ERK-MLCK Pathway Mediates The Mechanism Of Melatonin On The Tight Junction Of Esophageal Epithelium

Gastroesophageal reflux disease(GERD)refers to the disease that patients suffer from discomfort and(or)complications caused by the gastric reflux into the esophagus.The acid in gastric juice is regarded as a n important pathogenic factors of GERD.Esophageal mucosa l defense dysfunction is important in the pathogenesis of GERD.The esophageal mucosal barrier is mainly made up of the epithelial barrier,including epithelial membranes and tight junctions(TJ).The esophageal epithelium of patients with GERD is found to exist dilated intercellular spaces(DIS),which is considered to be the sign of esophageal epithelial barrier dysfunction.The transmembrane proteins(occludin,claudin)are fixed in cytoskeleton by scaffold protein ZO-1,forming "zipper" between adjacent cells.They constitute the basic structure of TJ,maintaining the integrity of the epithelial barrier.A large number of laboratory and clinical data suggest that the abnormal expression and distribution of TJ protein can be used as the objective indicators of GERD esophageal epithelial barrier dysfunction.However,there hasn’t yet been any research about the mechanism that the alteration of TJ proteins lead to the dysfunction of GERD esophageal epithelial barrier.Mitogen-activated protein kinases(MAPK)signal transduction pathway is an important signal system mediated biological reaction in eukaryotic cells.MAPK signaling system includes extracellular signal regulating kinase(ERK),c-Jun amino terminal kinase(JNK)and p38 MAPK.It is found that ERK,JNK and p38 MAPK can regulate the epithelial barrier in different types of tissues and cells.Among them,the most reports are about ERK.MAPK signaling molecules play important roles in regulating the epithelial barrier by influencing the expression and activity of MLCK.MLCK is a kind of calmodulin(Ca M)dependent kinase,which widely exists in the epithelial cells.MLCK contracts Adenosine Triphosphate(ATP)dependent actomyosin by phosphorylating myosin light chain(MLC).Cytoskeleton contracts and the distribution of TJ proteins are altered,these lead to DIS.This is considered to be the classical mechanism of the regulation of the epithelial barrier.MLCK can also regulate the expression of TJ proteins by influencing the concentration of intracellular Ca2+,then alter the epithelial barrier function.It’s reported that MAPK signaling molecules are mainly distributed in the cytoplasm of the resting cells.Under the appropriate stimulation,multiple members of MAPK family can be activated,then shift into the nucleus quickly and significantly.The activated MAPK exercise their cellular function by regulating the transcription of genes via activation of transcription factors.It was found in intestinal epithelial cells that ERK activation can prompt Elk-1(the downstream transcription factor of ERK1/2)activation and shift into the nucleus.This can trigger MLCK gene activation,m RNA transcription and protein synthesis,resulting in the dysfunction of epithelial barrier.But so far,there hasn’t been any report about the regulatory mechanism of the esophageal epithelial barrier.At present,proton pump inhibitors(PPIs)is considered to be the first-line therapy drugs for GERD because of its strong inhibitory effect of gastric acid.Due to the characteristics of GERD symptoms,which is chronic and relapsing,patients often need long-term use of PPI.However,a series of security problems are caused by the long-term use of PPI.In 2006,oral MLT was reported to relieve GERD symptoms in patients without side effects.In recent years,there are growing numbers of studies focus on the esophageal mucosal protective effect of MLT.A lot of experimental data support the view that MLT has a protective effect on GERD/RE esophageal mucosa.MLT,a kind of neuroendocrine hormone,has complex biological effects: regulation of sleep cycles,circadian rhythm,reproductive rhythm,blood pressure fluctuations,immune system activity;removal of oxygen free radicals;inhibition of tumor growth,etc.Exogenous MLT can also protect the digestive tract mucosa through a variety of ways.In a number of studies about retinal epithelial cells,renal tubular epithelial cells,intestinal epithelial cells and human umbilical vein endothelial cells,MLT was found to regulate TJ via adjusting the cytoskeleton,thus play a protective role on the epithelial barrier.MLT can also inhibit the expression and activity of MLCK up-regulated by MAPK signal transduction pathway,then protect human umbilical vein endothelial barrier.Besides,MLT can specifically interact with calmodulin(Ca M)and PKC,then inhibit the activity of MLCK;this is also considered as the mechanism by which MLT regulate the cytoskeleton and protect the epithelial barrier.Nowadays,more and more studies have paid attention to the protective role of MLT on GERD esophageal mucosa,however,the most of them has focused on the anti-inflammatory and antioxidant properties of MLT.As the defense function of esophageal mucosa can be reflected by the esophageal epithelial barrier,it’s of great significance to explore the mechanism that MLT effects on the esophageal epithelial barrier.The current research may offer an new perspective in GERD therapy,and lay a theoretical basis for the clinical application of MLT on GERD.Part 1 Dilated intercellular spaces and tight junction proteins in GERD esophageal epitheliumObjective: To explore the mechanisms that intercellular spaces dilated and tight junction proteins altered in GERD esophageal epithelium.Methods: According to the inclusion/exclusion criteria,patients were divided into control group,NERD group and RE group.The esophageal mucosal tissue,3-5cm above the dentate line and without erosion,were clipped by the disposable biopsy forceps and then preserved separately in liquid nitrogen,4% paraformaldehyde and 3% glutaraldehyde.Transmission electron microscope was used to observe DIS.The expression of TJ proteins were examined by immunohistochemical staining(IHC),real-time PCR and western blot.The expression and activity of MLCK,as well as the expression of MAPK molecules,were also examined by western blot.Results: DIS occurred in the esophageal epithelium of NERD and RE patients,and mainly exist in prickle cell layer.Compared with the control group,the expression of TJ proteins were significantly reduced in NERD and RE patients.Immunohistochemistry showed that the continuous expression of TJ proteins near the cell membrane were destroyed in N ERD and RE,and in some esophageal epithelial cells,the expression of TJ proteins were lost.In addition,the expression and activity of MLCK were significantly up-regulated in N ERD and RE.Among MAPK signaling molecules,the phosphorylation level of ERK1/2 raised significantly,while no obviously change happened in JNK and p38 MAPK.Correlation analysis showed that DIS was negatively correlated with TJ protein expression level,and positively correlated with the expression and activity of MLCK.Conclusion: The alteration of TJ proteins,MLCK and ERK1/2 may participate in the damage process of esophageal epithelial barrier in GERD.Part 2 Acid damage the esophageal epithelial tight junctions through ERK-MLCK pathwayObjective: To explore the mechanisms that acid effects on the esophageal epithelial tight junctions.Methods: The normal esophageal epithelial cells(Het-1A)were used in the present experiments.(1)Trypan blue was used to count the vitality of Het-1A cells under the different acid condition for different time.The epithelial resistance meter and fluorescence spectrophotometer were used respectively to detect trans-epithelium electrical resistant(TER)and the permeability of FITC-dextran in Het-1A monolayer.(2)After the treatment of acid for 1h and 6h,the expression and distribution of TJ proteins were detected by using western blot,real-time PCR and laser confocal immunofluorescence method.(3)Pretreat Het-1A cells with specific ERK inhibitors(PD98059)and MLCK inhibitors(ML-7),then expose Het-1A cells to acid(p H4.0)for 1h.The phosphorylation of ERK1/2 and expression/activity of MLCK were examined by western blot and real-time PCR.(4)Silence the expression of ERK1/2 by using si ERK1/2,then examine the effect of si ERK1/2 on MLCK by western blot and real-time PCR.Silence the expression of MLCK by using si MLCK,then examine the expression/distribution of TJ proteins and the barrier function of Het-1A by western blot,real-time PCR,laser confocal immunofluorescence,TER and the permeability of FITC-dextran.(5)Extract the nucleus proteins,then examine the activity of transcription factors by western blot.(6)Silence the transcription factors activated by acid,then examine the expression of MLCK by western blot and real-time PCR.(7)Pretreat Het-1A cells with PD98059,then expose the cells to acid(p H4.0),examine the effect of acid on the transcription factors by western blot.Results:(1)After treatment with acid(p H4.0)for 1h,TER of Het-1A monolayer was significantly reduced,while the permeability of FITC-dextran increased obviously.(2)The expression and distribution of TJ proteins were altered by acid.The dense,uniform,continuous expression of TJ proteins near the cell membrane turned out to be the cytoplasm expression,as well as a small amount and discontinuous membrane expression.(3)PD98059 can significantly inhibit the activation effect of acid on ERK1/2.ML-7 inhibited the activity of MLCK up-regulated by acid,without influence on the expression of MLCK.PD98059 could inhibit the increasing expression and activity of MLCK caused by acid.(4)Si ERK1/2 could significantly inhibit the up-regulation of MLCK caused by acid.Si MLCK could improve the effect of acid on the expression and distribution of TJ proteins,as well as the barrier function of Het-1A.(5)Acid could activate the transcription factors(Elk-1 and NF-κB)in Het-1A cells.(6)The increasing expression of MLCK caused by acid could be significantly inhibited by si Elk-1,but no obvious influence was noticed with si NF-κB.(7)The activity of Elk-1 was down-regulated when ERK1/2 was inhibited.Conclusion:(1)Acid damages the esophageal epithelial tight junctions through altering the expression and distribution of TJ proteins.(2)The mechanism that acid damage the esophageal epithelial tight junctions may be the activation of ERK1/2-Elk-1-MLCK signal transduction pathway.Part 3 MLT protect the esophageal epithelial tight junctions through ERK-MLCK pathwayObjective: To study the mechanism that MLT effect on the esophageal epithelial tight junctions.Methods:(1)Before treatment with acid,pretreat Het-1A monolayer with MLT(0.1 μM、1 μM、10 μM 和 20 μM)for different time(2h、4h、6h、8h、12h),thenexamine TER and the permeability of FITC-dextran.(2)Het-1A cells were divided into 4 groups: control,MLT,acid and MLT+acid.The expression and distribution of TJ proteins were examined by western blot,real-time PCR and laser confocal immunofluorescence.(3)Het-1A cells were divided into 5 groups: control,acid,PD+acid,MLT+acid and MLT+PD+acid.Western blot was used to examine the phosphorylation of ERK1/2,the expression/activity of MLCK and the expression of Elk-1.Results:(1)MLT could improve the effect of acid on TER and the permeability of FITC-dextran in Het-1A monolayer.(2)MLT could improve the alteration of TJ proteins caused by acid.(3)In the PD-acid-treated,MLT-acid-treated and MLT-PD-acid-treated Het-1A cells,the increasing expression and activity of MLCK caused by acid were obviously inhibited,as well as the activation of ERK1/2 and Elk-1.No significant difference was found between the three groups.Conclusion: MLT protects the esophageal epithelial tight junctions against acid through regulating the expression and distribution of TJ proteins via ERK1/2-Elk-1-MLCK signal transduction pathway.