| Objective: To investigate the dose-effect and time-effect relationship of delayed cardioprotective induced by ShenFu Injection preconditioning on cultured neonatal rat myocardiocytes subjected to Hypoxia/reoxygenation (H/R) and its possible mechanisms.Methods: Cultured neonatal rat myocardiocytes was used and H/R models were established in the present study. The myocardiocytes were preconditioning by using ShenFu Injection at different concentrations(100,200,400μl/ml) and different time points (6,12,24,48,72,96h). At the same time, we also use antisense HIF-1αand MAPKs (mitogen activated protein kinases) inhibitor PD 098059 (an upstream kinase inhibitor of ERKs) pretreating to observe its influence on the delayed cardioprotection induced by ShenFu Injection preconditioning. Cell viability, apoptosis ratio, lactate dehydrogenase (LDH) release and malondialdehyde (MDA) formation were measured to determine the protective effects against H/R injury. The detailed methods as follows: flow cytometry was used to test cell viability, and apoptosis ratio; LDH release were tested by automatic biochemistry analyzer and MDA formation were checked by Rat malondialchehyche Kit; finally, we use western blotting analysis technology to measure the expression of the protein HIF-1α.Results: Increased cell viability, decreased apoptosis ratio, lower LDH and MDA release were observes in cardiomyocytes treated with ShenFu Injection. This cardioprotective effects developed in the periods from 12 to 96 h in the concentration of 200 to 400μl/ml.The delayed protection was abolished by pretreating with eitherantisense HIF-1αor PD 098059.Conclusions: ShenFu Injection preconditioning can mimic the delayed cardioprotection in rat neonatal cardiomyocytes. The protective mechanisms is associated with upregulation of HIF-1αinduced by ERKs.
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