The Mechanism And The Effect That Induced By The Intracellular Helicobacter Pylori On Gastric Epithelial Cell

ObjectiveH. pylori (Helicobacter pylori, Hp) is generally viewed as an extracellular pathogen,in recent years, a large number of research results showed Helicobacter pylori caninternalize into gastric epithelial cells and can be breeding in the intracellular survival.This research investigated the cellular functions of the internalized H. pylori on AGScells which would contribute to exploring the pathogenesis mechanism of H. pylori.MethodsChoose four Hp strains of different invasion force from the Hp strains kept in ourlaboratory：two H.pylori standard strains(NCTC11637、SS1) and two clinical strains(T494、T049) invade AGS cells at a multiplicity of infection (MOI) of100. ThroughGentamicin protection assay investigated the cellular functions of the internalized H.pylori on AGS cells using: a. CCK8detection kit detect the rate of cell proliferation; b.Flow cytometry detect the rate of cell apoptosis; c. ELISA kit measure the secriton ofIL-8;d..Immunofluorescence detect the protein expression of AID;e.Real Time-PCRanalysis the difference of expression of AID;f.Firefly luciferase evaluation the activityof nuclear nuclear factor κ B (NF-κB).ResultsThe internalized H. pylori can: a. promote the proliferation of AGS cell, the rate ofcell proliferation of NCTC11637、T494（37.72%and36.51%）are stronger than SS1、T049（26.97%and26.17%）, and this effect is attenuated by pre-treatment withAnti-integrinβ1; b. does not affect the the apoptosis of AGS cell, the rate of cellapoptosis of NCTC11637、T494（2.79%and2.70%）and SS1、T049（2.51%and2.44%）are similar with the control(2.62%);c. induce the secretion of IL-8, the levelsof NCTC11637、 T494(9.94pg/ml and9.10pg/ml) are higher than SS1、T049(6.31pg/ml and6.30pg/ml), and this levels is down-regulated by pre-treatmentwith Anti-integrinβ1; d. activate the expression of the protein of AID, which locatedin endochylema, the mean fluorescence intensity value of NCTC11637、T494（14.48 and13.48） are higher than SS1、T049（11.07and9.96）; activate the expression ofthe mRNA of AID, the relative expression levels of NCTC11637、T494（1.57and1.55） are higher than SS1、T049（1.35and1.23）;Anti-integrinβ1can down-regulatethe expression of AID;e. activate NF-κ B pathway, the relative luciferase activityvalues of NCTC11637、T494（1.57and1.55）are higher than SS1、T049（1.35and1.23）,Anti-integrinβ1might suppress the activity of NF-κ B; SN-50, a NF-κ Binhibitor,can degrade the rate of cell proliferation and reduce the express of IL-8andAID.Conclusion1. The internalized H. pylori can promote the proliferation and does not effect theapoptosis of AGS cell，meanwhile induce the overexpress of IL-8and AID；2.The internalized H. pylori may participate in the pathogenic process throughactivate the pathway of NF-κ B;3.The effect of the cellular functions of the high invasion Hp strains on AGS cell arestronger than the low ones and the pathogenicity of the intracellular Hp is closelyrelated with the invasiveness of the strain itself.