赤芍川芎有效部位对波动高血糖介导血管内皮损伤过程中血小板活化及PKCβ1表达的影响

Hyperglycemia is an independent risk factor for atherosclerosis. Vascular endothelial injury is termed as one of the most important pathophysiologic basis of hyperglycemia. Compared to consistent high glucose, fluctuant high glucose is more likely to cause vascular endothelial injury. High blood hyperviscosity, a main hemodynamics change in diabetes patients, is regarded as a specific feature of 'blood stasis' according to the theory of Traditional Chinese Medicine. Our previous studies have shown that protein kinase C beta 1 (PKCβ1) as a target molecule of coronary heart disease with blood stasis syndrome plays a key role in pathogenesis of vascular endothelial injury induced by fluctuant high glucose. Meanwhile, high level of PKCβ1 expression is involved in platelet activation. Previous studies have shown that effective components of Chuanxiong Rhizome and Peony Root represent a robust inhibitory influence on platelet aggression and endothelial injury. In the current study, we therefore investigate PKCβ1-induced molecular events that might be responsible for the platelet activation in vascular endothelial injury induced by fluctuant high glucose using human platelet and umbilical vein endothelial cells in vitro and type 2 diabetes mellitus rats model in vivo. Meanwhile, the effects of effective components of Chuanxiong Rhizome and Peony Root on vascular endothelial injury and platelet activation are also investigated. We aim to explore the exact role of PKCβ1 in the vascular secondary changes induced by fluctuant high glucose and the mechanisms of promoting blood circulation and removing blood stasis drugs, meanwhile provide experimental evidences for their clinical uses in diabetes mellitus prevention and treatment.

高血糖是动脉粥样硬化的独立危险因素，血管内皮功能异常是其心血管病变的病理生理基础。波动高糖较稳定高糖更易引起血管内皮的损伤。糖尿病患者以高粘血症为特征的血液流变学改变是中医"血瘀"的具体体现之一。课题组前期发现PKCβ1作为冠心病血瘀证的目标分子之一，在波动高糖引起血管内皮损伤过程中扮演关键角色，同时发现PKCβ1高表达也参与了血小板活化过程。研究证实活血化瘀制剂芎芍胶囊具有抗血小板聚集、保护血管内皮功能的作用，在抗动脉粥样硬化方面疗效确定。本研究借助波动高糖培养人脐静脉内皮细胞与人血小板建立实验体系，体内以波动高糖大鼠模型为研究对象，以PKCβ1为切入点，观察在血管内皮损伤过程中血小板活化和PKCβ1的表达变化及赤芍川芎有效部位的干预效应，探讨PKCβ1在波动高糖继发血管病变中的作用及活血化瘀制剂的作用靶点，为活血化瘀药物防治糖尿病的临床应用提供实验依据。

糖尿病是心血管疾病的等危症，血管内皮功能异常是糖尿病心血管病变的病理生理基础。临床上高血糖多处于波动状态，研究波动性高血糖内皮损伤机制并寻求抗波动性高血糖造成内皮损伤的干预措施是该研究领域亟待解决的关键问题。糖尿病患者以高粘血症为主要表现的血液流变学改变是中医“血瘀”的具体体现之一。PKCβ1是冠心病血瘀证的目标分子，在冠心病血瘀证疾病过程中具有调控作用。赤芍川芎有效部位在抗血小板聚集、抗动脉粥样硬化及保护血管内皮等方面具有可靠疗效。本研究在前期波动性高血糖损伤内皮机制及冠心病血瘀证血小板活化基因组学研究的基础上，以PKCβ1为切入点，体外构建波动性高血糖诱导损伤人脐静脉内皮细胞实验模型和人血小板—脐静脉内皮细胞实验体系，观察赤芍川芎有效部位对波动性高血糖损伤血管内皮的保护效应以及PKCβ1表达变化的情况，探讨波动性高血糖诱导人脐静脉内皮细胞损伤培养液温育对人外周血血小板聚集的影响以及PKCβ1在其中所扮演的角色；体内动物实验通过引入“升糖指数”（Glucose Index，GI）的概念，构建2型糖尿病大鼠波动性高血糖和稳定性高血糖模型，观察赤芍川芎有效部位对血糖波动状态下2型糖尿病大鼠血管内皮功能、血小板聚集活化以及冠心病血瘀证关键调控分子PKCβ1表达的影响，探讨赤芍川芎有效部位拮抗波动性高血糖介导内皮损伤和血小板过度活化的相关机制。结果发现：① 波动性高血糖能够显著诱导并加重人脐静脉内皮细胞损伤和凋亡，且该效应与PKCβ1过表达密切相关；赤芍川芎有效部位对波动性高血糖状态下的血管内皮功能损伤具有明显的保护作用，该作用机制与其显著抑制PKCβ1表达进而减轻氧化应激和炎症反应密切相关。② 波动性高血糖诱导人脐静脉内皮损伤培养液温育具有显著促进健康人血小板聚集的作用，PKCβ1抑制剂及赤芍川芎有效部位对此均具有显著的拮抗效应，赤芍川芎有效部位的抗血小板聚集效应可能与其抑制PKCβ1表达相关。③ 赤芍川芎有效部位能够通过显著降低血糖波动2型糖尿病大鼠的炎症反应和氧化应激水平，发挥抗内皮损伤效应；同时能够显著抑制血糖波动状态所致的血小板过度活化，且上述效应与其显著抑制冠心病血瘀证目标分子PKCβ1的过度表达相关。该项为活血化瘀药物防治糖尿病代谢相关疾病的临床应用提供实验依据。