The Study Of Snake Venom Metalloproteinase Atrase B In Xenotransplantation

Part I Atrase B inhibits complement in vitro[Objective] To evaluate the inhibitory effect of atrase B on complement activation using a normal human serum(NHS)mediated complement dependent cytotoxicity(CDC)model with an immortalized porcine aortic endothelial cell line(i PECs)as the target.[Methods] Fresh non-anticoagulated peripheral blood was obtained from six healthy volunteers.NHS preincubated with different final concentrations of atrase B for various time,then incubated with i PECs.The lysis of i PECs and the deposition of xenonatural antibodies Ig G,Ig M and complement fragment C3 c,C4c and C5b-9 onto i PECs were detected by flow cytometry.At the same time,the immunofluorescence staining was used to verify the results.[Results] In the low concentration groups of atrase B,the iysis of i PECs decline slightly.When the concentration of atrase B was raised to 7?M,the lysis of i PECs was dramatically reduced,to a level close to that of the negative control.With the extension of pretreatment time,the inhibitory effect became more obvious.FACS analysis showed that both xenoreactive Ig G and Ig M could significantly bind to i PECs,and the degree of binding seen in the atrase B-treated group and the untreated group were not significantly different.Atrase B pretreatment had no impact on the deposition of C3 c or C4c(p>0.05 vs.the atrase B-untreated group),however,it potently suppressed the deposition of C5b-9(p<0.05 vs.the atrase B-untreated group).All these results were further confirmed by immunofluorescent staining.[Conclusion] 1.Atrase B inhibits human serum-mediated CDC in a dose and time dependent manner;2.Atrase B pretreatment potently suppressed the deposition of C5b-9,but not that of C3 c and C4 c,confirming that the inhibition occurred at the level of the MAC;3.Atrase B may have the potential to be used as a potent complement inhibitor in pig-to-human xenotransplantation.Part II Atrase B prolongs xenograft survival in a guinea pig to rat heart transplant model[Objective] To investigate the inhibitory effects of atrase B on complement activation and coagulation,as well as the effect on xenograft survival in a discordant xenotransplantation model.[Methods] Twenty Wistar rats were randomly divided into four groups(n=5 per group).Atrase B-treated groups were given different doses of atrase B via the tail vein(3mg/kg,6mg/kg,12 mg/kg).Normal group: the same volume of PBS was infused.Blood was collected from the inferior vena cava 2h after the treatment.Part of each blood sample(1.8ml)used to prepare plasma.The remaining blood was allowed to prepare serum.The prepared plasma was analyzed for PT,APTT,TT and FIB.The serum used to detect the complementary activity.Twenty one recipient rats were randomly divided into four groups:(1)The Sham group(n=6): operations were the same as the xeno group except that no transplantation was performed;(2)The Xeno group(n=6): rats administered the same volume of PBS 2h before reperfusion;(3)The Atrase B(endpoint)group(n=6): rats treated with a single dose of atrase B(6mg/kg)2h before reperfusion;(4)The atrase B(7min)group(n=3): rats treated with a single dose of atrase B(6mg/kg)was sacrificed at 7min after reperfusion.Heterotopic intraabdominal cardiac transplantation of guinea pig hearts to rats was done according to the method described previously(Qian-Yun Sun,Toxicon,2003).On cessation of heartbeat,rats were sacrificed to obtain the blood sample and graft tissue.[Results] As compared to the PBS-treated group,the atrase B-treated group showed a significantly reduced serum complement and coagulation activity in a dose dependent manner.The survival time of xenograft was significantly prolonged in the atrase B group.Histological examination revealed less erythrocyte extravasation,perivascular edema,and thrombus formation in the Atrase B group,and this phenomenon was more obvious in the samples taken at the same time as the control group(p<0.05).In addition,treatment with atrase B significantly prevented the formation of platelet microthrombi and the deposition of fibrin and C5b-9.[Conclusion] A single dose of atrase B treatment could achieve significant inhibition of both complement and coagulation at 2 h after administration.Atrase B treatment significantly prolonged xenograft survival and attenuated pathologic damage,the formation of platelet microthrombi,and the deposition of fibrin and C5b-9.Part ? Atrase B protects against renal ischemia reperfusion injury in mice[Objective] To investigate whether atrase B can provide protection against reversible renal injury from ischemia-reperfusion in mice.[Methods] Mice reversible renal ischemia reperfusion injury was induced by clamping left renal pedicles for 28 min at 32?.Atrase B(35mg/kg)or PBS was administered via intravenous infusion 12 h before the ischemia.The serum and renal tissue samples were obtained at 24 hours after reperfusion.The efficacy of atrase B was evaluated by detecting serum creatinine and urea nitrogen in mice.HE and immunohistochemistry were used to evaluate the pathological damage of renal tissues and complement deposition.we also detected the m RNA levels of tumor necrosis factors-?(TNF-?),interleukin-1?(IL-1?),and interleukin-6(IL-6)by RT-PCR.Eighteen mice were equally divided into three groups(n=6per group):(1)The PBS group: murine administered the same volume of PBS except for the untrapped kidney.(2)The IRI group: clamping left renal pedicles for 28 min with vascular micro clamps at 32?,then the offside kidney was removed.(3)The Atrase B group: reversible ischemia-reperfusion injury model was established 12 h after intravenous injection of Atrase B.[Results] The serum creatinine of IRI group was about 100?mol/L,which was consistent with the characteristics of the reversible ischemia-reperfusion injury model.The serum creatinine and urea nitrogen decreased significantly in the atrase B group compared with the IRI group.HE showed that atrase B treatment markedly reduced necrosis of the renal tubular epithelium cells and infiltration of inflammatory cell.The deposition of complement C5b-9 in renal was significantly decreased compared with IRI group.In addition,atrase B suppressed proinflammatory factors and chemokines expression in IRI.[Conclusion] Atrase B had a significant protect effect on reversible ischemia reperfusion injury through meliorate complement activation in mice.This suggests that atrase B contribute to reduce graft damage in transplantation.