| Background: Reperfusion therapy is the key factor of ischemic heart disease treatment when acute vascular jams happened. While, ischemia/reperfusion injury induced after reperfusion could increase myocardial injury and leading to deterioration,and even death. The mechanism of I/RI is complex and still not illustrate clearly by far. The interaction of endothelial cells and myocardial cells plays a crucial role in the regulation of cardiac function, especially the function of endothelial cells. Previous studies show that endothelial cells could promote the survival of cardiomyocytes and reduce the injury induced by I/RI. O ur preliminary studies have confirmed that the I/RI was closely related to the apoptosis of endothelial cells and cardiomyocytes and the apoptosis would be inhibited by insulin. However, the relationship between cardiac microvascular endothelial cells(CMECs) and myocardial cells during this process needs further research. As an epidermal growth factor, NRG-1 is synthesized and released from endothelialcells. It has various function such as promoting heart mature and protecting heart from injury. While, there is rare report that illustrate whether CMECs can released N RG-1 that promote cardiomyocytes survival. Therefore, this study will establish simulated ischemia reperfusion(SI/R) and CMECs-cardiomyocytes coculture models on the cellular level, and further observe whether CMECs can released NRG-1 that decrease the cardiomyocytes apoptosis induced by I/RI, and discussed the possible mechanism.Methods: 1.Cardiac myocytes were isolated from the hearts of neonatal rats and CMECs from the adult rats. CMECs-cardiomyocytes coculture model was established. Then cells were endured simulated I/R(4 h / 4 h). Collected CMECs culture medium during SI/R. The CMECs were randomized into three groups: Control group, SI/R + Ins(10-7mol/L) group and SI/R group. While the myocardial cells were randomized into following groups: Control group, SI/R + Coculture group, SI/R + Coculture + anti-Erb B2 group,SI/R + NRG-1 groups,SI/R + NRG-1 + anti-Erb B2 group and SI/R + NRG-1 + PD group. 2.The cell viability of cardiac myocytes was measured by MTT assay. The apoptosis of cardiomyocytes was detected by TUN EL method. The expression of secreted NRG-1 in CMECs culture medium, phosphorylation of Erb B2 and ERK and COX-2 in cardiac myocytes were analyzed by Western blot. Measured Caspase-3 activity reflected the apoptosis of cardiomyocytes.Results: 1. CMECs and cardiomyocytes were isolated successfully and the models of SI/R and CMECs- cardiomyocytes coculture were established. 2.The cardiomyocytes viability was impaired in the SI/R(4 h / 4 h) group(P < 0.01) while increased in the SI/R + Coculture group(P = 0.02). Within the groups of CMECs, SI/R(4h / 4h) induced the release of NRG-1(2.96 ± 0.30) and it can be increased by insulin(4.62 ± 0.26)( P < 0.01). Detecting the secretion of NRG-1 at different times of SI/R found that in the time of 4h / 4h N RG-1 was remarkable increased.Within the groups of cardiac myocytes, the apoptosis index and Caspase-3 activity of the cardiomyocytes were increased after SI/R when compared with the Control group(27.97 ± 0.90 vs. 3.58 ± 0.23, P< 0.01; 375.93 ± 11.76 vs. 100.00 ± 0.00, P < 0.01). While treated with Coculture or NRG-1 during SI/R could reduce the apoptosis index and Caspase-3 activity when compared with the SI/R group(16.82 ± 1.03 vs. 27.97 ± 0.90,P < 0.01;166.16 ± 15.15 vs. 375.93 ± 11.76,P < 0.01). Besides, they can significant up-regulate the level of phosphorylation of Erb B2 and ERK and the expression of COX-2(P < 0.01 vs. Control and SI/R). However, when cardiomyocytes were pretreated with Erb B2 or ERK1/2 inhibitor, the effect of Coculture and NRG-1 vanished(P < 0.01).Conclusion: In the process of I/RI, cytokine NRG-1 released from CMECs could protect cardiomyocytes against ischemic reperfusion injury through up-regulation of p Erb B2 and activation of downstream ERK/COX-2 signal. |
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