Effect And Mechanism Of The Class ?A PI3K Isoforms Inhibitors On Acute Rejection Of Heart Transplantation In Mice

Experiment 1 The establishment of abdominal heterotopic heart transplantation model in miceObjective To master rapidly the establishment of abdominal heterotopic heart transplantation model in miceMethod Establishment of mode was divided into several phases for some goals.In micromanipulation training earlier and later phase,We use Kun Ming mouse as donor or recipient,and use inbred line C57 and BALB/c mouse as donor and recipient respectively in mode stable phase.Operation procedure include,anaesthetizing simultaneously two mouses as as donor and recipient,harvesting donor heart and preserving,blocking both ends of abdominal aorta and postcava,connecting donors aorta with recipients abdominal aorta and connecting donors pulmonary artery with recipients postcava,opening the bloodstream and closing the abdominal cavity,and finally,rewarming and reviving after surgeryResult Surgical success rate in training earlier,later phase and mode stable phase were 16.00%,54.00% and 86.67%.Operation success rate was improved and every period of operation was shortened with practicing continuously.Recipients were dead due to artery anastomosis(42.00%)or Venous anastomosis(14.00%)in the training earlier phase,while Recipients were dead due to artery thrombosis(12.00%)in later phase.Conclusion Practicing purposefully in several phases contributes to mastering rapidly the establishment of model and avoiding a waste of experimental animals.Details has always been the key points of surgical success.Experiment 2 The role of Class ?A PI3 K Isoforms inhibitors in cardiac allograft rejection and possible mechanism.Objective To study the role of Class ?A PI3 K Isoforms inhibitors in cardiac allograft rejection and possible mechanism.Method Isogenic and allogenic heart transplantation modes were established,and western blot tested the class ?A catalytic subunits p110 protein expression in heart graft of two groups.We established allogenic heart transplantation modes and divided into four groups randomly.One group,as a control,was treated with intraperitoneal injection of 0.5% DMSO.Other three groups,named PIK group,TGX group and IC group,were treated respectively with intraperitoneal injection of PIK-75,TGX-211 and IC-87114.Observe and record the survival time of allograft in four groups.Cardiac allografts were harvested on days 7 after transplantation,and the tissue sections were stained,marked and graded.The Th1,Th2,Th17 Tfh and Treg cells of splenic were detected by flow cytometry.The concentration of IFN-?,IL-10,IL-17 A,IL-21 and TGF-? in recipients' sera were assayed with ELISA.Analysis of phosphorylation and expression of different proteins belonging to AKT/m TOR pathways was performed by Western blot.Result Compared with isograft group,the p110? protein levels of allograft group were significantly up-regulation on days 7 post-transplantation(p<0.05),while p110? and p110? in the allograft group increased significantly both on days 5 and 7 post-transplantation(p<0.05).compared with DMSO group,we found that treatment with PIK and IC significantly prolonged the survival of the allografts(p<0.01),and pathological grading was significantly decreased in the two groups(p<0.05),while TGX barely prolonged allograft survival(p>0.05)and altered pathological grading(p>0.05).compared with DMSO group,the percentage rate of Th1 and Tfh cells/monocytes in the spleens from the PIK group were lower(p<0.05);TGX reduced the rates of Th2 and Tfh cells/monocytes in the spleens after cardiac transplantation compared with control group(p<0.05).Compared with control group,the rates of Th1(p<0.01),Th2,Th17 and Tfh(p<0.05)cells/monocytes in the spleens from the IC treatment group were all down-regulation,while the percentage of Treg cells was remarkable increased(p<0.01).Compared with DMSO group,the expression of IFN-? and IL-21 was significantly decreased(p<0.01),and the level of IFN-? was also down-regulation(p<0.05),While the IL-10 level was significantly increased in the PIK group.The expression of IL-21 in TGX group was significantly decreased(p<0.01),and the level of IFN-? and IL-17 A was also downregulation(p<0.05).In IC group,the level of IL-17 A and IL-21 was declined(p<0.05)and the expression of IFN-? was significantly decreased(p<0.01),while the concentration of IL-10 and TGF-? was increased(p<0.05).The treatment with these three inhibitors has little effect on the expression of total AKT(AKT1/2/3),whereas effectively reduced AKT phosphorylation.IC significantly reduced the expression of P70 S6 Kinase? and 4E-BP1 protein,so did PIK and TGX although with less efficacy.Besides,intervention with IC and PIK has better efficacy in suppressing P70 S6 Kinase? and 4E-BP1 protein phosphorylationConclusion The small molecule inhibitors of p110? and p110? can suppress acute rejection of heart allograft in mice.These inhibitors maybe play a role in anti-rejection through impacting the phosphorylation and expression of proteins in AKT/m TOR pathways,which modulated CD4+T cell subsets of recipients and reduced proinflammatory factor or increased anti-inflammatory cytokines.