Effect Of EPO On High Glucose-induced In Vitro Cardiac Fibroblasts And The Role Of RhoA/ROCK Signaling Pathway

Objective:To observe the effects of erythropoietin(EPO) on neonatal rat cardiac fibrosis(CFs) induced by glucose in vitro and the association with RhoA/ROCK signaling pathway.Methods:(1) primary cell culture:CFs were isolated from neonate Sprague-Dawley rats by double enzyme digestion and differential speed adherent to depurate the neonate rats CFs which adherent earlier than cardiac myocyte. Then subculturing, using 2 to 3 generation cells which growth well in our experiment. (2) Identification of cells:Using immunohistochemistry to identify the CFs:The results of Fibronectin1 (+)、Vimentin (+)、a-actin (-) were confirmed CFs. (3)Different concentrations of glucose for CFs proliferation:With different concentrations of glucose culture 48h on CFs, with CCK-8 to be measured, the absorbance value to reflect the proliferation of CFs. (4)Drug intervention:The CFs were used to establish the model of fibrosis by high glucose (25mmol/L) in vitro. They were treated with EPO (20U/ml) or and RhoA/ROCK inhibitor Y27632 (10ummol/L). And then the same batch of cells were randomly divided into five group:blank control group (glucose:5.5mmol/L)、high glucose group(glucose:25mmol/L), high glucose+EPO group(25mmol/L of glucose+20U/ml EPO), high glucose+Y27632 group(25mmol/L of glucose+lOumol/L Y27632). high glucose+EPO+Y27632 group(25mmol/L of glucose+20U/ml EPO+10umol/L Y27632). (5) The protein levels of TGF-P1 and ROCK1 were analyzed by Western blot:The different drug-treated CFs were extracted total protein respectively. After purificating、electrophoresising-. transferring、antibody incubating、exposuring、developing and fixing, the relative protein expression levels of TGF-β1 and ROCK1 by Western blot. (6)The mRNA levels of TGF-β1 and I-procollagen were analyzed by quantitative real-time PCR:The different drug-treated CFs were extracted total RNA respectively, then reverse-transcribed into cDNA. The mRNA levels of TGF-β1 and I-procollagen were analyzed by quantitative real-time PCR.Results:(1) Primary cell culture CFs:The CFs growth rapidly than myocardial cells. After the primary cell culture 3 to 5 days, the CFs can be reached 80%-90% fusion state. The CFs were spindle-shaped with larger cell body, which had 1 to 2 nucleus, transparent cytoplasm and without spontaneous pulse. (2) immunohistochemistry to identify the CFs:The results of Fibronectinl (+)%Vimentin (+)、a-actin (-) were confirmed CFs. (3)With different concentrations^.5mmol/L^ 10mmol/L、25mmol/L、50mmol/L) glucose-stimulated 48h, with the increase of the glucose concentration, corresponding to a dose-dependent increase in absorbance; 25mmol/L group reached a peak,50mmol/L group decreased slightly, but compared the two groups of 50mmol/L and 25mmol/L group, (P=0.097) P>0.05, the difference was not statistically significant. (4)Western blot results showed that:① Compared with high glucose group, high glucose+EPO, high glucose+Y27632 group the expression of TGF-β1 was no significant difference; but EPO(20U/ml)+Y27632(10umol/L) can significantly suppressed high-glucose induced upregulation the expression of TGF-β1. ② Compared with high glucose group, high glucose+EPO,high glucose+Y27632 group the expression of ROCK1 was no significant difference; but EPO(20U/ml)+Y27632(10umol/L) can significantly suppressed high-glucose induced upregulation the expression of ROCK1. (5)RT-PCR results showed that:① Compared with high glucose group, high glucose+EPO, high glucose+Y27632 group the mRNA expression of ROCK1 was no significant difference; EPO(20U/ml)+Y27632(10umol/L) can reduce the level of mRNA expression of TGF-β1. ② Compared with high glucose group, high glucose+EPO, high glucose+Y27632 group the mRNA expression of I-procollagen was no significant difference; EPO(20U/ml)+Y27632(10umol/L) can reduce the level of mRNA expression of I-procollagen.Conclutionsr(1) High glucose can promote the proliferation of CFs, which induced further myocardial fibrosis. (2)Increasing the concentration of glucose in the cell culture can effectively induce the activation of CFs, which provide a platform for the study of cells of diabetic cardiomyopathy induced myocardial fibrosis research. (3)EPO can effectively inhibit the activation of CFs, reduce the expression of TGF-β1、I-procollagen and ROCK1. The preliminary confirmed the effects of anti-myocardial fibrosis might be associated with RhoA/ROCK signaling pathway.