| Background:Intestinal mucosal barrier,including mechanical barrier,chemical barrier,biologic barrier and immunologic barrier,plays a central role in maintaining the intestinal flora balance and resisting the invasion of external pathogens.Clinical conditions such as mesenteric vein thrombosis,small bowel transplantation,burn,trauma,shock and surgical stress lead to intestinal ischemia which cause damage of intestinal mucosa barrier due to the intestinal mucosal ischemia and hypoxia.The damage of mechanical barrier often lead to increased intestinal permeability which cause fatal condition including sepsis and multiple organ failure,resulting a high degree of morbidity and mortality.Mechanical barrier is the basic barrier of intestinal mucosa barrier.Once the mechanical barrier is destroyed,intestinal permeability will increase significantly.Water and electrolyte will enter the intestine from the tissue,resulting in loss of fluids,exacerbating shock.At the same time,the intestinal bacteria will largely translocate into other organs,inducing systemic inflammatory response.Claudin family,one of the tight junction protein,is the most important protein which mainly maintain the barrier function of the cell.In many intestinal diseases,the severity of the disease is often closely related to the expression of claudin protein.The expression of claudin protein will be a sharp decline once intestines occur ischemia,resulting in the defect of intestinal barrier function.Therefore,the regulation of claudin protein in the early stage of intestinal ischemia reperfusion is of great significance to reduce the damage of intestinal barrier.Silent information regulator 2(Sir2),widely existing in a variety of species genes,is found in the study of yeast transcriptional silencing.It plays an important role in repairment of DNA damage,silence of mating-type gene,chromatin stability and prolonging life.SIRT-1 which depends on the nicotinamide adenine dinucleotide(NAD+)owns histone deacetylase activity.Some studies have pointed out that SIRT-1 can be used to regulate the protein expression from the level of transcription via acetyling histone.However,the role of SIRT-1 involved in intestinal I/R remains largely unknown.It is essential to explore the variation of SIRT-1 during the reperfusion phase and its specific roles.More importantly,detailed mechanism by which SIRT-1 is regulated also requires further investigation.However,the mechanism of SIRT-1 regulates the tight junction protein claudin-4 and intestinal mucosal barrier function after intestinal ischemia-reperfusion is still not clear.Therefore,we propose the hypothesis that SIRT-1 in acute intestinal ischemia and reperfusion can regulation of tight junction protein claudin-4 expression and protection due to intestinal ischemia reperfusion induced mucosal barrier function injury.In this study,the effects of on the expression of claudin-4 in vitro and in vivo were discussed.The effects of hypoxia and re-oxygenation on the expression of SIRT-1 were discussed.Trans-epithelial resistance(TER)was used to evaluate the epithelial barrier resistance.The mechanism of SIRT-1 how to regulation intestinal epithelial barrier after ischemia reperfusion can provide new ideas for protection research of the intestinal mucosal barrier injury,and provide a new treatment direction about small intestinal ischemia reperfusion injury.Methods:1.Collect 5 cases about clinical mesenteric thrombosis.All of them were partial small bowel resection.The expression of claudin-4 protein in normal and necrotic intestinal mucosa was detected by Western blot.Intestinal ischemia reperfusion model was established to detect the expression of claudin-4 protein in vivo.2.Intestinal ischemia reperfusion model was established in vivo.The expression of claudin-4 in SIRT-1 was detected by Western blot and immunofluorescence assay after using Resveratrol,an activator of SIRT-1.Intestinal epithelial hypoxia re-oxygenation model in vitro was created by using Caco-2 cell line.The cell line was pretreated by SIRT-1 activation and inhibitor.The changes in the expression of SIRT-1 and trans-epithelial resistance were detected.3.Intestinal epithelial hypoxia re-oxygenation model in vitro was created by using Caco-2 cell line.Small interfereing RNA was used to knock-down SIRT-1 expression.Changes about SIRT-1 and claudin-4 expression were detected by Western blotting,immunofluorescence etc.methods.Results:1.The resected intestinal tissue from mesenteric thrombosis patients showed claudin-4 expression was significantly decreased compared with the normal tissue.In the mice intestinal ischemia and reperfusion model,claudin-4 expression of the intestinal mucosa showed the same tendency.2.The SIRT-1 expression together with claudin-4 expression was significantly increased after using resveratrol.In H&E staining visible tissue damage was significantly reduced and immunofluorescence showed intestinal mucosal claudin-4 expression increased.3.In vitro experiments,the expression of claudin-4 in model group compared with control group increased after using resveratrol activated SIRT-1 expression.Immunofluorescence revealed the increased expression of claudin-4.Epithelial resistance increased significantly.The expression of claudin-4 in model group compared with control group decreased significantly after using the EX-527 inhibited the expression of SIRT-1.Immunofluorescence revealed the expression of claudin-4 expression was reduced.Epithelial resistance decreased significantly.4.After being treated with si-RNA interference,the expression of claudin-4 in model group followed with SIRT-1 was lower than that in the control group.Conclusions:1.In the case of intestinal ischemia and reperfusion,the function of mucosal barrier decreased with the decrease of claudin-4 expression.The regulation of claudin-4 can obviously relieve the damage of mucosa after intestinal ischemia and reperfusion.2.After hypoxia and re-oxygenation,the up-regulation of SIRT-1 expression can increases the tight junction protein claudin-4 expression,and then improve the intestinal barrier function.Down regulate expression of SIRT-1 will reduce the tight junction protein claudin-4 expression,which makes the intestinal epithelial cell trans-epithelial resistance decreases,increasing the intestinal epithelial barrier damage. |
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