The Role And Mechanisms Of IL-35 In Myocardial Ischemia-reperfusion Injury

Part I IL-35 ameliorates myocardial ischemia-reperfusion injury and cardiomyocyte apoptosisObjective:Myocardial ischemia-reperfusion injury(MIRI)involves multiple pathological processes,with inflammation being a common feature.IL-35 is a new anti-inflammatory cytokine.However,the role of IL-35 in the treatment of MIRI is unclear.The purpose of this study was to find out the effect of IL-35 on MIRI.Methods:The model of myocardial ischemia-reperfusion injury was established by ligating the left anterior descending coronary artery of C57BL/6 mice heart for 30 minutes,followed by reperfusion.We injected the mice with 1?g of recombinant mouse IL-35 10minutes prior to reperfusion to study the potential effect of IL-35 in MIRI.In addition,to compare the effects of IL-35 treatment at different times,we examined the effects of treatment with IL-35 either 0 minute post-reperfusion or 1 hour post-reperfusion and evaluated cardiac function 24 hours after reperfusion.HL-1 cells(cardiac cell line)were pretreated with IL-35 and stimulated with H₂O₂ in vitro to induce apoptosis.The expression of cleaved caspase-3 in each group was detected by western blot to evaluate apoptosis.Results:We found that I/AAR in IL-35 group decreased,serum c Tn T decreased and EF and FS in IL-35 group increased compared with vehicle group.In addition,the protective effect of IL-35 treatment can be exerted during reperfusion.However,the protective effect disappeared when the IL-35 treatment was delayed to 1 hour after reperfusion.At 4 hours after myocardial ischemia-reperfusion,compared with vehicle group,the TUNEL positive rate of cardiomyocyte in IL-35 group was reduced,and the expression of cleaved caspase-3in cardiac tissue was reduced.In addition,the expression of cleaved caspase-3 in the H₂O₂+IL-35 group was lower than that in the H₂O₂ group,which proves that IL-35 can protect cardiomyocyte against apoptosis in vitro.Conclusion:IL-35 can reduce the infarct size and improve cardiac function in MIRI.At the same time,IL-35 can play a protective role in cardiomyocyte apoptosis in vivo and in vitro.Part II IL-35 exerts protective effects on MIRI by activating STAT3Objective: Like other members of the IL-12 family,IL-35 could exert its biological activity by activating the STAT family.At the same time,it has been reported that the gp130-STAT3 pathway can play a protective role on cardiomyocytes in MIRI.Whether IL-35 plays a role in MIRI through activation of the STAT family is unclear.The purpose of this study was to explore the signaling pathways activated by IL-35 in MIRI.Methods: In the experiment to detect the effect of IL-35 on the activation of STAT family in MIRI,mice were subjected to either sham operation or 30 minutes of ischemia followed by reperfusion for 0,5,15,30,60,or 120 minutes in the absence or presence of IL-35.The activation of STAT1,STAT3,STAT4,STAT5,and STAT6 was measured by western blot.We pretreated MIRI mice with STAT3 inhibitor S3I-201 and STAT5 inhibitor pimozide,respectively,and tested whether IL-35 still protected MIRI in this case.To further study the role of cardiac STAT3 in IL-35-mediated protection,we performed MIRI on wild-type and cardiomyocyte-specific STAT3 knockout mice(stat3 cko),and pretreated with IL-35 to observe the effect of IL-35 on cardiac function and apoptosis in stat3 cko.In the STAT3 activation experiment of HL-1 cells,IL-35 was used to stimulate HL-1,and STAT3 activation was detected by western blot at 0,5,15,30,and 60 minutes after stimulation.In addition,we used STAT3 inhibitor S3I-201 or stat3 si RNA to inhibit STAT3 in HL-1 cell,then we examine whether the pretreatment of IL-35 on HL-1 cells can protect against apoptosis induced by H2O2 when STAT3 was inhibited.The expression of cleaved caspase-3 in each group was measured by western blot to evaluate the apoptosis of HL-1 cells.Results: In the experiment to detect the effect of IL-35 on STAT family activation in MIRI,IL-35 can only further activate STAT3 and STAT5.In the in vivo experiment of the effect of the STAT3 inhibitor S3I-201 on the protective effect of IL-35,the reduction of infarct size by IL-35 was abolished by S3I-201,consistent with the elevated c Tn T levels and the deterioration of cardiac function.Then,we used pimozide to inhibit STAT5 phosphorylation in vivo.Unexpectedly,the STAT5 inhibitor pimozide had no influence on the effect of IL-35 on improving cardiac function in MIRI.At the same time,IL-35 also lost its protective effect on cardiac function in stat3 cko mice.In the experiments of STAT3 activation on HL-1 cells,STAT3 was tyrosine phosphorylated within 15,30,and 60 minutes of IL-35 stimulation.Meanwhile,under the administration of S3I-201 or stat3 si RNA,the decreasing expression of cleaved caspase-3 induced by IL-35,an indicator of apoptosis,was abolished.Conclusion: In vivo experiments and in vitro experiments showed that IL-35 protected MIRI and reduced the apoptosis in cardiomyocyte through STAT3 activation.Part III: IL-35 activates STAT3 through gp130 to exert protective effects on MIRIObjective: IL-35 is a member of the IL-12 cytokine family,and each member of the IL-12 family is a heterodimer,of which each member can activate downstream signals through pairing of corresponding receptor chains such as IL-12R?1,IL-12R?2,IL-23 R,gp130,and IL-27R?.The purpose of this study was to examine the potential receptors contributing to IL-35 binding and STAT3 activation in cardiomyocytes.Methods: We used knockout mice to study the receptors that IL-35 relies on to activate STAT3 in cardiomyocyte.In the in vivo experiments,30 minutes after the establishment of the MIRI model,proteins were extracted from the heart and the activation of STAT3 was detected by western blot.In the in vitro experiments,adult mouse cardiomyocytes isolated from wild-type mice and each receptor knockout mouse were cultured and stimulated with IL-35,and STAT3 activation was detected by western blot.In addition,HL-1 cells were treated with si RNA and neutralizing antibodies corresponding to each receptor,and stimulated with IL-35.STAT3 activation was detected by western blot.To investigate whether IL-35 relies on gp130 to mediate protective effect,we performed MIRI modeling on wild-type and myocardial-specific knockout gp130 mice(gp130 cko),and pretreated with IL-35 to detect cardiac function and apoptosis.At the same time,we used gp130 si RNA or gp130 neutralizing antibody to inhibit gp130 in HL-1 cell,then we examined whether the pretreatment of IL-35 on HL-1 cells could protect against apoptosis induced by H2O2 when gp130 was inhibited.The expression of cleaved caspase-3 in each group was measured by western blot to evaluate the apoptosis of HL-1 cells.In addition,we explored the exact receptor combinations comprised of gp130 after IL-35 stimulation in cardiomyocyte through co-IP.Results: In vivo experiments,gp130 cko mice showed less cardiac STAT3 activation under IL-35 stimulation,while STAT3 activation on other receptor knockout mice was consistent with STAT3 activation on wild-type mice.In addition,IL-35 had no effect on STAT3 on adult mouse cardiomyocyte of gp130 cko mice,but activated STAT3 on adult mouse cardiomyocyte of other receptor knockout mice.In addition,the use of si RNA to silence IL-12R?2,IL-27R?,or IL-12R?1 subunits on HL-1 cells did not affect IL-35-mediated STAT3 activation,while silencing gp130 completely inhibited this activation.And gp130 neutralizing antibodies can also inhibit IL-35-mediated STAT3 signaling.At the same time,IL-35 could not exert protective effects on cardiac function and apoptosis after MIRI in gp130 cko mice.Meanwhile,silencing of gp130 by gp130 si RNA or gp130 neutralized antibody blocked the ability of IL-35 to reduce cleaved caspase-3 expression in HL-1 cells,suggesting that gp130 is involved in STAT3-mediated anti-apoptosis by IL-35.In addition, the result of co-IP showed that IL-35 induced gp130-gp130 homodimer and gp130-IL-12R?2 heterodimer,but had no effect on the binding of gp130 to IL-27R? or IL-12R?1.Conclusion: IL-35 activates STAT3 through gp130 to protect MIRI and reduce cardiomyocyte apoptosis.And IL-35 may exert its protective effects through inducing the formation of gp130-gp130 homodimer and gp130-IL-12R?2 heterodimer.