| ObjectivesIntestinal epithelial barrier(IEB) is an important barrier structure against pathogen invasion, which mainly consists of the mechanical barrier, chemical barrier, biological barrier and immunological barrier. The mechanical barrier formed by the intestinal epithelial cells and the tight junction(TJ) structure between the intact intestinal epithelial cells(IEC) provides the fundamental structures of IEB. Recent studies indicated that enteric glial cells(EGC), the most abundant cell type in enteric nerve system(ENS), play a critical role not only in nutritional support and enteric neurone protection, but also in maintaining IEB functional stability. Researches have shown that abnormalities of EGC are closely correlated with a variety of intestinal diseases accompanied with increased intestinal mucosal permeability as well as IEB dysfunction. EGC knockout in mice may cause acute intestinal inflammation and IEB functional collapse.Acute ischemia reperfusion(I/R) injury caused by severe trauma, burns, and major surgery is recognized as a major problem affecting clinical treatment. Especially in recent years, the number of major surgeries, including major vascular surgery, combined organ transplantation, cardiopulmonary bypass and so on, is continuously growing, resulting in intestinal hypoperfusion and reperfusion accompanied by release of a large number of pro-inflammatory media and quite often acute intestinal I/R injury, which has become a critical factor affecting surgical prognosis and patient survival. Numerous studies have shown that acute intestinal I/R injury has become one of the vital reasons of common intestinal mucosal barrier injury, intestine failure and even multiple organ failure. Therefore, it is urgent to further elucidate the mechanism of IEB injury during I/R and to find ways to enhance intestinal protection.Previous studies by our group have shown that intestinal I/R injury rapidly induces obvious EGC activation, which significantly relieve damage to the barrier function of intestinal epithelial cess(IEC) caused by hypoxia reperfusion(HR), suggesting a protective role of EGC during intestinal I/R stimulation; however the specific molecular mechanism is unclear yet. So the major concern of this project is to further elucidate the mechanism how EGC participates in IEB protection of IEB function after I/R.Several studies have shown that S-Nitrosoglutathione(GSNO) could be a key molecule in the regulation of IEB function by EGC. As the major form of S-nitrosothiol(SNO) in vitro, GSNO may exert its biological function by regulating the storage and release of nitric oxide(NO). Studies have shown that GSNO reductase(GSNOR), a critical reductase in metabolism of GSNO, regulates intracellular NO level by reducing GSNO into oxidized GSH(GSSG). As an important source of intestinal GSNO, EGC-derived GSNO has been proved to be capable of alleviating the destructive effect on the intestinal epithelial cells and reducing inflammatory reaction upon infection by Shigella bacteria. This may at least partially explain the protective role of EGC for IEB.In summary, this study investigated the protective effect of EGC on IEB function in EGC-Caco-2 co-culture model under both physiological and HR conditions. We also established a mouse model of acute intestinal I/Ras well as an in vitro HR model to test the protective effect of GSNO pretreatment on intestinal mucosal permeability and IEB functions under I/R stimulation. si RNA was transfected into EGC by lentivirus to suppress GSNOR expression in EGC cells, and the dynamics of GSNO and its effect on IEB function was observed in reference with the in vitro I/R model.Methods1. Through GSNO pretreatment or EGC-Caco-2 co-culture, the effect of EGC and GSNO on epithelial permeability and tight junction proteins under physiological and HR conditions were investigated using transepithelial resistance(TER), immunofluorescence and western blot analysis respectively.2. A mouse model of acute intestinal I/R was established with GSNO pretreatment,;thechanges in mucosal injury, intestinal epithelial TJ proteins, and intestinal mucosa permeability were evaluated by Ussing chamber and immunofluorescence techniques seperately.3. GSNOR expressionwere suppressed by si RNA through lentiviral transfection, and change in GSNO concentration in EGC and the effect on IEB function under acute HR conditions were determined by high performance liquid chromatography(HPLC).Results1. Compared to the Caco-2 group, both of EGC-Caco-2 co-culture group and GSNO pretreatment can increase the value of transepithelium electrical resistant by 17.2% and 15.2%(P<0.05)2. HR stimulation strongly reduced transepithelial electrical resistance(TER) of the Caco-2 cell layer to 31.8%compared to control group(P < 0.05); the relative protein expression of occludin and ZO-1 in reduced by 49.1% and43.2% respectively(P<0.05).3.The structure of intestinal mucosal epithelia was damage serious after I/R stimulation that include the villus of intestine, intestinal permeability(156at include,P<0.05),score of Chiu’standard for evaluation(4-5). Meanwhile the damaging effect of I/R was effectively relieved after GSNO treatment. the relative the score of Chiu’ is 2-3,and it significantly increased TER of the I/R to 31.4%(P<0.05)。4. No matter the HR model in vitro or I/R stimulation vivo.The Levels of messenger RNA and expression of GSNOR proteins were also increased by 39.7% and 43.4%(P<0.05).However the concentration of GSNO in EGCwas decline by 45.5%(P<0.05)。5. As indicated by HPLC assay, GSNO concentration in GSNOR-/- group increased by 48.6%.In addition in the Caco-2 cells co-cultured with EGC model, Occludin and ZO-1 expression in increased by 35.1% and 38.9% respectively(P<0.05).Conclusions1. GSNO exerts a protective effect on IEB function under both physiological condition and acute pathological conditions such as HR and I/R through modulating the expression of TJ proteins of IEC.2. Acute HR or I/R stimulation may induce the overexpression of GSNOR, the key enzyme of GSNO metabolism, which lead to the significant decrease of intracellular GSNO concentration in EGC. This could be the potential mechanism to clarify the dysfuntion of EGC in protecting IEB functions under acute pathological stimulation... |
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