Protection Of Chitooligosaccharide On Intestinal Oxidative Stress Damage In Weaning Piglets

Chitooligosaccharide(COS)is alkaline oligosaccharides which have various biological activities and can eliminate ROS.However,there is a few reaserch for the COS used as antioxidant in vivo.This study aims to exaimne the protective effects of COS on intestinal oxidative damage and other reactions of piglets with long-term feeding normal dose of COS and short-term feeding high dose of COS.Exp.1 Protection of chitooligosaccharides on intestinal oxidative injury in pigletsThis study was conducted to explore whether dietary COS had a protective effect on the intestinal oxidative damage of piglets.A total of 24 weaned piglets Duroc×Landrace×Yorkshire(DLY)(24 days of age)with similar initial body weight 7.34±0.09 kg were divided into two groups randomly,feeding basal diet(CON)and basal diet with 50 mg/kg of COS(COS)respectively.After three weeks,the CON(n=12)and COS(n=12)groups were further subdivided into 2 groups accordding to their initial weight and average-day-gain.Pigs were injected intraperitoneally with diquat at a dose of 10 mg/kg body weight or the same amount of sterile 0.9%NaCl solution.After injection,all pigs kept feeding as before for another week.On day 29,blood samples were collected before slaughtered and jejunum samples were harvested from the small intestine to test the rodox status of jejunum mucosa,gut structure and function,apoptosis and inflammation related indicators.Results showed that:(1)Injection of diquat induced jejunum redox imbalance in piglet and then induced oxidative damage.Feeding with COS decreased the content of ROS siginificantly,and markly increased the ratio of GSH/GSSG,T-AOC and GSH-Px activity(P<0.05).At the same time,the increase of MDA,PCO and 8-OHdG was siginificant suppressed in diquat injection pigs.(2)Injection of diquat destroyed permeability of jejunum,the mRNA express of tight junction protein ZO-1,OCLN,CLDN and the mucous membrane protein MUC2 were markly down-regulated(P<0.05).Meantime,the activities of disaccharidases were reduced,and the mRNA level of nutrient transport carrier gene was down-regulated obviously(P<0.05).Dietary COS supplementation decreased serum diamine oxidase activity(P=0.05)and increased mRNA expression of MUC2(P=0.05),GLUT-2(P<0.05),SGLT(P<0.05)in jejunum,the activity of lactase,sucrsae,maltase were recoverd siginificantly(P<0.05).(3)Injection of diquat caused the increase of intestinal cell apoptosis,which showed a significant up-regulation in the mitochondrial pathway related genes bax,caspase3,caspase9 and the endoplasmic reticulum pathway marker genes GRP78 and CHOP(P<0.05),and down-regulation of bcl-2 gene expression(P<0.05).Dietary COS supplementation significantly inhibited the upregulation of bax,caspase9,caspase3,GRP78,CHOP genes,and significantly increased the expression of anti-apoptotic protein bcl-2 gene(P<0.05).(4)Injection of diquat caused intestinal immune response by reducing the content of mucosal secretion protein sIgA and anti-inflammatory cytokine IL-10 and TGF-β1 gene expressionincreasing the pro-inflammation cytokines IL-6,TNF-α,IL-1β,NF-κB gene expression in intestinal mucosa.Dietary COS supplementation effectively inhibited the pro-inflammation factor NF-κB gene expression(P=0,06)and significantly decreased IL-1β gene expression(P<0.05),enhanced the anti-inflammatory cytokine TGF-β1 gene expression(P=0.07),and significantly increased the secretion of jejunum sIgA in stressed piglets(P<0.05).In conclusion,these results revealed that,injection of diquat in piglets induced gut oxidative stress.Dietary COS supplementation at 50 mg/kg effectively alleviated diquat-induced jejunum injure by eliminating ROS and down-regulation of the expression of apoptosis and pro-inflammatory gene,improved intestinal permeability and gut defence,and increased nutrients absorbtion.Exp.2 Effects of high doses of COS on the intestinal oxidative stress induced by weaning and the intake levels of nutrients in pigletsThe purpose of this experiment was to study the protective effect against oxidative stress on weaning piglets or piglets had nutritional problems when feeding short term high dose of COS.30 piglets DLY(21 d of age)with average weight of 6.57±0.32 kg were randomly allotted to five groups.One group stayed in farm as breast feeding(BF)(n=6),the remaining 24 were divided into 4 groups by the level of nutrients intaking(ad libitum and restriction(feed 1/3 of ad libitum))and feeding of COS(0 vs.1000 mg/head/day).Groups listed as followed:ad libitum group(CF),restricted feeding group(LF),free feeding+COS group(CFC),limit feeding+COS group(LFC).The trial lasted for 7 days.Taking Jejunum samples were taken from the slaughtered piglets and the redox status and oxidative damage indicators were detected.It was found that,comparing with the breast feeding group,the free feeding weaned pigs decreased confrontation ·OH,T-AOC,SOD activity and the mRNA expression of GSH-Px significantly,and siginificantly increased the ratio of GSSG/GSH,as well as the content of 8-OHdG and the Nrf2 gene expression in jejunum mucosa(P<0.05).In weaned piglets groups,comparing with free feeding group,the restricted feeding group showed a further increaseing of oxdative injure and a reduction of confrontation·OH,T-AOC,GSH content and SOD activity,meanwhile,increased the ratio of GSSG/GSH,MDA and PCO(P<0.05).When supplement with COS in free feeding piglets,the level of T-AOC,GSH content,GSH-Px enzyme activity and GSH-Px mRNA expression were significantly increased(P<0.05),8-OHdG content was significantly decreased(P<0.05),and the activities of SOD had an increasing trend(P=0.06),while the expression of Nrf2 gene showed a downregulation(P=0.09).When supplement with COS in restricted feeding piglets,GSH content and GSH-Px gene expression significantly increased(P<0.05),while confrontation ·OH showed increasing trend(P=0.05),and decreased the level of MDA(P=0.06)and PCO(P=0.07).All evidences showed that weaning induced gut oxidative injure and further nutrients intake restrictededwould aggravate oxidative injure in piglets.Short-term feeding high doses of COS in free feeding piglets effectively improved the antioxidant system for alleviating the intestinal oxidative stress and oxidative damage.However,in restricted feeding piglet supplmented with high doses COS by short-term showed a certain degree of mitigative effect on oxidation injury.All above,this study showed that,dietary COS supplementation at 50 mg/kg by long-time can effectively alleviate diquat-induced jejunum injure,protect intestinal mucosa integrity and improve gut digestion and absorption function.Weaning induced gut oxidative stress in piglets,and supplement high doses of COS by short-term in ad libitum pigs can alleviate oxidative damage caused by weaning,however,supplement high doses of COS by short-term in restricted pigs had a little effect on aggravating oxidative injure.