Molecular Mechanism Of Apoptosis In Weaned Piglets And Nutrition Regulation

Weaning is a critical event for porcine weaning-associated disease, such aspost-weaning stress syndrome, which involves intestinal dysfunction, causes hugeeconomic losses in pig production. Evidences have proved that apoptosis disorder can leadto intestinal mucosal barrier dysfunction and injury the gastric bowel function, which hasalso been reported closely related to the disorder of the free radical metabolism andapoptosis. We have previously demonstrated that weaning induced free radicaldysfunction, increased the concentrations of NO and H2O2in serum in post-weaning pigs.However, the relationships among weaning induced free radical generation, apoptosis, andintestinal mucosal barrier damage were still unclear in pigs. N-acetyl cysteine (NAC), anantioxidant, has been demonstrated to be beneficial in various clinical treatments, but littleis known about the effect of NAC on the regulation of free radical generation, enterocyteapoptosis, and the dysfunction in intestinal barrier. Based on these,4studies weredesigned as follows.1Enterocyte apoptosis in weaned pigletsIn present study, concentrations of Caspase-3,8, and9were measured by ELISAmethod. Transmission electron microscopy, terminal deoxynucleotidyl transferasemediated dUTP nick-end labeling, and enzyme-linked immunosorbent assay methodswere conducted to characterize enterocyte morphological changes induced by weaning in pigs. Expressions of Caspase-3,8,9, Bcl-2, and Bax were also analyzed by quantitativereverse transcription-PCR (qRT-PCR) to study the molecular mechanism of apoptosis in21d weaned piglets. Results showed that, weaning caused the DNA fragmentation ofintestinal issue in weaned piglets. The activities of caspase-3, caspase-8, and caspase-9were strongly increased in the jejunum of weaning pigs as compared to suckling pigs (P <0.01). Histological examination of the jejunum revealed an increase in the number ofapoptotic cells (apoptotic nuclei are stained dark brown) in weaning pigs, with significantlocalization of apoptotic cells in the upper tip of the intestinal villi. Moreover, theapoptotic index was significantly higher in weaning pigs (P <0.01) than in suckling pigs.Electron microscopic examination revealed that some enterocytes showed necrotic injuriescharacterized by serious damage to the plasma membrane, complete loss of the junctionspecializations between adjacent cells, and swollen mitochondria without crests. Someother detached cells had characteristic features of apoptosis with vacuolated cytoplasm,cell compaction, condensed chromatin, and fragmented mitochondria. The microvilli andapical vacuoles were well developed in control pigs. No significant Bax (pro-apoptosisfactor) and Caspase-9expression differences were observed between the two groups,while weaning decreased the expression of Bcl-2(anti-apoptosis factor) in weaning pigs(P <0.01). The mRNA gene expression of caspase-3and caspase-8were increased inweaning pigs (P <0.05) than the control pigs. In addition to the increased caspaseactivities, a significant increase in Fas levels were observed in weaning pigs (P <0.01).Hence, results indicate that weaning caused DNA fragmentation, enhanced the contents ofapoptosis related enzymes, and increased enterocyte apoptosis by activating both theintrinsic and the extrinsic apoptosis pathways in post-weaning pigs, as well as thecoexistence of apoptosis and necrosis.2Relations among the free radical generation, apoptosis, and intestinal barrierfunction in weaned pigletsMeasure the contents of CAT, SOD, GSH-px, and MDA by visible light, xanthineoxidase, disulfide nitro benzoic acid, and thiobarbituric acid method, respectively. Concentrations of cortisol, HIF-1α and TNF-α were measured by ELISA method. Bystudying the antioxidant ability, intestinal permeability, and the expression of interginαvβ6and transmembrane protein Occludin-1and ZO-1in weaned piglets to study theintestinal barrier function in weaned piglets. The correlation analysis among free radicalgeneration, apoptosis and intestinal barrier function was established by multivariateanalysis to explore the relationship among free radical generation, apoptosis and intestinalbarrier function.Results found that cortisol, HIF-1α and TNF-α concentrations were increased (P <0.01) after weaning. Activities of SOD and GSH-Px in serum were significantly lower (P<0.05) in weaning pigs than in control pigs. Content of MDA, the product of lipidperoxidation was significantly increased (P <0.05) in pigs after weaning. While the serumability to inhibit hydroxyl radical were decreased (P <0.05) in pigs after weaning. Nosignificant difference of NO and H2O2(P>0.05) was observed after weaning. Asignificant increase in intergin αvβ6levels was observed in weaned pigs (P <0.01versusControl). Expressions of Occludin-1and ZO-1(P>0.05) were decreased in weaned pigs.Results indicate that weaning could decrease serum antioxidant ability, and tight junctionexpression, leading to the intestinal barrier dysfunction. The increased apoptosis inweaned piglets was associated with the intergin αvβ6expression and decrease ofantioxidant ability, as well as the increase of free radical metabolism.3Gene expression profile of differentially gene expression and apoptosis associatednetwork analysis in weaned pigletsTo gain new insight into the interaction between weaning stress and intestinalfunction, changes in genomic expression in the intestinal of weaned pigs, together withsuckling pigs were analyzed by microarray analysis to gain the target genes related toweaning stress. Keen interest was focused on differentially expressed genes related toapoptosis. Bioinformatics technologies were used to analyze the function of differentiallyexpressed genes and its relationship with apoptosis in weaned piglets. QRT-PCR was used to analyze the mRNA expression of target genes, maps of intestinal apoptosissignaling pathways were also drawn to clarify the mechanism of apoptosis induced byweaning stress.Four hundred and forty five genes showed altered expression after weaning treatment(up-regulated,286; down-regulated,159), among which,51up-regulated cell cycle processrelated genes,25down-related. Most of these altered genes are cellular process-relatedand regulators which may involve in the cell differentiation, cell migration, cell death, cellcycle, and so on. By using the KEGG database, the significant down-regulated pathwaysmainly contained immune response, while the up-regulated pathways contained cellapoptosis, signal transduction, and endocrine. The altered genomic expression of genesthat related to apoptosis was confirmed by quantitative real-time PCR. Of the genes tested,greater (P <0.05) expression of genes involved in apoptosis (CYCS, Bad, and ATM),pro-inflammatory signals (TNF and IL-8), and transcription factor (NFATC2and NOS2)were detected in weaned pigs compared with suckling pigs, but that cell cyclecontrol-related genes, such as E2F5and Smad4, and the signal transduction factor c-JUNwas less expressed in weaned pigs than suckling pigs. These selected genes likely indicatethat weaning induced cell cycle arrest, enhanced apoptosis, and inhibited cell proliferationvia the intrinsic and extrinsic apoptosis pathways. The results of this study provide a basisfor understanding the molecular pathogenesis of weaning treatment.4Effect of dietary N-acetylcysteine supplementation on intestinal barrier function inweaned pigletsThe aim of this study was to determine the effects of early administration of NAC toweaning piglets on serum antioxidant content and apoptosis regulation after weaning.Genes related to apoptosis and cell cycle control were selected for mRNA expression, tostudy the molecular mechanism of NAC on intestinal apoptosis regulation and intestinalbarrier repair.Results showed that feeding the NAC diet decreased the concentrations of cortisol andHIF-1α (P <0.05). No significant difference was observed in TNF-α contents after NAC addition (P>0.05). Supplementing the diet with NAC increased the activities of serumSOD and GSH-Px activities, and liver GSH-Px activities (P <0.05). No significantdifferences of serum and liver MDA contents, and liver GSH-Px activities were observed(P>0.05) after NAC supplementation. Villus height in the jejunum was significantlydecreased (P <0.01) after weaning, whereas villus width showed a small decrease (P <0.05) in the weaning pigs relative to the controls. Feeding the NAC diet increased (P <0.05) villus height and villus width (P <0.01), and the villus structure was relatively intactin the jejunum compared with the weaning group. The apoptotic index was decreased inthe pigs fed the NAC diet compared to the weaning group (P <0.05), and the microvilliand apical vacuoles were well developed. Compared with that in weaned pigs, the NACdiet decreased the expressions of Fas (P <0.05), expression of Bcl-2(anti-apoptosis) wasincreased in NAC-treated pigs (P <0.05), expressions of caspase-8and caspase-9showeda trend to decrease (P=0.141and P=0.206, respectively) in NAC-treated pigs. Greaterexpression of cell cycle control related gene E2F5and Smad4(P <0.05) were detected inNAC-treated pigs compared with weaned pigs. Expression of apoptosis promoter ATMwas increased, while wound healing gene αvβ6was less (P <0.05) in NAC-treated pigsthan weaned pigs. All these indicate that NAC has the potential to counteract weaningstress, enhance the antioxidant ability, inhibit cell death, and preserve epithelial integrity.The protection of NAC in weaned piglets was not only by regulate the cell apoptosispathway, it also involved in cell cycle regulation.In conclusion, the oxidative stress caused by early weaning could decrease theantioxidant ability, increase free radical metabolism, and induce enterocyte apoptosis byactivating both the intrinsic and the extrinsic apoptosis pathways in post-weaning pigs,which might involve in the intestinal barrier dysfunction. The apoptosis and necrosis wereconcurrence in weaned pigs. The enhanced enterocyte apoptosis was seriously associatedwith the decrease of antioxidant ability and increase of free radical metabolism afterweaning. Microarry analysis revealed that, after weaning, the cellular process was active,cell cycle control process was impressed. The NAC diet could alleviate intestinal mucosaldamage and inhibit cell death, preserve epithelial integrity. The protection of NAC inweaned piglets was not only by regulate the cell apoptosis pathway, it also involved in cell cycle regulation. Further studies should be conducted to more completely determine theregulation of intestinal mucosal function by NAC after weaning.