Erythropoietin Attenuates Cardiac Fibrosis In Vitro And The Association With TGF β₁-TAK1-p38MAPK Pathway

Objective:To observe the effects of erythropoietin (EPO) on angiotensin II (Ang II) induced neonatal rat cardiac fibrosis and the association with TGF β1-TAK1-p38MAPK signaling pathway.Methords:1、Primary cell culture:Cardiac fibroblasts were isolated from new-born Sprague-Dawley rats:the ventricular musclet were obtain from neonate rat (1～3day), then went throughing digesting、filtering and differential speed adherent to depurate the neonatal rat cardiac fibroblasts which adherent earlier than cardiac myocyte. After subculturing, the experiment using2to3generation cells which growth well.2、Drug intervention:The cardiac fibroblasts were used to establish the model of fibrosis by Ang Ⅱ(10-6mol/L) in vitro. They were treated with EPO (20U/ml) or/and p38MAPK inhibitor SB203580(15Um/L). And then the same batch of cells were randomly divided into five groups:blank control group, Ang II group, the EPO group, Ang Ⅱ+EPO group and Ang Ⅱ+EPO+SB203580group.3、Immunocytochemical method to detect the expression of fibrosis markers (a-SMA):After cardiac fibroblasts adherented, then they were treated with different drugs in groups.Detecting the protein expression of α-SMA as CFs fibrosis observed indicators. Observing the intracellular protein expression of a-SMA with Immunohistochemistry in each groups, and then compared the differences in the expression of α-SMA protein. 4、The mRNA levels of intracellular signaling molecules was analyzed by quantitative real-time PCR:The different drug-treated cardiac fibroblasts were extracted total RNA respectively, then reverse-transcribed into cDNA.The mRNA levels of transforming growth factor β1(TGF-β1) and p38mitogen activated protein kinase (p38MAPK) was analyzed by RT-PCR.5、The protein levels of intracellular signaling molecules were analyzed by Western blot:The different drug-treated cardiac fibroblasts were extracted total protein respectively. After purificating、electrophoresising、transferring、antibody incubating、exposuring、developing and fixing, the relative protein expression levels of a-SMA、TGF-β1、TAK1and p38MAPK and the phosphorylation of TAK1and p38MAPK were analyzed by Western blot.Results:1、Primary cell culture of CFs:The CFs growth rapidly than myocardial cells.After the primary cell culture3to5days, the CFs can be reached80%～90%fusion state. The CFs were spindle-shaped with larger cell body, which had1to2nucleus、transparent cytoplasm and without spontaneous pulse.2、Immunocytochemical method to detect the expression of fibrosis markers(α-SMA):Ang Ⅱ (10-6mol/L) can effectively induce the expression of a-SMA protein in CFs.20U/ml of EPO can effectively inhibit Ang Ⅱ-induced cardiac fibrosis, and also can reduce the intracellular protein expression of a-SMA.But the inhibition effect of expression of a-SMA were similar with adding the specific inhibitor SB203580.3、RT-PCR results showed that:EPO(20U/ml) can reduce the level of mRNA expression which were the CFs fibrosis-related signaling molecules,such as TGF-betal and P38MAPK. And the effects can be strengthened by SB203580.4、Western Blot results showed that:EPO(20U/ml) can significantly suppressed AngⅡ-induced upregulation the expression of TGF-β1, TAK1, p38MAPK and weaked the phosphrylation of TAK1and p38MAPK.And also the effects can be strengthened by SB203580.Conclusion:1、Ang II can induce CFs fibrosis and simulate the process of ventricular remodeling after myocardial infarction.The Neonatal rat CFs can be used as EPO anti-CFs Fibrosis cell platform.2、EPO can effectively inhibit Ang II-induced cardiac phenotypic switched into myofibroblasts, reduce myocardial fibrosis, also can reduce the signaling molecules expression of TGF-β1-TAK1-p38MAPK pathway. Then preliminary confirmed the effects of anti-myocardial fibrosis and reverse ventricular remodeling cardioprotective might be associated with TGF β1-TAKl-p38MAPK signaling pathway.