Experimental Study Of The Protective Effect Of Heme Oxygenase-1 On Intestinal Epithelial Barrier Under Hypoxia

1.Background and PurposeIntestinal tract is not only a vital organ with the function of digestion and absorption of nutrients,but also has the function of endocrine, immune regulation and mucosal barrier. Intestinal mucosal barrier is the basis of maintaining the normal function of intestinal tract.Under the conditions of severe trauma, acute pancreatitis, a large area of burnuing, abdominal surgery, hemorrhagic shock, severe peritonitis, the strueture and the barrier function of intestinal mucous membrane may be damaged seriously.And the permeability of intestinal mucous membrane increases,which may cause the intestinal mucosal barrier dysfunction, and then causes the iniestinal bacteria and endotoxin translocation, even induces the function damaged of mang organs of the body. Intestinal tract is not only the target organ of multiple organs dysfunction syndrome(MODS),but also the power site of MODS. Heme oxygenase-1 is a kind of enzyme. Stress caused by shock, hypoxia, infection, ischemia-reperfusion, heavy metal ions may lead to heme oxygenase-1 overexpress. Heme oxygenase-1 is the rate-limiting enzyme of heme and it breaks down heme to carbon monoxide, free iron ion and biliverdin, which is readily converted into bilirubin. Three isozymes of heme oxygenase have been discovered. Studies have confirmed that heme oxygenase-1 and its metabolites (carbon monoxide, free iron ion and biliverdin)involved in many physiological and pathological processes of the body, has anti-inflammatory, antioxidant, anti-apoptotic and cell protective effects.Thus, we hypothesized that heme oxygenase-1 also has has a protective effect on intestinal mucosal barrier. This study was to confirm the protective effect of heme oxygenase-1 on intestinal mucous membrane through observing the changes of the distribution and expression of the tight junction proteins(Occludin、ZO-1) in the model of epithelial mucosal barrier in vitro. So that we further elaborate the mechanisms of heme oxygenase-1,and provide new treatment strategies and theoretical basis for clinical treatment of intestinal diseases.2.MethodsEstablishing the model of intestinal mucosal barrier in vitro, in the hypoxia model, the cells were divided into four groups:control group, hypoxia for 4h, hypoxia for 8h, hypoxia for 12h. Eukaryotic overexpression vector pShuttle-CMV-HO-1 were transfected into Caco-2 cell. The cells were divided into four groups:normoxia group, hypoxia group, transfection group, negative control group. The mRNA and protein expressions of HO-1, Occludin and ZO-1 were detected by using Real Time-PCR and Western Blot. Immunofluorescence was used to analyze Occluding and ZO-1. Analyzing the experiment data with SPSS 17.0 statistical software.3.Results(1)Immunofluorescence staining showed that Occluding、ZO-1 in linear fluorescence was distributed around the cell membrane in normoxia group, the immunofluorescence staining was disrupted in both hypoxia group and negative control group, the immunofluorescence staining was not disrupted so seriously in transfection group.(2) Hypoxia can cause the expressions of mRNA and protein of Occludin and ZO-1 reduced, but heme oxygenase-1 could enhance the expressions of mRNA and protein of Occludin and ZO-1 under hypoxia.4.ConclusionHypoxia can cause the expressions of mRNA and protein of Occludin and ZO-1 reduced, heme oxygenase-1 has protective effect on intestinal mucosal barrier and it could enhance the expressions of mRNA and protein of Occludin and ZO-1 under hypoxia.