蕨麻正丁醇部位对缺氧心肌细胞μ-calpain表达的影响

Effect of n-butanol fraction of Potentilla anserine L. on the expression of μ-Calpain in myocardial cells exposed to hypoxia

【目的】研究蕨麻正丁醇部位对大鼠心肌细胞缺氧所致钙超载和细胞凋亡的抑制作用,并通过研究钙蛋白酶μ-calpain表达的变化,探讨其可能的机制。【方法】建立原代心肌细胞缺氧模型,设正常对照组、缺氧损伤组、维拉帕米组(5μmol/L)及蕨麻正丁醇部位高、中、低浓度组(250.0、62.5、15.6μg/ml)。采用荧光分光光度法检测心肌细胞内钙离子变化,Hoechst3342/PI染色检测心肌细胞凋亡,RT-PCR法检测μ-calpain m RNA水平,Western blotting方法和免疫组织化学方法检测μ-calpain蛋白。【结果】缺氧损伤组心肌细胞内游离钙离子的浓度较正常对照组明显增加(P<0.01),细胞凋亡严重,μ-calpain m RNA及蛋白表达显著上调(P<0.01),蕨麻正丁醇部位250.0、62.5μg/ml组心肌细胞内游离钙离子的浓度较缺氧损伤组明显降低,μ-calpain m RNA及蛋白表达均显著下调(P<0.01),细胞凋亡程度明显减轻。【结论】蕨麻正丁醇部位可明显抑制缺氧所致心肌细胞游离钙离子浓度增加及μ-calpain的上调,减少心肌细胞晚期凋亡和死亡,提示蕨麻正丁醇部位对心肌细胞缺氧损伤的保护作用可能与其抑制μ-calpain的表达有关。

【Objective】To study the inhibiting effect of the n-butanol fraction of Potentilla anserine L. on hypoxia-induced calcium overload and apoptosis of rat myocardiocytes and explore its underlying mechanisms by the changes of μ-calpain expression.【Methods】The hypoxic models of primary myocardial cells were established. There were 6 groups： control group, hypoxia injury model group, verapamil group（5 μmol/L）, intervention groups of n-butanol fraction of Potentilla anserine L.（250.0、62.5、15.6 μg/ml）. The intracellular dissociative calcium concentration in myocardial cells was determined with fluorospectrophotometry. The apoptosis was observed by Hoechst3342/PI staining. The level of μ-calpain m RNA was examined with RT-PCR method. The μ-calpain protein was detected with western blotting and immunochemistry assays.【Results】Compared with the control group,the dissociative calcium concentration in myocardiocytes increased obviously（P〈0.01）;a severe apoptosis was observed; the expressions of μ-calpain m RNA and its protein were significantly upregulated（P〈0.01）in the hypoxia injury model group. Compared with the hypoxia injury model group, the dissociative calcium concentrations in myocardiocytes treated respectively with n-butanol fraction of Potentilla anserine L. of 250.0 μg/ml and 62.5 μg/ml decreased obviously and apoptosis was attenuated. Meanwhile the expressions of μ- calpain m RNA and its protein were down regulated（P〈0.01）【Conclusion】n-Butanol fraction of Potentilla anserine L. could obviously inhibit calcium influx of myocardial cells and μ-calpain upregulation, and reduce late apoptosis and death of myocardiocytes. It was suggested that the protective effect of n-butanol fraction of Potentilla anserine L. on myocardiocytes exposed to hypoxia might be related with its down-regulating expression of μ-calpain.