Proteomics Analysis On The Intestinal Mucosa Of Weaning Piglets Using SWATH-MS

Weaning stress,leading to slow-growth and increased mortality of piglets,has been considered as a serious problem in the swine industry.It can cause irreversible damage the intestinal mucosal barrier and affect the function of the intestine.According to previous reports,weaning stress is closely related with the activation of hypothalamic pituitary adrenal axis,mast cell hyperplasia,as well as MAPKs pathway.Most studies focus on the physiological levels or single signal pathway.However,the underlying mechanisms is still not clear.In our study,21-day-old piglets were chosen as weaning animal model to establish proteomic analysis strategy suitable for intestinal mucosa.SWATH-MS technology combined with bioinformatics analysis were employed to systematically analyze the changes of intestinal mucosal proteins and related pathways in piglets.This study may provide an insight for reducing the injury of piglets'intestinal mucosa caused by weaning stress.This thesis involved three major sections:Experiment 1:Effect of 21-days-weaning on the intestinal barrier in piglets.A 2x3 completely random design was chose and the two factors including weaning?weaning and suckling groups?and age?22-day,24-day,and 28-day?.In this experiment,six litters of 21-day-old Yorkshire piglets were selected.Six piglets per litter were randomly divided into two groups,indicating that there were eighteen piglets in each group.At Day 22,24 and 28,six piglets were slaughtered to determine the production performance,intestinal morphology and barrier factors.The results are shown as below:1)both the daily gain and final body weight were significantly decreased when piglets were weaning at 21 days of age?P<0.01?.2)there were significant interactions of weaning and days of age for jejunal crypt depth,diamine oxidase?DAO?activity,the expression of Occludin and TNF-?,ileum villi height?P<0.05?.3)the jejunal crypt depth was significantly decreased?P<0.01?,and the expression of Occludin was increased and then decreased between 22 and 28 days?P<0.05?,while the ileum villi height was increased between 22 and 24 days of age?P<0.01?.4)Villi height,mucosal DAO activity and the expression of ZO-1,Occludin and IL-10 was significant decreased due to the weaning stress,while crypt depth and the expression of TNF-?increased?P<0.05?.These results demonstrated that the weaning at 21 days of age disrupt the structure of intestinal epithelial cells and the expression of tight junction in piglets,which causing shorten and shed of villi,increased intestinal permeability,inflammatory response and decreased daily gain.Experiment 2:Identification of intestinal mucosal proteome in piglet.The ileal mucosa from weaning and suckling group at 22 days of age were used to establish proteomic analysis suitable for studying the intestinal mucosa of piglets.We studied the protein extraction efficiency of three types of buffer solutions involving 50 mM TEAB,6 M Urea,Cocktail;50 mM Tris-HCl,4%SDS,50 mM DTT,pH 7.6;50 mM Na₂HPO₄,300 mM NaCl,Cocktail.Also the protein digested methods?acetone precipitation and Filter-aided sample preparation?and the separated time of liquid chromatography were optimized.Hence,the optimized protein extraction solution was 50 mM Na₂HPO₄,300 mM NaCl,Cocktail).In addition,the optimized methods to increase the number of identified proteins intestinal mucosa were acetone-precipitating method and the 90 min elution time.Using the optimized methods,there are 1611 of identified protein and 9498 of identified peptides.This work provided an optimized proteome strategy for intestinal mucosa analysis in the future.Experiment 3:Label-free quantitative proteomics study reveals the changes of intestinal mucosal proteins in weaned piglets base on SWATH technology.22-days of age of weaning and suckling piglets?n=6?from experiment 1 were slaughter and the ileal mucosal were collected.Profiled the changes of intestinal protein proteome in two groups using SWATH.In this study,a total of 4895 quantitative proteins were identified,in which 376 differentially expressed proteins related to weaning stress were obtained with P<0.05 and fold change>2.The expression of important differentially expressed proteins was verified by qRT-PCR.The results confirmed that the consistent of SWATH and qRT-PCR,indicating that quantitative proteomics is reliable.Further in-depth analyzing the biological function of differentially expressed proteins,preliminary results revealed the possible molecular mechanisms of intestinal injury induced by weaning stress.In summary,early weaning stress can seriously damage the structure of intestinal epithelial cells and the expression of tight junction in piglets,causing shorten and shed of villi,increased intestinal permeability,inflammatory response and decreased daily gain.The effects of injury intestinal mucosal barrier may be through the following ways:1)Unbalanced the material metabolism by down-regulating proteins in galactose metabolism and fat metabolism.Energy supply could not be guaranteed.The growth performance of piglets is reduced.2)Disrupted the intestinal epithelial barrier by up-regulating the negative regulator of epithelial cell and down-regulating the proteins associated with cytoskeleton.Increased intestinal permeability causing the process of bacterial invasion of epithelial cells.In addition,weaning stress disordered cell metabolism by inhibiting lysosome and Fc gamma R-mediated phagocytic signaling pathway.Eventually,the changes led to impaired of intestinal morphology and function.This study explored the regulation of mucosal proteins involved in weaning stress,and preliminarily the mechanism of intestinal mucosal barrier.It can provide a theoretical basis for the rational development of strategies to reduce weaning stress.