五首活血化瘀方改善血瘀证家兔内皮细胞功能差异

Differences of Five Blood-activating and Stasis-resolving Prescriptions on Endothelial Cell Function in Rabbits with Blood Stasis Syndrome

目的观察五首活血化瘀方改善血瘀证家兔内皮细胞功能的作用机制及疗效差异。方法选择清洁级新西兰兔70只,其中正常组6只,余下64只应用饥饿+高脂+肾上腺素复合造模方法构建兔血瘀证模型,造模成功的家兔按随机数字表法分为模型组(9只),血府组(10只),桃红组(10只),丹参组(9只),活络组(10只),失笑组(9只)。使用光镜观察主动脉病理学变化,电镜观察主动脉内皮细胞超微结构,血小板-内皮细胞黏附分子(CD31)和TUNEL双染检测主动脉内皮细胞凋亡,ELISA法检测血清内皮细胞损伤相关因子一氧化氮(NO)、内皮素-1(ET-1)、血管内皮细胞黏附分子-1(VCAM-1)、血管假血友病因子(vWF)、活性氧(ROS)、白细胞介素-1(IL-1)、肿瘤坏死因子α(TNF-α)水平,RT-PCR法检测ERK5、MEK5、NRF2、KLF2基因表达。结果与模型组比较,五首活血化瘀方组主动脉及血管内皮细胞结构均有所改善,内皮细胞凋亡数减少(P<0.01);除活络组外,其余4组NO水平较模型组升高,ET-1水平均较模型组降低(P<0.05),五首活血化瘀方均可降低vWF、VCAM-1、ROS水平(P<0.05),失笑组、血府组IL-1、TNF-α水平同时降低,丹参组、桃红组IL-1水平降低,活络组TNF-α水平降低(P<0.05);五首方均可有效降低ERK5、MEK5、NRF2、KLF2基因表达水平(P<0.01),但对不同基因表达水平的影响有差异(P<0.05)。结论五首活血化瘀方均可能通过调控MEK/ERK信号通路,改善血瘀证家兔血管内皮细胞功能,五首活血化瘀方在保护血管内皮细胞的作用力及作用靶点不同。

Objective To observe the mechanism and different effects of five blood-activating and stasisresolving prescriptions on endothelial cell function in rabbit with blood stasis syndrome.Methods Seventy New Zealand rabbits were selected and 6 in which as control group,the remaining 64 were given hunger+hyperlipidemia+adrenaline method to establish blood stasis syndrome.The successfully established model rabbits were divided into model group(n=9),Xuefu group(n=10),Taohong group(n=10),Danshen group(n=9),Huoluo group(n=10)and Shixiao group(n=9)according to a random number table.The pathological changes of aorta was observed by light microscope,the morphology of aortic endothelial cells was observed by electron microscope,the apoptosis of aortic endothelial cells was detected by CD31 and TUNEL double staining,and the levels of nitric oxide(NO),endothelin-1(ET-1),vascular cell adhesion molecule-1(VCAM-1),von Willebrend factor(v WF),reactive oxygen species(ROS),interleukin-1(IL-1)and TNF-αwere analyzed by ELISA,the expressions of ERK5,MEK5,NRF2and KLF2 genes were detected by RT-PCR.Results Compared with the model group,the aorta and the structure of vascular endothelial cells in five blood-activating and stasis-resolving prescriptions groups were all improved,and the numbers of apoptosis of endothelial cells were significantly reduced(P<0.01).Except for the Huoluo group,the levels of NO in the other four groups were higher and the levels of ET-1 were lower than that in the model group(P<0.05).The five blood-activating and stasis-resolving prescriptions could reduce the levels of vWF,VCAM-1 and ROS(P<0.05).The levels of IL-1 and TNF-αin Shixiao group and Xuefu group decreased at the same time,the levels of IL-1 in Danshen group and Taohong group decreased,and TNF-αin Huoluo group decreased(P<0.05).All the five prescriptions could effectively reduce the expression levels of ERK5,MEK5,NRF2 and KLF2 genes(P<0.01),but the curative effects of the five prescriptions on gene expression levels were different(P<0.05).Conclusion The five blood-activating and stasis-resolving prescriptions could all regulate the MEK/ERK signaling pathway and improve the function of vascular endothelial cells in rabbits with blood stasis syndrome.However,the five prescriptions have different curative effects and different targets on the protection of vascular endothelial cells.