The Effect Mechanism Of Helicobacter Pylori Infection Depends On NF-κB Pathway Mediated Inflammatory Supernatant Promotes EMT In Colon Epithelial Cells

Objective:In recent years,the detection rate of Helicobacter pylori in gastric and colon cancer has increased significantly.The possibility of normal colonic epithelial cells transforming into cancer cells is closely related to epithelial mesenchymal transformation.In previous studies,it has been confirmed that activation of NF-kappa B signaling pathway can induce EMT in colon epithelial cells,but the specific mechanism is still unclear.Therefore,the aim of this study is to elucidate the effect of inflammatory microenvironment induced by activation of NF-kappa B signaling pathway in U937 cells on EMT in colon epithelial cells and its mechanism,so as to provide a new direction for diagnosis and treatment of diseases caused by HP infection.Methods:1.Helicobacter pylori was cultured in Colombia solid medium combined with degreased fiber sheep blood and mixed antibiotics.HP was identified by bacterial staining and biochemical experiments(Gram staining,urease,oxidase,catalyst).2.U937 cells were cultured routinely,and HP was used to intervene U937 cells(HP bacteria: cells = 100:1).The experiment was divided into four groups: control group(Mock group),HP intervention group(8 h),HP intervention group(16 h),HP intervention group(24 h).The expression of NF-κBp65 protein in four groups of cells and the cytotoxicity of four groups of cells were detected by Western blotting and CCK-8 assay respectively.3.Cells and supernatants of U937 cells were collected after HP intervention.The expression of MIF,IL-1beta and NF-κBp65 factors in the supernatants and cells of four groups were detected by ELISA and RT-PCR respectively.4.FHC cells were cultured routinely and treated with HP inflammatory supernatant(volume ratio: cell culture medium = 1:50).The experiment was divided into four groups: control group(Mock group),HP inflammatory supernatant treatment group(12 h),HP supernatant treatment group(24 h),HP inflammatory supernatant treatment group(36 h).Mitochondrial membrane potential JC-1 kit and Western blotting were used to detect the early apoptotic level of FHC cells and the expression of related apoptotic proteins(Bcl-2 protein and Bax protein).5.Through step 4,the experiment was divided into four groups.The expression of EMT-related proteins(N-cadherin protein,E-cadherin protein and Vimentin protein)was detected by Western blotting and immunofluorescence.6.After PDTC treatment,an inhibitor of NF-kB signaling pathway,inhibited the NF-kappa B pathway,the expression changes of NF-kappa Bp65 protein,inflammatory factors,early apoptotic level,apoptotic protein(Bcl-2 protein,Bax protein),EMT-related protein(N-cadherin protein,E-cadherin protein,Vimentin protein)were detected by RT-PCR,ELISA,Western-blot and CJ-1.Results:1.After HP culture,Gram staining negative showed gull-like red or corynebacterium,urease test,oxidase test and catalyst test showed positive reaction.2.After HP intervention on U937 cells,the expression of NF-κBp65 protein in the intervention group(16 h,24 h group)was significantly higher than that in the Mock group(P<0.05);the cytotoxicity of the intervention group(8 h,16 h,24 h group)was significantly higher than that in the Mock group(P<0.05);the secretion of inflammatory factors MIF and IL-1beta in the intervention group(8 h,16 h,24 h group)was significantly higher than that in the Mock group(P<0.05).At the molecular level,RT-PCR results also showed that the expression of MIF,IL-1β and NF-κBp65 in the intervention group increased significantly compared with that in the Mock group(P<0.05).3.The early apoptotic level of FHC cells treated with HP inflammatory supernatant was significantly lower in the treatment group(12 h,24 h,36 h group)than in the Mock group(P<0.01),the expression of Bcl-2 in the treatment group(24 h,36 h group)was significantly higher than that in the Mock group(P<0.05),and the expression of Bax protein in the treatment group(24 h,36 h group)was significantly lower than that in the Mock group(P<0.01).The expression of E-cadherin protein in treatment group(24 h,36 h group)was significantly lower than that in Mock group(P<0.01);the expression of N-cadherin protein in treatment group(12 h,24 h,36 h group)was significantly higher than that in Mock group(P<0.05);the expression of Vimentin protein in treatment group(24 h,36 h group)was significantly higher than that in Mock group(P<0.01);the number of immunofluorescence of Vimentin protein in treatment group(12 h,24 h,36 h group)was significantly higher than that in Mock group(P<0.01).4.After PDTC inhibited the NF-κB pathway,the expression of NF-κBp65 protein in the intervention group(100 mM,200 mM,500 mM,1000 mM group)was significantly decreased(P<0.001);the cell activity intervention group(50 mM,100 mM,500 mM,1000 mM group)was significantly inhibited compared with the Mock group(P<0.05),there was no difference between the 5 mM and 200 mM groups and the control group;the secretion and gene expression of inflammatory factors in the intervention group were both significant.There was a significant decrease(P<0.05);Bcl-2,Bax,N-cadherin protein,E-cadherin protein and Vimentin protein did not change significantly after adding 200 mM PDTC inhibitor in advance compared with the control Mock group.The results showed that PDTC inhibitor itself had no effect on FHC(P>0.05).5.After treated with HP supernatant inhibited by PDTC,the early apoptotic level of FHC cells in treatment group(12 h,24 h,36 h group)was significantly higher than that in Mock group(P<0.05);the expression of Bcl-2 in treatment group(12 h,24 h,36 h group)was significantly lower than that in Mock group(P<0.05);the expression of Bax protein in treatment group(12 h,24 h,36 h group)was significantly higher than that in Mock group(P<0.05)There were no significant changes in N-cadherin and E-cadherin protein in treatment group(12 h,24 h,36 h group)compared with Mock group(P>0.05);Vimentin protein in treatment group(12 h,24 h,36 h group)was significantly lower than that in Mock group(P<0.05);the expression of N-cadherin and Vimentin protein in C57 mice treated with HP microenvironment supernatant was significantly higher than that in Mock group(P<0.01),and E-ca-cadherin and Vimentin protein in treatment group were significantly higher than that in Mock group(P<0.01).The expression of dherin protein decreased significantly(P<0.05),the expression of Bax protein decreased significantly,and the expression of Bcl-2 protein increased significantly(P<0.05).Conclusion:HP-induced inflammatory supernatant promotes the occurrence of EMT in FHC cells depending on the NF-kappa B signaling pathway of U937 cells.These results provide a new theoretical basis for exploring the development of EMT in HP-induced inflammatory microenvironment supernatant.