| Objectives: The study about the correlation between H.pylori infection and PTEN/ PI3K/Akt pathway in the development of gastric mucosa-related diseases was still blank.This investigation was to explore whether H.pylori was able to alter the apoptosis and cell cycle of gastric epithelial cells through the PTEN/PI3K/Akt pathway,cause the injury of gastric epithelium mucosae and result in gastric diseases by detecting the influence of H.pylori on the PTEN/PI3K/Akt pathway of gastric epithelial cells treated with H.pylori culture supernatant,thus establish a novel pathway for prevention and cure of H.pylori associated diseases.Methods: 1. The GES-1 cells were randomly divided into five groups according to the results of Methylthiazoy tetrazolium(MTT) assay: Experimental groups(group A,B,C ) were treated by 0.6mg/ml,0.3mg/ml and 0.15mg/ml H.pylori culture supernatant, respectively,group D as the negative control and group E blank,which were dealt with H.pylori medium and DMEM medium.2. The survival rate was detected by MTT assay; Flow Cytometry(FCM) was used to determine cell apoptosis and cell cycle;The expression of PTEN was examined by RT-PCR and Immunocytochemistry;The expression of phospho-Akt was assessed by Western blot.Results: 1. H.pylori culture supernatant inhibited growth of GES-1 cell in a time and dose-dependent pattern. The survival rate of group A was significantly lower than group B,C and D(P<0.01),without any difference among the latter three groups(P>0.05) after initial 24 h; After 48 and 72h,the survival rate of GES-1 cells in group A and B was notably lower than group D(P<0.05~P<0.01),and the survival rate of group A was strikingly lower than group B(P<0.05~P<0.01),while no diversity was found between group C and D(P>0.05);Meanwhile,there were significant differences in the survival rate of cells in group A among the time point of 24,48 and 72h(P<0.05~P<0.01).2. H.pylori culture supernatant induced apoptosis of GES-1 in a time and dose-dependent pattern.The initial 24h incubation with H.pylori culture supernatant had no significant effect on the cell apoptosis of group A compared to group D(P>0.05);After a further 24h,the cell apoptosis of GES-1 cells in group A and B was significantly higher than group C and D(P<0.01),and the apoptosis in group A was remarkably higher than group B(P<0.01),while no disparity was displayed between group C and D(P>0.05); Meanwhile,apoptosis of group A after 48h was significantly higher than that of after 24h(P<0.01).3. H.pylori culture supernatant induced cell cycle G0-G1 phase capture of GES-1 in a time and dose-dependent pattern. 24h later, the GES-1 cell proportionality at G0-G1 phase in group A was conspicuously higher than group D(P<0.01);The GES-1 cell proportionality at G0-G1 phase in group A and B after 48h was strikingly higher than group C and D(P<0.01),with a notable distinction between the former two groups(P<0.05),while there was no distinction between group C and D (P<0.05);Simultaneously,the cell proportionality at G0-G1 phase in group A after 48h was significantly higher than that of after 24h(P<0.05).4. H.pylori culture supernatant induced the expression of PTEN mRNA.After induction with H.pylori culture supernatant for 48h,the expression of PTEN mRNA in group A was strikingly higher than group D(P<0.01).5. H.pylori culture supernatant induced the expression of PTEN protein in a time and dose-dependent pattern.After 24h,the expression of PTEN protein in group A was significantly higher than group D(P<0.01);48h later, the expression of PTEN protein in group A and B was obviously higher than group C and D(P<0.01),and group A was notably higher than group B(P<0.01),while no difference was found between group C and D (P>0.05); Meanwhile,the expression of PTEN protein in group A after 48h was significantly higher than that of after 24h(P<0.05).6. H.pylori culture supernatant induced down-expression of phospho-Akt in a time and dose-dependent pattern.24h later,the expression of phospho-Akt in group A was significantly lower than group D(P<0.01);After 48h, the expression of phospho-Akt in group A and B was strikingly lower than group C and D(P<0.01),and group A was significantly lower than group B(P<0.05),without any difference between group C and D(P>0.05);Meanwhile, the expression of phospho-Akt in group A after 48h was obviously lower than that of after 24h(P<0.01).7. There was a negative correlation between the GES-1 cell proportionality at G0-G1 phase,cell apoptosis and survival rate,the expression of PTEN protein and phospho-Akt.After incubation with H.pylori culture supernatant for 48h,the survival rate was negatively correlated with the cell proportionality at G0-G1 phase and apoptosis of GES-1 in each group(r=-0.779~-0.874,P<0.01). Additionally,there was a negative correlation between the expression of PTEN protein and phospho-Akt(r=-0.931,P<0.01).Conclusions: 1. H.pylori culture supernatant can induce apoptosis and cell cycle capture at G0-G1 phase,thus cause growth inhibition of GES-1 in a time and dose-dependent pattern.2. H.pylori culture supernatant can up-regulate the expression of PTEN mRNA,and induce the expression of PTEN protein with the phosphorylation of Akt inhibited in a time and dose-dependent pattern.3. H.pylori culture supernatant can induce apoptosis and growth inhibition of GES-1 in a time and dose-dependent pattern through the PTEN/PI3K/Akt pathway,which might be one of the pathogenic mechanisms of gastric mucosa injury that resulted in H.pylori associated diseases. |
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