Interferon-gamma Regulated HIF-1α Pathway Involves In The Regulation Of Intestinal Epithelial Barrier Function In An Ischemia/reperfusion Model

BackgroundIntestinal ischemia/reperfusion (I/R) induces the dysorder of the intestinal epithelialbarrier function, increases the intestinal permeability, and often causes fatal conditionsincluding sepsis and multiple organ failure, resulting in a high degree of morbidity andmortality.Recent studies have shown that local intestinal mucosal hypoxia under differentpathological conditions is closely related to the regulation of intestinal mucosal barrierfunction, which due to the unique anatomic structure of the intestinal mucosa. Intstinalmucosal hypoxia may be one of the incentive factors resulting in the loss of intestinalbarrier function.Recent evidences show that Hypoxia-inducible factor-1(HIF-1) is the key factor inmucosal hypoxia induced intestinal mucosal barrier dysfunction. HIF-1is a keytranscription regulatory factor involved in a number of pathways responsible for theexpression of barrier protection factors, including mucin, intestinal trefoil factor (ITF),ecto-5’-nucleotidase (CD73) and vasodilator stimulated phosphoprotein (VASP).Clinical and experimental data have established that intestinal barrier functionundergoes the process of damage and repair after intestinal I/R, accompanied with a largenumber of cytokine release, including interferon-gamma (IFN-γ) and tumor necrosisfactor-alfa (TNF-α). Recent study shows that the increase of intraepithelial lymphocytes(IEL) derived interferon-γ (IFN-γ) expression caused by hypoxia results in the loss ofintestinal barrier function. Study has shown that intestinal I/R injury may also activate IELand increase the IEL derived IFN-γ expression.Lots of evidence suggest that these cytokine elevations are responsible for the observedbarrier dysfunction. First, in vitro studies have shown that IFN-γ can induce barrier dysfunction in cultured epithelial monolayers. In vivo, both in human patients and animalmodels, IFN-γ antagonism can diminish disease severity and restore barrier function.Although both in vitro and in vivo data confirm that IFN-γ can cause dysfunction of theepithelial barrier, the mechanisms of this effect remain unknown.Currently, there are several candidate pathways that might be involved incytokines-related signaling mechanisms of TJ assembly. Several lines of evidence havesuggested that phosphatidylinositol3-kinase (PI3K) is a key regulator for TJ assembly.Previous investigators have found that glutamine deprivation alters Claudin-1expressionvia a PI3K/Akt mediated pathway in cultured Caco-2cells. Rao et al have shown thatoxidative stress activated PI3K pathway in intestinal cell monolayers, and caused theup-regulation of transcellular permeability. Inhibition of PI3K prevented the increase inpermeability.However, the mechanism of IFN-γ regulates HIF-1is not cleare, we postulated that thePI3K/Akt pathway is activated at the same time when intestinal undergoesischemia/reperfusion injury, which may involved in IFN-γ mediated modulation of thejunction integrity. We also hypothesized that the activation of PI3K/Akt pathway involvedin regulating the expression of HIF-1under hypoxic conditions and thus participate in theregulation of intestinal barrier function.MethodsFirstly, adult C57BL/6J mice undergoing intestinal I/R injury, and the small bowel washarvested. The expressions of HIF-1α, PI3K and p-AKT were detected byimmunohistochemistry，RT-PCR and Western Blot method.Secondly, intestinal epithelial cells (Caco-2cells) were treated with recombinant IFN-γ,the expressions of HIF-1α、PI3K, p-AKT and tight junction proteins were measured withwestern blot and confocal microscopy analysis.Thirdly, Caco-2cells were treated with LY294002, the inhibitor of PI3K/AKT pathway,the expressions of HIF-1α, PHDs and tight junction proteins were measured with westernblot and confocal microscopy analysis. And the transepithelial resistance (TER) wasmesured use a Millicell ERS.Results and conclusion1． The expression of HIF-1α and IFN-γ was significantly increased after the intestinal I/R injury，and the activation of PI3K/Akt signaling pathway is increased.2． IFN-γ is involved in the loss of epithelial mucosal barrier function in hypoxia.3． The HIF-1α protein expression in the intestinal mucosa significantly increasedunder hypoxia condition, and the HIF-1αtranslocation into the nucleus increased.4． The administration of IFN-γ to Caco-2cells resulted in the decrease of tightjunction proteins and the loss of TER.5． IFN-γ administration significantly increased the expression of PI3K and AKTPhosPharylation, LY294002administration significantly attenuated the decreasing of TJexpression caused by IFN-γ.6． The administration of IFN-γ to Caco-2cells increased the expression ofHIF-1αand down-regulated the expression of PHD1and PHD2. LY294002administrationsignificantly attenuated the decreasing of PHD2expression caused by IFN-γ.