The effect of chronic restraint stress on esophageal visceral sensitivity and significance of stress-induced esophageal inflammation and oxidative stress in a murine model

Abstract

Objective: To discuss the effect of chronic restraint stress (CRS) on esophageal hypersensitivity and inflammation, as well as the impact of oxidative stress in a murine model. Methods: 20 male SPF mice were randomly divided into two groups, CRS and normal control(NC) group. Then the mice in CRS group were submitted to 2 h per day of restraint stress for a period of 14 days. For the rest of the time, mice in both groups were given free drinking and eating in the same environment. Histopathological changes of esophageal tissue were observed using HE staining and analyzed by histological damaging score. The esophageal expression levels of transient receptor potential vanilloid (TRPV-1) and nicotinamide adenine dinucleotide phosphate oxidase 4(NOX-4) were detected by immunohistochemical staining. Further more, protein expression level of TRPV-1 was also observed by Western blotting. In addition, mRNA expression levels of TRPV-1, oxidant/antioxidant enzymes and related cytokines in esophageal tissues were detected by real time PCR. Results: HE staining for esophageal tissues revealed that histopathological changes including basal cell hyperplasia, infiltration of neutrophils and plasmocytes were observed in stressed mice, while there was no distinct changes observed in non-stressed mice. Histological damaging scores for epithelial damage and submucosal inflammatory cells were higher in CRS group, and the differences were statistically significant(all P<0.05). Immunohistochemical staining revealed that positive cells for 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 TRPV-1 protein in CRS mice was higher than that of NC mice (1.0±0.0 vs 1.6±0.2; t=-7.06, P<0.001). RT-PCR showed that mRNA expression levels of TRPV1 and NOX-4 in CRS group were higher than those in NC group (1.0±0.0 vs 2.0±0.2, 1.0±0.0 vs 2.0±0.2; t=-13.44, -14.32, all P<0.001). Moreover, mRNA expression levels of antioxidant enzymes, such as Mn-SOD and Cu Zn-SOD, were higher in stressed mice (1.0±0.0 vs 0.7±0.1, 1.0±0.0 vs 0.7±0.1), and the differences were statistically significant (t=8.39,7.36, all P<0.05). The mRNA expression levels of important cytokines, such as MCP-1, IL-8, TNF-α, in stressed mice were overexpressed comparing to non-stressed mice (1.0±0.0 vs 2.4±0.4, 1.0±0.0 vs 1.8±0.2, 1.0±0.0 vs 2.5±0.4), and the differences were statistically significant (t=-10.06, -13.24, -12.40, all P<0.001). Conclusions: Chronic stress may result in esophageal hypersensitivity through activation of TRPV1, and may also cause esophageal inflammation. Stress-induced oxidative stress may contribute to the formation of esophageal hypersensitivity.