| Helicobacter pylori(H.pylori)infection is the main pathogenic factor of chronic gastric and peptic ulcer,which is closely related to the incidence of gastric mucosa-associated lymphoma and gastric cancer.It was generally believed that H.pylori was an extracellular pathogen,but more and more evidence showed that this bacterium can survived and proliferate in gastric epithelial cells,and the immune response of the body can clear the intracellular bacterium,leading to the occurrence of stomach diseases caused by persistent infection.Normally balanced microbial flora plays a vital role in establishing and maintaining the host immune balance.More and more animal and clinical studies show that gastrointestinal microbiota can significantly affect the clinical effect of immunotherapy for diseases.H.pylori infection can induce the body’s natural and acquired immune response and induce cellular and humoral immune responses.At present,the researches on the cytokines related to H.pylori infection mainly focus on the cytokines related to Th cells.According to different cytokine profiles and biological functions,T cells are dividedinto helper T cells(Th1,Th2)and regulatory T cells(Treg)subsets.Treg is a T cell subgroup with regulatory function,which is different from Th1 and Th2 effector T cells in immunosuppression,and plays an important regulatory role in a variety of immune diseases.In recent years,Treg has become a hotspot in the field of immunology.According to its surface markers,cytokines produced and mechanism of action,Treg can be divided into CD4+CD25+Treg,Treg1 and Th3.Gastric microbiota is an essential regulatory factor for gastric mucosal immunity.Specific members of the symbiotic flora,such as clostridium and bacteroides fragilis(pod polysaccharide A production),are strong inducers of natural forkhead box protein 3(Foxp3+Tregs).Foxp3+Tregs expressed CD4+CD25+Tregs,and its importance in regulating gastrointestinal immune homeostasis and promoting the establishment and maintenance of peripheral tolerance has been confirmed in both animal and human experiments.Previous studies have found that oligomonas has an immune stimulation effect,which can induce the expression of TNF-a and significantly promote the inflammatory response.Further studies showed that many of the less dominant gastric bacteria were significantly correlated with two major immunosuppressive cells.Most of these bacteria belong to proteobacteria and firmicutes,which regulate gastric mucosal immunity alone or together with bacterial metabolites.Most previous studies have been based on the intestinal environment,focusing on the interaction between microbiota and mucosal immunity,while relatively few studies have been conducted on these interactions in the stomach.H.pylori infection causes activation of Tregs and Th17 and changes in cytokines,which may cause gastritis and peptic ulcer.At present,it is not clear whether and how the gastric mucosa-related microbiota with H.pylori infection is related to the changes in Tregs.H.pylori infection can induce autophagy in gastric epithelial cells,which can degrade pathogens and protect cells.It was found that H.pylori-induced autophagy reduced the damage of gastric epithelial cells caused by H.pylori by degrading intracellular VacA.At the same time,it was observed that only part of H.pylori in autophagosome was cleared,while the other part could survive and proliferate in autophagosome.Currently,the autophagy process of H.pylori-activated host is still not completely clear.Although different bacterial and host factors are involved,different strains and host cell lines can produce different results.How does H.pylori regulate or block autophagy-mediated transport of pathogens to lysosomes in favor of its own survival and replication in autophagosomes?Studies have found that in addition to H.pylori,a variety of other bacteria can manipulate autophagy(such as salmonella,shigella,etc.),can regulate the surface of the cell membrane,and can also express effector and toxin,interfere with and manipulate autophagy,and increase the potential for intracellular replication and further invasion.So does H.pylori infection affect and alter the gastric mucosal microbiota?The study of adults reported that the gastric mucosal flora of H.pylori-infected patients was dominated by H.pylori,accompanied by relative depletion of other bacteria.There are few studies on the gastric mucosal microbial community of children infected with H.pylori.Foreign studies suggest that H.pylori occupies an absolute advantage in the gastric mucosal microbial community of children infected with H.pylori,but the sample size is small.The structural characteristics of gastric mucosal microbiota and its effect on mucosal immune function in Chinese children with H.pylori infection have not been reported.Based on the above,we made the following speculation:H.pylori infection affected the changes in the microbiota related to gastric mucosa,and the changes in the microbiota affected the synthesis and secretion of Tregs,thereby affecting the local immune function and inflammatory response of gastric mucosa.H.pylori infection affects the microbial community related to gastric mucosa.Does the altered microbial community affect the autophagy process of gastric epithelial cells?Therefore,the study on the regulation of autophagy by microbiota may reveal the pathogenesis of H.pylori chronic persistent infection.Therefore,this study intends to explore the relationship between changes in gastric mucosa-related microbiota of H.pylori infection and mucosal immune factors,mucosal autophagy and related pathways in combination with animal experiments,so as to prove the influence of H.pylori infection on gastric mucosa-related microbiota and the role and mechanism of regulating local immune function.Objectives:1.Investigate the changes of microbiota related to gastric mucosa and duodenal mucosa with H.pylori infection.2.Determine the effects of H.pylori infection on immune-related factors and cytokines in gastric mucosa.3.Elucidate the process and signal pathway changes of autophagy induced by H.pylori infection in gastric mucosa.Methods:From December 2016 to August 2017,122 children who were admitted to the children’s hospital,Zhejiang university school of medicine due to symptoms of"repeated abdominal pain,abdominal distension,dyspepsia,nausea and vomiting" were selected.There were three groups:57 children in the HP infection group(HP positive group),37 children in the HP negative gastritis group(HP negative group),and 28 children in the HP negative normal mucosa group(control group).Four gastric antrum mucosa biopsy samples and two duodenal bulb mucosa samples were collected for the following experiments.1.Influence of H.pylori infection on microbiota of gastric mucosa and duodenal mucosa in children.Total mucosal DNA was extracted from 1 piece of each group’s gastric antrum and duodenal ball mucosa,and 16S rDNA V4 region sequence was extracted by PCR.The diversity of microbiota related to gastric mucosa and duodenal mucosa in the three groups was analyzed by Illumina Miseq high-throughput sequencing platform.2.Study on immune regulation and mechanism of gastric mucosa in children induced by H.pylori infection.For the gastric antrum mucosa of the children in the HP positive group,the HP negative group and the control group,12 cases were randomly selected from each group.One piece of gastric antrum mucosa was taken from each group,and total RNA was extracted.The gene expression of FOXP3,IL-10,TGF-β,TNF-α,IFN-y,IL-1,IL-6,IL-8,IL-12,IL-17A,IL-21 and IL-23 was analyzed by real-time quantitative polymerase chain reaction.The expression of CD4+T cells and macrophages(CD68)was observed by immunohistochemical staining.The expression characteristics and correlation of CD4+T cells,CD68 cells,FOXP3,IL-10,TGF-β,IL-17A and IL-23 in children with HP infection were investigated.3.Study on the mechanism of autophagy regulation and persistent infection in gastric mucosa of children induced by H.pylori infection.One piece of each gastric antrum mucosa was taken from each group and used to extract total protein,and another 6 cases(2 cases in each group)were observed by 2.5%glutaraldehyde for fixed transmission electron microscope observation.Western blot was used to detect autophagy signature proteins,autophagy related proteins and related signaling pathways in gastric mucosa specimens infected with H.pylori,and transmission electron microscopy was used to detect autophagy vesicle formation in gastric mucosa specimens infected with H.pylori.Results:1.High-throughput bacterial sequencing analysis confirmed the existence of a large number of bacterial communities in the gastric and duodenal mucosa of children,with unique bacterial community characteristics and diversity.The presence of HP significantly affected the Alpha diversity and Beta diversity of gastric mucosa related microbioys,but not the diversity of duodenal mucosa related microbiota.There were significant differences in the structure and diversity of gastric mucosal bacterial community between HP positive group and HP negative gastritis group.The abundance level of 6 phyla,5 classes,9 orders,10 families and 8 genera in the HP positive group was significantly lower than that in the HP negative group,Only the proportion of helicobacter is more abundant.There was no significant difference in the structure and diversity of the duodenal mucosal bacterial community between HP positive group,HP negative gastritis group and HP negative mucosa normal group.There was no significant difference in the structure and diversity of the duodenal mucosal bacterial community between HP-positive group,HP-negative group and the control group.The presence of HP had no significant effect on the duodenal mucosal microbiota,and there was no significant difference in the abundance of multiple classification levels between the groups.2.The mRNA levels of FOXP3,TGF-1,IL-10,IL-17A and IL-23 in HP positive group were significantly higher than those in HP negative group and the Control group(p<0.01).In HP-positive group,the FOXP3 mRNA level was positively correlated with the mRNA level of TGF-1 and IL-10(r=0.836,p<0.01;r=0.711,p<0.01),and the FOXP3 mRNA level was positively correlated with the abundance of HP(r=0.833,p<0.001).The number of CD4+T cells and CD68 cells in HP positive group was significantly higher than that in HP negative group and the Control group(p<0.05),and the number of CD4+T cells was positively correlated with the inflammatory score(r=0.795,p<0.001).The mRNA ratio of IL-17A/FOXP3 was negatively correlated with HP abundance(r=-0.717,p<0.01).The expressions of IL-1,IL-6,IL-8,IL-12,TNF-α,IFN-γ,and IL-21 in HP positive group were not significantly different from those in HP negative group and the Control group(p>0.05).3.Western-blot analysis showed that the expression level of autophagy marker protein LC3 in HP positive group was significantly lower than that in HP negative group and the Control group,while the expression level of P62 was significantly higher.Beclin-1,Atg5 and Atg16L1 expression levels of autophagocyte-related proteins were significantly lower than those of the HP negative group and the Control group.The level of phosphorylated Akt protein was lower than that of HP negative group and Control group,while the level of phosphorylated mTOR was higher.Conclusions:1.The presence of HP significantly affected the gastric mucosa related microbiota,resulting in low abundance of multiple classification levels.Compared with HP negative group,HP infection had a greater effect on gastric microflora.2.The balance of IL-17A/FOXP3 in response to regulatory T cell bias favors the persistence of bacteria,leading to chronic active gastritis.3.HP infection may be involved in the inhibition of autophagy through the PI3K/Akt/mTOR signaling pathway. |
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