Protective Effects Of Chlorogenic Acid On Oxidative Damage Of Intestinal Epithelial Barrier In Weaned Pigs And Its Mechanisms

At weaning,pigs often suffer from various stresses,which induce overproduction of the free radicals and subsequently lead to damage of the intestinal epithelial cells.Impaired intestinal barrier can lead to the invasion of luminal pathogens and weaken the digestion and absorption of nutrients,which are responsible for the diarrhea and growth retardation,and then cause a considerable economic loss for pig production.Therefore,powerful and safe therapeutic approaches are urgently needed in this critical phase to alleviate the negative effects of weaning stress on intestinal barrier of pigs.Chlorogenic acid(CGA),formed by caffeic acid and quinic acid,is a phenolic compound and contains five hydroxy.The special molecular structure impels CGA to possess strong antioxidant effects by scavenging the free radical.At present,there is little literature about the application of dietary CGA supplementation in livestock.Also,until now,little information is available concerning the protective effect of CGA on alleviating the oxidative damage of intestinal epithelial barrier of weaned pigs.Therefore,in this study,we firstly explored the effects of CGA on growth performance and intestinal barrier in weaned pigs.Subsequently,we constructed oxidative stress model in vivo and in vitro using weaned pigs and jejunum epithelial cells IPEC-J2,respectively,to study the protective effects of CGA on intestinal epithelial barrier and the related signaling pathways in weaned pigs.This study explored the possible mechanism of CGA on relieving oxidative damage of the intestinal barrier in weaned pigs.This not only offers a theoretical reference for the rational application of CGA in weaned pigs,but also provides a new idea for further exploration of nutritional regulation measures on alleviating the weaning stress of pigs.Experiment 1 Different levels of chlorogenic acid on growth performance of weaned pigs Maintaining the healthy of piglets plays a key role in improving the economic efficiency of pig production.Weaning is an unavoidable stressful event,which can damage the healthy of piglets and lead to growth retardation.With banning the use of antibiotic,it is particularly important to seek nutritional regulation measures to alleviate the negative effects of weaning stress in pigs.CGA,extracted from natural plants,is a phenolic compound and possesses a variety of biological activities,including antioxidant,anti-inflammatory and so on,which seem to impel CGA to serve as a green feed additive for maintaining the health of animals.However,at present,there are few studies about the applications of dietary CGA supplementation in weaned pigs.Therefore,the aim of this study was to investigate the effects of different CGA levels on growth performance of weaned pigs and then explore the appropriate dietary addition dose.In this experiment,200 DLY weaned pigs were randomly divided into 4 treatments(5 replicates for each treatment and 10 replicates for each treatment)according to the initial body weight and sex,receiving either a basal diet or the basal diet supplemented with 250,500 and 1000 mg/kg CGA for 14 days.The results showed that,compared with the control group,250 and 500 mg/kg CGA supplementation had no significant influence on the growth performance of weaned pigs.However,dietary supplementation with 1000 mg/kg CGA decreased the F/G of pigs(P < 0.05),indicating that under the conditions of this experiment,the appropriate dietary addition dose of CGA in improving the growth performance of weaned pigs is 1000 mg/kg.Experiment 2 Effects of chlorogenic acid on intestinal barrier structure and function of weaned pigs Based on the results of Experiment 1,the aim of this experiment was to investigate the effects of CGA on nutrients availability and intestinal barrier function of weaned pigs.In this experiment,24 DLY weaned pigs were selected and randomly divided into 2 treatments(12 replicates for each treatment and 1 pig for each replicates)according to the initial body weight,receiving either a basal diet or the basal diet supplemented with 1000 mg/kg CGA for 14 days.The results are as follows:(1)Compared with the control group,the apparent digestibility of crude protein,crude fat and crude ash were increased(P < 0.05),and the diarrhea rate were decreased in pigs on the CGA group(P < 0.05).(2)Dietary CGA supplementation decreased the activity of diamine oxidase(DAO),the concentration of Dlactic acid,as well as the levels of TNF-? and IL-1? in serum(P < 0.05).In addition,the activities of serum GSH-Px and CAT were increased by CGA supplementation(P < 0.05).(3)Dietary CGA supplementation increased the villus height of jejunum,and the villus height/crypt depth of jejunum and ileum(P < 0.05).Higher claudin-1 protein abundance and DAO activity in the intestine were also observed in pigs fed CGA-supplemented diet(P <0.05).In addition,the number of tryptase positive mast cells,the levels of histamine and tryptase and the apoptosis rate of intestinal epithelial cells were decreased by CGA supplementation(P < 0.05).(4)Dietary CGA supplementation increased the activities of digestion and absorption related enzymes(sucrase,maltase and alkaline phosphatase)and antioxidant enzymes(GSH-Px and CAT)(P < 0.05),whereas decreased the concentration of MDA in intestine(P < 0.05).(5)The m RNA expression of pro-apoptotic genes(Fas,caspase-3 and caspase-9)and pro-inflammatory cytokines(TNF-?,IL-6 and IL-1?)(P < 0.05)were down-regulated,as well as the m RNA expression of anti-apoptosis gene(Bcl-2),digestion and absorption related gene(SGLT1,GLUT2,ZNT1 and DMT1)and antioxidant-related gene(Nrf2 and HO-1)were up-regulated by dietary CGA supplementation(P < 0.05).These results indicated that CGA could improve the nutrients digestibility,and maintain the integrity of intestinal barrier structure and function by increasing antioxidant capacity,reducing inflammatory reaction,and decreasing apoptosis and intestinal barrier permeability.Experiment 3 Chlorogenic acid alleviates diquat-induced oxidative damage of intestinal epithelial barrier in weaned pigs The results in Experiment 2 showed that CGA could improve the antioxidant capacity of weaned pigs and maintain the structural and functional integrity of intestinal barrier.However,under oxidative stress,the protective effects of CGA on alleviating oxidative damage of intestinal epithelial barrier in weaned pigs have not yet been studied.Diquat is a commonly used oxidative stress inducer and is widely used in in vitro and in vivo trials.Therefore,in this experiment,we constructed an intestinal oxidative stress model of weaned pigs induced by diquat to investigate the role of CGA in relieving oxidative damage of intestinal epithelial barrier.40 DLY weaned pigs were selected and randomly divided into 5 treatments(8 replicates for each treatment and 1 pig for each replicates)according to the initial body weight and sex: Control group(normal saline),Oxidative stress group(Diquat),CGA1 group(500 mg/kg CGA + Diquat),CGA2 group(1000 mg/kg CGA + Diquat)and CGA3 group(1500 mg/kg CGA + Diquat).During the first 14 days of the experiment,both the Control group and the Diquat group were fed basic diets,while the CGA1,2 and 3 groups were fed corresponding diets,respectively.In the morning of the 15 th day,except the Control group,all the other groups were intraperitoneally injected with diquat(10 mg/kg body weight),while the Control group was injected with sterilized saline of the same volume.The pigs were continued to be fed the corresponding diets for 1 week.The trial lasted for 21 days.The results are as follows:(1)Oxidative stress decreased the ADFI and ADG of weaned pigs(P < 0.05),but 500 and 1000 mg/kg CGA significantly improved the growth performance(P < 0.05).(2)Oxidative stress increased the contents of aspartate amino transferase,creatine kinase,cortisol and inflammatory factors(TNF-? and IL-6,IL-1?),as well as the levels of DAO,D-lactic acid and endotoxin in serum of weaned pigs(P < 0.05).The level of serum D-xylose and the activities of serum antioxidant enzymes(SOD,GSH-Px and CAT)were also decreased in Oxidative stress group(P < 0.05).However,dietary CGA supplementation decreased the levels of endotoxin and inflammatory factors,and increased the activities of antioxidant enzymes in serum(P < 0.05).(3)Oxidative stress decreased the villus height and villous height/crypt depth of small intestine(P < 0.05),reduced the claudin 1 protein abundance and improved the intestinal epithelial cell apoptosis rate(P < 0.05).In addition,oxidative stress decreased the activities of disaccharide enzyme(maltase,lactose,and sucrose),alkaline phosphatase and antioxidant enzymes(GSH-Px and CAT)(P < 0.05),and increased MDA content in intestine(P < 0.05).However,dietary CGA supplementation alleviated the structural and functional damage of intestinal barrier induced by oxidative stress in weaned pigs(P < 0.05).Moreover,this study found that CGA could decrease the expression levels of apoptosis-related genes(Fas,Bax,caspase-3 and caspase-9)and inflammatory factors(TNF-? and IL-1?)(P < 0.05),as well as increase the expression levels of absorption-related genes(SGLT1 and GLUT2)and key molecules of antioxidant pathway(Nrf2 and HO-1)in the intestine of weaned pigs(P < 0.05).These results indicated that oxidative stress induced severe damage to the intestinal epithelial barrier of weaned pigs,while CGA could maintain the integrity of intestinal barrier structure and function by improving the antioxidant capacity,reducing inflammatory response and decreasing apoptosis of intestinal epithelial cell,and then alleviate the oxidative damage of intestinal barrier in weaned pigs.Experiment 4 Chlorogenic acid alleviates diquat-induced intestinal epithelial barrier dysfunction in IPEC-J2 cells via the PI3K/Akt-Nrf2 signaling pathway Nrf2,an important transcription factors in oxidative stress,could protect the cell or tissue from oxidative stress damage by regulating the m RNA expression of antioxidant molecules and HO-1.PI3K/Akt pathway,an upstream regulator of Nrf2,is one of the key signaling pathways in regulating cell proliferation and apoptosis,and plays a dominant role in the initiation of cell defense system and resisting oxidative damage of cells.In addition,HO-1 could inhibit the inflammation damage induced by oxidative stress via inhibiting the activation of NF-?B pathway and the transcription of downstream inflammatory related genes.However,whether these two signaling pathways could mediate the effects of CGA on alleviating oxidative damage of intestinal barrier have not yet been reported.Therefore,the aim of this experiment was to explore the protective effect of CGA on intestinal epithelial barrier structure,cell apoptosis and the expression of antioxidant molecule,as well as whether PI3K/Akt-Nrf2 and NF-?B signaling pathways involved in these protection procession by an IPEC-J2 model induced by diquat.The results are as follows:(1)Diquat significantly reduced cell viability,promoted apoptosis and decreased the claudin-1 protein abundance(P < 0.05).However,CGA increased the cell viability and the abundance of claudin-1,as well as decreased the apoptosis of intestinal epithelial cells(P < 0.05).(2)Diquat increased the ROS and MDA level and SOD activity in intestinal epithelial cells(P < 0.05).However,CGA decreased the levels of ROS and MDA(P < 0.05),and the m RNA expression of MCP-1,TNF-? and IL-1? induced by diquat(P <0.05).(3)CGA increased the phosphorylation levels of Nrf2 and the expression levels of HO-1 protein(P < 0.05).However,after inhibiting the PI3K/Akt pathway,the phosphorylation of Nrf2 was significantly reduced(P < 0.05),while the level of ROS and apoptosis rate were also increased(P < 0.05).(4)CGA reduced the phosphorylation levels of NF-?B and I?Ba in the cell under oxidative stress(P < 0.05).However,after inhibiting the activity of HO-1 protein,the phosphorylation levels of NF-?B and I?Ba were significantly increased(P < 0.05).These results indicated that CGA could promote the phosphorylation of Nrf2 through PI3K/Akt signaling pathway,thus improving the antioxidant capacity of intestinal epithelial cells,inhibiting the production of inflammatory factors and apoptosis,maintaining the structural integrity of intestinal epithelial barrier,and thereby alleviating the oxidative damage of intestinal epithelial barrier.In addition,CGA could indirectly inhibit the activation of NF-?B signaling pathway by increasing the expression of HO-1 protein,thereby relieving the inflammatory injury of intestinal epithelial cells induced by oxidative stress.Experiment 5 Chlorogenic acid alleviates inflammation damage of intestinal epithelial cells via inhibiting the NF-?B signaling pathway Under oxidative stress,the expression of inflammatory factors,such as TNF-?,are significantly increased,leading to the inflammatory damage of cells and tissues.NF-?B signaling pathway is one of the most important pathways and can mediate the cellular inflammatory response.The results in Experiment 4 showed that CGA could inhibit the activation of NF-?B pathway by increasing the expression of HO-1 protein.Therefore,the aim of this experiment was to further explore the mechanism of CGA in relieving the inflammation damage of intestinal epithelial barrier induced by oxidative stress.In this experiment,we constructed the IPEC-J2 in vitro inflammation damage model induced by TNF-? to detect the effects of CGA on intestinal epithelial barrier,cell apoptosis,inflammatory factor expression and NF-?B signaling pathway.The results are as follows:(1)TNF-? decreased the cell viability and claudin-1 protein abundance(P < 0.05),while increased apoptosis of intestinal epithelial cells(P < 0.05).However,CGA significantly increased the cell viability and claudin-1 protein abundance,as well as decreased the apoptosis of intestinal epithelial cells(P < 0.05).(2)TNF-? increased the expression levels of inflammatory related genes(MCP-1,IL-1? and IL-6)(P < 0.05)and the phosphorylation levels of NF-?B and I?Ba(P < 0.05).However,CGA could inhibit the activation of NF-?B signaling pathway induced by TNF-?(P < 0.05).(3)After inhibiting the activity of HO-1 protein,CGA still decreased the phosphorylation levels of NF-?B and I?Ba under inflammatory stress(P < 0.05),and reduced the expression of inflammatory factors and the apoptosis of intestinal epithelial cells(P < 0.05).These results indicated that CGA could protect intestinal epithelial cells from inflammation damage by inhibiting the activation of NF-?B signaling pathway,decreasing the expression of inflammatory factors and reducing the apoptosis of intestinal epithelial cell.In conclusion,dietary CGA supplementation could significantly improve the growth performance and intestinal health of weaned pigs.Under oxidative stress,CGA could promote Nrf2 phosphorylation and antioxidant gene expression through PI3K/Akt signaling pathway,thus improving the antioxidant capacity of weaned pigs,inhibiting the production of inflammatory factors and apoptosis,maintaining the structural integrity of intestinal epithelial barrier,and thereby alleviating the oxidative damage of intestinal epithelial barrier.In addition,CGA can also inhibit the activation of NF-?B signaling pathway under oxidative stress,reduce the production of inflammatory factors,and then alleviate cellular inflammation damage.