The Role Of P38 MAPK In The Progression Of Gastroesophageal Reflux Diseases

Background and aim:Gastroesophageal reflux diseases(GERD) is one of the common gastrointestinal diseases that are increasingly recognized as a significant health problem with extensive global, social and economic impacts. RE is an inflammation of the lower esophagus due to the regurgitation of gastric acid, characterized by a burning pain in the chest (so-called heartburn) and regurgitation following eating. Since gastric acid plays a key role in the pathogenesis of reflux esophagitis, luminal pH control, including histamine type 2 antagonists and proton pump inhibitors, is considered of importance in the treatment of this disease. However, in spite of their marked therapeutic effect, a number of patients have suffered from incidences of relapse and shown incomplete mucosal healing, continued symptoms and complications. Therefore, the exact pathophysiological mechanisms of esophageal mucosal damage during gastro-esophageal reflux are not fully explained by acid reflux alone.The p38 mitogen-activated protein kinase (MAPK) pathway is activated in response to cell stress (e.g., ionizing irradiation, chemotherapeutic drugs, or osmotic stress) and inflammatory insults (e.g., Toll-like receptor ligands or cytokines). It was found that acidic deoxycholic acid, chenodeoxycholic acid, and bile acids induced activation of p38 MAPK in human esophageal epithelial cells. The esophagoduodenostomy (a model of duodenoesophageal reflux) in rats led to activation of p38 MAPK in esophageal samples. Recently, it was found that activity of p38 MAPK was higher in the esophageal epithelium of patients with Barrett’s esophagus than that in normal esophageal mucosa.This study was designed to investigate the role of p38 MAPK in human esophageal epithelial cells exposed to acid or trypsin in vitro, and in experimental chronic RE, induced by fundus ligation and partial obstruction of the pylorus in rats in vivo.Methods:Part 1:Human esophageal squamous epithelial cells were exposed pH 5.0,6.0, and 7.3 (Control) for 15 min. The p38 MAPK phosphorylation was determined by Western blotting analysis. Human esophageal squamous epithelial cells were pretreated with SB203580 for 1 hour, and then were exposed 7 times to pH 5 for 15 min over a 48-hours period. The mRNA levels of pro-inflammatory cytokines including IL-8, COX2, TNF-alpha were determined by RT-PCR method. The iNOS protein expression and NO content were determined by Western blotting analysis and Griess Method, respectively. The mRNA levels of tight junction proteins including claudin-1, occludin, and ZO-1 were determined by RT-PCR method.Part 2:Human esophageal squamous epithelial cells were exposed to trypsin (20,40, and 80μg/ml) for 4 hours. The p38 MAPK phosphorylation was determined by Western blotting analysis. Human esophageal squamous epithelial cells were pretreated with SB203580 for 1 hour, and then were exposed 3 times to trypsin for 4 hours over a 48-hours period. The mRNA levels of pro-inflammatory cytokines including IL-8, COX2, TNF-alpha were determined by RT-PCR method. The iNOS protein expression and NO content were determined by Western blotting analysis and Griess Method, respectively. The mRNA levels of tight junction proteins including claudin-1, occludin, and ZO-1 were determined by RT-PCR method.Part 3:Rats were divided into two groups including:sham-operated rats and RE rats.14 days post surgery, esophagus were removed for Immunohistochemical staining and Western blotting analysis for p38 MAPK and phosphorylated p38 MAPK.Experiment 2:rats were divided into three groups as follows:sham-operated rats, RE rats, and RE rats treated with SB203580. The SB203580-treated group rats were then treated with SB203580 via intraperitoneal injection (1 mg/kg/day). An equal volume of saline was injected as a vehicle control in sham-operated rats and RE rats.14 days later, the esophagus were collected for histological and biochemical detection.Results:Part 1:The p38 MAPK phosphorylation was enhanced in human esophageal squamous epithelial cells when exposed to acid stimulation. Pretreatment with SB203580 attenuated up-regulation of proinflammatory cytokines including IL-8, COX2, TNF-α, enhancement of iNOS expression and NO production, and down-regulation of tight junction proteins including claudin-1, occludin, and ZO-1 induced by acid exposure.Part 2:The p38 MAPK phosphorylation was enhanced in human esophageal squamous epithelial cells when exposed to trypsin stimulation. Pretreatment with SB203580 attenuated up-regulation of proinflammatory cytokines including IL-8, COX2, TNF-α, enhancement of iNOS expression and NO production, and down-regulation of tight junction proteins including claudin-1 and ZO-1 induced by trypsin stimulation.Part 3:Immunohistochemical staining and Western blotting results revealed the activation of p38 MAPK signaling in the esophagus mucosa 14 days post injury. Through gross and histological assessment, we found that inhibition of p38 MAPK activation by SB203580 attenuated esophageal mucosal damage in RE rats. Inhibition of p38 MAPK activation in RE rats attenuated esophageal barrier dysfunction, through enhancing expression of tight junction proteins and reducing expression of matrix matalloproteinases-3 and -9. Inhibition of p38 MAPK activation in RE rats reduced CD68-positive cells in esophagus mucosa and mRNA levels of TNFa, IL-6, and IL-1β in esophagus and protein levels of TNFa, IL-6, and IL-1β in serum. In addition, we found that inhibition of p38 MAPK activation in RE rats suppressed protein expression of inducible nitric oxide synthase and reduced formation of nitric oxide (NO) and 3-nitrotyrosin in esophagus.Conclusion:Inhibition of p38 MAPK activation attenuated acid or trypsin induced injury in esophageal squamous epithelial cells in vitro, and esophageal mucosal damage in RE rats in vivo, possibly by modulating esophageal barrier function and suppressing inflammation, and formation of cytokines and NO.