The Effect Of NF-κB Signaling Pathway On Delayed Xenograft Rejection Of Mouse To Rat Cardiac Xenografts

ObjectiveDelayed xenograft rejection(DXR) is the major barrier to succeed in xenotransplantation.In order to study the mechanism of DXR and to prevent it,mouse to rat cardiac transplantation was used as the model to investgate the effect of NF-κB signal pathway and its regulated factor on DXR. Methods and ResultsPART Ⅰ To establish a model to study DXR.NIH mice and Wistar rats were served as donors and recipient,respectively.Heart from the donor was transplanted heterotopically into the recipient.The aorta of the donor heart was anastomosed end-to-end to the right common carotid artery of the recipient and the pulmpponary artery was anostomosed end-to-end to the right external jugular vein by shef technique.Xenograft function was assessed by daily palpation;the day of rejection was defined as the day when no pulsations could be detected in the transplanted heart.The rejected xenografts were analysed histologically and immunohistochemically for C3,IgG and leukocyte marker CD68 to identify macrophages(MФ).The results revealed that the mean sulvival time of the xenografts was 2.00 ±0.59 days.20 times of transplantation were done with the successful rate of 90%.The histology of the rejected showed widespread intravascular thrombosis,less hemorrhage,associated with a large number of infiltrated inflammatory cells,focal ischemic infacts and coagulative necrosis.Immunohistochemistry showed no C3 were deposited in the xenografts,however,61%(11/18) xenograft had IgG deposition.Immunostaining for CD68 found Mф were the major cells of the onfiltrated imflammatory cells.This data suggest IgG and Mф play an important role during rejection of mouse to rat heart transplantation and the complements have little or no effect.So this model can be used to study DXR.PART Ⅱ Mouse to rat heterotopic heart transplantation was performed to the effect of NF-κB P65 protein and P65 DNA binding activity on DXR.The animal were divided 2 groups:group A(n=6),served as control group which did not receive any operation;group B(n=6),heterotopic transplantation group.The day of rejection was defined as the day when no pulsation could be detected in the transplanted heart.The mean survival time of the xenograts was 2.17 ±0.41 days.The expression of P65 were examined by Western blot and immunohistochemistry.Electrophoretic mobility shift assay(EMSA) was used to analyze the level of NF-κB P65 DNA binding activity.The results revealed that the level of P65, NF-κB P65 DNA binding activity in group B were higher than those in group A.These data suggest NF-κB may play a important role in DXR.PART Ⅲ This study was designed to investgate the immuosupresive CsA and Lef influence on NF-κB signal pathway in DXR.The animal were divided into 4 groups:groupsA(n=6),served as xenograft control and received no imminosuppression; groupB(n=6),received CsA 20mg/kg,every other day from day 0;group C(n=6),treated with leflunomide(Lef) 10mg/kg from day 0;group D(n=6),received CsA and Lef.The expression of IKK,P65,IκBα,ICAM-1 were examined by Western blot and immunohistochemisty,EMSA used to analyse the level of NF-κB P65 DNA binding activiy.The results revealed administration of CsA alone had no appreciable effect on xenograft survival time, however,treatment with Lef alone significantly prolonged xenograft suivival time to 4.17±1.33 days,(p<0.05 vs group A).Combination Lef with CsA achieved synergistic immunosuppressive effect to prolong the xenograft survival time to 6.50±1.52 days( p<0.05 vs group A,B and C).The level of IKK,P65, IκBα,ICAM-1 and NF-κB P65 DNA binding activiy have a significantly difference in each group(p<0.05).This data suggests the combination Lef with CsA can influence the NF-κB signal pathway in DXR.PART Ⅳ This study was designed to investgate the combination PDTC with immunosuppressive Lef and CsA influence NF-κB signal pathway in DXR.The animal were divided into 5 groups:group A(n=6),served as xenograft control and received no immunosuppression;group B(n=6),received PDTC,500mg/kg,tw...