Dietary Puerarin Supplementation Alleviates Oxidative Stress in the Small Intestines of Diquat-Challenged Piglets

animals Article Dietary Puerarin Supplementation Alleviates Oxidative Stress in the Small Intestines of Diquat-Challenged Piglets Meng Li 1,2,† , Daixu Yuan 3,† , Yanhong Liu 4 , Hui Jin 1 and Bie Tan 1,5, * 1 2 3 4 5 * † Laboratory of Animal Nutritional Physiology and Metabolic Process, Key Laboratory of Agro-ecological Processes in Subtropical Region, National Engineering Laboratory for Pollution Control and Waste Utilization in Livestock and Poultry Production, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha 410125, China; (M.L.); (H.J.) University of Chinese Academy of Sciences, Beijing 100008, China Department of Medicine, Jishou University, Jishou 416000, China; Department of Animal Science, University of California, Davis, CA 95616, USA; College of Animal Science and Technology, Hunan Agricultural University, Changsha 410128, China Correspondence: ; Tel.: +1-511-611-0998 These authors contributed equally to this work. Received: 11 March 2020; Accepted: 4 April 2020; Published: 7 April 2020



 Simple Summary: The oxidant stress which piglets suffer from during the weaning period has caused huge losses to the pig farm industry. It is important for scientists to find an effective way to alleviate the oxidant stress in weaned piglets. This study was designed to test the hypothesis that dietary puerarin supplementation alleviates oxidative stress in the small intestine of diquat-challenged piglets. Interestingly, dietary puerarin supplementation improved intestinal morphology, cell proliferation, barrier function, and increased Nrf2 and its downstream enzymes in diquat-challenged piglets, which shows that puerarin has potent protective effects against diquat-induced oxidative stress. These findings will be very beneficial to the pig industry, especially to the development of antibiotic-free diets, new anti-inflammatory drugs and the application of puerarin in piglets. Abstract: This study was conducted to demonstrate that dietary puerarin supplementation alleviates oxidative stress in the small intestine of diquat-challenged piglets. The results showed that puerarin administration markedly alleviated diquat-induced intestinal injury, which was indicated by the improvement of intestinal morphology, cell proliferation and barrier function. One of the potential mechanisms responsible for this was the decrease in oxidative stress, as evidenced by the increase in activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and total antioxidant capacity (T-AOC) in the small intestine. Puerarin increased the protein expression levels of NF-E2-related factor 2 (Nrf2) and its downstream enzymes, including heme oxygenase 1 (HO-1), glutamate–cysteine ligase catalytic and its modifier subunit (GCLc and GCLm) in the jejunal mucosa of diquat-induced piglets. Puerarin administration improved intestinal morphology, cell proliferation, and barrier function, and increased Nrf2 and its downstream enzymes. These findings indicate that the dietary supplementation of puerarin attenuates the oxidative stress involving Nrf2 signaling pathways in diquat-challenged piglets. Keywords: puerarin; diquat; piglets; oxidative stress; Nrf2 pathway Animals 2020, 10, 631; doi:10.3390/ani10040631 www.mdpi.com/journal/animals Animals 2020, 10, 631 2 of 13 1. Introduction Piglets are prone to oxidative balance disruption and oxidative injury during the weaning period. The previous study demonstrated that weaning is associated with oxidative injury of the lipid, protein, and DNA, as well as declines in the activities of the intestinal antioxidant enzymes under the p65 and NF-E2-related factor 2 (Nrf2) signals [1]. Under normal physiological conditions, the production of oxidants and antioxidants is balanced in biological systems [2]. Oxidative stress occurs when antioxidant systems cannot neutralize the excess reactive oxygen species (ROS) produced in cells and tissues [3]. As the products of mitochondrial metabolism of the eukaryotic cell, ROS are capable of initiating oxidation and play an important role in maintaining cell homeostasis and regulating signal transduction, gene expression, and enzyme activation at low levels [4,5]. The supplementation of exogenous antioxidants may help restore the pro-oxidative–antioxidative balance [6]. Puerarin, a natural isoflavone compound, has been reported to have strong antioxidant activities and exert a wide range of beneficial effects [7–9]. Puerarin can scavenge free radicals, inhibit pro-inflammatory cytokines, decrease inflammatory genes, up-regulate antioxidant enzymes, modulate transcription factors, and enhance the Nrf2 signaling pathway [10,11]. It has been reported that puerarin effectively inhibited inflammation in the kidney induced by both lipopolysaccharide and AGEs in mouse mesangial cells [12,13]. Puerarin also exerted renal protective effects in diabetic rats and renovascular hypertensive rats [14,15]. However, the protective effects of puerarin on oxidative stress in the intestines of weanling piglets have rarely been reported. Therefore, the current study was designed to explore the protective roles of puerarin in piglets suffering from oxidative stress. We hypothesized that puerarin would exert protective effects on oxidative stress in the intestines of diquat-challenged piglets, which may involve Nrf2 signaling pathways. 2. Materials and Methods 2.1. Animals and Experimental Design The animal experiments were approved by the Institutional Animal Care and Use Committee of the Institute of Subtropical Agriculture, Chinese Academy of Sciences (2013020). A total of 48 healthy piglets with body weight (BW) 7.26 ± 0.51 kg weaned at 21 days were randomly assigned to receive one of three treatments, with eight replicate pens per treatment, and two piglets per pen. The three treatments included a basal diet, a basal diet + diquat, and a 0.1% puerarin diet + diquat. (The puerarin dose was based on the growth performance of piglets in the preliminary experiment.) Diquat was purchased from Sigma-Aldrich (St. Louis, MO, USA). The basal diets were designed to meet the nutrient requirements for weaned piglets, as shown in Table 1. The piglets were housed in a room with hard plastic-slatted flooring. All piglets had free access to drinking water. After an adaptation period of seven days, the piglets were fed their respective diets three times per day for a 14-day experimental period. On day 7 after the initiation of treatment, the piglets on the basal diet + diquat and 0.1% puerarin + diquat treatments received an intraperitoneal injection of diquat at 8 mg/kg BW (a dose of 8 mg/kg BW was used according to the results reported by Yin et al) [16], while the piglets on the control treatment received the same volume of sterilized saline. On day 14, 24 piglets (one pig per pen) were randomly selected and slaughtered. The intestinal samples were collected from the center of the jejunum a (...truncated)