Mechanism And Effects Of Mesenchymal Stem Cells Affected By H. Pylori On Gastric Epithelial Cells

Objective Mesenchymal stem cells (MSCs) are multipotent adult stem cells that have been found in multiple tissues, they have another unique characteristic (tropism migration) which facilitates migration to damaged tissue sites with inflammation or wounds. Helicobacter pylori (H. pylori) is a main pathogenic factor resulting in active chronic gastritis and peptic ulcer, and is intimately associated with gastric carcinoma. Helicobacter-induced epithelial responses and damage is able to activate the migration properties of MSCs and be directly responsible for MSC recruitment and homing in the gastric mucosa. Therefore, MSCs, as a part of mesenchymal stromal cells, may be one of the important components in chronic gastritis microenvironment induced by H. pylori, however, little is known about the mechanism and means by which MSCs play a critical role(s) in gastric mucosal lesions or cancer initiation and progression induced by H. pylori so far. The aim of this study is to explore the effect of H. pylori on MSCs and the role of MSCs in the mechanism of gastric mucosal lesions or cancer initiation and progression mediated by H. pylori. This work will provide a new basis for intervention, diagnosis and treatment of the initiation, progression of gastric cancers infected with H. pylori.Methods Human umbilical cord MSCs (hucMSCs) were co-cultured with the H. pylori strain11673(multiplicity of infection (MOI)=100:1) for24h in vitro infection model. The morphology of cells was observed by invert phase contrast microscope. Cells were collected and prepared for Western blot and quantitative RT-PCR analysis, the culture supernatant were harvested, centrifuged for5minutes at3000rpm, and then, stored at-80℃until use. Three duplicate wells were prepared for each experimental condition. The expression of FAP, a-SMA, N-cadherin and Vimentin at both mRNA and protein levels in the infected hucMSCs with H. pylori were determined by Real-time RT-PCR and Western blot assay, respectively. The concentration of cytokines in the supernatant derived from the infected hucMSCs with H. pylori were detected by Luminex or ELISA. To investigate whether nuclear factor kappa B (NF-kB) is involved in hucMSCs underwent CAF-like myofibroblastic phenotype differentiation in response to H. pylori infection and in H.pylar-induced cytokines expression in hucMSCs. Western Blot was used to detect the level and activity of NF-kB p65and degradation of NF-kB inhibitory protein IkB-α using the specific antibodies. The NF-kB inhibitor, PDTC, was used for inhibitory assays in order to demonstrate the activation and effect of NF-kB in H. pylori induced hucMSCs into CAFs-like myofibroblastic phenotype and cytokines expression in the infected hucMSCs. HucMSCs were cultured in DMEM medium containing PDTC, and then were co-cultured with the H. pylori strain11673(MOI=100:1) in vitro infection model for24h. Human immortalized gastric epithelial cell line (GES-1cells) was exposed to normal medium RPMI-1640, hucMSCs conditioned medium (CM) and CM from the hucMSCs induced by H. pylori for48hours, respectively. The morphological changes of cells were observed by invert phase contrast microscope, and then cells were collected and prepared for Western Blot and quantitative PCR analysis. We detected the EMT-gene expression of E-cadherin, N-cadherin and Vimentin at both mRNA and protein levels in the treated gastric epithelial cells with three types of conditioned medium. Transwell invasion assay was used to investigate the effect of the CM derived from the infected hucMSCs by H. pylori on gastric epithelial cells invasion. Cell proliferation was assessed by using chromogenic methylthiazol tetrazolium bromide (MTT) dye and cell count, respectively. Plate clone forming assay for cell colony formation ability was performed to determine self-renewal ability in the cultured gastric epithelial cells with three types of CM. Flow cytometry assay for apoptosis in gastric epithelial cells and Western blot assay for apoptosis related proteins in the above GES-1cells. Several stem cell associated protein including Sox2, Nanog and BMI in GES-1cells treated with several CM above mentioned were detected by Western blot assay, and then, the expression of oncogene-associated proteins and tumor suppression gene-associated proteins in the treated GES-1cells with CM derived from the infected hucMSCs. The pretreated hucMSCs with PDTC were co-cultured with H. pylori strain11673(MOI=100:1) in vitro for24h, and then, the culture supernatant were harvested. The conditioned medium was co-culture with GES-1for48hours, which was set as the experimental group and the normal medium and H. pylori were used as control. All data were expressed as means±SD. SPSS software was used to analyze the data. The means of different treatment groups were compared by two-way ANOVA test or Student’s t-test. Statistical significance was assumed at a P value of less than0.05.Results HucMSCs acquire more typical spindle-shape phenotype of CAF after24hours co-cultured with H. pylori. Quantitative RT-PCR analyses showed that H. pylori promoted the gene expression of CAF markers Vimentin, N-cadherin, a-SMA and FAP in hucMSCs, In agreement with the mRNA expression, FAP, a-SMA, N-cadherin, and Vimentin protein levels were also increased in hucMSCs upon exposure to H. pylori. H. pylori can promote lots of cytokines secretion in the hucMSCs. Compared with the untreated hucMSCs, the level of NF-kB p65in the nucleus of infected hucMSCs by H. pylori was significantly increased, but was significantly decreased in cytoplasm. The level of phospho-NF-KB p65in the infected hucMSCs was also significantly increased, but IkB-α level was markedly decreased. PDTC (10μM) could inhibit lots of cytokines expression and the gene expression of CAFs markers Vimentin、N-cadherin、a-SMA and FAP in the treated hucMSCs with H. Pylori, these results indicate that hucMSCs undergo CAF-like myofibroblastic phenotype differentiation in response to H. pylori infection, some of which were mediated by NF-kB/IkB signal pathway; Gastric epithelial cells also acquire more typical spindle-shape phenotype of MSCs after48hours co-cultured with CM derived from the infected by H. pylori. In the present study, we found that the infected MSCs can upregulate the expression of the mesenchymal markers Vimentin and N-cadherin and suppress the expression of the epithelial marker E-cadherin in gastric epithelial cells. The results showed the infected MSCs by H. pylori damages gastric epithelial cells via converting gastric epithelial cells into EMT. The mesenchymal-like cells from gastric epithelial cells treated with the infected MSCs by H. pylori expressed significantly higher levels of stem cell markers, suck as Nanog, BMI and Sox2. Our findings showed that gastric epithelial cells might acquire more stem-like properties during EMT. The results also showed that the infected hucMSCs can promote and increase the expression of oncogene-associated proteins MUC2, anti-apoptotic protein Bcl-2, and inhibit the expression of tumor suppression genes-associated proteins p53, p21, a pro-apoptotic effector Bax in gastric epithelial cells via EMT in vitro. The infected MSCs by H. pylori also remarkably enhanced gastric epithelial cells colony formation ability and invasive ability. The gastric epithelial cells treated with CM derived from the infected MSCs by H. pylori also acquired some specific biological property that tumor cells possessed via EMT, whch plays an important role(s) in the initiation and progression of malignancies. Western Blot analyses showed that CM derived from PDTC-pretreated hucMSCs induced by H. pylori inhibited the expression of N-cadherin and Vimentin at protein levels in gastric epithelial cells, but expression of E-cadherin at protein levels was increased compared with the infected hucMSCs by H. pylori.Conclusions Our results indicate that H. pylori can induce MSCs to differentiate into CAF-like phenotype, to secrete some cytokines and become part of the chronic gastritis microenviroment infected with H. pylori. The infected MSCs destroys cell junctions, promotes cell invasive ability, enhances colony forming ability, act as a trigger to develop sternness capacity of cell and inhibits cell proliferation via converting gastric epithelial cells into EMT. Our findings suggests that H. pylori might contribute to progression and lesion/malignancy of gastric epithelial cells by converting MSCs into CAF-like myofibroblastic phenotype within gastritis infected with H. pylori, which might play an important role in the genesis, development and metastasis of gastric carcinoma, some of which are NF-kB dependent. This work paves the way to a better understanding of the role and mechanism of MSCs in the initiation and progression of gastric cancers infected with H. pylori, which will provide a new potential target for initiation, metastasis and clinical treatment to H. pylori mediated Cancer.