| Background and objectives:EPO is a member of the cytokine I family, which is named by its promoting the erythropoiesis. EPO induces the proliferation and differentiation of the erythroid progenitor cells and then increases the number of circulating erythrocytes and the concentration of hemoglobin, which has been used to treat anemia caused by renal failure, chemotherapy, surgery and so on. However, expression of EPOR has been found in many non-hematopoietic tissues and EPO has been proved to have many different non-hematopoietic biological effects, in which cytoprotective effect of EPO has drawn much attention from the researchers. EPO was first found to have neuroprotective effects and the following studies further demonstrated that EPO might be a general cytoprotective cytokine, which has protective effects in the injury of kidney, vascular endothelial cells, vascular smooth muscle cells, skin, small intestine and so on.Cardiac protective effect of EPO is a new topic in the recent two years. Juul found that cardiac myocytes of human embryo could express EPOR gene, and then Tramontano proved that the neonatal rat ventricular cardiac myocytes could express EPOR protein with immunostaining and Western blot. Cai found that the adult rat myocardium could express EPOR mRNA with RT-PCR. However, the distribution of EPOR protein in mature myocardium is still unclear. It needs further research to identify whether mature cardiac myocytes can express EPOR protein. Expression of EPOR in myocardium suggests that EPO can directly act on cardiac myocytes. However, there are still few reports on the cardiac protective effects of EPO at present.The protective effects of EPO pretreament has been reported in the I/R injury of brain and kidney, but in myocardium there are few reports. Therefore, in this study, rats and cultured cardiac myocytes were treated with H/R injury and the cardiac protective effect of EPO pretreatment was investigated. And then, the signal transduction mechanismassociated with NF-kB was studied. NF-kB activation has been reported to be related to ischemic preconditioning and pretreatment of some drugs such as adenosine, and when activation of NF-kB during preconditioning is inhibited, the myocardial protective effect is significantly weakened or completely abolished. Therefore, the signal transduction mechanism associated NF-kB was studied in this study. NF-kB may be activated by the signal molecules including PI3K/Akt, JAK2/STAT5 and MAPKs, which may participate in EPO's tissue protective effect. Therefore, NF-kB is probably the congenerous way of these signal pathways.Methods:In the first part of this study, rat model with myocardial H/R injury was built, and 60 SD male adult rats were randomly divided into 3 groups. Rats in control group was treated with saline through peritoneal injection and without H/R; rats in EPO pretreatment group (EPO group) was treated with 5000U/kg RHuEPO through peritoneal injection and after 24 hours, were treated with H/R; rats in H/R group were treated with saline and H/R injury. Serum myocardial enzyme activity, myocardial MDA, HE staining, myocardial ultrastructure, myocardial apoptosis and cardiac function were detected in rats.Then 40 rats were divided into 4 groups, including control group, EPO pretreament group (EPO group), EPO pretreatment group with H/R injury (EPO-H/R group), H/R group, and the treatment was the same. Myocardial EPO protein expression was identified with immunostaining and western blot. The expression of bcl-2, bax and TNF-α in myocardium was investigated with immunostaining. NF-kB activity in myocardium was analyzed with EMSA.In the second part, neonatal rat ventricular myocytes were cultured and identified. Then the cardiac myocyte model with H/R injury was built according to the previous report. All the myocytes were divided into 4 groups, including control group, EPO pretreatment group (EPO group) (RHuEPO 10U/ml culture medium), EPO+PDTC pretreatment group (EPO+PDTC group) (RHuEPO lOU/ml and PDTC 5μg/ml medium) and PDTC pretreatment group (PDTC group) (PDTC 5μg /ml medium). 24 hours after the pretreatment, cardiac myocytes were treated with H/R injury (hypoixa for 120 min and reoxygenation for 30 min). Before and after H/R, morphological changes of myocytes were observed with inverted microscope, LDH activity in medium was analyzed, the survival rate of myocyteswas detected by MTT chromatometry, and the apoptosis was analyzed with TUNEL and flow cytometry.In the third part, the grouping and treatment of cultured cardiac myocytes were described in the second part. NF-kB activity in myocytes was analyzed with EMSA before and after H/R. The expression level of I-kB(x protein in myocytes was detected by Western blot before and after H/R. The expression of bcl-2 and TNF-α in myocytes was analyzed by RT-PCR and Western blot before and after H/R.Results:In the first part:1. H/R injured myocardium markedly, including the increasing of serum myocardial enzyme activity and myocardial MDA, the elevation of apoptotic rate, injury of myocardial ultrastructure and the decreasing of cardiac function. EPO pretreatment minimized the myocardial injury after H/R.2. Immunostaining and Western blot proved that adult rat cardiac myocyte could express EPOR protein, and immunostaining demonstrated that the expression of EPOR protein in adult rat myocardium was lightly positive, and EPOR protein was mainly expressed in the cytoplasm of cardiac myocytes and vascular endothelial cells. EPO pretreatment and H/R had no significant influence on the expression of myocardial EPOR protein(P>0.05).3. Immunostaining of bcl-2 in myocardium demonstrated that EPO pretreatment upregulated the expression of bcl-2 before H/R, compared to the control(P<0.01). After H/R, the expression level of bcl-2 in myocardium of H/R group and EPO-H/R group increased, compared to before H/R (P<0.01, PO.05), and the expression level of bcl-2 was higher in EPO-H/R group than in H/R group (P<0.05).4. Immunostaining of bax in myocardium showed that EPO pretreatment had no marked influence on the expression of bax before H/R(P>0.05). After H/R, the expression of bax in myocardium of H/R group and EPO-H/R group was much higher than that of control group (P<0.01). There was no significant difference in the expression of bax between H/R group and EPO-H/R group (P>0.05).5. EPO pretreatment significantly raised the bcl-2/bax in myocardium before H/R, compared to control (P<0.01). After H/R, bcl-2/bax in H/R group and EPO-H/R groupdecreased markedly, compared to control group and EPO group (P<0.01, P<0.05). bcl-2/bax in EPO-H/R group was higher than that in H/R group (P<0.05).6. Immunostaining of TNF-α in myocardium showed that EPO pretreatment had no significant influence on the expression of TNF-α before H/R (P>0.05). After H/R, the expression of TNF-α in EPO-H/R group and H/R group was higher than that in control group (P<0.01). The expression in H/R group was higher than that in EPO-H/R group (P<0.05).7. EPO pretreatment increased the NF-kB activity in myocardium before H/R, compared to control (P<0.01). After H/R, the NF-kB activity in myocardium of EPO-H/R group and H/R group increased markedly, compared to before H/R (P<0.05, P<0.01), and the NF-kB activity was higher in H/R group than in EPO-H/R group (P<0.05).In the second part:1. After H/R, LDH activity in medium of all the groups increased significantly compared to the control group before H/R(P<0.01, P<0.05), and LDH activity of EPO group was lower than that of the other 3 groups(P<0.05).LDH activity of EPO+PDTC group was not markedly different from that of control group and PDTC group(P>0.05).2. After H/R, survival rate of myocytes markedly decreased in all the groups compared to the control group before H/R(P<0.01, P<0.05), and apoptosis rate of myocytes increased significantly (P<0.01). After H/R, survival rate of EPO group was higher than that of the other 3 groups(P<0.05) and apoptosis rate was lower than that of the other 3 groups(P<0.01). Apoptosis rate and survival rate of EPO+PDTC group were not significantly different from those of the control group and PDTC group, after H/R.In the third part:1. EPO pretreatment increased the NF-kB activity in myocytes before H/R, compared to the control (P<0.01), and the increasing of NF-kB activity followed the time-dependent mode. At 6, 12, 24 hour after the administration of EPO, NF-kB activity increased markedly, compared to control (P<0.05 , P<0.01). The administration of PDTC during EPO pretreatment abolished the activation of NF-kB (P<0.01). After H/R, NF-kB activity in myocytes of different groups increased markedly, compared to control group before H/R (P<0.01), and NF-kB activity was lower in EPO pretreatment group than in the other three groups (P<0.05). There was no significant difference in NF-kB activity among the otherthree groups before and after H/R (P>0.05).2. EPO pretreatment decreased the expression of I-KBa protein in myocytes before H/R, compared to control (P<0.01), and the decreasing of the I-KBa expression followed the time-depent mode. At 6, 12, 24 hour after the administration of EPO, the I-KBa expression level decreased markedly compared to control (P<0.05 , P<0.01). The administration of PDTC during EPO pretreatment abolished this change (P<0.01). After H/R, I-KBa expression level in myocytes of different groups decreased markedly compared to the control group before H/R (P<0.01), however, I-KBa expression level was higher in EPO pretreatment group than in the three other groups (P<0.05). There was no significant difference in I-KBa expression among the three other groups before and after H/R (P>0.05).3. EPO pretreatment upregulated the expression of bcl-2 protein and mRNA in myocytes before H/R, compared to control (P<0.01) and this upregulation followed the time-dependent mode, too. Administration of PDTC significantly weakened the upregulation of bcl-2 during EPO pretreatment (P<0.01). After H/R, the expression level of bcl-2 protein and mRNA in myocytes of different groups increased, compared to control group before H/R (P<0.01), and the expression level of bcl-2 was higher in EPO pretreatment group than in the other three groups (P<0.01). There was no significant difference in bcl-2 expression among the other three groups before and after H/R (P>0.05).4. Myocytes in control group before H/R could express low level of TNF-α mRNA and protein, and EPO pretreatment had no significant influence on TNF-α expression before H/R (P>0.05). After H/R, the expression of TNF-α was markedly upregulated in myocytes of different groups compared to control before H/R (P<0.01), and the TNF-α expression level was lower in EPO pretreatment group than in the other three groups (P<0.05, P<0.01). There was no significant difference in TNF-α expression among the other three groups before and after H/R (P>0.05).Conclusions:1. Adult rat myocardium could express EPOR protein, and the expression was lightly positive. EPOR protein was mainly expressed in cytoplasm of cardiac myocytes and vascular endothelial cells. EPO pretreatment and H/R didn't affect the expression of EPOR protein in myocardium.2. EPO pretreatment had the protective effects on rat hearts with H/R, which includedreduction of the leakage of myocardial enzyme, reduction of oxygen-derived free radicals, protection of myocardial ultrastructure, inhibition of apoptosis, better recovery of cardiac function during reoxygenation.3. EPO pretreatment promoted the survival, inhibited the apoptosis and reduced leakage of LDH in cultured cardiac myocytes with H/R injury, which was related to the activation of NF- k B during EPO pretreatment.4. EPO pretreatment activated NF-kB in cardiac myocytes before H/R, which then upregulated the expression of I-kBα, inhibited the decreasing of I-kBα protein expression after H/R, and inhibited the further activation of NF-kB after H/R depending on the negative feed-back mechanism of NF-kB activation.5. EPO pretreatment inhibited the further activation of NF-kB in cardiac myocytes after H/R through the negative feed-back mechanism of NF-kB activation, which reduced the upregulation of TNF-α expression in myocytes after H/R.6. NF-kB was activated in cardiac myocytes during EPO pretreatment, which upregulated the expression of bcl-2 in cardiac myocytes and protected the cardiac myocytes against H/R injury.
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