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The cardiac protective role of a novel erythrocyte-derived depressing factor (EDDF) on spontaneous hypertensive rats (SHR), calcium overload (CaO) rats and Wistar rats and its mechanism was evaluated. Mean artery pressure (MAP), heart rate (HR) and LVdp/dtmax were measured by physiological recorder. The effect of EDDF on the Ca2+-ATPase activity in myocardial sarcoplasmic reticulum (SR) of CaO rats was determined by inorganic phosphate assay. Calcium transport in myocytes was measured by 45Ca2+ radioactive isotope measurement. The phosphorylation levels of extracellular signal-regulated protein kinases (ERK1/2) in myocardial tissue of SHR and CaO rats were measured by Western blot method. And the ultrastructures of cardiac muscle cells were observed with the transmission electron microscope. The results indicated that EDDF could significantly decrease MAP, HR and LVdp/dtmax in a dose dependent manner (P < 0.05). It seems that the mechanism might relate with activating the Ca2+-APTase, enhancing the uptake and release of Ca2+ from SR (P < 0.05), decreasing the phosphorylation levels of ERK1/2 of myocytes (P < 0.01) and lightening the ultrastructural lesion of cardiac muscle cells. In CaO rats, the Ca2+-ATPase activity decreased clearly compared to control (64.99 ± 7.16 vs 94.48 ± 7.68 nmol·min-1·mg-1 protein, P < 0.01), while EDDF (100 μg/mL) could significantly increase the activity (87.93 ± 9.54 vs 64.99 ± 7.16, P < 0.05, n = 7). Both uptake and release rate of Ca2+ (μmol 45Ca2+/g protein/min) from myocardial SR of CaO rats remarkably decreased compared to control (32.40 ± 2.70 and 15.46 ± 1.49 vs 61.09 ± 10.89 and 25.47 ± 4.29, P < 0.05); EDDF (100 μg/mL) could significantly stimulate their activities (50.48 ± 6.76 and 21.76 ± 2.75 vs 32.40 ± 2.70 and 15.46 ± 1.49, P < 0.05). EDDF could evidently down-regulate the phosphorylation of ERK1/2 in myocardial tissue from SHR and CaO rats (P < 0.01), lighten the ultrastructural lesion of cardiac muscle cells of SHR as well. It is concluded that EDDF seems to play protective roles on both structure and function of heart, which closely related with amelioration of Ca2+ transport and inhibition of Ca2+-MAP kinase pathway.