Expression And Regulatory Mechanisms Of DNA-damage Inducible Transcript 4 In Helicobacter Pylori-associated Gastritis

BackgroundsHelicobacter pylori（H.pylori）,a gram-negative bacillus,consistently colonizes on the gastric mucosa.Persistent H.pylori infection leads to a series of problems,including the pathologic change of gastric mucosa cells and the change of the local microenvironment,resulting in a series of stomach diseases and evolution,such as chronic superficial gastritis developing to chronic atrophic gastritis and eventually developing to gastric cancer.It is reported that H.pylori infection is vital factor for gastric adenocarcinoma.H.Pylori infection is characterized by wide infection range,high infection rate and large infection population.Therefore,it is of vital clinical significance to clarify the pathogenesis of H.pylori infection so as to provide targeted treatment.Although the mechanisms of developing H.pylori-associated gastritis remain unclear,the interaction between gastric epithelial cells（GECs）and bacteria may be a vital determinant,especially the inflammatory effects followed by this interaction induced by bacterial virulence factor cytotoxin associated gene A（cag A）protein.After cag A entering host cells,it will lead to a series of pathological reactions,which gradually leading to changes in local microenvironment,bacterial colonization and inflammation.A large number of studies have shown that cag A plays an vital role in H.pylori infection.DNA damage-induced transcription 4（DDIT4）is a conserved,ubiquitous protein and is made up of 232 amino acids.DDIT4 characterized as inhibiting rapamycin（m TOR）signaling pathway of the mammal,and the function of DDIT4 is various,such as regulating cell growth,leading to apoptosis,regulating mitochondrial function,and oxidative stress.Importantly,DDIT4 plays various roles in different inflammatory diseases.However,virtually nothing is known about the expression,regulation and function of DDIT4 in H.pylori-associated gastritis.Objectives1.To research the correation between H.pylori’s infection and DDIT4’s expression;2.To explore the regulatory mechanisms of DDIT4 expression in H.pylori infection.Methods1.To research the correation between H.pylori’s infection and DDIT4’s expression Clinical specimensCollection of specimens:Gastric mucosal tissue and peripheral blood samples of patients were collected from gastritis patients.Screened into groups:H.pylori infection was preliminarily determined by ¹⁴C respiratory test and rapid urease test of gastric mucosal tissue,and subsequently ensured through RT-PCR for 16S r DNA and detection of peripheral blood for specific anti-H.pylori antibodies（Abs）.The gastritis specimens of H.pylori infection patients were graded according to pathological evaluation criteria,and were successively divided into mild,moderate and severe gastritis according to the degree of inflammation,and the correlation between DDIT4 expression and gastritis degree was analyzed and evaluated.Mouse modelInoculating SPF C57 mice with H.pylori.According to the experimental plan,mice were sacrificed painlessly at the specified time point and then gastric mucosal tissue samples were collected for detection and analysis,DDIT4 expression in gastric mucosa of mice was detected by RT-PCR,immunohistochemistry and Western blot.In vitro H.pylori infectionThe human and mice’s gastric mucosa without H.pylori infected were collected and infected by WT H.pylori orΔcag A H.pylori.DDIT4 expression was detected by RT-PCR,immunohistochemistry and Western blot.Using different H.pylori MOI or times to infect GECs including Human gastric epithelial cell lines and human or mice primary gastric epithelial cells as well.2.To explore the regulatory mechanisms of DDIT4 expression in H.pylori infectionFirstly,using signaling pathway inhibitors to treat AGS cells to block specific signaling pathway.Secondly,AGS cells were infected by H.pylori.Lastly,The expression of DDIT4and signal molecules were evaluated.The changes of DDIT4 promoter activity in gastric epithelial cells after H.pylori infection were analyzed by dual-lucifase report assay.The binding site of NF-κB in the DDIT4 promoter region was analyzed by PROMO,and Chromatin Immunoprecipitation（Ch IP）assay was used to verify whether p65 directly binds to the DDIT4 promoter region and directly regulates the expression of DDIT4.Results1.The expression of DDIT4 in gastric mucosa and gastric epithelial cells during H.pylori infectionDDIT4 expression in gastric mucosa was significantly increased in patients with H.pylori infection,and the increase of DDIT4 expression in gastric mucosa was more obvious in cag A⁺H.pylori compared to cag A^-H.pylori infection.Besides,the expression of DDIT4 was higher in more severe gastritis within gastric mucosa of patients infected with H.pylori.Similar results were got by detecting mice’s gastric mucosa at 7w after H.pylori infection.It is found that DDIT4 expressed in the higher level after H.pylori infection in AGS,BGC-823,GES-1,HGC-27,SGC-7901 cells,human and mouse primary gastric epithelial cells.Besides,it was more significant with WT H.pylori infection compared toΔcag A H.pylori infection.DDIT4 expression was up-regulated with the increase of WT H.pylori MOI and infection time.In other words,The expression of DDIT4 was dependent on infection time and MOI in AGS cells.2.The regulatory mechanisms of DDIT4 expression in H.pylori infectionMAPKp38 pathway was significantly activated after WT H.pylori infection of AGS cells,and the phosphorylation level of P38 was significantly increased.When inhibited MAPKp38 pathway by SB203580,the up-regulation of DDIT4 by WT H.pylori was obviously abolished.In addition,inhibiting cag A phosphorylation could down-regulated the p38 phosphorylation and decrease the DDIT4 expression.Furthermore,the activation of DDIT4 promoter was detected by double luciferase report analysis,it was found that DDIT4promoter could be activated after AGS cells were infected with WT H.pylori,while DDIT4promoter activity was significantly decreased after MAPKp38 signaling pathway inhibited.Ch IP test showed that P65 directly binds to the DDIT4 core promoter sequences and mediates DDIT4 expression.Conclusion1.H.pylori increases DDIT4 expression in gastric mucosa and gastric epithelial cells;2.H.pylori induced gastric epithelial cells to express DDIT4 via the phosphorylated cag A that activated MAPKp38 pathway to mediate NF-κB directly binding to DDIT4promoter.SignificanceIn this study,we found a new molecule in the occurrence and development of H.pylori infection-related gastritis and its expression regulation mechanism by studying the pathogenesis of H.pylori infection-related gastritis,.Besides,This study aims to provide theoretical guidance for the clinical treatment of H.pylori infection-related gastritis,and provide a new direction for in-depth exploration of the mechanism of H.pylori infection-induced gastritis and treatment of H.pylori infection-related gastritis.