The Role Of ROS And PI3K/Akt Signaling Pathway In Gastric Epithelial Cells DNA Damage By H.pylori

Objective:Helicobacter pylori (Hp) is the causative agent of a variety digestive diseases, and this diversity is closely related with its affect the gastric epithelial cells apoptosis and proliferation. Hp infect the gastric epithelial cells can increase the reactive oxygen species (ROS) and DNA damage. ROS is a series of derivatives, which generated in the aerobic cells metabolic process, participate in and influence a range of cellular signal transduction’s biological effects. Hp plays an anti-apoptotic role by activate the PI3K/Akt signaling pathway, but with the activation of PI3K signaling pathway, Hp mainly induce the gastric epithelial cells apoptotic, it may be relate with the activation of Akt, which can increase the ROS. Thus we assume that Hp activate Akt signaling pathway in gastric epithelial cells, determines the fate of cell proliferation or apoptosis is closely related with the ROS levels. The relationship among the ROS level and the PI3K/Akt signaling pathway is not clear. So we use Hp infect GES-1, and use the PI3K signaling pathway inhibitor Ly294002and the ROS inhibitor N-acetylcysteine, through detect the change of ROS, DNA damage, and the expression of Akt signaling pathway proteins, then try to explore the molecular mechanism.Methods:1. GES-1cell culter:GES-1cell culture medium contained10%fetal bovine serum,2%penicillin-streptomycin and high gloucose DMEM medium. Before the Hp infect GES-1cells, the medium replace with no antibiotic high gloucose DMEM medium, which only contain10%fetal bovine serum.2. Hp culture and live bacteria suspension:Select Hp standard strains ACTC43504(CagA+, VacA+), cultured three days in the solid agar palte which contain10%sheep blood, then identified, and prepare the live bacteria suspension.3. Establishment of the Hp infect GES-1cell model:Use logarithmic growth phase Hp infect GES-1cells,(1) Pre-experiment:Design the control group (0,1h,3h, 6h,12h),3h MOI (0:1,25:1,50:1,100:1,200:1,300:1),6h MOI100:1(0, Hp+GES-1, Hp+DMSO+GES-1, Hp+Ly294002+GES-1, Hp+NAC+GES-1);(2) Take bacterial cells ratio100:1for0,1h,3h,6h,12h;(3) Use the different Hp concentration infect GES-1cells(MOI0:1,25:1,50:1,100:1,200:1,300:1) in6h;(4) Design6h MOI300:1(0, Hp+GES-1, Hp+DMSO+GES-1, Hp+Ly294002+GES-1, Hp+NAC+GES-1);4. Detection of intracellular ROS level:NAC pretreatment GES-1for1h, then Hp infect GES-1for6h, through load the fluorescent probe DCFH-DA into GES-1cells, then use Live Cell Imaging System observe the change of intracellular ROS and use versatility microplate (Bio-Rad680) quantitative detect the intracellular ROS level.5. The detection of PI3K/Akt signaling pathway protein:Use Ly294002, NAC, DMSO pretreatment GES-1cells, then infect Hp, through Western Blot detect protein expression.6. DNA damage detection:Use ROS scavenger NAC pretreatment GES-1cells, then infect Hp for6h, use single cell gel electrophoresis comet assay detect DNA damage.Results:(1)The infection of ROS levels on Hp infected GES-1by NAC:Use a fluorescent microplate reader quantitative detect the ROS levels, we found that the relationship of ROS level and Hp concentration is proportional, the ROS level is the highest when the MOI300:1, various concentrations of NAC could significantly inhibit the generation of ROS, compared with the control group, the difference was statistical significance.(2)The impact of Akt signaling pathway proteins on Hp infected GES-1by different pharmacological preconditioning:A. In the control group, the express of Akt signaling pathway proteins (Akt, p-Akt308, GSK3-β, p-GSK3-β) does not change at different time. B. At6h, total protein Akt and GSK3-β does not increase with the Hp concentration change, the difference was not statistically significant (P>0.05); the express of p-Akt and p-GSK3-β were gradually increased when the Hp concentration increased, the difference was statistically significant (p<0.05); C. When the MOI100:1, the express of p-Akt and p-GSK3-β were gradually increased as the Hp infected time increased. D.Use Ly294002pretreatment, significantly inhibited the express of p-Akt and p-GSK3-β, compared with the Hp group, the difference was statistically significant (p<0.05), Hp group compared with the DMSO group, the difference was not statistically significant (P>0.05); User NAC pretreatment, the express of p-Akt and p-GSK3-β are significantly lower than the Hp group, the difference was statistically significant (P<0.05), Hp group compared with DMSO group, the difference was not statistically significant (p>0.05).(3) The impact of DNA damage on Hp infected GES-1by different pharmacological preconditioning:Compared with the control group, the values of Tail length, Comet length, Tail moment, Olive tail moment are obvious increased in the Hp group, the difference was statistically significant (P<0.05); After DMSO pretreatment, the associate DNA damage indicators are all declined, but the difference compared with the Hp group was not statistically significant (P<0.05); User NAC pretreatment, compared with the Hp group, the values of Tail length, Comet length, Tail moment, Olive tail moment has a clear downward trend, the difference was statistically significant (P<0.05).Conclusion:The vitro experiments showed that Hp infect GES-1cells could increase intracellular ROS level, result in DNA damage, activate Akt signal pathway. Inhibit the the generation of ROS could reduce DNA damage and weaken the activation of Akt signal pathway. It showed that the ROS level and Akt signal pathway is closely related in the process of Hp infection.