| Background:The previous studies have proven that acute intestinal ischemia reperfusion(I/R)caused by major operation as well as acute traumatic injury has become a common cause of intestinal epithelial barrier(IEB)injury and intestinal failure,even one of the crucial reasons of multiple organ dysfunction.It is an urgent problem to clarify the molecule mechanisms under the intestinal I/R injury-associated IEB dysfunctionand establish new strategy to protect the small intestine from acute I/R injury.Adenosine 2a receptor(A2AR),a kind of G protein coupled receptor,can contribute to the production of cAMP by combining with adenosine through activating adenylate cyclase.Recent evidence has shown that A2 AR activation can ameliorate organ injury by regulating inflammatory mediators and relieving the inflammatory reaction.The activation of A2 AR play a protective function in heart,lung and liver under I/R stimulation.However,the function of A2 AR in the intestinal inflammation remains still unknown.Our present study was designed to identify the potential protective role of A2 AR under aucte I/R stimulation by using A2 AR knock-out mice as well as the co-cultured model of enteric glial cell(EGC)-intestinal epithelial cell(IEC).Methods and Research:1.By using EGC-IEC-6 co-culture system,the barrier functions of IEC-6 monolayers were assessed with transepithelial resistance(TER)measurements as well as the assessment of TJ protein expression.The agonists and antagonists of A2 AR were used to intervene the activity of A2 AR in EGC to investigate the role of A2 AR in the barrier-modulating mechanisms of EGC.At the same time,q PCR technology was used to detect the changes of IL-1 beta,TNF-alpha of EGC cells at the m RNA level.2.Twelve 6-8 weeks C57BL/6 male mice were divided into sham group and I/R group.Twelve 6-8 weeks C57BL/6 background A2 AR gene knock out male mice(A2AR-/-)were divided into A2AR-/-+sham group and A2AR-/-+I/R group.The mouse intestinal I/R model was established by the occlusion of the superior mesenteric artery temporarily for 30 min,following by reperfusion for 6 hour.Histological changes in the small intestinewere observed by hematoxylin-eosin staining(HE).The tight junction protein ZO-1 expression was evaluated by immunofluorescence assay and western blotting.Results:1.Compared with IEC-6 group alone,the exression level of ZO-1 protein as well as TER value in IEC-6 cells co-cultured with EGC cells significantly increased(p<0.05);while the treatment of A2 AR agonist CGS21680 on EGC cells further upregulated the expression of ZO-1 protein and TER values of IEC-6 cells(p<0.05).Meanwhile,hypoxia reoxygenation(HR)treatment significantly inhibited the increase of ZO-1 protein expression as well as TER value of IEC-6 cells when co-cultured with EGC cells as compared with normoxia group(p<0.05).The pretreatment of A2 AR agonist CGS21680 on EGC cells significantly reversed the decrease of ZO-1 protein expression as well as TER value of IEC-6 cells(p<0.05);while A2 AR antagonist ZM241385 exacerbated the decline in ZO-1 protein expression level as well as TER value of IEC-6 cells under HR stimulation(p<0.05).2.The detection of proinflammatory cytokines incuding IL-1? and TNF-?by q PCR analysis revealed that,HR stimulation induced the significant upregulaton of both IL-1? and TNF-? at the mRNA level when compared with control normoxia group(p<0.05);The treatment of A2 AR agonist CGS21680 significantly blocked the upregulation of the expression of IL-1? and TNF-? of EGC cells under HR treatment.while A2 AR antagonist ZM241385 treatment promoted the upregulation of IL-1? and TNF-? mRNA induced by HR stimulation(p<0.05).3.HE staining results showed both the sham group and the A2AR-/-+sham group displayed theintegral intestinal mucosa with no dropsy;no difference was observed between these two groups;while the intestinal mucosa from I/R group and A2AR-/-+I/R group showed edema?fractured,and detached partly,and the A2AR-/-+I/R group is more severe than that of I/R group(p<0.05).4.Compared with the sham group,TER values of intestinal mucosa decreased by 46% and 62% in the I/R group and the A2AR-/-+I/R group respectively(p<0.05).Compared with the I/R group,the TER value of the A2AR-/-+I/R group was obviously decreased(p<0.05).5.Immunofluorescence staining results displayedthat tight junction protein ZO-1 expressed by IEC is characterized by continuous liner distribution in the sham group.While the fluorescence intensity of ZO-1 protein staining in the sham groupwas significantly higher than that of the A2AR-/-+sham group(p<0.05),indicating that ZO expression in IECs was downregulated by A2 AR knock out.Compared with the sham group,the fluorescence intensity in the A2AR-/-+I/R group and the I/R group were significantly weaker.Moreover,the A2AR-/-+I/R group was significantly lower than the I/R group in fluorescence intensity(p<0.05).6.EGC-specific marker GFAP staining analysis showed that GFAP protein is mainly distributed in the intestinal villi within the basal layer of the punctate expression withfluorescent red,mainly expressed in the cytoplasm of EGC.Compared with A2AR-/-group,the fluorescent intensity of GFAP protein staining increased significantly in A2AR-/-+ sham group(p<0.05);while,the fluorescent intensity of GFAP protein staining in A2AR-/-group was significantly increased as compared with the sham group(p<0.05).7.Western blot results showed that the expression of ZO-1 in the sham group was significantly higher than that of the A2AR-/-+I/R group(p<0.05),indicating that A2 AR is capable of regulating the expression of ZO-1 protein at the protein level.While compared with the I/R group,the expression of ZO-1 protein in the A2AR-/-+I/R group was significantly higher,suggesting that A2 AR knockout exacerbates the IEB damage induced by I/R stimulation(p<0.05).Conclusions:1.A2 AR is involved in the crosstalk between EGCs and IECs.Via A2 AR signaling pathway,EGCs could protect the IEB functions against hypoxia reoxygenation stimulation through upregulating the expression of TJ protein ZO-1.2.The knockout of A2 AR significantly exacerbates the musical damage and the down regulation of ZO-1 protein in the intestinal mucosa induced by acute intestinal I/R injury,indicating that A2 AR might be playing a protective role in the IEB function under acute intestinal injury stimulation.Interventing the activity of A2 AR in EGC might be a new target for the intestinal barrier protection.3.The expression of GFAP protein in the intestinal mucosa of the A2 AR knockout mice was significantly higher than that of the wild type group,suggesting that A2 AR may be involved in the regulation of IEB function through modulating the activity of EGC. |
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