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目的:研究六味地黄汤含药血清对过氧化氢(H2O2)损伤的人脐静脉血管内皮细胞(HUVECs)的保护作用,并阐明其可能的机制。方法:六味地黄汤药物煎剂浓缩为生药2g/ml,以人临床用量×动物等效剂量比值×血清稀释度为参考浓度给家兔灌胃(35g/kg),每天2次,连续3天,正常对照组给予等量生理盐水。于末次给药后1 h(灌药前禁食不禁水12 h)颈动脉取血,分离血清。本实验采用400μmol/L H2O2复制HUVECs损伤模型,六味地黄汤含药血清分为3个剂量干预组(体积分数为5%、10%、20%),MTT法测定细胞活力,电子显微镜观察细胞内超微结构的改变,PI单染法流式细胞仪检测细胞凋亡及细胞周期的变化,激光共聚焦检测细胞内钙离子荧光强度的改变。结果:体积分数5%、10%、20%六味地黄汤含药血清均能促进H2O2损伤的血管内皮细胞增殖;电镜和流式细胞仪检测结果均显示体积分数为5%、10%、20%的六味地黄汤含药血清可减少血管内皮细胞凋亡;激光共聚焦结果提示六味地黄汤抑制H2O2所致的血管内皮细胞凋亡可能是通过降低细胞内钙离子浓度实现的。结论 :六味地黄汤含药血清可以抑制H2O2诱导的细胞凋亡,这种保护作用是通过改变细胞内钙离子浓度实现的。
Objective: To study the protective effect of Liuwei Dihuang decoction containing serum on human umbilical vein endothelial cells (HUVECs) injured by hydrogen peroxide (H2O2) and to elucidate its possible mechanism. Methods: Liuweidihuang Decoction decoction was concentrated to 2g / ml crude drug. The rabbits were gavaged (35g / kg) twice a day for three days at the dose of human clinical dose × the equivalent dose of animal x serum dilution The normal control group was given the same amount of saline. 1 h after the last administration (before fasting can not help but water 12 h) carotid blood, serum was isolated. In this study, 400μmol / L H 2 O 2 was used to replicate HUVECs injury model. The serum containing Liuwei Dihuang decoction was divided into three groups (5%, 10% and 20%, respectively). The viability was measured by MTT assay. Ultrastructural changes were observed by PI staining and flow cytometry. The changes of cell fluorescence and fluorescence intensity were detected by confocal laser scanning microscopy. Results: The drug serum containing 5%, 10%, 20% of Liuweidihuang decoction could promote the proliferation of vascular endothelial cells injured by H2O2. The results of electron microscopy and flow cytometry showed that the volume fraction was 5%, 10%, 20% Liuwei Dihuang decoction containing serum could reduce the apoptosis of vascular endothelial cells. Confocal laser scanning results suggested that Liuweidihuang Decoction could inhibit the apoptosis of vascular endothelial cells induced by H2O2 by decreasing the intracellular calcium concentration. Conclusion: Liuwei Dihuang decoction containing serum can inhibit H2O2-induced apoptosis, this protective effect is achieved by changing the intracellular calcium concentration.