| Objective:Helicobacter pylori is one of the most common chronic bacterial infections affecting at least half of the world’s population. Of the infected individuals,approximately 15-20% and 1% will develop peptic ulcer and gastric cancer. Studies have found that Hp infection can increase the content of reactive oxygen species(ROS) in tissues or cells.ROS is a series of active oxygen molecules produced in the process of metabolism.High doses of ROS Play the role of oxidative stress damage,it can damage cellular lipids, roteins, DNA, RNA,etc.And it can cause a range of diseases and even tumors.N- acetyl cysteine(NAC) is an antioxidant, which can protect the cells by scavenging oxygen free radicals when the tissue is injured by oxidative stress. PI3K/Akt signaling pathway has anti apoptosis and promoting proliferation.Previous studies of this subject by in vitro experiments confirmed that Hp infected GES-1 could increase intracellular ROS level, result in DNA damage,and activate Akt signaling pathway. NAC could reduce the DNA damage by removing ROS, also could inhibit the activation of Akt signal pathway.Because there are differences between the vitro experiment and the whole system.In vitro experiment lack the repair process of cell and tissue and the influence of the host immune system.So we focused on the results of preliminary research,further confirmed by animal experiments.With Hp infection Balb/c mice to establish animal model of chronic gastritis,and using N- acetylcysteine(NAC) inhibit the generation of ROS.By detecting the change of ROS,MDA、GSH content,DNA damage, and the expression of Akt signaling pathway proteins in the gastric tissues of mice in each group, then try to explore the protective effect of NAC on the chronic gastritis induced by Hp infection and its effect on the Akt signaling pathway.Methods:一、Establishment of chronic gastritis and NAC intervention model in Balb/c mice infected with H.pylori1.A total of 60 conventional Balb/c mice were randomly divided into 5groups:group A as normal control group, giving Brucella broth and distilled water;group B as Hp model group, giving Hp suspension + distilled water; group C as NAC drug control group, giving Brucella broth and NAC;group D as Hp+NAC intervention group,given Hp suspension, while the model of the first 12 weeks to give the NAC; group E as Hp+NAC treatment group,given Hp suspension, while after 12 weeks of modeling to give the NAC until the end of the experiment.All mice were sacrificed in Week 24 after the Hp final gavage,and gastric tissue samples taken for testing.2.Detection of Hp colonization:Gimesa staining and PCR quantitative detection.3.HE staining for histopathological analysis.4.DCFH-DA probe to detect the ROS content in gastric mucosa of all groups.5.According to Hp positive, Chronic inflammatory response and ROS level decreased after NAC intervention, suggesting that the successful establishment of Hp infection related chronic gastritis and NAC intervention animal model.二、The change of ROS levels in mice gastric tissue and oxidative damage1.TBA method to detect the content of MDA in gastric mucosa tissue;2.Beutler modified method to detect the contents of antioxidant GSH in gastric mucosa tissue;3.Immunohistochemical method to detect the content of 8-OHd G in gastric mucosa tissue.三、The detection of the expression of Akt signaling pathway proteins Immunohistochemistry combined with western-blot method to detect the expression of Akt signaling pathway proteinsResults:一、Chronic gastritis and NAC intervention model in Balb/c mice infected with H.pylori1.The colonization of Hp: normal control group and drug control group were not detected in H pylori.Hp model group, Hp+NAC intervention group, Hp+NAC treatment group of 12 mice were detected by H pylori.2.The results of Histopathology:The gastric mucosal tissues in the normal control group and the NAC drug group without or with a few scattered lymphocytes.The mice in the Hp model group had inflammatory response,HE staining showed that there were many lymphocytes and eosinophils in the gastric mucosal Lamina propria and submucosa.The mice in Hp+NAC intervention group and Hp+NAC treatment group also had inflammatory reaction,and there were several or more lymphocytes in the gastric mucosal Lamina propria and submucosa.The inflammation score of Hp model group was higher than that of normal control group,the difference was statistically significant(p<0.05).Compared with Hp model group, the Hp+NAC intervention group and Hp+NAC treatment group had no significant difference(p>0.05).3.The content of ROS:Compared with the normal control group, the level of ROS in gastric tissue of Hp model group was increased, the difference was statistically significant(p<0.05); there was no significant difference in ROS level between the normal control group and the NAC drug group((p>0.05).Compared with the Hp model group, Hp+NAC intervention group and Hp+NAC treatment group ROS levels were decreased, the difference was statistically significant(p<0.05).4.According to the above results, it was suggested that the model is successful.二、The effects of NAC on Hp gene copy number, MDA, GSH content and DNA damage in mouse gastric mucosa1.The results of PCR quantitative detection:Compared with the normal control group, the ure gene copy of gastric mucosa in Hp model group was significantly higher(P<0.01), and there was no significant difference in the ure gene copy(p>0.05)between the normal control group and the NAC drug group.Compared with the Hp model group, the ure gene copy of gastric mucosa in Hp+NAC intervention group and Hp+NAC treatment group were all decreased, and the difference was significant(p<0.01).2.The content of MDA:Compared with the normal control group,the level of MDA in gastric tissue of Hp model group was significantly higher, and the difference was statistically significant(p<0.01);there was no significant difference in the level ofMDA between the normal control group and the NAC drug group((p>0.05).Compared with the Hp model group, Hp+NAC intervention group and Hp+NAC treatment group MDA levels were decreased, the difference was statistically significant(p<0.01).3.The content of GSH:Compared with the normal control group, the level of GSH in gastric tissue of Hp model group was decreased, the difference was statistically significant(p<0.01); there was no significant difference in GSH level between the normal control group and the NAC drug group((p>0.05).Compared with the Hp model group, Hp+NAC intervention group and Hp+NAC treatment group GSH levels were increased, but the difference was no statistically significant(p>0.05).4.Expression of DNA damage product 8-OHd G:Immunohistochemistry showed that 8-OHd G was mainly localized in cytoplasm, and it was expressed in different degrees in each group.The expression of 8-OHd G in the normal control group and drug control group was mainly weak positive.The expression score of 8-OHd G in Hp model group was higher than that in normal control group, the difference was statistically significant(p<0.05);The expression score of 8-OHDG in Hp+NAC intervention group and Hp+NAC treatment group were lower than that in Hp model group, the difference was statistically significant(p<0.05).三、The effect of NAC on the expression of PI3K/Akt signaling pathway related protein in mouse gastric mucosa1.Western blotting showed:The expression of total protein Akt and GSK3-β did not change with the different factors, and there was no significant difference among groups(p>0.05).The expression of p-Akt and p-GSK3-β in Hp model group was higher than that in normal control group, the difference was statistically significant(p<0.01).There was no statistical difference in p-Akt and p-GSK3-βexpression between the normal control group and the NAC drug group(p>0.05).Compared with Hp model group, the expression of p-Akt and p-GSK3-β in Hp+NAC intervention group and Hp+NAC treatment group were decreased, the difference was statistically significant(p<0.01).2.Immunohistochemistry showed: Akt and GSK3-β were mainly located in the cytoplasm, and they were expressed in different degrees in each group.But the expression was not obvious,there were no significant difference in the expression scores among groups(p>0.05).p-Akt and p-GSK3-β were mainly located in cytoplasm, which were expressed in different degrees in each group.There was no significant difference in the expression of p-Akt and p-GSK3-β between the normal control group and the NAC drug group(p>0.05).The expression of p-Akt and p-GSK3-β in Hp model group was significantly higher than that in normal control group,the difference was statistically significant(p<0.05).Compared with Hp model group, the expression of p-Akt and p-GSK3-β in Hp+NAC intervention group and Hp+NAC treatment group were decreased, the difference was statistically significant(p<0.05).Conclusion:Through animal experiments, we confirmed that Hp infected Balb/c mice could increase the gastric mucosal tissue ROS, causing cell lipid peroxidation damage and DNA damage.Hp infection could also activate the Akt signaling pathway. NAC could reduce lipid peroxidation damage and DNA damage, but also inhibit the activation of Akt signaling pathway by inhibiting the generation of ROS. |
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