The role of Nox-4 in gastric mucosal inflammation induced by chronic restraint stress in mice

Objective To investigate the expression of NADPH oxidase Nox-4 induced by stress in gastric mucosa and its role in inflammation. Methods Twenty male SPF Kunming mice were randomly divided into chronic restraint stress group(stress group) and control group.Stress mice were restrained in self-made restraint device for 2 hours each day.The rest of the time, the mice in the two groups had free access to food and water normally, experiment lasted 14 days.The histopathological changes of gastric mucosa were assessed by HE staining under light microscope.The expression of Nox-4 in gastric mucosa of mice was carried out by immunohistochemical method.The relative expression levels of Nox-4, antioxidant protein(Mn-SOD, GSH, Catalase) and inflammatory factors(IL-8, IL-1β, TNF-α) in gastric mucosa were detected by real-time quantitative RT-PCR and ELISA. Results Basal cell proliferation, neutrophil, eosinophil and plasma cell infiltration and inflammatory changes were observed in the lamina propria and glandular epithelium of stress mice, while no obvious abnormalities were found in control mice.The expression of Nox-4 in stress group was deeper and more abundant than that in control group, mainly expressed in lamina propria and glandular epithelium.The mRNA expression levels of Nox-4 in gastric mucosa of stress group was(2.42±0.51) times higher than that of control group, and blood concentration of stress group was(2.23±0.67) times higher than that of control group(t=-46.32, P<0.001). The RT-PCR of antioxidant proteins in gastric mucosa showed that the transcription levels of Mn SOD, GSH and Catalase in stress group were significantly lower than that of control group(Mn-SOD: 0.59±0.10, GSH: 0.58±0.11, Catalase: 0.57±0.09), and there were significant differences between the two groups(t=13.57, 11.67, 15.01, P<0.01). RT-PCR results showed that the transcription levels of IL-8, IL-1β, TNF-α in stress group were significantly higher than those in control group(IL-8: 1.47±0.34, IL-1β: 1.48±0.42, TNF-α: 1.51±0.37), and there were significant differences in two groups(t=-18.45, -19.14, -20.85, P<0.01). ELISA results showed that the serum levels of inflammatory factors in stress group were significantly higher than those in control group(2.25±0.37, 3.59±0.45, 3.41±0.34), and the differences were statistically significant(t=-47.11, -79.36, -96.32, P<0.01). Pearson correlation analysis showed that there was a positive correlation between serum concentration of Nox-4 and inflammatory factors(IL-8, IL-1β, TNF-α) in stress group(r=0.97, 0.99, 0.98, P<0.01). Spearman rank correlation analysis showed that the grade of gastric mucosal inflammation was positively correted with serum levels of Nox-4 and inflammatory factors(IL-8, IL-1β, TNF-α)(r=0.96, 0.92, 0.91, 0.94, all P<0.01) Conclusion Stress may lead to gastric mucosal lesion by overexpression of proinflammatory factors through destroying the balance of oxidation/antioxidant system in gastric mucosa. Key words: Chronic restraint stress; Gastric mucosal lesion; Nox-4; Anti-oxidant proteins; Inflammatory cytokines; Mice