Chronic Restraint Stress Induces Gastric Mucosal Inflammation with Enhanced Oxidative Stress in a Murine Model.

BACKGROUND: Although the underlying mechanisms of chronic stress are still unknown, this condition has been related to the pathophysiology of gastric mucosal inflammation, whose development is accelerated by oxidative stress. The present study investigates how chronic stress influences gastric mucosal oxidative stress and inflammation. METHODS: Eight-week-old C57BL/6J male mice were subjected to two-week intermittent restraint stress. The expressions of CD11b (a specific for monocyte/macrophage), monocyte/macrophage cell surface markers (CD68 and F4/80), NADPH oxidase-4 (Nox-4) and 8-hydroxy-2'-deoxyguanosine (8-OHdG, a sensitive biomarker of oxidative stress) were determined using immunohistochemistry, RT-PCR, and enzyme-linked immunosorbent assay, respectively. The expressions of antioxidant enzymes, such as superoxide dismutase, catalase, and glutathione peroxidase, were examined by RT-PCR and Western blotting. The expressions of proinflammatory cytokines, including monocyte chemoattractant protein-1 (MCP-1), interleukin-1ß (IL-1ß), and tumor necrosis factor-α (TNF-α), were determined using immunohistochemistry and RT-PCR, respectively. RESULTS: Chronic stress increased the lymphocytic infiltration and inflammation within the gastric mucosa of mice. Stress remarkably increased the expression levels of CD11b and mRNA expression levels of CD68 and F4/80 in the mucosa of the stomach of stressed mice. Stress remarkably increased both mRNA and plasma concentrations of Nox-4 and 8-OHdG; and markedly reduced gastric mRNA and protein expression levels of antioxidant enzymes such as superoxide dismutase, catalase, and glutathione peroxidase. The expressions of proinflammatory cytokines (MCP-1, IL-1ß, and TNF-α) were predominantly observed in the gastric mucosal layers of the stressed mice. Furthermore, stress remarkably elevated the gastric mucosal mRNA expression levels of MCP-1, IL-1ß, and TNF-α. CONCLUSION: Two weeks of restraint stress induced gastric inflammation in the murine model with enhanced oxidative stress and reduced anti-oxidative system.

The expression of NADPH oxidase-4 and acid sensitive receptors in the esophagus of chronic restraint mice / 中国医师杂志

Objective:To investigate the chronic restraint stress induced expressions of acid sensitive receptors and its role in the esophageal inflammation and oxidative stress.Methods:Twenty male specific pathogen free (SPF) Kunming mice were randomly divided into two groups: stress group and control group (each group, n=10). Stress mice were restrained in self-made restraint device for 2 hours per day and lasted for total 14 days. The histopathological changes of esophageal mucosa were observed by hematoxylin eosin (HE) staining under light microscope. The expression of nicotinamide adenine dinucleotide phosphate (Nox-4) was detected by immunohistochemistry, real time fluorescent quantitative polymerase chain reaction (qRT-PCR) and enzyme linked immunosorbent assay (ELISA). The mRNA expressions of acid sensitive receptors were detected by qRT-PCR. Results:HE staining showed that stress mice had obvious infiltrations of neutrophils and eosinophils, and also showed inflammatory change in esophgus, while no significant abnormality was found in the esophagus of control mice. The inflammotory scores in stress group were significantly higher than that in control group ( P<0.001). Immunohistochemistry showed that Nox-4 was mainly expressed in the lamina propria, mucosa and submucosa of esophagus. The mRNA expression levels of Nox-4 in stress group was (2.67±0.62) times higher than control group, with statistically significant difference ( P<0.001). In addition, the plasma concentration of Nox-4 in stress group was significantly higher than that of control group [(0.42±0.01)ng/ml vs (2.13±0.35)ng/ml, P<0.001]. The transcription levels of acid sensitive receptors in stressed mice, such as transient receptor potential vanilloid-1 (TRPV-1), TRPV-4, acid-sensing ion channel-1 (ASIC-1), ASIC-2 and ASIC-3 were significantly higher than those in the control group, with statistically significant difference ( P<0.001). Pearson correlation analysis showed that there was a positive correlation between Nox-4 mRNA expression and TRPV-1, TRPV-4, ASIC-1, ASIC-2, ASIC-3 mRNA expression in stress group ( r=0.97, 0.94, 0.98, 0.95 and 0.99, P<0.01). Conclusions:Stress may increases the expression of acid sensitive receptors and result in an esophageal inflammation and oxidative stress, which may contribute to the formation of esophageal hypersensitivity.

Chronic restraint stress induces esophageal fibrosis with enhanced oxidative stress in a murine model.

Although the underlying mechanism of stress remains unknown, it has been associated with the pathophysiology of gastroesophageal reflux diseases, the development of which appear to be accelerated by oxidative stress and fibrosis. The aim of the current study was to investigate the effect of chronic restraint stress on esophageal oxidative stress and fibrosis, as well as the impact of oxidative stress in a murine model whereby 8-week old C57BL/6J male mice were subjected to intermittent chronic restraint stress for a two-week period. The current study demonstrated that chronic restraint stress significantly reduced the body weight of mice compared with the control group. Although chronic restraint stress did not significantly alter the levels of triglycerides or cholesterol, free fatty acid concentration was significantly increased compared with the control group. Furthermore, chronic restraint stress significantly upregulated the expression levels of several fibrotic biomarkers including collagen type I, transforming growth factor ß-1, α-smooth muscle actin and SMAD-3 compared with the control group. In addition, the expression levels of the reactive oxygen species (ROS) NADPH oxidase-4 and malondialdehyde were significantly increased, while the expression levels of nuclear factor erythroid 2-related factor 2 and heme oxygenase-1 were significantly decreased in esophageal tissue from mice in the chronic restraint stress group compared with the control group. In conclusion, chronic restraint stress may induce esophageal fibrosis by accumulating ROS and increasing fibrotic gene expression in a murine model.

Chronic stress augments esophageal inflammation, and alters the expression of transient receptor potential vanilloid 1 and protease­activated receptor 2 in a murine model.

Stress is a pivotal factor for inflammation, reactive oxygen species (ROS) production and formation of visceral hypersensitivity (VH) in the process of gastroesophageal reflux disease (GERD). In the present study, the effects of stress on esophageal inflammation, oxidative stress and VH were investigated in a chronic restraint stress mouse model. C57BL/6J male mice were subjected to 2 weeks of intermittent restraint stress, and histopathological analysis revealed that stress induced esophageal inflammation and fibrosis, while no distinct changes were detected in non­stressed control mice. In addition, increased NADPH oxidase 4 expression was observed in the plasma and esophagus of stressed mice, indicating accumulation of ROS. The expression levels of antioxidants, including Mn­superoxide dismutase (MnSOD), Cu/Zn­SOD, catalase and glutathione peroxidase, were also analyzed using reverse transcription­quantitative polymerase chain reaction (RT­qPCR). In addition, transient receptor potential vanilloid 1 (TRPV­1) and protease­activated receptor 2 (PAR­2), which are crucial receptors for VH, were measured by immunohistochemistry and RT­qPCR. The results demonstrated that stress markedly reduced antioxidant expression, while it significantly upregulated TRPV­1 and PAR­2 expression levels in the mouse esophagus. Finally, 2 weeks of restraint stress significantly increased the esophageal and plasma levels of inflammatory cytokines, including interleukin (IL)­6, IL­8, interferon­Î³ and tumor necrosis factor­α. Taken together, the present study results indicated that stress­induced esophageal inflammation and ROS generation involves VH.

The role of chronic restrain stress induced Nox-4 expression and its significances in adipose inflammation / 中华内分泌代谢杂志

Objective To investigate stress induced Nox-4 expression and to explore its role in adipose inflammation. Methods Twenty male Kunming mice were randomly divided into two groups ( n=10 each) , chronic restraint stress group and control group. Stress mice were restrained in self-made restraint device for 2 hours per day for 14 days. HE staining, immunohistochemistry, RT-PCR, and ELISA were used to analyze the expression of Nox-4, CD11b, antioxidant protein ( Mn SOD, GSH-Px, Catalase), adipocytokines ( adiponectin, MCP-1, IL-6, TNF-a). Results White adipose tissue (WAT) of stress mice inguinal fat pad significantly shrank compared to control group. HE staining showed that there were a large number of mononuclear cells, neutrophils, eosinophils, and cell infiltration reactions and inflammatory changes in WAT of stress mice. The stress significantly increased CD11b-positive cells and the expression of mF4/80, CD68. The concentration of serum FFA in stress group increased significantly, nearly twice of the control group ( P<0.01) . Nox-4 positive staining cells in stress WAT were deeper and more abundant. The level of Nox-4 in stress WAT was significantly higher than that of control group(P<0.01). The levels of antioxidant proteins such as Mn-SOD, GSH-Px, and catalase in stress WAT were significantly lower than those of control group (P<0.01). The expression levels of adiponectin in stress WAT were significantly reduced as compared to control group ( P<0.01) . The levels of MCP-1, IL-6 and TNF-α in stress WAT were significantly higher than those in control group (P<0.01). Conclusion Stress may lead to imbalance of adipose oxidation/antioxidant system and abnormal expression of adipocytokines, which may result in adipose inflammation.

Role of MDA in the pathogenesis of GERD esophagus mucosal inflammatory injury / 中国医师杂志

Objective To investigate the expression of malondialdehyde ( MDA) in esophageal mu-cosa of different types of gastroesophageal reflux disease ( GERD) patients and its role in the esophageal in-flammation. Methods According to the inclusion and exclusion criteria, 42 patients hospitalized in the the Xinjiang Uygur Autonomous Region People's Hospital from December 2017 to October 2018 were selected as the research group. 8 healthy subjects completed physical examination were set up as healthy control group. GERD completed GERDQ score, 24 h pH monitoring, and taken 3 cm on the dentate line of the esophagus as a specimen. The study group was divided into non-erosive reflux disease (NERD) group (17 cases) and Ero-sive reflux disease [erosive esophagitis (RE)] group (25 cases). Then hematoxylin-eosin (HE) staining, immunohistochemistry, real-time polymerase chain reaction ( qPCR ) , enzyme-linked immunosorbent assay (ELISA) methods were used to detect inflammation, oxidative stress (MDA), antioxidant enzyme [manga-nese superoxide dismutase (Mn SOD), glutathione (GSH), catalase (CAT)], and proinflammatory cyto-kines [monocyte chemotactic protein-1 (MCP-1), interlukin-8 (IL-8), tumor necrosis factorα(TNF-α)]. Results There was no significant difference in body mass index ( BMI ) between the three groups ( P >0. 05). 24 h pH monitoring of esophagus showed that the indexes of weak acid reflux (4<pH<7), acid re-flux ( pH<4 ) , esophageal near end acid reflux (％) and DeMeester score in RE group were significantly higher than those in NERD group, with statistical significance between the groups (P<0. 05). There was no significant difference in esophageal pressure between high resolution groups (P>0. 05). In RE group , the infiltration of immune cells (neutrophils, eosinophils), nipple lengthening, edema and other inflammatory changes were found in the esophageal mucosa, and the inflammation score reached the peak, which was signif-icantly higher than that in NERD group, with statistical significant difference (P<0. 001). The positive ex-pression of MDA in the two groups ( NERD, RE) was higher than that in the control group, and the MDA ex-pression in the RE group was almost covered with the full layer esophagus. The serum MDA concentration in the NERD and RE groups was significantly higher than that in the control group (P<0. 001). Compared with the NERD group, the serum MDA in the RE group reached the peak (P<0. 01). The relative expression of mRNA ( Mn SOD, GSH and CAT) in NERD group and RE group was significantly decreased, and there was a significant difference between the three groups (P<0. 001). Compared with the NERD group, the mRNA expression level of Mn SOD and CAT in RE group was significantly decreased (P<0. 01). The relative ex-pression of mRNA (MCP-1, IL-8, TNF- α) increased significantly in the two groups (NERD, RE), and there was a statistically significant difference between the three groups ( P <0. 001 ) . Compared with the NERD group, the expression of its inflammatory factors in the RE group significantly increased (P<0. 01). Conclusions The expression level of MDA in different types of GERD is significantly higher, which may be closely related to esophageal inflammation induced by acid reflux.

The role of Nox-4 in gastric mucosal inflammation induced by chronic restraint stress in mice / 中华行为医学与脑科学杂志

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 selfmade 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-αt) 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.