Effect Of ?7nAchR Agonist On Enteric Glia Cells After Cardiopulmonary Bypass In Rats

Objective:To observe the changes of enteric glia cells in intestinal barrier injury after cardiopulmonary bypass in rats,then to explore the effect and possible mechanism of?7nAchR agonist on enteric glia cells.Methods:Ninty-six clean male SD rats weighting of 400~500g were randomly divided into four groups(n=6): sham group(group S),CPB group(group C),PHA568487+CPB group(group P),MLA+ PHA568487 +CPB group(group M).Each group was divided into four subgroups according to the time of blood sampling,six rats were in each subgroup.The rats couldn' t eat but drink from six hours before operation,anesthesia by intraperitoneal injection of 10% chloral hydrate(300mg/kg).In group S,the left femoral artery,caudal artery,right femoral vein and right internal jugular vein were punctured and intubated after anesthesia.In group C,we built CPB model after anesthesia and intubation,which last for one hour.In group P,PHA568487 was given by intraperitoneal injection(0.8mg/kg)after anesthesia and intubation,thirty minutes before building CPB model.In group M,PHA568487(0.8mg/kg)and MLA(6mg/kg)were given in turn by intraperitoneal injection after anesthesia and intubation,thirty minutes before building CPB model.Then respectively at T0(CPB instantly),T1(CPB 60min),T2(after CPB 2h),T3(after CPB 6h),we collected blood samples by right internal jugular vein,and recorded the changes of hemodynamics and blood gas analysis results.ELISA was used to detect the changes of serum IL-6,TNF-?,D-lactic acid and I-FABP.Respectively in each time point,rats were put to death by spinal dislocation method.The intestinal tissues were used to make pathological slices.The morphological changes of each group were observed by HE staining.Western blotting method was used to detect the expression levels of ZO-1,occludin,GFAP and S-100.The changes of EGCs were observed by immunohistochemical method.The signification of experimental data was Mean±SD(X ±s),T-test was used as a method of comparison among groups,make statistical analysis by SPSS 19.0,p<0.05 represents significant difference.Results:1.The morphological changes of intestinal tissuesHE staining revealed the intestinal mucosa in group S hadn't no obvious changes.In group C,the intestinal mucosa appeared different degrees of damage,villi shortened.The intestinal mucosa had hemorrhage and edema.The inflammatory cells infiltrated and intestinal epithelial cells degenerated and necrosed.In group P and M,the damage of intestinal mucosa was reduced.In group P,the damage was least,only mild intestinal mucosa glands expanded.The intestinal epithelial cells degenerated a little and a few inflammatory cells released.2.The content changes of IL-6 and TNF-? in serumCompared with group S,the contents of IL-6 and TNF-? at T1~T3 were all significantly increased in group C,P and M(p<0.05).Compared with group C,the contents of IL-6 and TNF-? at T1~T3 were both significantly decreased in group P and M(p<0.05).Compared with group P,the content of IL-6 and TNF-? at T1~T3 was significantly increased in group M(p<0.05).3.The changes of serum D-Lac and I-FABP levelsCompared with group S,the levels of D-Lac and I-FABP at T1~T3 were all significantly increased in group C,P and M(p<0.05).Compared with group C,the levels of D-Lac and I-FABP at T1~T3 were both significantly decreased in group P and M(p<0.05).Compared with group P,the level of D-Lac and I-FABP at T1~T3 was significantly increased in group M(p<0.05).4.The expression changes of ZO-1 and occludin by western-blot detectionCompared with group S,the expressions of ZO-1 and occludin at T1~T3 were all significantly decreased in group C,P and M(p<0.05).Compared with group C,the expressions of ZO-1 and occludin at T1~T3 were both significantly increased in group P and M(p<0.05).Compared with group P,the expression of ZO-1 and occludin at T1~T3were significantly decreased in group M(p<0.05).5.The expression changes of GFAP by western-blot detectionCompared with group S,the expression of GFAP at T1~T3 was all significantly increased in group C,P and M(p<0.05).Compared with group C,the expression of GFAP at T1~T3 was both significantly increased in group P and M(p<0.05).Compared with group P,the expression of GFAP at T1~T3 was significantly decreased in group M(p<0.05).6.The expression changes of S-100 by western-blot detectionCompared with group S,the expression of S-100 at T1~T3 was all significantly increased in group C,P and M(p<0.05).Compared with group C,the expression of S-100 at T1~T3 was both significantly decreased in group P and M(p<0.05).Compared with group P,the expression of S-100 at T1~T3 was significantly increased in group M(p<0.05).7.The positive expression changes of GFAP by IHCThe immunohistochemical results revealed that GFAP existed in the enteric glial cells of the ileum in rats.The granules dyed brown were the cells of GFAP positive expression.Compared with group S,the positive expression of GFAP in group C,P and M was all increased.Compared with group C,the positive expression of GFAP in group P and M was both increased.Compared with group P,the positive expression of GFAP in group M was decreased.Conclusion:1.The activity of enteric glia cells can be affected by cardiopulmonary bypass,which may reduce the expression of tight junction between intestinal cells to damage the intestinal barrier.2.?7nAchR agonist plays an important protective role in intestinal barrier injury induced by cardiopulmonary bypass through activating enteric glia cells and inhibiting inflammatory response.