六味地黄汤含药血清对H_2O_2致HUVECs损伤的保护作用

Effects of liuweidihuang Decoction medicated serum on H_2O_2-injured human umbilical vascular endothelial cells

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 observe the effect of Chinese Medicine Liuweidihuang Decoction（ LWDHD） medicated serum on H2O2-injured human umbilical vascular endothelial cells（ HUVECs） and elucidate its possible mechanism. Methods： HUVECs were injured by H2O2with concentration of 400 μmol /L. LWDHD medicated serum was divided into three groups（ volume fraction was 5%,10%,20%）. Cell viability was determined using MTT assay. The changes of cell ultrastructure were observed by electron microscope. Apoptosis and cell cycles were determined using flow cytometry. The intracellular calcium ion concentration [Ca2 ＋]was detected by laser scanning confocal microscope. Results： Treatment with LWDHD medicated serum（ volume fraction was 5%,10%,20%） significantly increased the proliferation of HUVECs and reversed cell apoptosis. The [Ca2 ＋]was clearly diminished and the apoptosis decreased significantly. Conclusion： LWDHD medicated serum protected HUVECs against apoptosis induced by H2O2,might be due to its ability to decrease the the intracellular calcium ion concentration.