The paraventricular nucleus of the hypothalamus contributes to stress responses induced by foot shocks(2). Foot shocks promotes escape behavior in mice, resulting in compelled acute exercise. Compelled acute exercise may induce non-specific immune responses in the body, including diarrhea, abdominal pain, even GI mucosal injury and bleeding. It is estimated that 30–65% of long-distance runners experience exercise-related GI mucosal injury(28). Compelled acute exercise causes oxidative stress and sports anemia(29), leading to erythrocyte damage(30), decreased SOD level and increased MPO activity in the serum. Moreover, pro- and anti-inflammatory cytokines (IL-1β, IL-6, IL-10, TNF-α, and iNOS) may play a role in the initiation of GI mucosal injury/bleeding induced by AUE caused by foot shocks.
To investigate whether the model was related to GI mucosal injury and bleeding, we observed the morphological changes of gastric and duodenal mucosa after stress. Acute GI mucosal injury and bleeding following stress indicated that AUE was involved in this process. Previous studies confirmed the correlation between exercise-induced visceral hypoperfusion and GI diseases(31). It was consistent with the data obtained from animal models and patients, showing that GI cell damage was caused by AUE and ischemia(32). The decreased RBC count, hemoglobin concentration and hematocrit is a marker of anemia(33). Our results suggested that anemia was induced by exercise. The exercise-induced oxidative stress was determined by the activities of SOD and MPO in the serum. Oxygenated free-radicals play a fundamental role in regulating the damage associated with GI mucosal injury and bleeding (34). The decreased serum level of SOD indicates the occurrence of GI mucosal injury and bleeding. MPO is a key player in oxidative process and the regulation of pro-inflammatory cytokines(31, 35). In our study, the increase of MPO matched with the decrease of SOD. The activity levels of SOD and MPO in the serum indicated that this model was involved in oxidative stress.
Physiological systems of the body respond to stress stimuli to promote defense and survival. In the present study, the animal model was considered as a special stressor that might cause GI mucosal injury/bleeding and oxidative stress. The release of inflammatory cytokines during stress induces the alterations in brain neuroendocrine functions. We explored the effects of pro- and anti-inflammatory cytokines (IL-1β, IL-6, TNF-α, iNOS, and IL-10) on the hypothalamus in male mice. Results showed that the expressions of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α, and iNOS) after stress were significantly increased compared to the control group. After the activation of IL-1β, the elevated HPA activity is related to the increased release of norepinephrine (NA) and 5-hydroxytryptamine (5-HT) in the brain(36). It has been found that the level of IL-1β is significantly increased in the hypothalamus of rat with restraint stress(37). In addition, IL-6 and TNF-α produce corticotropin-releasing GABA by stimulating HPA axis(38). Hypothalamic paraventricular nucleus repairs colon injury by regulating the expressions of TNF-α and IL-1β(39). Clinical studies found that the activity of NOS in patients with celiac disease was higher than that in other patients(40). Animal studies showed that IL-1β was the key mediator of nitric oxide after endotoxin exposure. It promoted microglia to produce vasodilator and neuromodulator nitric oxide by increasing the biosynthesis of iNOS(41). Our findings were in line with previous studies, which suggested a mutual activation relationship of pro-inflammatory cytokines. Our data on the expression of IL-10 was also consistent with that in the excessive eccentric exercise model, in which the levels of IL-1β and IL-6 were increased, while the expression of IL-10 was decreased(42).
Next, we measured the expressions of well-known inflammatory markers in the duodenum. Pro-inflammatory cytokines (IL-1β, IL-6, TNF-α, and iNOS) were significantly increased as compared to the controls after stress. Recent evidence has shown that IL-1β increases the intestinal permeability(43). In intestinal cells, IL-1β partially increased the intestinal permeability by reducing the expression and redistribution of occludin(23). IL-6 is mainly distributed in intestinal monocytes and the activation of IL-6 promotes the proliferation and repair of intestinal epithelium after injury(44). It has been reported that TNF-α induces the apoptosis and inflammation of intestinal epithelial cells, and damages the intestinal mucosal barrier. TNF-α plays an essential role in regulating GI diseases and its expression in intestinal cells is closely correlated with intestinal barrier defect(45, 46). NOS produces excessive nitric oxide (NO) in the progression of various intestinal inflammatory diseases. The synthesis of NO by iNOS is associated with a variety of pathophysiological processes, including inflammation. Clinical data showed that the activity of iNOS in duodenal epithelial cells was increased in patients with celiac disease(47, 48). Consistently, we found that the pro-inflammatory cytokines (IL-1β, IL-6, TNF-α, and iNOS) in the duodenum were activated, resulting in GI mucosal injury/bleeding and the activation of immune responses in mice. IL-10 is a key anti-inflammatory cytokine produced by intestinal macrophages(49). As shown in an in vitro study, IL-10 exhibited opposite effects on cellular functions when compared to TNF-α, IL-1β, and IL-6(45). Here, we found that IL-10 was not activated in the animal model, which was consistent with previous findings.
As shown in Fig. 7, IRS activated the hypothalamic CRH system, resulting in the secretion of ACTH into the peripheral circulation and the induction of glucocorticoids. Then the brain-gut axis activates mucosal mast cells, increases the expressions of pro-inflammatory cytokines, and promotes the activity of endocrine gland(9, 50). The intestinal homeostasis is maintained by the neural connections through the brain-gut axis, together with the regulation of reactive oxygen metabolites and pro-inflammatory cytokines, such as IL-1 β, TNF-α, and IL-6. The increase of MPO activity and the decrease of SOD activity in inflamed mucosa is associated with gastric ulcers(34). A study of intestinal cells found that the overexpression of pro-inflammatory cytokines increased GI permeability and induced GI bleeding(35). In this study, the expressions of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α, and iNOS) in hypothalamus and duodenum were significantly increased following AUE. Our model of AUE induced-GI mucosal injury/bleeding was consistent with the results of previous studies.