| Gastro-esophageal reflux disease(GERD) is a common condition that impacts many.millions of people worldwide,and is gaining more clinical recognition as a gastrointestinal(GI) disease.Lifestyle factors,such as obesity,high-fat diet,and physical inactivity,boost the incidence of GERD[1].GERD occurs when stomach and duodenal contents reflux into the esophagus.Reflux-induced damage to and inflammation in the esophageal mucosa are referred to as reflux esophagitis(RE).The pathogenic process of RE is highly complex.However,the current consensus among scientists is that RE mainly results from damage induced by reflux of the defending esophageal mucosa system.An imbalance between offensive and defensive factors in the esophagus leads to the development of RE.The epithelial defense system consists of three parts:pre-epithelial defense,epithelial defense and post-epithelial defense.In 2002,the German cellular biologist L.Langbein[14]reported that in addition to desmosomes,TJs could be found in the esophageal epithelium;and both were involved in maintaining the cellular barrier.Since then,tight junctions and desmosomes are considered to be important contributors to the epithelial defense of esophageal mucosa.Other contributors include intercellular lipids,mucin and epithelial transportation system(such as Na-H exchanger and Na-dependent Cl-HCO3 exchanger).Recent studies showed that TJs created a circular substance-blocking barrier between adjacent cells.TJs also function in cell adhesion,cell polarity and permeability,and in signal transduction resulting in cell proliferation and differentiation. Intercellular TJs consist mainly of claudins,occludins,JAM and ZOs(Figure 1).In 2003,Calabrese et al.[15]discovered in their studies that the intercellular space of the lower esophageal epithelium in patients with GERD was three times as wide as that in normal controls,and suggested 0.74μm as the threshold between normal and abnormal intercellular space.In 2005,Daisuke Asaoka[16.17]reported for the first time that expression of TJ proteins was altered during esophagitis;the expression level of claudin-3 decreased while that of claudin-1 increased.Such changes have a determinant effect on the permeability of the esophageal epithelium.Therefore,further investigtion warrants experiments testing the hypothesis that altered expression of TJ proteins and desmosome proteins results in failure of TJs and desmosomes,and thus leads to widened intercellular space and contributes to RE.In the investigation into reflux esophagitis,the use of human samples has methodological limitations.Hence, we created animl models of RE in rats,and sought to analyze the changes in intercellular space,changes in distribution and expression of TJ proteins and desmosome proteins and thier correlation with the expression level of IL-6 in the pathogenic process of RE.Moreover,some rats were treated with aluminum phosphate to determine its efficacy in treating RE.To explorer the role of tight junction(TJ) and desmosome proteins in the pathogenesis of RE,we established three animal models of RE on the Wistar rat background.We performed three types of surgical procedures on these rats,and measured the distributions and expression profiles of claudin 1,occludin, ZO-1,JAM-1,DSG-1 and IL-6 in the esophageal mucosa of the various RE rats, untreated and treated with aluminum-phosphate.220 8-week-old male Wistar rats were randomly divided into 4 groups.For control group(10 rats),rats underwent laparotomy without any further operation.To establish animal models of acid reflux esophagitis,alkaline reflux esophagitis and mixed reflux esophagitis,rats in group A,B and C were treated with partial pyloric ligation+cardiomyotomy,total gastroetomy + end-to-side esophago-duodenal anastomosis and cardiotomy + cardia ligation + end-to-side esophago-duodenal anastomosis,respectively.At day 3,6,9 and 14 after the procedures,part of the rats were executed and middle-low esophagi of these rats were dissected for evaluation of the success rates.Severity of the damage in esophagi was examined according to diagnostic criteria of RE by Chinese Medical Association Digestive Endoscopy Society in 1999.At 14 day after the procedure,the rest of the rats started aluminum-phosphate treatment,0.5g twice a day for 15 successive days.At day 30 or day 60 after treatment started,they were executed.2.Transmission electron microscope(TEM) examinationAt days 30 or 60 following aluminum-phosphate treatment initiation,the rats were sacrificed and a transmission electron microscope(TEM;2600 J EME2000X,Hitachi) was used to observe the morphological features of tight junctions and desmosomes in the esophageal epithelium.Samples were obtained from the lower esophagus at 5 cm from the lower end,fixed in glutaraldehyde solution at 4℃,and then clarified, dehydrated,embedded and sectioned into ultra-thin slices.Ten slices,prepared with samples obtained from different sites of the same esophagus,were selected for each esophagus and then observed and pictured under TEM.The LEICA image analyzing system was used to measure the intercellular space and calculate the number of desmosomes.Ten pictures were taken for each sample.In each picture,an intact cell was selected and an intercellular space was calculated by detecting the vertical distance between the selected cell and its adjacent cells at 10 randomly selected directions,with each direction at least 1.0 um apart.100 intercellular spaces were measured in 10 pictures and average width of intercellular space was calculated.We counted the number of desmosomes in the intercellular spaces of the 10 selected cells,and summed and averaged these values.3.Gross and microscopic observations for evaluating the damage severity in the esophagusAt post-procedure days 3,6,9 and 14,the lower esophagi were dissected for mucosal damage evaluation based on the Los Angeles Classification of Erosive Esophagitis(1994),and epithelial thickness was measured and evaluated as an indication of damage.Esophageal tissues were then fixed in 10%buffered-formalin, shaped into 4 mm tissue masses,dehydrated with gradient ethanol,clarified with xylene,embedded in paraffin and sectioned into 4 um slices,which were then stained with hematoxyline-eosine(HE) solution.The stained slices were then observed under the optical microscope,and a diagnosis was made based on the pathological features according to diagnostic criteria of RE by Chinese Medical Association Digestive Endoscopic Society(1999)revealed the incidence of RE in these animal models4 Immunohistochemical assayImmunohistochemical methods.were used to measure the expression levels of claudin 1,occludin,ZO-1,JAM-1,DSG-1 and IL-6.For these assays,the middle-lower esophagus was obtained and fixed in 10%buffered-formalin,shaped into 4.0 mm tissue masses,dehydrated with gradient ethanol,clarified with xylene,embedded in paraffin and sectioned into 4.0 um slices,which were then dewaxed with xylene,debenzolized with anhydrous ethanol and dehydrated with gradient ethanol,and then treated with 3.0%hydrogen peroxide and rinsed with water.After being restored at high pressure and high temperature,blocking agent(10%goat normal serum) was added to dissolve the slices at room temperature.Thirty min later,rabbit anti-IL-6,claudin 1,ZO-1, JAM-1 and DSG-1 antibodies were added at dilution of 1:80,and goat anti-occludin antibody at 1:100 dilution at 4℃.The slices were then incubated overnight and washed with phosphate-buffered saline(PBS) 3X,10 minutes each,the next day.At room temperature,general secondary antibody was added,and the slices were incubated at 37oC for 30 minutes.The slices were washed with PBS 3X-,for 10 min each time. Streptavidin-peroxidase(SP) was added at 37 oC.Thirty minutes later,the slices were washed again with 3X PBS for 10 min each time.The slices were then stained with 3,3-Diaminobenzidine(DAB) for 5 min,rinsed with water,and then stained with hematoxylene for another 5 min and rinsed with water again.The slices were then treated with acidic ethanol,and washed with running water for 30 min,dehydrated in gradient ethanol,and clarified with xylene,and sealed with gum.These slices of mucosa of RE were observed under the microscope for the measurement of expression of IL-6,claudin 1,occludin,ZO-1,JAM-1 and DSG-15.Western BlotWestern blots were used to measure the expression levels of claudin 1,occludin, ZO-1,JAM-1,DSG-1 and IL-6(all from Santa Cruz Biotechnology,Santa Cruz,CA). For these assays,the esophageal tissue(100 mg) was obtained and added to 1.0 ml of cell lysis buffer[Tris/HCL(100 mmol/L,Ph7.5),NaCl(100 mmol/L),0.5%sodium deoxycholate,ethylene diamine tetra-acetate(EDTA,1 mmol/L),1%NP40,0.1% sodium dodecyl sulfate(SDS) and protease inhibitor].Total protein was extracted,and the concentration was measured by the Lowry method.The extracted protein(50μg) was added to 15%SDS-polyacrylamide gel(SDS-PAGE) for electrophoresis(100V for 1.5 hours).When the bromophenol blue reached the bottom of the gel,the protein was blotted onto a nitrocellulose membrane.The blots were immersed in 5.0%milk (prepared with milk powder) for 2 hours.Then at 4℃,rabbit anti- IL-6,claudin 1, ZO-1,JAM-1 and DSG-1 antibodies at dilution of 1:80,and goat anti-occludin antibody at dilution of 1:100 were added to the blots,which were then incubated overnight.The next day,the membrane was washed,and the corresponding alkaline phosphate(AP)-labeled secondary antibodies were added at room temperature.The membrane was then stained with O-Dianisidine Tetrazotized andμ-Naphthyl acid phosphate,and analyzed with an electrophoresis gel imaging system.Using the grey scale measurement of the electrophoresis gel imaging system,the expression levels of IL-6,claudin 1,occludin,ZO-1,JAM-1 and DSG-1 were calculated and expressed as percentages relative to the(β-actin control(absorbance of these proteins/absorbance ofβ-actin).The ata were reported as x±s.6.RT-PCRRT-PCR was employed for cognate mRNA detection and quantitation.Total RNA was extracted with a Trizol Kit,(Invitrogen,Carlsbad,CA),measured,and verified - with an ultra-violet spectrophotometer.Primers were designed by Primer Premier 5.0 (Premier Biosoft International,Palo Alto,CA) according to mRNA sequences(by GenBank) of IL-6,claudin 1,occludin,ZO-1,JAM-1,DSG-1 and P-actin(as control). The primer sequences are shown in Table 1.RT-PCR was performed using the Takara RNA LA PCR kit(Takara,Kyoto,Japan) according to the manufacturers' instructions. RT-PCR was catalyzed under the following temperatures:5 min at 94oC;35 cycle of 30s at 94 oC,30s at 59 oC and lmin at 72 oC;finally 5 min at 72 oC.5μg of the PCR product was added to 2μl loading buffer,and electrophoresed in a 1.5%agarose gel at 100V for 1.5 hours.Analysis was carried out using a gel imaging system(Glyko, Novato,CA,USA).7 Statistical analysisData were processed with a statistical package SPSS 10.0(SPSS incorporated, Chicago) The means between two groups were compared for differences using the student's t-test;comparison of the ratio between two samples was tested with the chi-square test.A 95%confidence interval for the ave3rage weight of rats was calculated. In the analysis of correlation between expression of proteins,differences were considered significant at the P<0.05 level.1.Observation on experimental animalsAt post-procedure day 14,the RE model was established in all group A-,B- and C-sacrificed rats.By gross and microscopic observation,we ascertained that the mucosa was damaged and thickened as the disease progressed.Figure 2 helps illustrate the establishment of animal models with the three modified procedures.In the modified RE models,the survival of group A was 50%at day 14 after procedures(n=35/70); 90%(n=63/70) for group B,and 70%(n=49/70) for group C.At 14 day after the procedures,the RE models were established in all the rats(n=39/39).At day 3,6 and 9 after the procedures,the average weight of rats in the model groups continuously decreased as compared to the control group.In aluminum phosphate treated rats,the average weight -returned as treatment started and was comparable to the control group at one month afterward.2.Gross observation and histological assessmentA normal esophagus and RE-affected esophagus obtained within 2 weeks after the procedures are shown in Figure 5.In RE models,erosions of various sizes could be seen in the mucosa,while no erosion was detected in the control group.In most cases, esophagitis occurred in the middle-low part of esophagus.Histologically,the normal esophagus revealed a thin epithelial layer with squamous cells.In experimental RE models,the epithelium was markedly thickened and the lamina propria papillae were elongated into the epithelium.Basal cell hyperplasia and inflammatory cell infiltration was prominent in the lamina propria.And the thickness of the esophageal epithelium increased with time in RE models.In the control group,no expression of IL-6 was detected;claudin-1,occluding,ZO-1 and JAM-1 could be seen on the cellular membrane of the esophageal epithelial cells,mostly in the spinous and granular layers,while DSA-1 and JAM-1 could be seen on the cellular membrane of all mucosal layers.Among these proteins,DSG-1 had the highest expression level.In model rats, expression of IL-6 was only seen in inflammatory cells;the expression levels of claudin-1,occludin,ZO-1,JAM-1 and DSG-1 increased in both the cellular membrane and cytoplasm of spinous and granular layers in the mucosa around erosions,and these proteins were seen in cytoplasm as well as in membrane.However,as hyperplasia continued the in basal cells,expression of claudin-1,occludin,ZO-1,JAM-1 and DSG-1 decreased in individual cells of the spinous and granular layers.One month after treatment started,the histological changes of RE disappeared in acid reflux esophagitis rats,with comparable expression levels of these proteins to the control group.Treatment was not effective for alkaline reflux esophagitis rats,in which hyperplasia and elevated expression of IL-6 continued and vesicles could be seen.But expression of these proteins was significantly lower in individual cells as compared to control group3.Changes in ultra-micro structures under transmission electron microscope(TEM)As shown with TEM,the nucleus and cellular membrane were generally intact. However,a swollen cellular membrane within the intercellular space was noted; intercellular space was significantly wider in some cells,with fewer or even no desmosomes in these widened spaces.The number of desmosomes per unit area decreased as well.In RE rats,the width of the intercellular space was(2.39±0.42)μm. As for the control rats,the width was(0.63±0.21)μm,which was significantly smaller as compared to that in the RE rats(P<0.05).The average number of desmosomes was (0.124±0.044) /μm2,which was also significantly smaller than that in the control group[(0.221±0.031) /μm2](P<0.05)).4.Western blot and RT-PCRWhen compared to that in the control group,The Western blots revealed that the expression levels of IL-6,claudin-1,occludin,ZO-1,JAM-1 and DSG-1 increased with time in the RE groups.In the aluminum-phosphate-treated acid RE rats,the expression levels of claudin-1,occludin,ZO-1,JAM-1 and DSG-1 gradually decreased as treatment started and reverted to normal levels 30 days after treatment initiation.As for the mixed RE rats,the expression levels of IL-6,claudin-1,occludin,ZO-1,JAM-1 and DSG-1 returned to normal within 60 days after treatment initiation,while the expression levels of these proteins in the alkaline RE rats was markedly high at all time points during the study.The results of the Western blots and RT-PCRs suggested that as the expression level of IL-6 increased,those of claudin-1,occludin,ZO-1,JAM-1 and DSG-1 increased as well and became more prominent as the disease progressed. Within 30 days after initiation of the aluminum-phosphate treatment,theWestern blot and RT-PCR results indicated that the expression levels of IL-6,claudin-1,occluding, ZO-1,JAM-1 and DSG-1 in the acid RE rats(group A) decreased gradually and was comparable to that in the normal control group.Hyperplasia and widening of the intercellular spaces were restored as observed by microscopy.At day 60,the Western blot and RT-PCR results suggested that the expression levels of these proteins decreased gradually in the mixed RE rats(group C),but increased dramatically in the alkaline reflux group(group B). Conclusion1.Elevated expression of claudin 1,occludin,ZO-1,JAM-1 and DSG-1 occurs in the pathogenesis of RE,which can be considered an early molecular event of the disease.2.Aluminum-phosphate significantly protects esophageal mucosa by enhancing its defense against damage.However,efficacy of the drug is related to PH value of the reflux.It's effective in treating acid reflux esophagitis rather than alkaline reflux esophagitis3.In the pathogenesis of RE,positive regulation and synergism might occur in the expression of claudin 1,occludin,ZO-1,JAM-1 and DSG-14.CREB and tau protein are probably new targets of the research in recovering the neuronal function and cerebral ischemia therapy.4.while IL-6 is an inflammatory factor in the development and progression of RE. |
PDF Full Text Request