Studies On In Vitro Mechanism Of NLRC5 Mediated Helicobacter Pylori Gastritis/Intestinal Metaplasia

Objective To investigate the in vitro mechanism of NLRC5 mediated helicabacter pylori related gastritis/intestinal metaplasia by establishing a model with helicabacter pylori infected human gastric epithelial GES-1 cell.Methods 1.To set up the helicabacter pylori standard home-made strain(CagA+,VacA+)NCTC1 1637 infected human gastric epithelial GES-1 cells in vitro as helicabacter pylori infection group,and no helicabacter pylori as the blank control group,the level of IL-8 and IL-4 was detected by ELISA method,mRNA expression levels of NLRC5,CDX2,MUC2,SOX2 and MUC5AC genes were detected by qRT-PCR method,the expression levels of NLRC5,CDX2,MUC2,SOX2 and MUC5AC protein were determined by Western blot method.2.24 hours after using siRNA technology to silence NLRC5 genes on helicabacter pylori infection GES-1 cells,the effect of silencing NLRC5 gene on helicabacter pylori infected GES-1 cells was detected by qRT-PCR,and the effects of the gene change of the CDX2,MUC2,SOX2 and MUC5AC were detected.The effects of helicabacter pylori infected the GES-1 cells induced gastrointestinal mucosal markers of CDX2,MUC2,SOX2,and MUC5AC.Results 1.When helicabacter pylori infection group was infected with GES-1 cells after 12,24,48,72 hours respectively,the inflammatory factor IL-8 concentration was significantly higher than that of the blank control group,and the difference were statistically significant(P<0.05).12 hours after helicabacter pylori infected the cells,the IL-8 concentration reached the peak and gradually decreased thereafter.When helicabacter pylori infection group was infected with GES-1 cells on 12,24,48,72 hours,the inhibitory factor IL-4 concentration was significantly lower than that of the blank control group,and the difference was statistically significant(P<0.05).The IL-4 concentration gradually decreased with the time of infection.2.The mRNA expression levels of the NLRC5 genes and the intestinal mucosal markers CDX2 and MUC2 genes were significantly higher in helicabacter pylori group than that of the blank control group 24 hours after helicabacter pylori infected GES-1 cells,and the difference was statistically significant(P<0.05).The mRNA expression of the gastric mucosal marker SOX2 and MUC5AC genes was significantly lower in helicabacter pylori infection group than that of the control group,and the difference was statistically significant(P<0.05).3.The protein expression levels of NLRC5 and the intestinal mucosal markers CDX2 and MUC2 were higher in helicabacter pylori infection group than that of the control group 24 hours after the helicabacter pylori infected the GES-1 cells,and the difference was statistically significant(P<0.05).The protein expression level of the gastric mucosal markers SOX2 and MUC5AC in helicabacter pylori infection group was significantly lower than that of the control group 24 hours after the helicabacter pylori infected the GES-1 cells,and the difference was statistically significant except the SOX2(P<0.05).4.Compared with helicabacter pylori infection group + negative control group and helicabacter pylori infection group the mRNA expression levels of the NLRC5 genes and the intestinal mucosal markers CDX2 and MUC2 genes were significantly lower while the mRNA expression levels of the gastric mucosal markers SOX2 and MUC5AC was significantly higher in helicabacter pylori infection group + siRNA group when silencing the NLRC5 genes in the GES-1 cells with helicabacter pylori being infected for 24 ours,and the difference was statistically significant(P<0.05).Conclusion 1.It could promote the cells to secret proinflammatory cytokine IL-8 and inhibit the cells to secret the inhibitory factor IL-4 after the GES-1 cells with the helicabacter pylori were infected,thus eliciting inflammation.2.It could induce the expression levels of the genes and the proteins of NLRC5 and the gastrointestinal mucosa protein CDX2,MUC2,SOX2 and MUC5AC after the GES-1 cells with the helicabacter pylori were infected,and proved that the helicabacter pylori could promote the expression of NLRC5,CDX2 and MUC2 and inhibit the expression of SOX2 and MUC5AC in cells.3.NLRC5 may be involved in regulating the changes of inflammation and gastrointestinal mucosal markers induced by helicobacter pylori in the gastric mucosal intestinal metaplasia caused by helicobacter pylori infection.