| BackgroundThe oxidative stress damage is defined as the injury of cell and tissue induced by functional inbalance of oxidation system and antioxidation system, resulting from overproduction of oxygen free radicals in the presence of pathological stimuli or during the process of removing senescent cells. Recent studies demonstrated that oxidative stress damage was a major mechaniam for many cardiovascular system diseases, such as myocardial ischemia-reperfusion injury, atherosclerosis, myocardial infarction, heart failure, hyperlipidemia and hypertension. Therefore, how to alleviate the oxidative stress damage in cardiovascular diseases has become one hotspot of research.Erythropoietin (EPO) is a cytokine primarily secreted by the renal juxtaglomerular cells and it is essentially one kind of glycoprotein hormone belonging to the cytokine superfamily. The main function of EPO is to promote the differentiation and proliferation of hematopoietic cells and erythroid cells in bone marrow and to facilitate the mature of red blood cells. The production of Recombinant human erythropoietin (rhEPO) is based on the recombinant DNA techniques and its amino acid sequence and biological activity are identical to endogenous EPO. rhEPO is previously used for clinical treatment of anemia induced by chronic diseases such as kidney disease and cancer, due to its effect on the increase of hemoglobin in the hematopoietic system. EPO receptors is widely distributed in the body. It can be expressed not only in the endothelial cells, macrophages, retinal cells, myocardial cells, the adrenal cortex and medulla cells, but also in some important organs such as kidney, liver, heart, pancreas, brain, uterus, lung and spleen. Because of the continuous progress in the study of EPO, the effect of EPO on other tissues and organs has gradually been found. Recent studies have shown that EPO can protect a variety of tissues from injury, and especially it has a significant protective effect on the heart. Although the protective mechanism of EPO for cardiovascular system is still obscure, it shows a great application prospect in the prevention and treatment of cardiovascular diseases.Objective:In this research, we aim to study the role and mechanism of rhEPO in protecting cardiomyocytes from oxidative stress damage by investigating the effect of rhEPO on myocardial apoptosis and detecting the expression of apoptosis-related proteins in the oxidative stress injury model.Methods:Neonatal rat cardiomyocytes were randomly divided into three groups:(1) control group:myocardial cells of this group was cultured without any stimulation;(2) H2O2group:to establish myocardial cell oxidative stress damage model, H2O2was added in the culture process;(3)rhEPO group:cells of this group were pretreated with rhEPO before the establishment of oxidative stress model. The apoptosis rate of myocardial cells was detected by MTT assay and flow cytometry to investigate the effect of rhEPO on myocardial cell apoptosis. At the same time, Western blotting was used to detect the expression levels of caspase-3,Bax,and Bcl-2in myocardial cells of three groups.Results:1. The results of myocardial cell viability detected by MTT assayThe OD value of myocardial cells in H2O2group (0.31±0.06) and rhEPO group (0.51±0.07) were both significantly reduced comparing with control group (0.64±0.09), and the difference was statistically significant (P<0.05). This result showed that oxidative stress could lead to cardiac myocyte apoptosis. The OD value of myocardial cells in rhEPO group was significantly higher than that of H2O2group (P<0.05), which showed that the survival rate of myocardial cells in oxidative stress injury was significantly increased by pretreatment of cells with rhEPO.2. The results of myocardial cell apoptosis detected by Flow cytometryFlow cytometry demonstrated that apoptosis rate of myocardial cells in rhEPO group (5.6%±0.8%) was significantly higher than that of the control group (4.1%±0.7%), but was lower than that of H2O2group (10.7%±0.5%), and the differences were statistically significant (P<0.05). This result suggested that pretreatment with rhEPO can inhibit the myocardial cell apoptosis induced by oxidative stress injury.3. The expression levels of caspase-3, Bax, and Bcl-2in myocardial cells detected by Western blottingThe results of Western blotting showed that: When compared with the control group, the expression level of caspase-3and Bax protein in H2O2group and rhEPO group increased significantly (P<0.05), while the Bcl-2expression decreased significantly (P<0.05). This result revealed that oxidative stress could promote cell apoptosis by downregulation of the anti-apoptotic protein and upregulation of pro-apoptotic proteins. When compared with H2O2group, the expression level of caspase-3and Bax protein in rhEPO group decreased significantly (P<0.05), while the expression level of apoptosis inhibition protein Bcl-2increased significantly (P<0.05). which demonstrated that rhEPO could protect myocardial cells from apoptosis in oxidative stress injury by inhibiting the expression of pro-apoptotic proteins and increasing the expression of anti-apoptotic proteins.Conclusion:1. Pretreatment with rhEPO can inhibit apoptosis of myocardial cells induced by oxidative stress injury.2. rhEPO may inhibit myocardial cells apoptosis induced by oxidative stress injury by downregulating the expression of caspase-3and Bax protein and upregulating the expression of Bcl-2protein. |
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