The Interplay Of BMP4 And IL-7 Regulates The Apoptosis Of Intestinal Intraepithelial Lymphocytes Under Ischemia-reperfusion Conditions

Background:Intestinal ischemia-reperfusion(I/R)injury is one of the common tissue and organ damages in the process of severe infection,trauma,shock,heart and lung dysfunction,organ transplantation and so on.The intestinal mucosal barrier is a complex barrier structure,mainly composed of mechanical barrier,immune barrier,biological barrier and chemical barrier.Among those,the mechanical barrier and the immune barrier are most important in maintaining the intestinal barrier function.The mechanical barrier is mainly composed of intestinal epithelial cells(IEC)and intestinal intraepithelial lymphocytes(IEL).IELs are composed of a group of special cells,especially T lymphocytes.IEC interact with IEL by secreting protein factors,thus regulating their own proliferation,differentiation,apoptosis and so on.Therefore,studying the interaction of IEC and IEL in depth will contribute to clarify the pathogenesis of intestinal mucosal barrier dysfunction under stress conditions.The function of intestinal mucosal barrier in maintaining intestinal homeostasis and prevent endotoxin transfer are limited because of the great damage of mechanical barrier and the immune barrier which are composed of IEC and IEL when the intestinal tracts are subjected to I / R injury.Previous studies found that the expression of BMP4 protein in intestinal mucosal epithelial cells was increased under the stimulation of parenteral nutrition,hypoxia,ischemia and reperfusion.BMP4 promote the release of inflammatory factor by activating the downstream signaling pathway,thus aggravating the injury of intestinal mucous membrane barrier function.It has been reported that BMP4 from thymic epithelial cells could affect the maturation of CD34+ precursor cells through Smad signal pathway,then inhibiting the differentiation of T lymphocytes in the thymus,thereby decreasing the body’s immune function.This effect can be counteracted by IL-7.IELs as special intestinal immune cells,play an important role in the maintenance of intestinal mucosal immune barrier function.Under the stimulation of TPN and I/R,the function and the number of IEL are decreased and the apoptosis of IEL are increased,which lead to the deterioration of intestinal mucosal barrier dysfunction.In this research,we will study BMP4 and IL-7 derived from IEC interplay regulating the apoptosis of intestinal intraepithelial lymphocytes in ischemia-reperfusion injure to provide an possible experimental basis of IEC-IEL dialogue in ischemia reperfusion.Methods:1.Mouse intestinal I/R injury model was made,6 hours after cervical dislocation,the mouse was sacrificed and the upper part of the small intestine jejunum tissue was removed to paraffin sections and frozen sections,HE staining was used to observe morphological changes of intestinal villi,The expression of BMP4 and IL-7 protein in IEC was detected by IF.2.Killed the mouse by cervical dislocation,cut the full of intestinal,isolated normal mouse IEL,using RT-PCR and FCM to confirm expressio n of BMP signaling protein in IEL.3.After 6 hours of intestinal I/R in mouse,the intestinal epithelial lymphocytes were extracted,Sham was used as control group,and the expression of BMP and IL-7 signaling pathway proteins were detected by FCM in IEL.4.Isolated and cultured IEL,with stimulation of exogenous BMP4 and IL-7 protein,FCM was used to observe the apoptosis of in IEL.5.Western-bolt and FCM detect the change of BMP signaling pathway after treat ing the IEC-6 and IEL with exogenous IL-7 for 6 hour.6.Stimulated by exogenous BMP4 protein in IEC-6 and IEL,the expression of IL-7 and IL-7R/CD127 in the cells was detected by Western-blot and FCM.Results:1.After 6 hours of intestinal I/R,we found that some of intestinal mucosa broken,the cell loss and the partial structure disappeared under light microscope.The expression of BMP4 protein in intestinal IEC was significantly increased,but the expression of IL-7 protein was significantly decreased in the villi.2.The BMP receptor(BMPRIA,BMPRIB)and N-Smad protein(Smad1,Smad5,Smad8),p-NF-k B protein has been found in normal mouse IEL by RT-PCR and FCM.3.Under condition of mouse intestinal I/R model,the expression of BMPRIA,BMPRIB,P-Smad1/5 and p-NF-k B in IEL was significantly higher than that in Sham group.However,the expression of P-STAT5 and IL-7R/CD127 was significantly decreased.4.Exogenous BMP4 stimulation increased apoptosis rate of IEL,under the condition of NOGGIN,PDTC,IL-7 combined stimulation,the IEL apoptosis rate a nd Control group no significant difference.5.Under stimulation of IL-7,the expression of BMP4 reduced in IEC,BMP signaling pathway was suppressed in IEL.6.Under condition of exogenous BMP4 stimulation,the expression of IL-7 protein was decreased in IEC-6,and the expression of IL-7R/CD127 and P-STAT5 protein also decreased in IEL.Conclusion:1.Under the stimulation of I/R damage,the intestinal barrier function was greatly damaged including the partial villi,cell detachment and structure disappear ed,the expression of BMP4 protein increased and the expression of IL-7 decreased.2.The expression of BMP signaling pathway was present in IEL,and the BMP signaling pathway in IEL was activated under the stimulation of intestinal I/R damage.3.BMP combined with BMP receptor,activated Smad,NF-k B signaling pathway,and further induced the apoptosis of IEL.4.The exogenous IL-7 inhibited the transmission of BMP signaling pathway in IEC and IEL,and the role of BMP4 in promoting the apoptosis of IEL was inhibited.5.The expression of IL-7/CD127 signaling pathway protein decreased after I/R,and BMP4 can inhibit the IL-7/CD127 signaling pathway.which further aggravated the damage of intestinal mucosal barrier function.