| Objective: It is often used reperfusion therapy to recover the blood supply of the ischaemic myocardial cell. Myocardial cell apoptosis in myocardial ischemia-reperfusion injury is very common, and apoptosis is the main deadly forms in the early stage of ischemia-reperfusion. Studying the anti-apoptosis mechanisms after ischemia-reperfusion could provide a potential way to reduce the apoptosis induced by reperfusion.In recent years, it is recognized the important role of EPO(recombinant human erythropoietin) in cardiovascular diseases. Calvillo found that adding EPO in cultured hypoxic injury rat myocardial cells, apoptosis decreased by 50% and confirmed that EPO has the anti- apoptosis function. But the EPO on apoptosis genes is less clear. Bcl-2 protein gene family is critical in apoptosis genes, include anti-apoptosis gene Bcl-2 and pro-apoptosis gene Bax,but it is not clear that the anti-apoptosis function of EPO is created via Bcl-2 protein gene family, so studying the Bcl-2 protein gene family is helpful for explaining the protecting function of EPO. Study found that the expression of VEGF (vascular endothelial growth factor) may make it easier to angiogenesis, and can reduce long term myocardial cell apoptosis. The mechanisms of VEGF may increase the expression of anti-apoptotic protein Bcl-2, reduce the expression of pro-apoptotic protein Bax, thereby reducing apoptosis. So EPO inhibit apoptosis may be by influencing Bcl-2 protein family, and then improve the cardiac function after acute myocardial ischemia-reperfusion.In this study, a animal model of IR(ischemia-reperfusion) was used, and followed treatment with recombinant human EPO (EPO). The expression of Bcl-2 protein, Bax protein, VEGF protein, Bcl-2 mRNA, Bax mRNA were obtained after IR, and then to investigate the protective mechanismss of EPO. Methods: 54 healthy male Sprague Dawley rats (12-15 weeks and 210-260g) were recruited. Rat models of IR were induced by blocking left anterior decending coronary artery. The 54 rats were divided into 3 groups randomly: sham-operated group, IR group and EPO group (recombinant human EPO 3000U·kg-1·d-1 intraperitoneal injection, for 3 days). The Bcl-2 protein, Bax protein, VEGF protein, were quantified by immunohistochemical method. RT-PCR was used to observe the gene expression of Bcl-2 mRNA, Bax mRNA. All data were analyzed with SPSS version 17.0 statistical software. The comparison among groups was analyzed by One-Way ANOVA and S-N-K test.Results: (1) The effect of EPO on the expression of Bcl-2 protein in border areas: Compared with sham-operated group, the expression of Bcl-2 protein detected in border areas significantly decreased in IR group and EPO group at 48h, 2w and 3w (P<0.01); Compared with IR group, the expression of Bcl-2 protein detected in border areas raised 27.7%, 31.4%, 25.9% respectively in EPO group at 48h, 2w and 3w (P<0.01). (2) The effect of EPO on the expression of Bax protein in border areas: Compared with sham-operated group, the expression of Bax protein detected in border areas significantly increased raised in IR group and EPO group at 48h(P<0.01); Compared with IR group, the expression of Bax protein detected in border areas lowered 20.9%, 18.0%, 16.7% respectively in EPO group at 48h, 2w and 3w (P<0.01). (3) The effect of EPO on the ratio of Bcl-2 and Bax protein in border areas: Compared with sham-operated group, the ratio of Bcl-2 and Bax protein was significantly decreased in IR group and EPO group at the different time (P<0.05); Compared with IR group, the ratio of Bcl-2 and Bax protein was in EPO group at the different time (P<0.05); (4) The effect of EPO on the expression of Bcl-2mRNA in border areas: Compared with sham-operated group, the expression of Bcl-2mRNA detected in border areas decreased in IR group and EPO group at 48h, 2w and 3w (P<0.01); Compared with IR group, the expression of Bcl-2mRNA detected in border areas raised 61.0%, 32.7%, 39.9% respectively in EPO group at 48h, 2w and 3w (P<0.01). (5) The effect of EPO on the expression of BaxmRNA in border areas: Compared with sham-operated group, the expression of BaxmRNA detected in border areas significantly increased in IR group and EPO group at 48h, 2w and 3w (P<0.01); Compared with IR group, the expression of BaxmRNA detected in border areas lowered 11.4%, 7.7%, 34.2% respectively in EPO group at 48h, 2w and 3w (P<0.01).(6) The effect of EPO on the ratio of Bcl-2 and Bax mRNA in border areas: Compared with sham-operated group, the ratio of Bcl-2 and Bax mRNA was significantly decreased in IR group and EPO group at the different time (P<0.05); Compared with IR group, the ratio of Bcl-2 and Bax mRNA was significantly increased in EPO group at the different time (P<0.05); (7) The effect of EPO on the expression of VEGF protein in border areas: Compared with sham-operated group, the expression of VEGF protein detected in border areas significantly increased in IR group and EPO group at 48h, 2w and 3w; (P<0.01 or P<0.05); Compared with IR group, the expression of VEGF protein detected in border areas raised 22.1%, 21.4%, 20.5% respectively in EPO group at 48h, 2w and 3w (P<0.01).Conculsion:(1) This study showed that the lower expression of Bcl-2 protein and mRNA, higher expression of Bax protein and mRNA in border areas after myocardial ischemia-reperfusion. (2) EPO could increase the expression of Bcl-2 protein ,VEGF protein and Bcl-2 mRNA, reduce the expression of Bax protein and Bax mRNA, increase the ratio of Bcl-2/Bax in border areas after myocardial ischemia-reperfusion. It is possible that EPO may bring cardiac protective function by regulateing the expression of Bcl-2 and Bax to inhibit the ischemia-reperfusion injury.
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