| Objective:To investigate the effect of the supernatant of Helicobacter pylori (H. pylori) with different genotypes on human gastric epithelial cells (GES-1),the changes of cell morphology,apoptosis and proliferation,as well as the expression of P21WAF1, Bax,PTEN,PI3Kand p-Akt.To elucidate the effect and pathogenic mechanisms of H. pylori on GES-1,and establish new target and direction for further researches on treatment of H. pylori correlation diseases.Methods:According to the pre-experimental results,GES-1 were co-cultured with the supernatants of H. pylori, and divided into the following groups:(1)Experimental group of NCTC11637(CagA+, VacA+): groups(group A,B,C ) were treated with different concentrations of H. pylori supernatants(1.3875mg/ml,2.775 mg/ml,5.55 mg/ml);(2)Experimental group of NCTC11639(CagA+, VacA-): groups(group A,B,C) were treated with different concentrations of H. pylori supernatants(1.875mg/ml,3.75mg/ml, 7.5 mg/ml);(3)Group D treated with H. pylori medium as the negative control. Each group GES-1 were co-cultured with the supernatants of H. pylori at different time points 24h,48h and 72h.The cell morphology was observed with inverted microscope and transmission electron microscope. The inhibition rates of cell growth were determined by MTT assay. The level of mRNA was determined by RT-PCR. The level of protein was determined by immunocytochemistry.Results:1) The experimental group[0]s (groupNCTC11637,groupNCTC11639) of the GES-1 Were observed to presented the typical morphological change of apoptosis.2) The inhibition rates of cell growth of groups treated with NCTC11637 H.pylori supernatants were significantly higher than those of control group(P<0.001), and these inhibition rates were concentration-dependent. The inhibition rates of experimental groups B,C were significantly higher than experimental group A at 24h (P<0.01); The inhibition rates of experimental group C were significantly higher than experimental groups A,B,the inhibition rates of experimental group B were significantly higher than experimental group A ,at 48h and 72h(P<0.001~0.05).3) GES-1 inhibition rates were significantly affected with the treatment time of NCTC11637 H. pylori supernatants. In the concentrations range of1.3875mg/ml, 2.775 mg/ml,and 5.55 mg/ml,the GES-1 inhibition rates were significantly affected with the treatment time of H. pylori culture supernatants(P<0.001). In experimental groups B,C,the GES-1 inhibition rates at 48h and 72h were significantly higher than those at 24h(P<0.001~0.05),and the inhibition rates at 72h were the highest,and the inhibition rates at 48h and 72h have no differences (P>0.05);In lower concentration group(experimental groupA),no differences were found at each time poin(tP>0.05).4) The inhibition rates of cell growth of groups treated with NCTC11639 H. pylori culture supernatants were significantly higher than those of control group(P<0.001),and these inhibition rates were concentration-dependent. The inhibition rates of experimental groups B,C were significantly higher than experimental group A at 24h(P<0.01);The inhibition rates of experimental group C were significantly higher than experimental groups A,B,the inhibition rates of experimental group B were significantly higher than experimental group A,at 48h and 72h(P<0.001~0.05).5) GES-1 inhibition rates were significantly affected with the treatment time of NCTC11639 H. pylori culture supernatants. In the concentrations range of1.875mg/ ml,3.75mg/ml,and 7.5 mg/ml,the GES-1 inhibition rates were significantly affected with the treatment time of H. pylori culture supernatants(P<0.001). In experimental groups A,B,the GES-1 inhibition rates at 72h were significantly higher than those at 24h and 48h(P<0.001),and the inhibition rates at 24h and 48h have no differences (P>0.05). In experimental group C,the inhibition rates at 72h were significantly higher than those at 24h and 48h(P<0.001~0.05),the inhibition rates at 48h were significantly higher than those at 24h(P<0.05).6)The expression of P21WAF1 mRNA in GES-1 was significantly higher than that of control group when treated with NCTC11637(2.775 mg/ml) and NCTC11639 (3.75mg/ml)H. pylori culture supernatants for 48h.(P<0.001~0.01).7) The expression of Bax mRNA in GES-1 was significantly higher than that of control group when treated with NCTC11637(2.775 mg/ml) and NCTC11639 (3.75mg/ml)H. pylori culture supernatants for 48h(.P<0.01~0.05). The expression of Bax protein in GES-1 of experimental groups was significantly higher than that of control group,The Score of positive cells were more in experimental group than that of the control group(P<0.05).8) The expression of PTEN mRNA in GES-1 was down-regulated when treated with NCTC11637(2.775 mg/ml) and NCTC11639(3.75mg/ml)H. pylori culture supernatants for 48h,but was not significantly different with that of control group(P>0.05).The expression of PTEN protein in GES-1 of experimental groups was significantly lower than that of control group. The Score of positive cells were lower in experimental group than that of the control group(P<0.05).9) The expression of PI3K protein in GES-1 was significantly higher than that of control group when treated with NCTC11637(2.775 mg/ml) and NCTC11639 (3.75mg/ml)H. pylori culture supernatants for 48h. The Score of positive cells were more in experimental group than that of the control group(P<0.05).10) The expression of p-Akt protein in GES-1 was significantly higher than that of control group when treated with NCTC11637(2.775 mg/ml) and NCTC11639 (3.75mg/ml)H. pylori culture supernatants for 48h. The Score of positive cells were more in experimental group than that of the control group(P<0.01).Conclusion:1) The growths of human normal gastric epithelial cell line GES-1 were inhibited by both NCTC11637 and NCTC11639 H.pylori culture supernatants in time and concentration-dependent patterns, which may be one of the mechanisms of H. pylori causing gastric mucosa impairment.2) Both NCTC11637 and NCTC11639 H.pylori culture supernatant can upregulate the expression of P21WAF1 and Bax, which indicated that H.pylori may activate P21WAF1and Bax ,and play a role in promoting apoptosis.3) Both NCTC11637 and NCTC11639 H.pylori culture supernatant can downregulate the expression of PTEN, upregulate the expression of PI3K, as well as the phosphorylation of Akt ,which indicated that they can active PI3K/Akt signaling pathway in GES-1, that might be one of the pathogenic mechanisms of gastric mucosa injury that resulted in H.pylori associated diseases even gastric cancer. |
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