The Protective Effects Of Resveratrol On The Intestinal Health In Piglets Under Deoxynivalenol Exposure

Deoxynivalenol(DON),also known as vomitoxin,is the most contaminated mycotoxin in feedstuffs,causing great trouble to swine production.Resveratrol(RES),as a plant extract polyphenols,has antioxidant,anti-inflammatory and anti-bacterial activities.However,whether RES can alleviate DON-induced health damage in piglets has not been reported.In this study,the effect of RES on DON-induced intestinal injury in piglets were studied both in vivo and in vitro,using weaned piglets and IPEC-J2(porcine intestinal epithelial cell line),respectively.This study included of the following four parts:Experiment 1: This study was used to investigate the effect of RES supplementation on growth performance and intestinal barrier function of piglets fed with DON contaminated diet.Mildewed corn was prepared by inoculating Fusarium graminearum and used to formulate a contaminated diet with DON concentration of 3.79 mg/kg.Sixty-four21-day-old weaned piglets were randomly divided into four treatment groups: Control group(basal diet),DON group(DON contaminated diet),RES group(basal diet + 300mg/kg RES)and DON + RES group(DON contaminated diet + 300 mg/kg RES).Each group contained eight repetitive columns with two piglets per replicate.The experiment lasted 28 days.(1)In compared with the Control group,DON contaminated diet decreased the ADG and ADFI,and increased F/G of piglets(P < 0.05).Supplementation of RES in DON contaminated diet increased the ADG and decreased F/G of piglets(P < 0.05).(2)Supplementation of RES in DON contaminated diet effectively alleviated DON-induced liver and spleen injury,and increased the villus height,decreased the crypt depth in small intestine of piglets.(3)Supplementation of RES in DON contaminated diet effectively inhibited DON-induced elevation of diamine oxidase and D-lactic acid in plasma(P < 0.05),and increased gene and protein expression of tight junction proteins(such as ZO-1,Occludin,Claudin-1)in jejunal mucosa of piglets.Transmission electron microscopy analysis showed that supplementation of RES effectively alleviated DON-induced damage of tight junction structure and mitochondrial injury in jejunal epithelial cells of piglets.Collectively,supplementation of RES in DON contaminated diet effectively alleviated DON-induced growth performance decline,liver and spleen damage,intestinal morphology and barrier function damage in piglets.Experiment 2: Effects of RES on intestinal oxidative stress induced by DON contaminated diet in piglets were investigated.(1)DON contaminated diet increased the content of MDA,and decreased the activities of antioxidant enzymes in plasma,liver,jejunum and ileum mucosa of piglets.Supplementation of RES in DON contaminated diet could effectively reduce oxidative stress,increase the activity of antioxidant enzymes and decrease the content of MDA in piglets.(2)DON contaminated diet markedly inhibited nuclear accumulation of Nrf2,decreased gene and protein expression of Nrf2-regulated antioxidant enzymes(such as SOD1,GCLC,GCLM).In compared with the DON group,supplementation of RES in DON contaminated diet could effectively improve Nrf2 signal pathway activity and downstream antioxidant enzymes gene and protein expression in jejunal mucosa of piglets.(3)In compared with the DON group,supplementation of RES in DON contaminated diet effectively decreased TUNEL positive rate of jejunal epithelial cells induced by DON in piglets(P < 0.05).(4)Supplementation of RES in DON contaminated diet effectively inhibited the release of mitochondrial cyt-c,decreased the contents of Bax and active caspase-3,and increased Bcl-2 abundance.Collectively,supplementation of RES in DON contaminated diet effectively improved the antioxidant capacity of piglets,and alleviated the intestinal oxidative stress and apoptosis induced by DON.Experiment 3: Effects of RES on intestinal mucosal immunity by DON contaminated diet in piglets were investigated.(1)Supplementation of RES in DON contaminated diet effectively inhibited the DON-induced decrease in the number of goblet cells in jejunum of piglets(P < 0.05).(2)DON contaminated diet increased the gene and protein expression of pro-inflammatory factors(such as IL-1β,IL-6,TNF-α)in plasma and intestinal mucosa of piglets.In compared with the DON group,supplementation of RES in DON contaminated diet effectively inhibited the inflammation in plasma and intestinal mucosa of piglets.(3)Supplementation of RES in DON contaminated diet effectively increased the transcript abundance of antibacterial peptides and mucins(p BD-1,p BD-2,MUC-2)in intestinal mucosa of piglets.(4)Supplementation of RES in DON contaminated diet effectively increased the content of MUC-2 and s Ig A in intestinal mucosa of piglets(P < 0.05).Collectively,supplementation of RES in DON contaminated diet effectively alleviated DON-induced inflammation and increased innate immune function in intestinal mucosa of piglets.Experiment 4: The IPEC-J2 cells were used to investigate the protective effect and mechanism of RES on alleviating the cytotoxicity induced by DON.Cells were incubated with 15 μmol/L RES for 24 h and then exposed to DON for another 24 h.(1)In comparison with DON-only-treated cells,pretreatment with RES increased the cell viability,improved Ed U-positive cells,inhibited cell-cycle arrest in G0/G1 phase,stabilized mitochondrial-membrane potential,deceased accumulation of intracellular reactive oxygen species(ROS),prevented apoptosis induced by DON,and increased the content of Nrf2 protein in the nucleus and the transcript abundance of Nrf2-regulated antioxidant genes,SOD1,GCLC and GCLM.(2)Transfection with Nrf2 si RNA abrogated the protective effect of RES against DON-induced cytotoxicity,accumulation of intracellular ROS,apoptosis,and the contents of SOD1,GCLC and GCLM protein.(3)Transfection with Nrf2 si RNA abrogated the protective effect of RES against DON-induced release of mitochondrial cyt-c,increased the contents of Bax and activecaspase-3,and decreased Bcl-2 abundance.Collectively,RES protected IPEC-J2 cells against DON-induced damage at least partly via the Nrf2 signaling pathway.In conclusion,the results indicated that DON contaminated diet inhibited growth performance,impaired intestinal barrier function,induced intestinal oxidative stress and epithelial cell apoptosis,decreased innate immune function and increased inflammation in intestinal mucosa of piglets.Supplementation of RES in DON contaminated diet effectively alleviated DON-induced growth performance decline and attenuated the intestinal health damage of piglets.The protective mechanism of RES was mainly associated with the activation of the Nrf2 signaling pathway,thus improving intestinal antioxidant capacity,inhibiting oxidative stress and intestinal damage,and maintaining intestinal mucosal immune balance in piglets.