The Effect And Mechanism Of NRAGE On Intestinal Ischemic Reperfusion Injury

BackgroundIn clinical practice,intestinal ischemia reperfusion?I/R?is widespread.It was occured during the course of many diseases such as severe trauma and shock as well as some surgeries and clinical operations such as intestinal transplantation,abdominal vascular surgery and hemodialysis.I/R would threaten to patient's life if it was severe.Therefore,further studies should be done on the mechanism of intestinal I/R.The neurotrophin receptor-interacting melanoma antigen gene protein?NRAGE?was found by Salehi in 2000.It is a protein that can interact with p75NTR and plays an important role in the apoptosis induced by p75 NTR.NRAGE can be detected in many tissues,such as brain,heart,liver,kidney,muscle and gut.But the mechanisms on intestinal have not been clarified.This paper makes some researches on mechanism of NRAGE during ischemic reperfusion injury.Methods1.Culture cells under the oxygen deficit and reoxygenation conditions,collect total proteins and total RNA of IEC-6 cells after 6 h of hypoxia?1%O₂,5%CO₂?and after 6 h of hypoxia?1%O₂,5%CO₂?and 1 h of reoxygenation?20%O₂,5%CO₂?.The rats with I/R injury were cut via a midline abdominal incision after anesthesia,the superior mesenteric artery?SMA?was interrupted for 30 min and then recovered for 6 h,the tissue cut from jejunum washed in physiological saline and made to paraffin section.Then we used western blot,RT-PCR and immunohistochemical?IHC?to observe the change of NRAGE protein and mRNA in IEC.2.Add the overexpress NRAGE lentivirus suspension?Lv-NRAGE group?,interference NRAGE lentivirus suspension?sh-NRAGE group?,a control lentivirus suspension?Lv-control group?,and set another group without lentivirus suspension?NC group?to IEC-6 cells.After 48h after transfection,extracted total cells,total proteins and total RNA.Then we used western blot,RT-PCR to detect whether the transfection was succesful.3.After 48 h post-transfection,FCM was used to analyzed the early apoptosis rate of the IEC-6 cells and western-bolt was used to detect the change of the tight junction protein occludin.4.Add the interference NRAGE lentivirus suspension?sh-NRAGE group?and a control lentivirus suspension?Lv-control group?to IEC-6 cells.After 72h post-transfection,the medium without serum was displaced for 8 h,added to exogenous BMP2 and noggin for30 min,extracted total proteins.The level of p-NF-?B was detected by western blot.Results1.Hypoxia results to the increase of the level of NRAGE protein and mRNA in IEC-6 cells.Meanwhile,NRAGE protein was also significantly increased in IEC after 6 h of I/R compared to the Sham group by IHC analysis.2.After transfection for 48 h,NRAGE protein and mRNA of Lv-NRAGE group were increased compared to Lv-control group.On the contrary,NRAGE protein and mRNA of sh-NRAGE group were redunced.The transfection is succesful.3.Compared to Lv-control group,the early apoptosis rate was raised and the level of the tight junction protein occludin was reduced in Lv-NRAGE group,the early apoptosis rate was reduced and the expression of occludin protein was increased in sh-NRAGE group.4.Exogenous BMP2 upregulated the levels of NRAGE protein and p-NF-kB,and noggin,a antagonist of BMP,weakened the effect.After interfering NRAGE,the level of p-NF-kB was downregulated and block increase of p-NF-kB caused by exogenous BMP2.Noggin could reverse the increase of NRAGE protein caused by intestinal I/R.Conclusions:1.hypoxia and ischemia reperfusion could increase NRAGE in IEC.The NRAGE protein of sham group was expressed on membranes,while the increase in I/R group was on menbranes and cytoplasm.2.NRAGE promoted the apotosis of IEC,downregulated the expression of occludin protein,and then destroyed intestinal mucosa barrier.3.NRAGE may paly a role through noncanonical BMP2 signaling pathway in ischemic reperfusion.