xiao,1 Daixiu Yuan,2 Bihui Tan,1,3 Jing Wang,3 Yanhong Liu,4 and Bie Tan 1,3 1 College of Animal Science and Technology, Hunan Agricultural University, Changsha, 410128 Hunan, China Department of Medicine, Jishou University, Jishou, 416000 Hunan, China 3 National Engineering Laboratory for Pollution Control and Waste Utilization in Livestock and Poultry Production, Key Laboratory of Agro-Ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha, 410125 Hunan, China 4 Department of Animal Science, University of California Davis, Davis, 95616 CA, USA 2 Correspondence should be addressed to Bie Tan; Received 20 June 2019; Revised 30 July 2019; Accepted 7 August 2019; Published 2 September 2019 Academic Editor: Alin Ciobica Copyright © 2019 Dingfu Xiao et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Eucommia ulmoides flavones (EUF) have been demonstrated to alleviate oxidative stress and intestinal damage in piglets, but their effect target is still poorly understood. NF-E2-related factor 2 (Nrf2) pathway plays a very important role in the defense mechanism. This study was designed to investigate the regulation of EUF on the Nrf2 pathway and inhibition of Nrf2 on oxidative stress in the intestine of piglets. An in vivo study was conducted in weaned piglets treated with basal diet, basal diet+diquat, and 100 mg/kg EUF diet+diquat for 14 d to determine Nrf2 and Keap1 protein expressions, as well as downstream antioxidant gene mRNA expression. An in vitro study was performed in a porcine jejunal epithelial cell line to investigate the effect of inhibiting Nrf2 on cell growth and intracellular oxidative stress parameters. The results showed that the supplementation of EUF decreased the oxidized glutathione (GSSG) concentration and the ratio of GSSG to glutathione (GSH) but increased the protein expressions of nuclear Nrf2 and Kelchlike ECH-associated protein 1 (Keap1) as well as mRNA expression of heme oxygenase 1 (HO-1), NAD(P)H:quinone oxidoreductase 1 (NQO-1), and glutamate cysteine ligase catalytic subunit (GCLC) in the small intestinal mucosa of diquat-challenged piglets. When Nrf2 was inhibited by using ML385, cell viability, cellular antioxidant activities, expressions of nuclear Nrf2 and Keap1 protein, and downstream antioxidant enzyme (HO-1, NQO-1, and GCLC) mRNA were decreased in paraquat-treated enterocytes. These results showed that the Nrf2 signaling pathway played an important role in EUF-regulating oxidative stress in the intestine of piglets. 1. Introduction The intestinal tract of piglets is not fully developed and highly susceptible to stress due to its special vascular anatomical structure and convective oxygen exchange mechanism [1]. Oxidative stress is a key factor in the occurrence and development of intestinal injury [2]. Stress can induce the production of a large number of toxic reactive oxygen species (ROS) metabolites in the enterocytes and affect the stability of DNA and RNA as well as the activities of enzymes, resulting in intestinal mucosal damage [3]. Yin et al. found that early weaning at the age of 14 d damaged the oxidation bal- ance of piglets, and the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in plasma were significantly reduced, especially at 3 days after weaning [4]. Wang et al. also found that 21-day-old weaning could reduce glutathione (GSH) content by 25% and increase the ratio of oxidized glutathione (GSSG) to GSH by 59% in the jejunum of piglets [5]. Because of the rich xanthine oxidase in the intestinal tissue, the oxidative damage in the mucosal cells is more significant, and oxidative damage occurs the earliest, while the recovery is the slowest [6]. Long-term stress will further lead to inflammatory bowel disease (IBD), including ulcerative colitis and Crohn’s disease. In the inflammatory 2 bowel disease model, neutrophils were activated and released a large number of oxygen metabolites and proteases, resulting in the intestinal tissue damage [7]. Flavonoids have been reported to exhibit strong antioxidant activities that could directly scavenge free radicals and inhibit ROS, nitric oxide, and proinflammatory cytokine productions [8]. The previous study has indicated that dietary supplementation with Eucommia ulmoides flavones (EUF) alleviated the growth performance impairment, oxidative stress, inflammatory response, and intestinal damage induced by diquat in piglets [9]. However, the preciseness of targets of EUF-regulating oxidative stress in porcine enterocytes still needs to be elucidated. Most of the flavonoids are poorly absorbed through the gut barrier [10], so the intestine is the major site of antioxidant defense afforded by flavonoids [11]. NF-E2-related factor 2 (Nrf2) is a key factor in the oxidative stress response and highly expressed in the gastrointestinal tract. It plays an important role in mediating oxidative stress in the small intestine and stomach [12, 13]. If Nrf2 is disabled or absent, the expression level of downstream antioxidant enzymes is reduced, and the toxicity of oxidative stress cannot be resisted, leading to cell dysfunction, apoptosis, or necrosis. An activated Nrf2 signaling pathway can inhibit ubiquitinmediated degradation of Nrf2 protein and enhance the transcriptional activity of Nrf2 protein [14]. Many polyphenols can induce antioxidant response element (ARE) activation and enhance Nrf2 expression or nuclear translocation [15]. Therefore, the present study was conducted to investigate the regulation of EUF on the Nrf2 pathway in the intestine by using a diquat-induced oxidative stress piglet model. Meanwhile, a specific inhibitor ML385 was used to inhibit Nrf2 and investigate its effects on cellular antioxidant activities and downstream antioxidant enzyme mRNA expression in paraquat-treated enterocytes. 2. Materials and Methods 2.1. Animals and Experimental Design. The animal experiments were approved by the Institutional Animal Care and Use Committee of Hunan Agricultural University, Hunan, China. A total of 24 piglets (Duroc×Landrace×Large Yorkshire) weaned at 21 days were randomly assigned to receive 1 of 3 treatments with 8 replicate pens/treatment: basal diet, basal diet+diquat, and 100 mg/kg EUF diet+diquat, respectively. The basal diet was formulated to meet the nutrient requirements for weanling piglets, and the dose of 100 mg/kg EUF was based on the results showed in the previous study [9]. EUF powder that contained 83.61% total flavones was prepared a (...truncated)