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目的探讨骨碎补总黄酮(DTF)对血管内皮细胞功能和增殖、凋亡的影响及其机制。方法将培养的人脐静脉内皮细胞(HUVEC)随机分为4组:正常对照组(10%FBS),血清饥饿组(0%FBS),低剂量DTF组(0%FBS+20 mg·L~(-1)DTF)和高DTF组(0%FBS+100 mg·L~(-1) DTF),处理不同时间后,以MTT法检测细胞增殖,流式细胞术和Hoechst 33258检测细胞凋亡率和凋亡形态变化;Western blotting检测GRP78,CHOP,Caspase-12和Cyt.c凋亡相关蛋白表达变化;JC-1染色检测线粒体膜电位变化;分光光度法检测细胞内Caspase-3,8,9的活性,ELISA法检测内皮素(ET)和Griess法检测一氧化氮(NO)的表达变化。结果各个浓度的骨碎补总黄酮能促进血管内皮细胞的增殖,具有时间和剂量依赖性;骨碎补总黄酮能显著降低血清饥饿诱导的内皮细胞凋亡率,抑制GRP78,CHOP,Caspase-12和Cyt.c蛋白表达和线粒体膜电位的下降;降低Caspase-3,9的活性;并在抑制ET表达的同时增强NO的表达。结论骨碎补总黄酮可通过阻断内质网和线粒体途径,抑制血管内皮细胞凋亡,调节血管内皮细胞功能。
Objective To investigate the effects of total flavonoids of Rhizoma Drynariae (DTF) on the function, proliferation and apoptosis of vascular endothelial cells and its mechanism. Methods Human umbilical vein endothelial cells (HUVECs) were randomly divided into four groups: normal control group (10% FBS), serum starvation group (0% FBS), low dose DTF group (-1) DTF group and high DTF group (0% FBS + 100 mg · L -1 DTF). Cell proliferation was detected by MTT assay after different time. Flow cytometry and Hoechst 33258 were used to detect apoptosis Western blotting was used to detect the expression of GRP78, CHOP, Caspase-12 and Cyt.c.Cell was detected by JC-1 staining. The changes of intracellular Caspase-3, 9 activity, ELISA assay endothelin (ET) and Griess method detection of nitric oxide (NO) expression changes. Results Total flavonoids of Rhizoma Drynariae could promote the proliferation of vascular endothelial cells in a time-and dose-dependent manner. Total flavonoids of Rhizoma Drynariae could significantly reduce the rate of apoptosis induced by serum starvation, and inhibit the expressions of GRP78, CHOP, Caspase-12 And Cyt.c protein expression and mitochondrial membrane potential decreased; reduce the activity of Caspase-3,9; and inhibit the expression of ET while enhancing the expression of NO. Conclusion Total Flavonoids of Rhizoma Drynariae can inhibit the apoptosis of vascular endothelial cells and regulate the function of vascular endothelial cells by blocking the pathways of endoplasmic reticulum and mitochondria.