| After Helicobacter pylori (H. pylori) was identified in human gastric mucosa by Warren and Marshall, this bacterium was found to be associated with gastritis, peptic ulcers, gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma. In particular, H. pylori infection is a major causal factor in peptic ulcer, and recurrence is virtually prevented by eradicating this bacterium. Although there is a high probability of H. pylori infection in peptic ulcer cases, such infection is not sufficient on its own for peptic ulcer to develop: many infected people do not develop ulcers, and some gastric ulcer patients are H. pylori-negative.These findings suggest that other factors such as use of NSAIDs, stress, diet or smoking may contribute to the pathogenesis of peptic ulcer. Among these factors, psychological stress has been shown to trigger many ulcers and impair the response to treatment; it has been estimated that psychological factors contribute to 30-65% of ulcers. A dramatic increase in the incidence of gastric ulcers, especially bleeding ulcers, after the Hanshin-Awaji earthquake in Japan was a strong indicator of the role of psychological stress in these conditions.Some studies have shown a synergistic relationship between H. pylori infection and psychological stress in the formation of ulcers. Animal studies and clinical data have shown that H. pylori infection affects the development of stress-induced gastric mucosal injury. However, can psychological stress influence H. pylori infection of the gastric mucosa? What mechanism underlies the synergistic relationship between H. pylori infection and psychological stress? These questions have not been investigated to date.It is well known that H. pylori infection of the gastric mucosa follows a complicated course that depends on various factors such as the microenvironment of the stomach, the host immune system and the condition of the microorganism. Many experimental studies have indicated that psychological stress alters the inflammatory and immune response in the gastric mucosa. The mucosal immune system is regulated by the central nervous system through the hypothalamic-pituitary-adrenal (HPA) axis and glucocorticoid (GC) production, and psychological stress can affect the HPA axis, increasing GC production. We therefore hypothesized that psychological stress may affect the mucosal immune system via the neuroendocrine system, and then influence H. pylori infection of the gastric mucosa.RU486 is a commonly used, nonspecific antagonist of both the type II GC receptor and the progesterone receptor which can block the effect of GC. So, we used RU486 to test whether psychological stress affect the H. pylori colonization by changing the GC production. We therefore examined colonization of the stomachs of BALB/c mice by H. pylori to evaluate the level of infection. Using realtime PCR, we found that psychologically stressed mice were more heavily colonized by H. pylori than control mice after inoculation with the same amounts of the bacterium. Furthermore, we administered the GC receptor antagonist RU486 to mice before exposure to psychological stress to block the neuroendocrine pathway. This treatment decreased the colonization by H. pylori significantly in comparison to the psychological stress group mice.Objectives1. Using methods of electric shocks to establish psychological stress model in BALB/c mice, observing the behavior of mice, endocrine changes to evaluate the validity of psychological stress model.2. Infect mice with H. pylori after establish psychological stress model in BALB/c mice, detect the colonization of Helicobacter pylori in stomach, changes in gastric mucosal lesions, as well as the psychological stress on immune funtion in mice.Methods1. Establishment of psychological stress model in mice:The communication box paradigm consists of two types of compartments, A and B. A total of nine compartments are arranged like a checkerboard and are separated by transparent plastic walls. In the present study, parallel stainless steel electric grids were set into the floors of all compartments. However, the floors of the B compartments were covered with electrically insulating plastic panels. Mice were individually placed in each compartment and intermittent electric shocks were delivered through the grid floor by a shock generator. The animals in the A compartments (senders) were subjected to electric shocks to their feet through the electric grids on the floor, while the animals in the B compartments (responders) were exposed to emotional stress by seeing and hearing the struggle, jumping, and/or vocalizations of the sender mice in the adjacent compartments. The responder mice were exposed to emotional stress once a day for half an hour between 9:00 and 12:00; this treatment was repeated for 14 days. The unstressed control mice were placed individually in the compartments of the control isolation box, with no electric grid in the floor and without exposure to the senders, for the same period as the stressed mice. In 1 week and 2 weeks after the end of psychological stress, respectively, through the open-field tests, observing the behavior changes of normal group and psychological stress group mice, and detect the level of serum corticosterone changes. Behavior and endocrine evaluation in mice was as indicators of psychological stress model we established in this study.2. The effects of psychological stress on the immune function of BALB/c mice:Collect peripheral blood of psychological stress and control groups BALB / c mice respectively, detect serum IL-2, IL-4 level using ELISA method.3. The effects of psychological stress on infection of Hp in stomach of BALB/c mice:Seventy-two mice were divided into control group (C), the psychological stress group (PS), Hp infection (HI), psychological stress + drug group (RP), psychological stress + Hp infection (PH) and psychological stress + Hp infection + drug group (RPH). Each group of mice were subjected to psychological stress and (or) Hp infection and oral drug to deal with. Detect the amount of colonization of Hp of gastric tissue in mice by realtime PCR and analysis the degree of gastric mucosal damage in pathological examination.Results(一) The establishment of psychological stress model1. Animal behavior: When the psychological stress group mice were placed into the communication box, mice had a strong desire to explore, forepaw upright to nose sniffing the walls. The mice were jumping, screaming and closed eyes being electrical stimulation. All the mice were carried out to open field test after 1 week and 2 weeks stimulation. Both horizontal activity (P<0.05) and rearing score (P<0.05) of the mice were significantly decreased on weeks 1 and 2 after psychological stress.2. Determination of serum corticosterone: Before stress, the serum corticosterone levels of the three groups showed no significant difference (P>0.05). After 1 week, the level of serum corticosterone of PS and RP was increased significantly compared to control group (P<0.05), and reached a more higher level after 2 weeks (P<0.05). Whereas there was no significant differenc between the PS and RP group (P>0.05).3. Determination of serum IL-2,IL-4: After the psychological stress procedure, we killed all the mice and collect the peripheral blood, detect the serum level of IL-2,IL-4. The level of IL-2 of mice in PS group was decreased significently compared to control group (P<0.05). The level of IL-4 of mice in PS group was increased significently compared to control group (P<0.05).(二) Effects of Psychological Stress on H. pylori infection inBALB/c mice1. Colonization of stomach tissue by H. pylori: When the psychological stress model was established successfully, we infected the psychological stressed mice with H. pylori. Five weeks after H. pylori infection, we collected the gastric tissue. The H. pylori infection rate was 100% in HI, PH and RPH groups confirmed by bacterial culture,Urea test and H. pylori staining in stomach tissue sections. Realtime PCR was performed to quantify 16s rDNA in the H. pylori genome, which is specific to the bacterium; one copy of 16s rDNA represents one H. pylori. The H. pylori copy in the stomach tissue of the PH group was greater than that of the HI group (P<0.05), and the H. pylori copy in the RPH group, which were pretreated with the RU486 before psychological stress, was significantly lower than that of the PH group (P<0.05).2. Gastric mucosal injury caused by H. pylori and psychological stress: The stomach tissue was sent for pathological examination at 5 weeks after inoculation with H. pylori. Each sections with HE staining of the groups was carried out for gastric Microscopy, compared with representative section of each group. There were no significant changes in the stomach tissue in the Control and PS groups, and there were slight inflammation in the RPH group. And gastric mucosal defects appeared in the HI group, When the process continued, ulcers appeared in the stomach tissue, as in the PH group. We also found atrophy of gastric mucosal glands, bleeding in gastric mucosa and formation of folliculus lymphaticus in the PH group.The inflammation scores represent the severity of inflammation. Data were analyzed by Kruskal-Wallis tests, showing that there were statistical significance in the five groups (P<0.05). Comparing between every two groups by Mann-Whitney U test, the inflammation scores of the PH group were significantly higher than the other groups (all P<0.05). The scores of PS had no significant difference with the C group (P>0.05), and the scores of HI and RPH were significantly higher than the C group (P<0.05).These data showed that psychological stress alone did not significantly affect the histological examination, but it aggravated the adverse effect of H. pylori infection.Conclusion1. Establish pure psychological stress model of BALB/c mouse successfully, and evaluate the model through behavioral and endocrine indicators agree with psychology standards. Since this animal model of physiological factors or to the body is minimal, Therefore, the model could imitate human emotion betterly.2. The serum level of IL-2 in mice was decreased and the serum level of IL-4 in mice was increasing. We can draw the conclusion that pure psychological stress could inhibit the cellular immune function in mouse.3. The data of study showed that psychological stress alters the colonization of H. pylori in the mouse gastric mucosa and aggravated the adverse effect of Gastric mucosal injury caused by H. pylori infection. The mechanism of this phenomenon may be due to psychological stress by activating the HPA axis, the release of a large number of GC, lead to immune system suppression.significanceThe present study is the frist to demonstrste that repeated exposure to psychological stress increases the colonization of the gastric mucosa of BALB/c mice by H. pylori and aggravates the mucosa injury caused by this infection, and that this effect may be mediated by excess GC. Study of psychological stress will provide new ideas for clinical anti-Hp infection in the treatment of peptic ulcer disease. In addition, the stress - infection model will break through the traditional"single pathogen"theory.
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