The Sodium And Calcium Exchange Subtype 1 Mediated The Upregulation Of CDX2 To Promote The Development Of Barrett Esophagus

Objective:Barrett's esophagus?BE?is a pathological phenomenon in which squamous epithelium in the lower esophagus junction area is replaced by a single layer of columnar epithelium,with or without intestinal metaplasia.It is recognized as the most important precancerous lesion of esophageal adenocarcinoma.Studies have confirmed that bile reflux can upregulate the expression of intestinal differentiation marker tail-type homeobox transcription factor-2?CDX2?,thereby promoting the development of Barrett's esophagus,but the specific mechanism is unclear.Previous studies reported that the intracellular calcium increase in the normal esophageal epithelial cells after application of hydrochloric acid and deoxycholic acid.This result indicated that the ion channel plans an important role in the process,and the activition of intracellular calcium signaling involved in the regulation of CDX2.Preliminary experimental results have found that sodium-calcium exchanger subtype 1?NCX1?,which has bidirectional regulation of Ca²⁺,plays an important role in bile acid-stimulated intracellular calcium regulation.And some studies have reported that DCA membrane receptor TGR5 can activate ion channels to induce intracellular calcium signal transmission under DCA stimulation.The purpose of this study was to explore the mechanism of DCA-stimulated activation of TGR5 on NCX1 channels and the potential regulatory mechanism of activated NCX1-mediated intracellular calcium signal on intestinal metaplasia of esophageal cells.It is hoped to provide a new theoretical basis for the prevention and treatment of Barrett's esophagus with NCX1 as the target.Methods:?1?Immunohistochemistry and western blot to detection and comparison of the expression changes of TGR5,NCX1,p-P65 in human normal esophageal mucosa tissue and BE esophageal mucosa tissue,normal esophageal cell HEEC and esophageal adenine OE19,statistical analysis of the correlation of TGR5,NCX1,p-P65 expression levels.?2?The calcium ion imaging system is used to compare the difference of[Ca²⁺]cyt between normal esophageal cells and esophageal adenocarcinoma cells under DCA stimulation.Reveal the role of NCX1 from normal to the development of BE from a functional perspective.?3?Western bolt technology was used to detect and compare the differences in the downstream related signal pathways between normal esophageal cells and esophageal adenocarcinoma cells after NCX1 activation,and to screen the most important signal pathways and target genes.?4?Reversely verify the important role of NCX1 and its downstream regulatory pathways in promoting the development of BE through NCX1 overexpression and signaling pathway inhibitors.Results:?1??1?NCX1 is expressed in human normal esophageal mucosa tissue,BE esophageal mucosa tissue,normal esophageal epithelial cells,and esophageal adenocarcinoma cells,and its expression is significantly up-regulated in BE esophageal mucosa tissue?P<0.05?.The expression of NCX1 in esophageal adenocarcinoma cells was also significantly higher than that in normal esophageal cells?P<0.05?.?2?TGR5receptor protein is expressed in human normal esophageal mucosa tissue,BE esophageal mucosa tissue,normal esophageal epithelial cells,and esophageal adenocarcinoma cells,and its expression is significantly up-regulated in BE esophageal mucosa tissue?P<0.05?,It is mainly expressed in cell membrane and cytoplasm,and is up-regulated in esophageal adenocarcinoma cell lines,and there is significant difference in expression compared with normal esophageal cell lines?P<0.05?.?3?p-P65 protein was expressed in normal human esophageal mucosa tissue and BE esophageal mucosa tissue.Compared with normal esophageal mucosa tissue,p-P65 protein expression was significantly up-regulated in BE esophageal mucosa tissue?P<0.05?,and mainly expressed in the nucleus.?4?CDX2protein was expressed in BE esophageal mucosa tissue and esophageal adenocarcinoma cells?P<0.05?.?5?In BE tissues,TGR5 was positively correlated with NCX1 and CDX2expression?P<0.05?,positive correlation between levels of NCX1 and CDX2 in BE?P<0.05?,and p-P65 was positively correlated with CDX2 expression?P<0.05?.?2??1?In the presence of external Ca²⁺,[Ca²⁺]_cytyt in normal esophageal cells and esophageal adenocarcinoma cells increased significantly after DCA and PMA stimulation,and DCA stimulation was concentration-dependent.The increase rate was greater in esophageal adenocarcinoma cell lines?P<0.05?.?2?In the absence of external Ca²⁺,DCA and PMA stimulation did not cause changes in intracellular[Ca²⁺]cyt.?3?After being administered with store-operated calcium channel blockers 2-APB and SKF-96365 in OE19,[Ca²⁺]_cytyt change in OE19 induced by DCA stimulation were not affected.?4?In the presence of a specific sarcoplasmic reticulum Ca²⁺-ATPase inhibitor CPA,DCA can still induce an increase in intracellular[Ca²⁺]_cytyt after CPA-induced depletion of stored calcium.?5?After the blocking agents tiamterene,KB-R7943,and G?6976 treated esophageal cells,DCA or PMA-induced increase[Ca²⁺]_cytyt in esophageal cells was inhibited to varying degrees?P<0.05?.?6?In the absence of external Na⁺[Ca²⁺]_cytyt in normal esophageal cells and esophageal adenocarcinoma cells increased significantly after administered with LiCL PSS?P<0.05?,after treatment with KB-R7943,the calcium elevation was significantly suppressed?P<0.05?.Under the same stimulation,[Ca²⁺]_cytyt change induced by 0 mM Na⁺stimulation in OE19 cells were more significant than that in HEEC cells?P<0.05?.?3??1?DCA stimulation can enhance the expression of TGR5,NCX1,p-P65 and CDX2proteins in cells?P<0.05?.?2?The blocking agent tiamterene could reverse the expression of TGR5,NCX1 and CDX2 protein induced by DCA in esophageal cells?P<0.05?.?3?KB-R7943 significantly inhibited the expression of NCX1 and CDX2 proteins induced by DCA?P<0.05?.?4?BAPTA-AM can reverse the expression of NCX1 and CDX2 proteins induced by DCA in esophageal cells?P<0.05?.?5?SN50 inhibits the expression of p-P65protein induced by DCA in esophageal cells?P<0.05?.Conclusion:This study first confirmed the role and related mechanism of NCX1 in the occurrence and development of BE.DCA-induced NCX1 activation is mediated by TGR5/PLC/PKC signaling,which mediates the increase of calcium in esophageal adenocarcinoma cells,and thereby activates the downstream NF-kB?P65?signaling pathway to promote CDX2 expression in esophageal adenocarcinoma cells.Our results not only explain the new mechanism of bile acid triggering the occurrence and development of BE,but also provide a new theoretical basis for the prevention and treatment of Barrett's esophagus with TGR5 and NCX1 as targets.