【作者】 胡静；

【导师】 王四旺；

【作者基本信息】 第四军医大学 ， 药理学， 2013， 博士

【摘要】 研究背景及目的：糖尿病大血管并发症指心、脑及外周血管病变，包括冠心病、脑卒中和间歇性跛行与下肢坏疽，是糖尿病致死、致残的主要原因，约占糖尿病病人死亡原因的60-70%[1]。尤其是糖尿病可引起脑动脉血管功能障碍以致动脉粥样硬化，从而造成大脑长期相对供血不足，导致缺血缺氧，甚至脑萎缩。其缺血性脑卒中发生率更是非糖尿病人群的2-4倍。目前，临床上对于糖尿病大血管病变的治疗仍以西药为主，但西药虽然疗效较好确常伴发较明显的毒副作用。中药因其低毒、多靶点等特点，对于糖尿病大血管病变的治疗有望带来新的突破。糖尿病血管病变中医病理机制在于痰浊瘀血阻滞于经脉和络脉系统，这些有形之邪在脉道蓄积留滞的过程中不断沉积，导致脉壁增厚，管腔狭窄；同时，沉积物对脉壁不断刺激，对脉壁组织产生侵蚀、灼伤等病理作用，最终导致脉壁结构损伤[2]。因此，活血化瘀在中医糖尿病血管病变治疗中具有重要意义。双丹方出自《施今墨对药》，由丹参、牡丹皮两味中药组成。具有活血化瘀、通络止痛的功效。其制剂双丹口服液分别收载于2005、2010年版《中国药典》（一部），为中药标准制剂。丹皮酚（Pae）为牡丹皮中主要的活性成分之一[3-6]。同时在丹参中提取的水溶性化合物丹参素（DSS）在丹参药效方面发挥重要作用[7-9]。据相关文献报道，丹皮酚和丹参素分别对动脉血管具有舒张作用[10,11]，同时具有血管内皮保护的功效[12,13]。近些年发现丹参素联合丹皮酚具有良好的抗缺血再灌注损伤以及抗氧化的功效[14]。我们近期观察到丹参素联合丹皮酚可提高丹皮酚在心脏和脑组织中的分布[15]，同时能够增强在心肌缺血再灌注损伤过程中的药物功效[16]。是否丹皮酚联合应用丹参素同样可以起到明显的血管保护作用目前尚未见研究报道。本课题拟将分别来自丹参的丹参素和来自牡丹皮的丹皮酚，二者配伍组成成分组方，并与各单一成分及双丹口服液进行了药效学比较研究。糖尿病状态下血管内皮细胞发生损伤，甚至发生凋亡，从而发生内皮依赖性动脉舒张功能的障碍[17,18]。与此同时，动脉平滑肌在糖尿病状态下可表现为高反应性，导致动脉血管的张力增加[19,20]。其中很重要的一个原因为糖尿病抑制平滑肌细胞钾通道的活性，从而导致大量Ca²⁺内流促使平滑肌处于高张力状态[21,22]。据此本课题提出丹参素联合丹皮酚有可能具有抗糖尿病血管功能衰竭的作用，且药物效果要优于单独给药；同时探讨丹皮酚配伍丹参素对糖尿病大鼠基底动脉损伤保护作用及其机理。研究方法：本实验分别借助实验性糖尿病动物模型和高浓度葡萄糖环境下动脉血管内皮/平滑肌细胞来评价丹参素联合丹皮酚的血管保护作用。采用离体血管环灌流、PI-Hoechst细胞凋亡染色、MTT法、蛋白免疫印迹（Western blot）等实验方法，观察：①丹参素联合丹皮酚对糖尿病大鼠动脉血管功能的作用；②丹参素联合丹皮酚抗糖尿病血管内皮细胞损伤的作用及相关机制；③丹参素联合丹皮酚对糖尿病血管平滑肌细胞损伤的作用及相关机制。研究结果：1、丹参素联合丹皮酚可使大鼠基底动脉血管产生非内皮依赖性舒张作用：伴随着丹皮酚+丹参素浓度剂量的增加，对内皮完整与去内皮动脉环的舒张作用均呈明显加强，且呈浓度依赖性。内皮完整与去内皮动脉环对丹皮酚+丹参素的反应无显著性差异。说明丹皮酚+丹参素所诱导的基底动脉舒张作用为非内皮依赖舒张作用。2、长期给予丹参素联合丹皮酚对糖尿病大鼠基底动脉内皮依赖性舒张功能的影响：长期给予丹参素联合丹皮酚可增加乙酰胆碱（ACh）诱导的糖尿病大鼠基底动脉血管环舒张作用。糖尿病组ACh诱导的血管环舒张作用明显减小，与对照组存在显著性差异（p <0.01）。各药物干预组不同程度地加大ACh诱导的血管环舒张反应。丹皮酚+丹参素组动脉血管环舒张作用较各单一成分给药组明显增强（p <0.05），与双丹口服液组无显著性差异。3、长期给予丹参素联合丹皮酚处理后对去氧肾上腺素（PE）致糖尿病大鼠基底动脉环收缩作用的影响：长期给予丹参素联合丹皮酚处理后可降低因去氧肾上腺素（PE）诱导的糖尿病大鼠基底动脉血管（内皮完整/去内皮）收缩作用。糖尿病组动脉环收缩反应明显强于对照组（p <0.01）。糖尿病大鼠给予丹皮酚+丹参素以及各单一成分灌胃8周处理。PE诱导各药物处理组血管环收缩反应均弱于糖尿病组动脉血管环。其中以丹参素联合丹皮酚共同处理组大鼠动脉环（内皮完整/去内皮）收缩程度最弱（p <0.01）。同时，各实验组内皮完整和去内皮大鼠动脉血管环对PE诱导的收缩增加幅度分别存在显著性差异。其中，丹皮酚+丹参素处理组血管环内皮完整与去内皮状态下收缩幅度差异最为明显（p <0.01），幅度差异大于丹皮酚和丹参素单独给药组（p <0.05）。与双丹口服液组无显著性差异。4、长期给予丹参素联合丹皮酚对糖尿病大鼠基底动脉SOD活性和TBARS水平的影响：各药物治疗组（丹皮酚，丹参素，丹皮酚+丹参素）动脉组织样本SOD活性均明显升高（p <0.05），丹参素联合丹皮酚组SOD活性最高（p <0.01）。而TBARS水平在给予药物治疗组均较糖尿病组有所降低（p <0.05），以丹参素联合丹皮酚组最为显著（p <0.01）。5、丹参素联合丹皮酚急性干预后对CaCl₂致基底动脉环收缩作用的影响：丹皮酚+丹参素预孵正常与糖尿病大鼠去内皮血管环后，随即CaCl₂诱导浓度依赖性血管环收缩反应。通过血管环灌流实验发现，经过丹皮酚+丹参素孵育后正常与糖尿病大鼠去内皮血管环均表现出显著的相对性张力减低反应。以糖尿病大鼠血管环表现最为明显（p <0.01）。提示丹皮酚+丹参素在一定程度上抑制平滑肌细胞内Ca²⁺内流，从而降低了血管对CaCl₂诱导的收缩反应。6、钾通道阻断剂对丹参素联合丹皮酚舒张血管作用的影响：TEA（非特异性钾通道阻断剂）预处理可部分阻断丹皮酚+丹参素诱导的血管平滑肌舒张反应（p <0.01νs Pae+DSS组；p <0.05νs DM+Pae+DSS组）。提示丹皮酚+丹参素在一定程度上抑制平滑肌细胞内Ca²⁺内流是通过活化血管平滑肌K+通道来实现的。7、丹参素联合丹皮酚对高浓度葡萄糖诱导的血管内皮细胞凋亡的影响：丹参素联合丹皮酚可有效抑制高浓度葡萄糖诱导的血管内皮细胞凋亡（p <0.01），且观察到p-p38蛋白表达减少（p <0.01）。8、丹参素联合丹皮酚对高糖环境下血管平滑肌细胞BK_Ca-β1蛋白表达的影响：丹参素联合丹皮酚可有效增强高浓度葡萄糖环境下血管平滑肌细胞BK_Ca-β1的蛋白表达（p <0.05）。研究结论：1、国际上首次发现了丹参素联合丹皮酚具有抗糖尿病动脉血管内皮功能障碍作用，能显著抑制血管内皮细胞因高糖所致的凋亡，且此保护作用与丹参素联合丹皮酚抑制p38MAPK信号转导通路相关。2、国际上首先经实验初步证实丹参素联合丹皮酚具有抗糖尿病动脉血管平滑肌功能障碍作用，且此保护作用可通过对血管平滑肌上BK_Ca功能的保护来实现。更多还原

【Abstract】 Background：Diabetes mellitus （DM） causes multiple dysfunctions such as vascular dysfunction.Diabetic vascular complications refers to heart, brain and peripheral vascular disease,including coronary heart disease, stroke and intermittent claudication and lower limbgangrene, is a major cause of diabetic death and disability, accounting for60-70%ofcauses of death in diabetic patients. Diabetic patients with ischemic stroke incidence aretwo to four times that of the normal population. Modern medicine effective therapy fordiabetic vascular but has obvious side effects. Chinese medicine because of its lowtoxicity characteristics and multiple targets, in the treatment of diabetes vascular lesions isexpected to bring new breakthrough. Diabetes vascular lesions of traditional Chinesemedical science pathological mechanism is phlegm turbid and blood stasis block in themeridians and collaterals.In traditional Chinese medicine for activating blood circulationin treating diabetes vascular lesions is of great significance. Shuang-Dan prescription combines the use of Cortex Moutan （root bark of Paeoniasuffruticosa Andrew） and Radix Salviae miltiorrhizae （root and rhizome of Salviamiltiorrhiza Bunge）, which are famous herbs widely used in traditional Chinese medicine.In clinical practice, the Shuang-Dan prescription is often used for treating cerebrovascularand cardiovascular diseases.Paeonol （Pae,20-hydroxy-40-methoxyacetophenone） is a major phenolic componentin Cortex Moutan, whereas danshensu （DSS,3-（3,4-dihydroxyphenyl） lactic acid） is awater-soluble active component isolated from Radix Salviae miltiorrhizae. Similar to othernatural compounds, several studies showed that Pae and DSS elicit relaxation of isolatedrat aorta and protection of endothelial cells, respectively. We previously reported that thecombined use of Pae and DSS has synergistic protective effects on focal cerebralischemia-reperfusion injury in rats. Moreover, Pae combined with other hydrophilicphenolics of Radix Salviae miltiorrhizae could attenuate oxidative stress, protect vascularfunctions, and synergistically protect against myocardial ischemia in rabbits. Recently, wefound that the co-administration of DSS increases the concentration of Pae in heart andbrain tissues and increases pharmacological activity in treating cerebrovascular andcardiovascular diseases. However, the mechanism of the interactions of representativeactive components in the protection of vascular function is not well understood.Vascular dysfunctions are one of the major causes of morbidity and mortality inpatients with DM. Previous studies reported that forearm blood flow responsive toacetylcholineis reduced in type2diabetes, suggesting endothelial dysfunction. Moreover,vascular smooth muscle （VSMC） exhibits hyper-reactivity, hypertrophy, and apoptosis indiabetes. One of the pathogenesis of diabetic vascular dysfunction is oxygen derived freeradicals, which are significantly elevated under DM. Diabetic vascular dysfunction is alsorelated to increased Ca²⁺influx and inhibited vascular K+channels. Previous studiesshowed that the inhibition of vascular K+channels increases Ca²⁺influx, which leads todepolarization and vasoconstriction. Therefore, the aim of this study is to investigate theeffects of Pae+DSS on diabetes-induced dysfunction of cerebral arteries compared withthe individual effects of Pae or DSS. Methods：We assessed the role of the endothelium and smooth muscle in the responses to Pae+DSS, on cerebral artery （basal artery, Willis’ circle, and middle cerebral artery） fromdiabetic rats and on cultured endothelial/smooth muscle cells damaged by high glucose.Pae+DSS-induced effect was evaluated in vitro and in vivo. Experimental analyses wereperformed by using wire myograph, colorimetric method，PI-Hoechst dyeing，MTT andWestern blot, et al.Results：1. Pae+DSS Induced Relaxation in Rat Cerebral ArteryPae+DSS induced a strong relaxation on arterial rings obtained from rats in adose-dependent manner. The effect of Pae+DSS in endothelium-intact and endothelium-denuded rat arterial rings was investigated to identify the role of VSMC on Pae+DSSinduced vasorelaxation. The vascular relaxation induced by Pae+DSS was not abolishedin the endothelium removed rings of rats.2. Effect of Chronic Pae+DSS Administration on ACh Relaxation ResponseWhen the PE-induced contraction reached a plateau, ACh （109M to105M） wasadded cumulatively. The capability of the concentration-dependent relaxation induced byACh, which had a maximum response of105M, was significantly weaker in arterialsegments obtained from diabetic rats than those from normal rats. After chronicadministration of Pae+DSS, Pae, and DSS, the capability of ACh-induced relaxation inthe arterial segments of diabetic rats was enhanced significantly. In addition, the degree ofACh-induced relaxation in the DM+Pae+DSS group was stronger than that of Pae andDSS treated diabetic rats. No marked changes were observed concerning the degree ofACh-induced relaxation between the DM+Pae+DSS group and SDOS group.3. Effect of Chronic Pae+DSS Administration on Phenylephrine （PE） ContractionResponseWe also found that treatment with Pae+DSS significantly reduced the maximumcontraction of rings from diabetic rats. Meanwhile, the enhancement rates of contractileresponses to PE in all groups were significantly different in E+and E rings. Particularly, the enhancement rate of contractile responses in Pae+DSS treated diabetic rats was lowerthan that Pae and DSS treated diabetic rats. No marked changes were observed concerningthe degree of contractile responses to PE between the DM+Pae+DSS group and SDOSgroup.4. Effects of Pae+DSS on SOD Activities and TBARS Content in the CerebralArtery from Diabetic RatsThe enhanced generation of reactive oxygen species is induced by oxidative stress.The superoxide dismutase （SOD） activities and thiobarbituric acid reactive substances（TBARS） concentrations in the arterial tissues of all groups at the end of the study. Alltreated groups （Pae, DSS, and Pae+DSS） exhibited increased SOD activities anddecreased TBARS concentrations. Moreover, the Pae+DSS group exhibited morereduction in oxidative stress compared to Pae and DSS groups.5. Influence of Pae+DSS on CalciumIn Ca²⁺free solutions, pretreatment of Pae+DSS attenuated the CaCl₂inducedcontractions of denuded cerebral arteries from normal and diabetic rats.6. Influence of Pae+DSS on Potassium ChannelWe examined the transient vasoconstrictor PE response in the presence of thenon-selective K+channels blocker, tetraethylammonium （TEA）. In the presence of TEA,the vasorelaxation effect of Pae+DSS was partially inhibited when Pae+DSS was addedafter the PE contraction reached a plateau.7. Pae+DSS inhibited vascular endothelial cell apoptosis in high glucose.Cell apoptosis were attenuated in the Pae+DSS–pretreated cells.And, Pae+DSSprotects cells by the p-38signaling pathway.8. Pae+DSS can effectively enhance the protein expression of BK_Ca-β1in vascularsmooth muscle cells exposed to high glucose.Conclusions：1. Pae+DSS can attenuate endothelial dysfunction associated with diabetes andprotect high glucose-induced damage of endothelial cells.The p38MAPK pathway issuppressed for the protective effect of Pae+DSS.2. Pae+DSS can attenuate vascular smooth muscle dysfunction associated withdiabetes.The BK_Cais required for the protective effect of Pae+DSS.更多还原