The Effect Of Helicobacter Pylori On Toll Like Receptors Signal Pathway In Gastric Epithelial Cell

Objective:To investigate the effect of Helicobacter pylori (H. pylori) on Toll-like receptors (TLRs) signal pathway in gastric mucosa and its role in H. pylori pathopoiesis. To elucidate the mechanisms of recognization of gastric mucosa to H. pylori and the causation of inflammatory reaction. To establish experimental basis for further researches on prevention and treatment of H. pylori infection and correlative diseases through regulation of TLRs signal pathway, and provide theoretical and experimental evidences for the prevention and treatment of H. pylori correlative diseases.Methods:⑴The changes of TLR4 signal pathway in gastric mucosa of patients with H. pylori infection:①The protein expressions of TLR4 in 189 biopsies gastric mucosa samples were determined by streptavidin-perosidase immunohistochemistry. 189 biopsies samples included: Chronic Non-Atrophic Gastritis(CNAG) 75 (H. pylori–positive 50, H. pylori-negative 25), Chronic Atrophic Gastritis(CAG)24 (H. pylori–positive 13, H. pylori-negative 11), Intestinal Metaplasia and Dysplasia 90 (H. pylori-positive 45, H. pylori-negative 45). Twenty gastric mucosa samples of subjects with no evidence of H. pylori infection and with normal histological structure were used as control group.②30 BALB/c Mice were divided into three groups: the control group; H. pylori infection group with no treatment; H. pylori eradication group: omeprazole 0.06mg/mice, amoxicillin 2.9mg/mice, chitosan 12.5mg/mice, twice a day for 2 weeks. At 4 weeks after the last administration, mice were sacrificed and gastric mucosa were taken for quantitatively culture and Giemsa staining to detect H. pylori infection, and mRNA and protein expressions of TLR4 in gastric mucosa were determined by RT-PCR and immunohistochemical method.⑵The effect of H. pylori on TLRs signal pathway in human normal gastric epithelial cell line GES-1:①GES-1 were coincubated with the culture supernatants of H. pylori NCTC11637(CagA+, VacA+) or viable organisms and divided into the following groups: experimental groupA: groups treated with different concentrations of H. pylori culture supernatants(0.630μg/μl, 0.315μg/μl, 0.158μg/μl, 0.079μg/μl), at different time points 24h,48h and 72h, respectively; experimental group B: groups treated with different concentrations of H. pylori viable organisms(4.00×107CFU/ml, 2.00×107CFU/ml, 1.00×107CFU/ml, 0.50×107CFU/ml), at different time points 24h,48h and 72h, respectively; control group: just added H. pylori liquid medium.②The inhibiting rate of cell growth were determined by MTT assay.③The mRNA expressions of TLR2, TLR4and MyD88 were determined by RT-PCR.Results:⑴The changes of TLRs signal pathway in gastric mucosa of patients with H. pylori infection:①In various diseases groups, expressions of TLR4 in gastric epithelial cells as well as in inflammatory celsl were significantly higher in patients with H. pylori infection than those of subjects with no evidence of H. pylori infection(P<0.05~0.001).②In patients with H. pylori infection, the TLR4 expressions in gastric epithelial cells and inflammatory cells of the patients with CagA+ strain infection were significantly higher than those of subjects with no evidence of H. pylori infection and those of patients with CagA- strain infection(P<0.01~0.001). In inflammatory cells, the TLR4 expressions of the patients with CagA- strain infection were higher than those of subjects with no evidence of H. pylori infection(P<0.05). Whereas in gastric epithelial cells, the TLR4 expressions of the patients with CagA- strain infection and those of subjects with no evidence of H. pylori infection were not significantly different(P>0.05).③In H. pylori infection mice models, TLR4 protein and mRNA expressions in gastric epithelial cells of the mice with H. pylori infection were significantly higher than those of control group(P<0.01), whereas after H. pylori eradication TLR4 expression were significantly lower(P<0.01), with no difference with control group(P>0.05).⑵The effect of H. pylori on TLRs signal pathway in human normal gastric epithelial cell line GES-1:1) The inhibiting rates of cell growth of groups treated with H. pylori culture supernatants were significantly higher than those of control group(P<0.001), and these inhibition rates were concentration-dependent. At each time point, the inhibition rates of 0.630μg/μl group were significantly higher than those of other concentration groups(P<0.001); the inhibition rates of 0.079μg/μl group were significantly lower than those of 0.315μg/μl at 24h(P<0.01); the inhibition rates of 0.079μg/μl group were significantly lower than those of 0.315μg/μl and 0.158μg/μl groups at 48h and 72h(P<0.001~0.01); the inhibition rates of 0.158μg/μl group were significantly higher than those of 0.079μg/μl group at 72h(P<0.01).2) GES-1 inhibition rates were significantly affected with the treatment time of H. pylori culture supernatants. In the concentrations range of 0.315μg/μl,0.158μg/μl and 0.079μg/μl, the GES-1 inhibition rates were significantly affected with the treatment time of H. pylori culture supernatants(P<0.001~0.05). In 0.315μg/μl,0.158μg/μl H. pylori culture supernatants groups, the GES-1 inhibition rates at 48h and 72h were significantly higher than those at 24h(P<0.001~0.01), and the inhibition rates at 48h were the highest;In 0.079μg/μl group, the GES-1 inhibition rates at 48h were significantly higher than those at 24h(P<0.01). With different concentrations, the inhibition rates at 48h and 72h were not significantly different, which indicated that the inhibition rates were at the peak at 48h. In high concentration group (0.630μg/μl), no differences were found at each time(P>0.05).3) The inhibition rates of experimental groups treated with H. pylori viable organisms were significantly higher than those of control group(P<0.001), and these inhibition rates were dose-dependent. In 16h and 24h groups, the inhibition rates of 4×107CFU/ml groups were significantly higher than those of other concentration groups(P<0.001), and the inhibition rates of 0.5×107CFU/ml group were significantly lower than those of 2×107CFU/ml group; there were significant differences among every concentrations in 48h group(P<0.001~0.05).4) H. pylori viable organisms inhibited cell growth of GES-1 in a time pattern. In 16h group, the inhibition rates of all of the concentrations were significantly lower than those in 24h and 48h groups(P<0.001~0.01); furthermore,in the concentrations rang of 2×107CFU/ml and 1×107CFU/ml, the inhibition rates in 24h group were significantly lower than those in 48h group(P<0.01~0.05).5) The expressions of TLR4 mRNA in GES-1 were significantly different when treated with different concentration of H. pylori culture supernatants(P<0.001~0.05). At each time point, the expressions of TLR4 mRNA of 0.315μg/μl and 0.158μg/μl groups were the highest, which were significantly higher than those of control group and 0.630μg/μl groups(P<0.05~0.001), furthermore, at 48h and 72h, these also were significantly higher than those of 0.079μg/μl group(P<0.001~0.01). At 24h, the expressions of TLR4 mRNA were significantly higher of 0.158μg/μl group than those of 0.630μg/μl group(P<0.05), and at 48h, expressions of the TLR4 mRNA were significantly higher of 0.079μg/μl group than those of 0.630μg/μl group(P<0.05); nevertheless, the expressions of TLR4 mRNA in GES-1 were not significant different among the time points of 24h, 48h and 72h(P>0.05).6) The expressions of TLR4 mRNA in GES-1 were significantly different when treated with different concentrations of H. pylori viable organisms(P<0.001~0.05).At each time point, the expressions of TLR4 mRNA were significantly higher of 4×107CFU/ml,2×107CFU/ml and 1×107CFU/ml groups than those of control group(P<0.001~0.05). Furthermore, at 16h, the expressions of TLR4 mRNA were significantly higher of 2×107CFU/ml group than those of 1×107CFU/ml and 0.5×107CFU/ml groups(P<0.05). At 24h, the expressions of TLR4 mRNA were significantly higher of 0.5×107CFU/ml group than those of control group(P<0.05). At 48h, the expressions of TLR4 mRNA were significantly higher of 4×107CFU/ml and 2×107CFU/ml groups than those of 0.5×107CFU/ml group(P<0.05). Nevertheless, the expressions of TLR4 mRNA in GES-1 were not significant different among the time points of 16h, 24h and 48h(P>0.05).7) The expressions of TLR2 mRNA in GES-1 were significantly different when treated with different concentrations of H. pylori culture supernatants(P<0.001). At each time point, the expressions of TLR2 mRNA of 0.315μg/μl and 0.158μg/μl groups were the highest, which were significantly higher than those of control group,0.630μg/μl and 0.079μg/μl groups(P<0.001), but the TLR2 mRNA expressions in GES-1 were not significant different between 24h and 48h(P>0.05).8) The expressions of TLR2 mRNA in GES-1 were significantly different when treated with different concentrations of H. pylori viable organisms(P<0.001~0.05). At each time point, the expressions of TLR2 mRNA were significantly higher of 4×107CFU/ml, 2×107CFU/ml and 1×107CFU/ml groups than those of control group (P<0.001~0.05). Furthermore, the expressions of TLR2 mRNA were significantly higher of 4×107CFU/ml groups than those of 0.5×107CFU/ml group(P<0.001~0.05). At 48h, the expressions of TLR2 mRNA were significantly higher of 2×107CFU/ml group than those of 0.5×107CFU/ml group(P<0.01), but the expressions of TLR2 mRNA in GES-1 were not significant different between 24h and 48h(P>0.05).9) The expressions of MyD88 mRNA in GES-1 were significantly different when treated with different concentrations of H. pylori culture supernatants(P<0.05).At 24h, the expressions of MyD88 mRNA were significantly higher of 0.630μg/μl and 0.315μg/μl groups than those of control group and 0.079μg/μl group, respectively(P<0.01~0.05). At 48h, the expressions of MyD88 mRNA were significantly higher of 0.630μg/μl group than those of control group, 0.315μg/μl and 0.158μg/μl groups(P<0.01);In 0.158μg/μl group, the expressions of MyD88 mRNA were significantly higher at 48h than those at 24h(P<0.01),but in other concentration groups the expressions of MyD88 mRNA were not significant different between 24h and 48h(P>0.05).10) The expressions of MyD88 mRNA in GES-1 were significantly different when treated with different concentrations of H. pylori viable organisms(P<0.001~0.05).At 24h, the expressions of MyD88 mRNA were significantly higher in 4×107CFU/ml and 2×107CFU/ml groups than those in 0.5×107CFU/ml group and control group(P<0.01~0.05).At 48h, the expressions of MyD88 mRNA were significantly higher in 4×107CFU/ml and 2×107CFU/ml groups than those in control group(P<0.01),and the expressions of MyD88 mRNA were significantly higher in 4×107CFU/ml group than those in 1×107CFU/ml and 0.5×107CFU/ml groups(P<0.01~0.05);but the expressions of MyD88 mRNA were not significant different between 24h and 48h(P>0.05).11) The expressions of TLR2 and TLR4 mRNA were significantly positive correlated in GES-1 treated with H. pylori culture supernatants(P<0.001~0.01); but both of them had no correlation with the expressions of MyD88(P>0.05). When GES-1 were treated with H. pylori viable organisms,the expressions of the mRNA among TLR2,TLR4 and MyD88 were significantly positive correlated(P<0.001~0.05).Conclusion:⑴T he expressions of TLR4 in the gastric mucosa of patients and mice with H. pylori infection were significantly upregulated, which were lower after H. pylori eradication, indicating that H. pylori may upregulate the expressions of TLR4 and may induce inflammatory reaction through TLR4 signal pathway.⑵The growths of human normal gastric epithelial cell line GES-1 were inhibited with both H. pylori culture supernatants and viable organisms in time and concentration-dependent patterns, which may be one of the mechanisms of H. pylori causing gastric mucosa impairment.⑶The expressions of TLR2, TLR4 and MyD88 mRNA in human normal gastric epithelial cell line GES-1 were upregulated with both H. pylori culture supernatants and viable organisms, which indicated that they can actactivable TLRs signal pathway in gastric epithelial cell, and may be one of the mechanisms of gastric mucosa recognizing H. pylori and causing inflammatory reaction.