橙皮素通过抑制钙超载减轻低氧/复氧诱导的心肌 H9c2细胞凋亡

Hesperetin attenuates hypoxia/reoxygenation-induced apoptosis in H9c2 cells via inhibition of calcium overload

目的:本研究旨在探讨橙皮素对低氧/复氧(hypoxia/reoxygenation,H/R)诱导的心肌H9c2细胞凋亡的影响及其可能机制。方法:建立心肌H9c2细胞H/R模型,使用橙皮素进行预处理,利用CCK-8和乳酸脱氢酶(lactate dehydrogenase,LDH)试剂盒分别测定细胞活力和损伤情况;Hoechst 33258染色和流式细胞术检测细胞凋亡情况;采用Fluo-3AM孵育后观察细胞内的钙离子荧光强度;细胞Ca^(2+)-ATP酶活性与三磷酸腺苷(adenosine triphosphate,ATP)水平测定利用相应的ELISA试剂盒;利用JC-1染色检测线粒体膜电位;采用Western blot测定Bcl-2、Bax和细胞色素C(cytochrome C, Cyt-C)的蛋白表达水平。结果:橙皮素预处理可明显逆转H/R所致心肌H9c2细胞活力降低,并且减少细胞的凋亡率,降低细胞内钙离子的荧光强度,提高细胞Ca^(2+)-ATP酶活性,抑制线粒体膜电位去极化并提升ATP的水平(P<0.05),同时抑制Cyt-C蛋白从线粒体释放入胞浆并增加线粒体凋亡相关蛋白Bcl-2/Bax的比值(P<0.05);而给予钙离子拮抗剂尼莫地平后,与H/R组相比,线粒体膜电位去极化程度减轻,ATP获得提升并且线粒体凋亡相关蛋白表达降低(P<0.05)。结论:橙皮素可抑制H/R诱导的心肌H9c2细胞凋亡,其机制可能与减轻钙超载从而改善线粒体功能有关。

AIM:To investigate the effect of hesperetin on hypoxia/reoxygenation(H/R)-induced apoptosis in the H9c2 cells and to clarify the underlying mechanism.METHODS:The H/R model was established and the H9c2 cells were pretreated with hesperetin for 4 h.The cell viability and cell damage were measured by CCK-8 assay and lactate dehydrogenase(LDH)detection.The apoptosis was analyzed by Hoechst 33258 staining and flow cytometry.The intracellular calcium fluorescence intensity was measured by fluorescence microscopy and flow cytometry.The calcium-ATPase activity and the level of adenosine triphosphate(ATP)were measured by ELISA.The mitochondrial membrane potential was measured by JC-1 staining.The protein expression levels of Bcl-2,Bax and cytochrome C(Cyt-C)were determined by Western blot.RESULTS:Hesperetin reduced the apoptosis of the H9c2 cells induced by H/R,decreased intracellular Ca 2+fluorescence intensity,elevated Ca 2+-ATPase activity,inhibited the mitochondrial membrane potential depolarization and increased the level of ATP(P<0.05).In addition,hesperetin significantly reduced the release of Cyt-C protein from mitochondria to cytoplasma and increased the Bcl-2/Bax ratio(P<0.05).After using the calcium ion inhibitor nimodipine,the percentage of the cells with mitochondrial membrane depolarization was decreased,the ATP level was increased and the protein expression of mitochondrion-related apoptosis molecules were decreased(P<0.05).CONCLUSION:Hesperetin reduces the apoptosis of the H9c2 cells induced by H/R,which may be related to inhibition of calcium overload and improvement of mitochondrial function.