| Objective:No reflow,as a high incidence of phenomenon after PCI,is closely associated with a variety of poor prognosis,can seriously reduce the reperfusion therapy effect.GDF-15(Growth differential factor-15)is a distant member of the TGF-?superfamily,not only play an important role in cell growth,proliferation and differentiation,but also participate in a variety of pathophysiological process.GDF-15 was identified as a myocardial protective cytokine induced by kinds of stress,and could attenuate ischemia/reperfusion injury and inflammation via PI3K/Akt,ERK1/2 pathway.GDF-15 is also closely related to no reflow phenomenon.However,whether GDF-15 can reduce no reflow damage and which target molecule GDF-15 can regulate throughout no reflow is not clear.ZFP580,a novel C2H2(Cys2-His2)zinc-finger transcription factor,was cloned by our laboratory.Previous studies showed that ZFP580 could play an anti-apoptotic roleas a downstream target of ERK1/2 signaling.Meanwhile,ZFP580 could up-refgulate eNOS expression and stimulate the proliferation and migration of endothelial cells,and also participate in H2O2 induced ROS stress and inflammation.Then,can GDF-15 attenuate ischemia/reperfusion induced no reflow injury by regulating ZFP580/ZNF580?We aimed to explore the role and mechanism of ZFP580/ZNF580 in GDF-15 attenuating ischemia/reperfusion induced no reflow by I/R model in rats and sI/R model in HUVECs.Methods:1 Rats were subjected with 1h of LAD(left anterior descending)coronary arteryocclusion,followed by different reperfusion time(0h,0.5h,1h,2h,4h)to establish an I/R model,no reflow area and infaction area were detected by using Thioflavin S and TTC;GDF-15 levels in plasma and myocardial tissue were measured by ELISA;2 Simulated ischemia/reperfusion model was done for HUVECs in vitro;the cell viability was evaluated by MTT;tube formation in vitro was used to evaluate the change of endothelial function;3 ZNF580 overexpression and interference expression in HUVECs were achieved by transfecting with lentivirus setor;the mRNA expression level of GDF-15,ZFP580/ZNF580 and ICAM1 were detected by Real-time PCR;the protein expression levels of GDF-15,ZFP580/ZNF580,ICAM1,ERK1/2,p-ERK1/2 were determined by Western Blot.Results:1 The I/R model in rats increased the incidence of no reflow and infaction,upregulated the expression of GDF-15 and ZFP580Rats were subjected with 1h of LAD occlusion,followed by different reperfusion time(0h,0.5h,1h,2h,4h),sham group underwent the same procedure expect that the suture was passed without ligation.Results showed that all I/R groups encountered ECG abnormal change,no reflow and infaction expect for sham group.There were no signigicant difference in ischemia area of all I/R groups.RT-PCR and Western Blot showed that GDF-15 and ZFP580 levels are much higher in I/R groups than sham group(P<0.05).ELISA also presented higher GDF-15 levels in I/R groups than sham group(P<0.05).2 Simulated ischemia/reperfusion reduced cell viability and induced the expression of GDF-15 and ZNF580HUVECs were subjected to simulated ischemia with ischemia buffer in the hypoxia chamber,followed by simulated reperfusion with normal DMEM in the incubator.MTT asssay showed that the vialibity in s I/R groups were lower than Control(P<0.05).Meanwhile,the GDF-15 and ZNF580 levels were higher than Control(P<0.05).3 GDF-15 apparently upregulated the expression of ZNF580 and attenuated simulated ischemia/reperfusion induced injuryHUVECs cells were given different doses of GDF-15(1,10,20,50,100ng/mL)for 1h,it has shown that GDF-15 could upregulate ZNF580 compared with Control,especially when the concentration was 20ng/mL.Then HUVECs were given GDF-15 of 20ng/m L for continuous period(15min,30 min,1h,2h,4h),it showed that ZNF580 level increased most obviously at 1h,so the GDF-15 stimulus condition was set as 20ng/mL for 1h.Pretreat HUVECs with GDF-15 and then deal with simulated ischemia/reperfusion,it was shown that GDF-15 could further promote ZNF580 expression on the basis of sI/R,and could reduce sI/R induced ICAM1 overexpression.At the same time,GDF-15 could attenuate sI/R induced cells viability decrease and angiogenic ability in vitro.4ZNF580 played an important role in the protective effect of GDF-15 against sI/R-induced injuryTransfect HUVECs with lentivirus vector Lenti-ZNF580 or Lent-siZNF580 in order tooverexpress or silence ZNF580,using Lenti-NC or Lenti-si-scrambled as negative control.There was no significant difference among Lenti-NC,Lenti-si-scrambled and Control.However,Lenti-ZNF580 could obviously overexpress ZNF580 and Lenti-si-scrambled could apparently silence ZNF580.Pretreat Zfp580-deficient cells with GDF-15,then deal with simulated ischemia/reperfusion,it shown that silence of ZNF580 further enhanced sI/R induced ICAM1 overexpression and reduced cells viability and angiogenic ability in vitro.However,pretreatment with GDF-15 could partly eliminate the above effects.Overexpression of ZNF580 could also attenuate sI/R induced overexpression of ICAM1,decrease of cells viability and reduction of angiogenic ability in vitro.5 GDF-15 could attenuate simulated ischemia/reperfusion injury by regulating ZNF580 via ERK1/2 pathwayWe further explored the mechanism of ZNF580 involved in the GDF-15/ERK1/2 signaling pathway against sI/R-induced injury.There was a significant upregulation of p-ERK1/2 after treating HUVECs with GDF-15,but total ERK1/2 had not similar change.So we used PD98059,an inhibitor of ERK1/2 signaling pathway to cut off ERK1/2.It was shown that PD98059 could partly eliminate GDF-15 induced upregulation of ZNF580 and attenuate the protective effect of GDF-15 against sI/R.Conclusions:After I/R in rats heart,there was a significant upregulation of GDF-15 and ZFP580,and increaseof no reflow,infarct incidence;GDF-15 could obviously attenuate ischemia/reperfusion injury,and ZNF580 was involved,so to speak,GDF-15 attenuated ischemia/reperfusion injury by regulating ZNF580;ERK1/2,as an important signaling pathway related with cells proliferation and differentiation,was involved in GDF-15 regulating ZNF580. |
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