HO-1Enhenced Intestinal Mucous Membrane Barrier Resistance To Injury And Repair Capacity By Regulating Claudin-4Expression

Background and objective:Ischemia-reperfusion injury (IRI) refers to the clinical syndrome of organ function further deterioration with cellular functions and metabolic disorders and structural injury after blood flowing to restore from tissue ischemia for a period of time. Studies have shown the early changes of ischemia-reperfusion injury that the intestinal barrier is destroyed, with intestinal mucous membrane permeability increasing. The tight junction between epithelial cells is an important part of the intestinal mucosa barrier to prevent the harmful substance such as toxins or bacteria in the gut through the intestinal mucosa into other tissues and organs in the body and blood circulation. The injury of intestinal mucosa due to a variety of causes lead to the disorder of internal environment in body and the trigger of systemic infection. So the intestinal barrier function has become an important index to estimate the prognosis of critically ill patients.Intestinal tract is not only the target organs of multiple organ dysfunction syndrome (Multiple organ dysfunction syndrome,MODS), but also the starter of MODS. And it is well known that the hemoglobin oxidase1(heme oxygenase-1, HO-1) is the only hemoglobin oxidase can be induced and is the speed limit in heme decomposition in body. Three kinds of products from Heme degradation with play an important role in maintaining body’s internal environment. Studies have shown that HO-1and its metabolism play a important role in anti-inflammatory and anti apoptosis and cell protectionin in various oxidative stress and inflammation in body Therefore some scholars think that HO-1is the important target for the treatment of gastrointestinal diseases. This research observed the changes of tight junction protent Claudin-4among intestinal epithelial cells and HO-protein expression through the establishment the model of ischemia-reperfusion injury in rats and in vitro models of intestinal mucosa, to further reveal the pathogenesis of intestinal ischemia-reperfusion injury.Methods:Building the model of ischemia-reperfusion injury in rats by "ligating the superior mesenteric artery for60min" and establishing the colon cancer cell line with HO-1gene expression after building people HO-1gene expression vector. Animal experiment object is divided into6groups:control group,3、6、12、24and48h group after ischemia-reperfusion with five rats in each group.Cell experiment is divided into the experimental group Caco-pS/HO-1and blank control group Caco-pS2. Detecting Claudin-4and HO-1expression by using SYBR Green relative quantitative reverse transcription polymerase chain reaction (QRT-PCR)、protein immunoblot method (Western Blot) and immunohistochemical at every point after ischemia-reperfusion and cell experiment; CCK8cell proliferation experiment and immunofiuorescence observating the distribution of HO-1molecule in cell. Analyzing the experiment data with SPSS18.0statistical software.Results:1.RT-PCR and Western Blot result that the tight junction protein claudin-4expression increased at3h after ischemia reperfusion and reached the highest at12h, then gradually returned to normal;2. Double enzyme digestion and two-way sequencing confirmed the carrier builded successfully, and it was confirmed in the Caco-2cell with WB and PT PCR.Observing transfected caco-2cell in immunofluorescence with C-Y3labeled antibody by confocal laser microscope, we found HO-1molecules are mainly distributed in the cytoplasm and cell membrane; 3.CCK8results that the experimental group is much higher than the control group at the fifth day (Caco-pS:3.2500±0.2477VS Caco-pS/HO-1:5.4300±0.2497P<0.05);Cell scratch experiment:the experimental cells healed within48hours after the scratch scratch, but the control group don’t heal. After48hours, the experimental group and control group in cell scratch average width is respectively (17.25±7.31) vs (126.37±11.14) μm(P<0.05);4.Western Blot results that the tight junction protein claudin-4expression significantly increased after HO-1expression vector transfection (1.04±0.15) vs (0.50±0.07)(P<0.05).Conclusions:1. Successfully building the eukaryotic expression vector of HO-1gene and establishing colon cancer cell line with HO-1gene expression;2.Successfully setting up animal model of rat small intestine ischemia-reperfusion injury;3.Tight junction protein Claudin-4increases before it is back to normal after ischemia-reperfusion injury;4.HO-1gene promotes intestinal mucosal cell proliferation to enhance mucosa injury and repair by regulating Claudin-4expression.