The Significance Of Aberrant Expression Of PAR-2 And TRPV-1 Induced By Chronic Restraint Stress On Esophageal Oxidative Stress And Inflammation

Objective:To investigate the expression level of proteinase-activated receptor-2(PAR-2),transient receptor potential vanilloid-1(TRPV-1)and reactive oxygen species(ROS)in mouse esophagus by establishing chronic restraint stress(CRS)model,and the role they play in esophageal hypersensitivity and inflammation,as well as oxidative stress in gastroesophageal reflux disease(GERD).Methods:20male Kunming mice were randomly divided into two groups,CRS and normal control(NC),with 10 mice in each group,Mice in CRS group were submitted to 2h per day of restraint stress using home-made device for a period of 14days.The esophageal expression levels of PAR-2.TRPV-land nicotinamide adenine dinucleotide phosphate oxidase 4(NOX-4)were detected by using immunohistochemical staining.Furthermore,protein expression level of PAR-2 and TRPV-1were also observed by western blotting.In addition.mRNA expression levels of PAR-2,TRPV-1,oxidant/antioxidant enzymes and related cytokines in esophageal tissues were detected by real time polymerase chain reaction(RT-PCR).Results:Compared to NC mice,body weight increment of mice in CRS group is significantly decreased(P<0.001);HE staining for esophageal tissues revealed that histopathological changes which include basal cell hyperplasia,infiltration of neutrophils and plasmocytes were observed in stressed mice,while there was no distinct changes observed in non-stressed mice;In addition,histological damaging scores for epithelial damage and submucosal inflammatory cells were statistically higher in CRS group(all P<0.05),while the figures for submucosal edema in both groups were not distinctive(P>0.05).Immunohistochemical staining revealed that positive cellsfor PAR-2,TRPV-1 and NOX-4 were observed in majority of samples,but the number of positive cells and density were higher in CRS mice.Western blotting showed that the expression levels of PAR-2 and TRPV-1 protein in CRS mice were higher than that of NC mice(all P<0.001).RT-PCR resulted in mRNA expression levels of PAR-2,TRPV1 and NOX-4 in experimental group were markedly higher in CRS group,comparing to NC group(all P<0.001);Moreover,mRNA expression levels of antioxidant enzymes,such as Mn-SOD and Cu Zn-SOD,were distinctively higher in stressed mice(all P<0.05).In addition,the mRNA expression levels of important cytokines,such as IL-1?,IL-8,TNF-?,in stressed mice were over-expressed comparing to non-stressed mice(all P<0.001).Conclusions:(1)The level of PAR-2 and TRPV-1 in the esophagus of mice under chronic restraint stress were over expressed,this may indicate the formation of esophageal hypersensitivity;(2)The over expression level of ROS(Nox-4)and reduction of anti-oxidants(Mn-SOD,Cu Zn-SOD)in the esophagus of mice under chronic restraint stress may indicate that the accumulation of ROS and imbalance of oxidant/anti-oxidant system,which may result in oxidative stress;(3)Stress induced oxidative stress may take part in the formation of esophageal hypersensitivity.