The Role Of ZFP580 In ROS Mediated The Process Of Myocardial Oxidative Stress Injury

Objective: Oxidative stress is an adaptive response which is caused by the imbalance of reactive oxygen species（ROS）and antioxidant system.In humans,oxidative stress is involved in many diseases,such as Parkinson’s disease and Alzheimer’s disease.In the cardiovascular system,oxidative stress is also widely involved in myocardial ischemia reperfusion injury（myocardial ischemia/reperfusion injury,MI/RI）,atherosclerosis and hypertension.etc.ROS,as the core component of oxidative stress,can directly cause cell damage,but also can induce some biological effects indirectly through correlative signal transduction pathway such as,MAPK pathway,NF-KB pathway.Recently,the function of MAPK pathway in oxidative stress has been paid more attention.Previously,when we screened human aortic cDNA library,our group found a new zinc finger protein gene ZNF580 which cDNA was 1726 bp in length.This gene encodes a protein of 172 amino acids,containing proline at its amino terminal and three repeated tandem C₂H₂-type zinc finger motifs at its carboxyl terminus.Its protein domain is similar to the Sp/KLF family.ZFP580,as the murine homologue of ZNF 580,was cloned subsequently in our lab.Previously,our group identified that H₂O₂ could induce the higher expression of ZNF580,furthermore,the ZNF580 was regulated by NF-KB and P38 MAPK pathway.The purpose of this experiment is to explore the role of ZFP580 in ROS mediated oxidative stress injury of myocardial cells through H₂O₂ stimulating rat myocardial cell lines（H9C2）Methods: Different doses of H₂O₂ stimulated H9C2 cells in different time to find the best stimulating concentration and time point,and control group were treated with equal amounts of DMEM culture medium.Cell viability was determined with MTT cell viability assay;the activity of LDH in the supernatant was detected;Realtime-PCR detected ZFP580 mRNA,IL-6 mRNA and TNF-αmRNA expression level.Western-blot detected the protein expression levels of ZFP580,phospho-P38 MAPK,P38 MAPK,Caspase3 and cleaved-caspase3.Detecting related index after lentiviral overexpressed ZFP580 or using SB303580 inhibitors.Annexin V-PE/7-AAD staining for detection of apoptosis.Results:（1）According to the literature,cells were treated with 200 μmol/L H₂O₂ for 30 min,1h,2h,4h,6h and control group were treated with equal amounts of DMEM culture medium.MTT assay found that cell viability was significantly reduced between 2 to 6 hours（P<0.05）.For this reason,the time point 2h was utilized in the next experiments.After the determination of the time point was 2h,H9C2 cells were treated with 100μmol/L,200μmol/LL,400μmol/L,800μmol/L H₂O₂ for 2h,cell viability decreased as the concentrations of 200μmol/L,400μmol/L,800μmol/L increasesd（P<0.05）.Furthermore,LDH release assay was carried out after cells were treated with 200μmol/L H₂O₂ for 15 min,30min,1h,2h.The results found that,compared with the control group,cells were damaged after 15 min stimulationcan.All results showed that 200μmol/L H₂O₂ was enough to cause cells damage wthin 2h.So it was adopted in the next experiments.（2）Control expression level as a reference,the unit was 1.The levels of ZFP580 gene expression were markedly increased between 15 min and 2 h after H₂O₂ stimulating.（P<0.05）ZFP580 mRNA was found to maximize at 15 min,expression increased 14 folds.A significant increase of ZFP580 protein was similarly compared with the changes in gene expression at any time point reaching its peak value at 30 min and increased 3 folds（P<0.05）.（3）Control expression level as a reference,the unit was 1.H₂O₂ could cause phospho-P38 MAPK higher expression at different time points however,the expression of P38 MAPK had no significant difference.Phospho-P38 MAPK / total P38 MAPK was used to reflect P38 MAPK phosphorylation.It gradually increased with stimulation time prolonged,reaching its peak at 30 min.Pearson correlation analysis showed that changes in phospho-P38 MAPK / total P38 MAPK were similar to ZFP580（r=0.818,P <0.01）.（4）Cells were incubated with either DMSO or the inhibitors alone or with the inhibitors followed by exposure to 200μmol/L H₂O₂ for 30 min.We observed that pre-treatment of H9c2 cells with SB203580 markedly reduced H₂O₂-stimulated P38 MAPK phosphorylation（P < 0.05）and ZFP580 increasing（P<0.05）.（5）Cells were transduced with Lenti-negtive control or Lenti-ZFP580.Lenti-negtive control expression level as a reference,the unit was 1.Lenti-ZFP580 compared with Lenti-negtive control caused a significant increase in ZFP580 expression（P < 0.05）.Cells overexpressed ZFP580 followed by exposure or not exposure to H₂O₂,we observed a marked augment of IL-6 mRNA and TNF-αmRNA by RT-PCR（P<0.05）.（6）Control expression level as a reference,the unit was 1.H₂O₂ 200μmol/L stimulated H9C2 cells for 15 min,30min,1h,2h.Results showed that TNF-α mRNA expression was statistically significant increase at stimulating 30 min,1H,2h expression（P<0.05）.IL-6 mRNA expression was statistically significant increase at stimulating 15 min,30min（P<0.05）.Cells were pre-treated by SB203580 and followed by exposure to 200μmol/L H₂O₂ for 30 min or 1h.The results show that SB203580 markedly reduced H₂O₂-stimulated TNF-αmRNA and IL-6 mRNA increasing（P<0.05）.（7）Cells were transduced with Lenti-negtive control or Lenti-ZFP580.Lenti-negtive control.And cells overexpressed ZFP580 followed by exposure 200 μmol/L H₂O₂ for 2h.Then cells were further performed by annexin V/7-AAD staining.Cytofluorimetric analysis revealed that overexpression of ZFP580 significantly reduced apoptosis induced with 200 μmol/L H₂O₂ for 2h（P<0.05）.Furthermore,Western-blot detected the expression of the caspase 3 and cleaved-caspase 3,results showed both total caspase-3 and cleaved caspase-3 expression of cells overexpressed ZFP580 were markly decreased compared with Lenti-negtive control cells（P<0.05）.Conclusion: ROS upregulated the expression of ZFP580 in myocardial cells,and this process is regulated by the P38 MAPK pathway.The higher expression of ZFP580 could induce the expression of TNF-α mRNA and IL-6mRNA,and reduce the apoptosis induced by ROS.