| The protease-activated receptor-2 (PAR-2) is a member of the G-protein-coupled receptor family. Studies have shown that PAR-2 has a wide tissue expression in the gastrointestinal tract of animals and sugested to be anti-inflammatory and pro-inflammatory. PAR-2 has an important regulatory role in the gastrointestinal diseases. Diarrhea of weaning piglets (PWD) is a combination of factors caused diarrhea during the weaning period. PWD have characteristics of high morbidity and mortality, this characterixeics are severely detrimental to the porcine industry. In this study, we linked the PAR-2 and PWD, represented the relationship between PAR-2 and the gastrointestinal mucosal barrier of weaned piglets, understanded the role of PAR-2 in the diarrhea pathological process, got a new way for healing the PWD. The following two aspestes have been studied and analyzed in this study.The investigative object were 30 Duroc piglets aged 25-28 days and weighing (10±0.3)kg,18 piglets were infected with Enterotoxigenic E.coli (ECET) 5.0×1011 CFU oral dose.12 piglets with diarrhea were selected as a diarrhea group,12 healthy piglets as normal group. The piglets were abataged and collected the samples in the diarrhea 1th,3th,5th and 7th days(three healthy and three diarrhea piglets every time). Conventional methods were used to get the gastrointestinal tissues and blood from healthy and diarrhea piglets. HE staining was used to survey pathological changes in GI tracts Immunohistochemistry,Western Blotting and SYBR Green real-time RT-PCR were used to observe expression difference of PAR-2,Claudin-1,Occludin and ZO-1 at the level of genes and proteins; ELISA was used to test the concentration of CGRP,IL-6 and TNF-αin blood plasma; Conventional biochemical experiments were usd to test the concentration of phosphatide,aminohexose,endotoxin and DAO.The results showed that most pathological changes showed desquamation and denaturation in the epithelium mucosae, smooth muscle thinning of bowels, infiltrated neutrophil and eosinophil in the lamina propria, and lymph nodules were hyperplasia in submucosa. PAR-2 was expressed in all GI, there were significant differences between healthy and diarrhea group at the 3th day(P<0.05). Strong positive reaction occurs mainly in the epithelial cells of the lamina propria mucosa at the top of the epithelial cells of the blood vessels, rinsing basal parts, eosinophilic and neutrophils etc, all of the control group had positive reaction. There had been a marked increase of PAR-2 mRNA at the 3th,5th and 7th days, especially in ileum and colon. With the extension of diarrhea time, Claudin-1,Occludin and ZO-1 had obvious changes in the level of protein and gene, especially in intestinal tract, these results suggested that as the extension of diarrhea time, the more serious damage of the gastrointeseinal mucous, the ETEC major damage gut. In the third day, the density of aminohexose was decresed markly in the diarrhea group, and had significant difference compared with the normal group(P< 0.05), but the disparity was not signigicant in the 1th,5th and 7th days. Phospholipid also was decresed,signigicant difference in the 3th and 5th days, but not signigicant in the 1th and 7th days. With the extension of diarrhea time, the levels of endotoxin,TNF-α,IL-6 and DAO were signigicantly or very significantly increased in the plasma from diarrhea. In the 3-7th days, the above indexes difference were signigicantly comparaed with the control group(P<0.01), although the density of endotoxin and DAO were increased, but no signigicant differences(P>0.05). Compared with the control group, the density of CGRP were decreased, significant difference in the 5th and 7th days(P< 0.05), no dignificant difference in the 1th and 7th days(P>0.05). This part of the experimental results suggested that the was a negative correlation between PAR-2 and gastrointestinal mucosa system.The small intestine tissue were separated from newborn piglet, and cultured the intestinal epithelial cells(IEC) using enzymatic digestion, then got the cell monolayer after purification. A variety of stimulants were added in the medium of IEC, which included trypsin,soybean protease inhibitor,synthetic PAR-2 agonist SLIGRL-NH2,LRGILS-NH2,LPS and LT. In the action time was 2h,14h and 24h, ELISA was used to test the concentration of IL-6 and IL-8 in supernatant of cells; SYBR Green real-time RT-PCR was used to observe expression difference of Claudin-1,Occludin and ZO-1 mRNA. The data shown that Non-activated IECs produced negligible amounts of IL-6 and IL-8. Incubation of IECs with a PAR-2 agonist resulted in a low, but significant increase in IL-6 and IL-8 expression compared with controls. To examine the possible synergy between PAR-2 agonists and LPS+LT on IEC activation, IECs were incubated with trypsin and SLIGKV-NH2 in the presence concentrations of LPS+LT Incubation of IECs with the PAR-2 agonists in the presence of LPS+LT resulted in marked potentiation of IL-6 and IL-8 production at all concentrations tested. The change in IL-8 expression was more pronounced to that of IL-6. In the PAR-2 agonist stimulation 24h, the expression of Claudin-1,Occludin and ZO-1 mRNA was significantly lower than the group of control,soybean protease inhibitor and LRGILS-NH2, the difference was significant(P<0.05) which Claudin-1 change was evident.Above results suggested that PAR-2 had a negative regulation role on gasrointestinal mucosal barrier, and had a certain medium in the pathogenesis of diarrhea in weaned piglets. PAR-2 can promote the production of inflammatory cytokines after activation. The high wxpression of inglammatory factors can make neutrophils,eosinophils and other inflammatory cell migration to mediate inflammation in the gastrointestinal tract. Changes in the concentration of inflammatory cytokines can make the of expression decreased and shift of the three TJ proteins of Claudin-1,Occludin and ZO-1, resulting in structural damage of TJ and influenced the barrier function of epithelial cell.
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