Altered Expression Of Claudin 4 In The Patients With Gastro-esophageal Reflux Disease And The Study Of The Pathogenesis

Gastro-esophageal reflux disease (GERD), including non-erosive esophageal reflux disease (NERD), reflux esophagitis (RE) and Barrett Esophagus (BE) is a prevalent chronic disease, and could develop into lethal esophageal adenocarcinoma. As the pathogenesis remains unclear, which , to some extent, affects its diagnosis and therapy. Claudins (CLDNs), the recently identified multigene transmembrane protein family, are thought to be the major constituents of tight junctions (TJ), the most apical components of cell sealing complexes responsible for cell–cell adhesion, cell polarity, and control of paracellular ion transport. The expression of claudin 3 and 4 protein were not observed in the esophageal mucosal layers, but signifcantly increased in the epithelial cell of BE and tissue of esophageal adenocarcinoma. Although NERD and RE are the two main sub-types in our country, the changes of their expression and their impacting factors remain unclear, as well as the signal mechanism. Therefore, the following experiments were performed in this study.1. By the methods of RT-PCR, western blot and immunohistrochemistry, the expression of claudin 3 and 4 in normal esophageal, NERD, RE and BE were detected , by which we could explore their significance in the diagnosis and therapy of GERD.2. With the simultaneous ambulatory 24 hour oesophageal pH monitoring meter and 24h bilirubin monitor meter, pH value and bilirubin concentration in lumina of NERD and RE patients were mornitored. In the meanwhile, by the methods of RT-PCR, the mRNA expression of claudin 4 in the mucous membrane of esophageal was detected.3. By the method of MTT, the proliferation of human small intestine carcinoma cell line(HIC) was analyzed. The effects of hydrochloric acid, chenodeoxycholic acid(CDCA) and TNF-αon the cell line were evalued. Based on the above results, the suitable experimental parameters were chosen. The mRNA and protein expression of were investigated by the methods of real time PCR and Laser scanning confocal microscop(LSCM). By which, the effects of the injury factors such as gastric acid, CDCA and TNF-αon claudin 4 expression were investigated.4. With the drug tool of p38 kinase specificity antagonist--SB203580, the expression of claudin 4 protein was detected before and after p38 kinase signal activated, which could explore the role of p38 kinase signal in the gastric and bile acid induced claudin 4 protein expression.The main results were as followings:1. Results of 80 cases of GERD patients showed that claudin 3 did not or mildly express in the mucous membrane of normal esophagus, NERD and RE, but increased signifcantly in BE group.The mRNA and protein expression of claudin 4 was weak in normal esophageal group. While in NERD, RE and BE group, they increased gradually. The results of immunohistochemistry showed that both claudin 3 and claudin 4 positive stain was mainly in the cellular membrane of epithelial cells in NERD and RE group, while in the BE group, the positive stain both in the cellular membrane and cytoplasm.2. Results of monitoring gastric acid and bilirubin in 48 cases of GERD patients showed that there were no reflux in most NERD patients, while in RE group was mainly mixture reflux, and the percent of constitutent differed significantly. Significant differences were also observed between NERD and RE groups in the six parameters such as percent of total time the pH<4, percent supine time pH<4, No. of acid reflux episodes, DeMeester score, percent supine time the bilirubin absorbance level≥0.14 and number of bilirubin reflux episodes (p<0.05). The mRNA expression of claudin 4 in RE group was significantly higher than that in NERD group (p<0.05). The mRNA expressions of claudin 4 in the gastric acid reflux alone, bilirubin reflux alone and mixture reflux group were significantly higher than that in no reflux group, respectively (p<0.05), no difference between in the gastric acid reflux alone and in bilirubin reflux alone group. The expression was highest in the mixture reflux group, significantly higher than that in other group (p<0.05). The mRNA expression of claudin 4 in GERD patients was in correlation to percent supine time pH<4,No. of acid reflux episodes,DeMeester score, percent supine time the bilirubin absorbance level≥0.14 and number of bilirubin reflux episodes. 3. Results of MTT experiments showed that pH value ranged 5.5～7.4 and concertration of CDCA ranged between 50μM～200μM had no significant toxicity on the proliferation of HIC cells, which could be chosen as the suitable experimental parameters. Results of confocal showed that the mRNA and protein expression of claudin 4 increased with the time and concentrations of CDCA and hydrochloric acid. The increasing expression of claudin 4 protein distributed diffusely in the HIC cells in the presence of stimulation. No difference was observed in the mRNA and protein expression of claudin 4 after stimulation of TNF-αin different stimulus duration.4. P38 kinase could be actived by either acid hydroc, CDCA alone or the two combination in the HIC cell line. And the amount actived by CDCA with acid hydroc was significant higher than that actived by acid hydroc or CDCA alone. Meanwhile, the expression of claudin 4 inreased significantly. SB203580, as the p38 kinase signal specificity antagonist, inhibited the activation of p38 kinase and the expressions of claudin 4 induced by hydrochloric acid and bile acid alone or both.We could draw conclusions from the above results.1.The level of claudin 4 expression was closely associated with the severity of pathological changes in the mucous membranes. The high level of claudin 4 expression could be the new marker of the injury of tight conjunction in the mucous membrane. Be the new targets of diagnosis and therapy of BE, The specificity of claudin 4 in the diagnosing BE was lower than that of claudin 3.2. With the increasing of supine time and nubmer of reflux of acid and bilirubin ,RE would be more easily induced. And the effect of the combined stimulation was more obvious. Bile acid alone could induce high expression of claudin 4, therefore, which could be the independent factor of injurying esophageal mucous membrane. The synergism effect of gastric acid and bile acid on the claudin 4 expression and pathopoiesis was more obvious.3. With time and dose-dependent, bile acid and/or gastric acid could affect the expression of claudin 4 from the level of gene transcription and protein expression. And the effect of bile acid was more stronger than that of gastric acid, the combined reflux the strongest. Although TNF-αhad no effect on the expression of claudin 4, its role on the pathegensis of barrett esophageal need be further studied.4. Gastric acid and bile acid could induce the activation of p38 kinase of BE directly and then positively regulate the expression of claudin 4. Besides of p38 kinase signal, there would be other regulatory mechanisms involved in the increasing expression of claudin 4 protein.