The Attenuate Effects Of Arginine On The Intestinal Mucosal Injury Of Broiler Chickens And The Related Mechanisms

Six experiments were conducted to study the effects of L-arginine supplementation on the intestinal mucosal injury induced by the intestinal pathogenic bacteria in broiler chickens and the related mechanisms.Exp.1 was conducted to investigate the development of the intestinal physical barrier-related gene expression in the posthatch broiler chickens.The jejunums of birds were sampled at days(d)1,7,14,21,28 and 35.The results showed that the mRNA expression of tight junctions and adherin junctions significantly changed with time.The claudin-1 mRNA expression at d 1 was much higher than the other days while there were no differences among the other days.The claudin-2 mRNA expression gradually increased from d 1 to d 14,then sharply dropped from d 14 to d 21,and had a platform period from d 21 to d 35.The claudin-3 mRNA expression was steady from d 1 to d 7,gradually rose from d 7 to d 21,and gradually declined from d 21 to d 35.The claudin 5 mRNA expression gradually increased until d 21,gradually decreased from d 21 to d 28,and was steady during the last 7 days.The developmental patterns of occludin and ZO-1 were similar.Their mRNA expression gradually elevated in the first 14 days,and was steady in the rest of the days.The ZO-2 mRNA expression rose gradually in the first 28 days,and then gradually dropped from d 28 to d 35.The developmental patterns of ZO-3 and E-cadherin were similar.Their mRNA expression increased in the first 21 days but decreased in the last 14 days.There were significant quadratic responses on the expression of claudin-3,claudin-5,occludin,ZO-1,ZO-2,ZO-3 and E-cadherin with age.Significant positive correlations were also observed between them.These results suggest that most jejunal physical barrier-related gene expression increased during the first 14 days,and were steady from d 28 to d 35.The correlations of most physical barrier-related gene expression were probably due to their structure interactions.Exp.2 was conducted to investigated the effects of dietary L-arginine level and feeding duration on the intestinal immune and barrier functions of broiler chickens under necrotic enteritis.The experiment was designed as a factorial arrangement of(3 dietary treatments × 2 challenge statuses).Broilers were fed a basal diet(containing 1.25%L-arginine)or a high-arginine diet(containing 1.87%L-arginine),or the basal diet for the first 8 days and the high-arginine diet from d 9-28.Birds were coinfected with or without Eimeria and Clostridium perfringens(CP).Samples were collected at d 21 and d 28.The results showed that the co-challenge led to intestinal injury,as characterized by lower plasma D-xylose concentration,higher ileal paracellular permeability,and higher numbers of Escherichia coli and CP in caecal digesta.However,these situations could be alleviated by L-arginine supplementation.Besides,the decrease of jejunal slgA level on d 28 caused by the co-challenge was alleviated by L-arginine supplementation.The intestinal claudin-1 and occludin mRNA expression levels were decreased after the co-challenge,which were reversed by L-arginine supplementation.Moreover,the co-challenge upregulated intestinal claudin-2,IFN-?,TLR-2,and NOD1 mRNA expression,and L-arginine supplementation elevated IFN-?,IL-10,and NOD1 mRNA expression.In conclusion,L-arginine supplementation could inhibit CP overgrowth and alleviate intestinal mucosal injury by promoting innate immune responses and maintaining intestinal barrier function.Diets containing 1.87%L-arginine fed for 28 days could get better results.Exp.3 was conducted to investigate the effects of dietary L-arginine supplementation on the intestinal inflammation of broiler chickens challenged with CP and the related mechanisms.The experiment was designed as a 2 × 2 factorial arrangement with two dietary arginine concentrations(1.42%and 1.72%)and two challenge states(with or without CP challenge).Samples were collected at d 21 and d 28.The results showed that CP challenge significantly increased serum procalcitonin and urea nitrogen levels.Arginine supplementation significantly decreased serum diamine oxidase and myeloperoxidase activities,and nitric oxide levels.CP challenge reduced serum arginine and ornithine levels,which was reversed by arginine supplementation.CP challenge led to higher intestinal lesion score and jejunal morphological disruption,and elevated jejunal mucosal activities of lysozyme and iNOS,which was reversed by arginine supplementation.Infected chickens had higher mRNA expression of pro-inflammatory cytokines(IL-1?,IL-6 and IFN-?)and anti-proinflammatory cytokines(TGF-P3)in the jejunum,whereas their expression was lowered by arginine supplementation.CP challenge increased Cathelicidins(CATH)1 and CATH3 mRNA expression on d 21,but decreased avian beta-defensin(AvBD)1,AvBD2,CATH3 and liver-expressed antimicrobial peptide(LEAP)-2 mRNA expression on d 28 in the jejunum.Arginine supplementation decreased jejunal AvBDl,AvBD2,CATH1 and CATH3 mRNA expression on d 21.CP challenge significantly increased the jejunal mRNA expression of cationic amino acid transporter(CAT)-1,-2,-3 and increased the arginine catabolic enzymes inducible nitric oxide synthase(iNOS),arginase2(ARG2),arginine decarboxylase(ADC),and arginine:glycine amidinotransferase(AGAT);whereas their expression was lowered by arginine supplementation.Arginine supplementation alleviated the CP challenge-induced increases in jejunal Janus kinase(JAK)1,JAK2,JAK3,signal transducer and activator of transcription(STAT)1 and STAT6 mRNA expression.In conclusion,dietary L-arginine supplementation prevented C.perfringens challenge-induced circulated arginine deficiency,normalized arginine transport and metabolism,down-regulated the activated JAK-STAT signalling pathway,thus alleviated the intestinal inflammation and mucosal injury of broiler chickens challenged by CP.Exp.4 was conducted to investigate the effects of dietary L-arginine supplementation on the gut bacterial community composition and function of broiler chickens challenged with CP.This experiment was a one-factor completely random design with three groups:Control(CTL),C.perfringens-challenged(CP),and C.perfringens-challenged and fed diet supplemented with 0.3%L-arginine(ARGCP)groups.The infected birds were challenged with CP from d 14 to d 20,and ileal digesta were collected individually on d 21.The results showed that the three treatments harbored distinct microbial communities,but the microbial composition of CTL and ARGCP was more similar.CP challenge reduced the relative abundance of Firmicutes,but increased the relative abundance of other seven phyla,such as Proteobacteria.These situations were reversed by L-arginine supplementation.At the genus level,24 taxa were significantly more abundant in the CP group than in the CTL group,of which 15 taxa were belonged to the Proteobacteria.The abundance of 23 genera were significantly decreased by L-arginine supplementation.The abundances of only 3 genera were different between the CTL and ARGCP groups.LEfSe analysis showed that multiple classes belonged to the Proteobacteria were more abundant in the CP group.CTL group was rich in Ruminococcaceae and Lactobacillus gasseri.ARGCP group harbored more Clostridiales and Lactobacillales.Predictive functional profiling of microbial communities by PICRUSt showed that arginine supplementation could alleviate most of the changed microbial pathways induced by CP challenge.CP challenge upregulated the pathways belonged to metabolism,human diseases,and cellular processes,and downregulated the pathways belonged to environmental information processing and genetic information processing,which could be reversed by arginine supplementation.In conclusion,L-arginine supplementation could promote the potential probiotics such as Clostridiales and Lactobacillales,and reduced the pathogens affiliated with Proteobacteria.Moreover,it could attenuate the C.perfringens challenge-induced more abundant pathways belonged to metabolism and human diseases.Arginine supplementation normalized the ileal microbiota of C.perfringens-challenged chickens to resemble that of unchallenged controls in terms of microbial composition and functionality.Exp.5 was conducted to investigate the effects of dietary arginine on the intestinal injury of the broiler chickens challenged with Salmonella typhimurium(ST).The experiment was designed as a 2 × 2 factorial arrangement with two dietary arginine concentrations(1.42%and 1.72%)and two challenge states(with or without ST challenge).Samples were collected at d 17 and d 23.The results showed that arginine supplementation decreased the ST challenge-induced increases on serum diamine oxidase activity and procalcitonin level.ST challenge led to serum arginine deficiency and arginine supplementation reversed this situation.In ST infected birds,arginine supplementation increased serum ornithine and urea nitrogen concentration.Arginine supplementation alleviated the intestinal morphological damage caused by ST challenge.ST challenge increased jejunal IL-17 mRNA expression,but decreased NF-?B,IFN-? and IL-10 mRNA expression on d 17.Arginine supplementation elevated jejunal IFN-y mRNA expression but reduced IL-22 mRNA expression on d 17.ST increased jejunal IL-1?,IL-10,IL-17,IL-8 and IL-22 mRNA expression on d 23.Arginine supplementation upregulated IFN-? and IL-10 mRNA expression but downregulated IL-22 mRNA expression on d 23.ST challenge reduced the jejunal mRNA expression of multiple antimicrobial peptides on d 17 and LEAP2 on d 23,and increased trefoil factor(TFF)2 mRNA expression on d23.Arginine supplementation increased jejunal TFF2 mRNA expression on d 17,decreased jejunal mRNA expression of multiple antimicrobial peptides,and increased LEAP2 mRNA expression on d 23.ST challenge increased jejunal CAT-1,-2,-3 mRNA expression,which were further elevated by arginine supplementation.ST challenge decreased ARG2 mRNA expression on d 17,but increased ADC and AGAT mRNA expression on d 23.Arginine supplementation further increased the ADC mRNA expression on d 23.In conclusion,arginine supplementation attenuated the ST-induced circulated arginine defiency and inflammation,and improved the intestinal morphology.Arginine may increase the circulated arginine concentration via the elevated mRNA expression of CATs.Arginine supplementation could modulate the mRNA expression of cytokines and antimicrobial peptides,particularly upregulating the IFN-y expression,which may promote the clearance of ST.Compared the results of this experiment with that of Exp.3,we found that both CP challenge and ST challenge could induce circulated arginine defiency,systemic inflammation and intestinal injury,and also stimulated the expression of CATs.Arginine supplementation increased the circulated arginine concentration and alleviated the intestinal injury in both of these pathogen infections.However,the pathological processes of CP challenge and ST challenge were different.CP challenge induced the intestinal inflammation in the early stage post infection and the intestinal inflammatory responses were attenuated during the late stage post infection.Whereas ST challenge reduced the expression of IFN-?.NF-?B and multiple antimicrobial peptides in the early stage post infection,which may be attributed to the immune evasion mechanism of ST.ST challenge induced intestinal inflammation in the late stage post infection.There were also differences in the regulation mechanisms of arginine.In CP challenge,arginine supplementation reversed the CP challenge-induced the expression of arginine catabolic enzymes,pro-inflammatory cytokines,anti-inflammatory cytokins and antimicrobial peptides.These regulations were consistent,which may result from the feedback inhibition of intestinal arginine transport by increased circulated arginine concentration.However,in ST challenge,the regulations of arginine on different molecules varied a lot.Arginine played a protective role probably due to promoting the IFN-y production,and then elevating the number and activity of T lymphocytes,which needs further studying.Exp.6 was conducted to investigate the effects of arginine on the inflammatory response in CP-infected chicken primary intestinal epithelial cells.The results showed that CP challenge resulted in cellular cytotoxicity,and increased the mRNA expression of IL-1?,IL-6,IL-8 and iNOS in dosage-dependent and time-dependent manners.The optimum infection dose and time were multiplicity of infection(MOI)= 1 and 4h,respectively.Fifty and/or 400 ?M of arginine significantly attenuated the CP challenge-induced the cellular cytotoxicity and the elevated mRNA expression of CAT-1,CAT-3,iNOS and inflammatory cytokines(IL-1??IL8 and IL10).Besides,arginine further increased the CP challenge-induced IFN-? and ARG2 mRNA expression.The attenuation of inflammatory responses with 400?M arginine was better than 50?M.In conclusion,the cellular cytotoxicity,and the increased mRNA expression of CATs,iNOS and inflammatory cytokines induced by CP challenge was reversed by arginine supplementation,which was consistent with the results of Exp.3.In summary,arginine could alleviate the intestinal pathogenic bacteria-induced intestinal mucosal injury of broiler chickens by increasing the circulated arginine concentration,modulating the intestinal arginine transport and metabolism,regulating the expression of intestinal cytokines and antimicrobial peptides,improving the intestinal physical barrier function and modulating the microbial composition and functionality.The arginine supplemented diet fed during the whole period exhibited more beneficial effects than that only fed during the infection stage.