Study On The Role And Mechanism Of The IL-2/Anti-IL-2complex In Myocardial Ischaemia-reperfusion Injury

Part ?.Treg Cells Become Activated in Response to MIRIObjective:To oberserve the Tregs number of heart-draining lymph nodes and heart after Myocardial Ischemia/Reperfusion Injury(MIRI)in mice.Methods:Mice were subjected to 30 min of left anterior descending(LAD)ischemia followed by varying periods of reperfusion.The male mice with eight to ten-week-old were randomly divided to:(1)Sham d7 group(given with operation,but without ischemia,heart-draining lymph nodes and heart were harvested at day 7);(2)MIRI d3 group(given with ischemia,heart-draining lymph nodes and heart were harvested at day 3);(3)MIRI d7 group(given with ischemia,heart-draining lymph nodes and heart were harvested at day 7);(4)MIRI d14 group(given with ischemia,heart-draining lymph nodes and heart were harvested at day 14).The mice were killed through anesthesia and orbital venous blood,heart-draining lymph nodes and heart were isolated.The number of CD4+CD25+Foxp3+ Tregs in heart-draining lymph nodes was examined using flow cytometry analysis(FCAS).The Foxp3+ cells were stained by immunohistochemistry.Results:The number of CD4+CD25+Foxp3+ Tregs in heart-draining lymph nodes was significantly increased in MIRI group in contrast with sham group,and peaked in MIRI d7 group(12.24% in MIRI d3 group,20.85% in MIRI d7 group,13.50% in MIRI d14 group,9.30% in sham d7 group).Similiarly,the number of Foxp3+ Tregs in heart was significantly increased in MIRI group in contrast with sham group,and also peaked in MIRI d7 group(12.80/mm2 in MIRI d3 group,23.80/mm2 in MIRI d7 group,8.40/mm2 in MIRI d14 group,2.20/mm2 in sham d7 group).Conclusion:We first observed that activated Treg cells appear in response to MIRI.Part ?.IL-2/anti-IL-2 complex(IL-2C)alleviated Myocardial Ischemia/Reperfusion InjuryObjective: To observe and investigate whether IL-2C could affect MIRI,and its mechanism.Methods: Mice were subjected to 30 min of left anterior descending(LAD)ischemia followed by varying periods of reperfusion.To investigate the effect of IL-2C treated before operation on MIRI,the male mice with eight to ten-week-old were randomly assigned to three groups:(1)Sham group(given with operation,but without ischemia);(2)PBS group(given with PBS);(3)IL-2C after group(given with IL-2C).IL-2C or PBS was intraperitoneally administered to mice for 3 consecutive days,beginning 5 days before MIRI.To investigate the effect of IL-2C treated after operation on MIRI,The male mice with eight to ten-week-old were randomly assigned to two groups:(1)PBS group(given with PBS);(2)IL-2C after group(given with IL-2C).IL-2C or PBS was intraperitoneally administered to mice for 3 consecutive days,beginning 3 days after MIRI.Infarct area,cardiac function,Tregs number,cardiac apoptosis,lymphocyte proliferation response,inflammatory response and histological analyses were performed on days 1,5 and 14.Results: IL-2C induced significant expansion of Foxp3+CD25+CD4+ Tregs in both spleen and heart on day 5.Next,we found that IL-2C treatment could ameliorate MIRI,smaller infarct size/AAR,improved cardiac function and lower s Tn T were observed.Compared to control group,IL-2C treatment could inhibit cardiac apoptosis,following by reduced infiltration of neutrophils,macrophages,cardiac fibrosis,and increased microvessel and myocyte proliferation,in addition,decreased splenocyte proliferation and Th1,Th17 were found though no change of NKT and DCs.RT-PCR revealed that TNF-?,IFN-g,IL-12,and IL-17 A m RNA were down-regulated on day 7 after IL-2C treatment.However,anti-inflammatory cytokines TGF-b1 and IL-10 and M2-induced IL-13,Arginase 1,and CD206 were all up-regulated by IL-2C compared to control mice.At last,we found that IL-2C treatment after MIRI improves myocardial fibrosis and cardiac function,but not infarct area.Conclusions:IL-2C could ameliorate MIRI through inhibiting apoptosis,inflammation,cardiac fibrosis.IL-2C treatment after MIRI improves myocardial fibrosis and cardiac function,but not infarct area.Part ?.The Role of Tregs in protection function of IL-2C on MIRIObjective: To observe and investigate whether Tregs deleption,TGF-b1 neutralization,or IL-10 neutralization could alter the effects of IL-2C.Methods: Mice were subjected to 30 min of left anterior descending(LAD)ischemia followed by varying periods of reperfusion.To investigate the effect of Tregs deleption on MIRI,the male mice with eight to ten-week-old were randomly assigned to two groups:(1)IL-2C group(given with IL-2C);(2)IL-2C/PC61 group(IL-2C group receiving PC61,an anti-CD25 antibody).To investigate the effect of TGF-b1 and IL-10 on MIRI,the male mice with eight to ten-week-old were randomly assigned to three groups:(1)IL-2C group(given with IL-2C);(2)IL-2C+anti-TGF-b1 group;(3)IL-2C+anti-IL-10 group.Infarct area,cardiac function,and inflammatory response were performed on days 1 and 14.Results: When anti-CD25 antibody(PC61)was administered shortly after injection of IL-2C,expansion of Tregs in the spleen was completely abrogated.In addition,infarct size/AAR was significantly larger in IL-2C/PC61 than the IL-2C group.In parallel,we observed depressed cardiac function,as indicated by elevated EF,in IL-2C/PC61 mice after myocardial I/R compared with IL-2C alone.However,we found that neutralizing anti-IL-10 R or anti-TGF-? treatment did not abrogate the beneficial effects of IL-2C.Conclusions: Tregs deleption but not neutralizing IL-10 or TGF-? abrogated the beneficial effects of IL-2C.