Effect Of CEPO On High Glucose-induced In Vitro Cardiaomyocytes And The Role Of GSK-3β/Smad3 Signaling Pathway

Objective:In this study,we want to demonstrate a physiological and a possible pathologic statement of cardiomyocytes(CM)in a special environment by mimics a high glucose environment in vitro which is similar to internal environment of DM patients.And to observe the effects of carbamylated erythropoietin(CEPO)on neonatal SD rat cardiomyocytes and the role of GSK-3β/Smad3 in this special environment.In this way,we could find out whether the CEPO can protect cardiomyocytes from the hypertrophy or not,and put forward the feasible basis for the future treatment of diabetic cardiomyopathy.Methods:cell culture:CM were isolated from neonate Sprague-Dawley rats by double enzyme digestion and differential speed adherent to depurate the neonate rats CM which adherent later than cardiac fabric myocytes.(2)The erythropoietin(EPO)was carbamylated to CEPO by reaction of EPO and potassium cyanate.The product of CEPO digested with end proteinase Lys-C was identified by using sodium dodecyl sulfate polyacrylamide gel electrophoresis.(3)Myocardiocytes hypertrophy model:hypertrophy model is established by high glucose culture.After confirmed hypertrophy,set Six gropes:Control group(A group,glucose:5.5mmol/L),high glucose group(B group,glucose:25mmol/L),control+CEPO treatment group(C group,5.5mmol/L of glucose+20U/ml CEPO),high glucose+CEPO treatment group(D group,25mmol/L of glucose+20U/ml CEPO),high glucose+EPO treatment group(E group,25mmol/L of glucose+20U/ml EPO),high glucose+SB216763 group(F group,25mmol/L of glucose+3 μmol/L SB216763).(5)The protein levels of GSK-3 β,Smad3 and p-GSK-3 β were analyzed by Western blot.(6)The mRNA levels of GSK-3β and Smad3 were analyzed by quantitative real-time PCR.Results:(1)Primary cell culture of CM:After the primary cell culture 3 to 5 days,the CM can be impulse simultaneously and automatically.(2)immunohistochemistry to identify the CM:The results of Fibronectinl(-)、Vimentin(-)、α-actin(+)were confirmed CM.(3)Western blot results showed that:①Compared with high glucose group,in high glucose +CEPO group and control group,the protein expression levels of Smad3 and p-GSK-3 β were reduced,the protein expression levels of GSK-3β was raised.② Compared with high glucose group,in high glucose + SB216763 group,the expression of Smad3 was significantly raised;③ Compared with high glucose +EPO,in high glucose +CEPO group,the Smad3 expression was much lower and GSK-3β was higher than EPO group.(5)RT-PCR results showed same results to Western blot results.Conclutions:(1)CEPO and EPO can reduce the myocardiocyte hypertrophy in vitro.(2)Smad3 and GSK-3β mediates diabetic cardiac hypertrophy,and GSK-3p can directly inhibit the expression of Smad3.GSK-3β is a negative regulate factor of cardiac hypertrophy.(3)CEPO is better than EPO in reduce the diabetic cardiac hypertrophy.