【作者】 以敏；

【导师】 邓家刚；

【作者基本信息】 广西医科大学 ， 药理学， 2012， 博士

【摘要】 血液循环障碍(blood circulation disorder, BCD)是各种原因导致的局部或全身血液循环不足,造成组织器官缺血缺氧等损伤,以及机能、代谢的障碍或衰竭,甚至威胁生命的一种病理生理状态。微循环障碍(microcirculation disturbance, MCD)则主要指局部微血流与微血管水平发生的功能或器质性紊乱,从而造成局部血液灌注的障碍。血液循环障碍相关疾病一直是临床治疗中较为棘手的问题。中医寒凝血瘀证(cold stagnation and blood stasis syndrome)简称寒证(HS),瘀热互结证(heat stagnation and blood stasis syndrome)简称热证(RS)。研究发现,寒证和热证与一些BCD相关疾病关系密切。中药在治疗寒证和热证中医表征的同时,还有明显改善其BCD的效果。因此,寻找安全、有效的中药防治BCD相关疾病,研究中药的相关药效特性、作用机制具有重要意义。中药桃仁(Taoren)为蔷薇科植物桃的种仁,已被证明有扩张血管、增加器官血流量、抑制血小板聚集、抗凝血、抗血栓、促纤溶等作用。本课题组前期研究表明,桃仁等中药能治疗不同病理环境下(寒证和热证)的中医表征,其药效可能与不同致病因素所致的BCD不同有关,但尚未对其具体不同特点深入研究。目前也尚未见对不同致病因素所致BCD的中药研究报道。因此,为了进一步认识寒证和热证大鼠模型全身及局部BCD的特点,认识桃仁对两证BCD的药效特性、作用机理及不同特点,我们对两证模型及桃仁干预的影响进行了深入研究及对比分析。研究分三部分：第一部分：寒凝血瘀证和瘀热互结证模型血液循环障碍的特点研究目的：研究寒证和热证大鼠模型全身及局部血液循环障碍的不同特点。方法：将实验大鼠随机分为4组,每组10只：寒证正常组、寒证模型组、热证正常组,热证模型组。以-18±2℃冰柜冷冻,2小时×2次／天,连续7天建立寒证大鼠模型；腹腔注射角叉菜胶溶液连续6天,第7天皮下注射活性干酵母溶液,建立热证大鼠模型。造模第8天对各组动物观察指标：(1)观察动物中医表征,确立造模成功；(2)用微循环检测仪观察耳廓微循环血流速(Fve)、血流态(Fsc)变化；(3)用血流变检测仪检测腹主动脉血粘度(Vis)、血纤维蛋白原含量(Fib)；(4)用组织病理学(HE、PASM、改良PTAH)及病理图像分析法,观察及测量心、肺、肝、肾、脾微小动脉管径(Adia、s-Adia)、微小静脉管径(Vdia、s-Vdia)、肾血管球充盈面积(Gare)、血栓形成率(Trat)、器官实质损伤严重程度评分(Isco)。结果：(1)两证模型均出现了相应中医表征；(2)血流速两证均明显降低,血流态评分仅有热证明显降低；(3)全血粘度两证均明显增加,纤维蛋白原含量仅有热证明显增加；(4)小动脉管径仅有寒证明显增大,肾血管球充盈面积两证均明显增高；微动脉、微静脉、小静脉管径两证均无明显变化；血栓形成率两证均明显增高；实质细胞损伤评分除热证无明显肺损伤外,两证器官损伤评分均明显增高。结论：间歇低温冷冻法和角叉菜胶复合注射法可成功建立寒凝血瘀证和瘀热互结证模型。寒证和热证均存在血液循环障碍,其全身表现为血流速降低、血粘度增加；局部表现为微小血管径变化、血栓形成增加、器官实质细胞损伤。寒证与热证血液循环障碍的不同之处：血流态评分(寒证无明显变化,热证明显降低)、纤维蛋白原含量(寒证无明显变化,热证明显增高)、小动脉管径(寒证明显扩张,热证无明显变化)和器官损伤的范围(寒证有明显肺损伤,热证无明显肺损伤)等方面。第二部分：桃仁对寒凝血瘀证和瘀热互结证血液循环障碍的药效研究目的：研究桃仁对寒证和热证大鼠血液循环障碍的药效特点。方法：将实验大鼠分为两批,第一批随机分为4组,每组10只：寒证正常组、寒证模型组、寒证桃仁治疗组、寒证川芎对照组；第二批大鼠随机分为4组,每组10只：热证正常组、热证模型组、热证桃仁治疗组、热证丹参对照组。第一批大鼠按照第一部分方法建立寒证模型；同时寒证桃仁治疗组、寒证川芎对照组每天灌胃给予相应药液,连续7天。第二批大鼠按照第一部分方法建立热证模型；同时热证桃仁治疗组、热证丹参对照组每天灌胃给予相应药液,连续7天。第8天各组动物观察指标：(1)微循环检测仪观察耳廓血流速(Fve)、血流态(Fsc)变化；(2)血流变仪检测血粘度(Vis)、血纤维蛋白原含量(Fib)变化；(3)组织病理学及病理图像分析法,观察各器官小动脉管径(Adia)、肾血管球充盈面积(Gare)、血栓形成率(Trat)、器官实质损伤严重程度评分(Isco)等指标的变化。结果：桃仁治疗后,两证治疗组血液循环(1)血流速两证均明显增快,血流态评分两证均无明显变化；(2)血粘度两证均有明显降低,纤维蛋白原含量两证均无明显变化；(3)小动脉管径寒证明显减小,热证明显增大；肾血管球充盈面积寒证无明显变化,热证明显减小：血栓形成率两证均无明显变化；器官实质损伤评分除脾脏两证均无明显变化外,肾损伤评分两证均明显降低,心、肺、肝损伤评分寒证均明显降低,热证均无明显变化。结论：桃仁对寒凝血瘀证和瘀热互结证的血液循环障碍有改善作用,表现为使两证全身血流速加快、血粘度降低,使局部血管舒缩状态改变、使肾的损伤降低。桃仁改善两证循环障碍作用的不同之处是,使寒证小动脉收缩,使热证小动脉扩张；保护寒证心、肺、肝实质细胞,而对热证心、肺、肝实质无明显保护作用。第三部分：桃仁改善寒凝血瘀证和瘀热互结证血液循环障碍的相关分子机制研究目的：研究寒证和热证的分子表达特点及不同之处、桃仁干预后两证相关分子表达的变化及不同之处,探讨其可能的机制。方法：利用第二部分大鼠器官标本(心、肺、肝、肾、脾),共8组(分组同第二部分),进行相关分子病理学检测(免疫组化、TUNEL细胞凋亡、原位杂交)。检测的分子包括：(1)血小板／内皮细胞粘附分子CD31、血管内皮生长因子VEGF; (2)血管内皮细胞凋亡、凋亡抑制蛋白Bcl-2、凋亡诱导蛋白P53；(3)核因子NFκB p65及mRNA、抑制物IκB-a及mRNA;(4)肝CD68阳性巨噬细胞及其蛋白Caspase-1 p20; (5)血管周细胞a-SM-actin、血管平滑肌细胞AT1、ADRB2表达情况。结果：两模型的分子表达：(1)血管内皮细胞：CD31表达寒证无明显变化,热证明显增高；VEGF表达两证均无明显变化；细胞凋亡两证均明显增多；Bcl-2表达寒证不变而热证减弱；P53(突变型)表达寒证减弱而热证不变；NFκB蛋白表达寒证减弱且无核内活性,但其mRNA表达增强,而热证有核活性增强,但其mRNA表达不变；IKB蛋白表达寒证不变且无核活性,但其mRNA表达增强,而热证有核活性,但其mRNA表达不变；(2)肝巨噬细胞数量寒证明显减少,热证不变；Caspase-1 p20表达两证均无明显变化；(3)血管壁a-SM-actin、AT1表达均不变,ADRB2表达两证均减弱。桃仁干预后分子表达的变化：(1)血管内皮细胞：CD31表达寒证明显增强,热证不变；VEGF表达寒证不变,热证明显减弱；细胞凋亡两证均不变；Bcl-2表达寒证不变,热证明显增强；P53(突变型)表达两证均无明显变化；NFκB蛋白寒证出现明显核内活性,但其蛋白及mRNA表达不变,而热证核活性不变,其蛋白及mRNA表达均增强；IKB蛋白表达寒证减弱且无核活性,其mRNA表达不变,而热证其核活性减弱,但其蛋白及mRNA表达均不变；(2)肝巨噬细胞数量寒证明显增多,而热证不变；Caspase-1 p20表达强度两证均无明显变化；(3)血管壁a-SM-actin、ADRB2表达均无明显变化,AT1表达寒证明显增强,热证表达不变。结论：(1)与寒证血液循环障碍关系密切的分子有P53(突变型)、NFκB、IκB、ADRB2,且P53诱导的内皮细胞凋亡增多、巨噬细胞活性降低导致的修复能力下降可促进寒证发生发展；而反复的低温-复温条件所致的血管内皮、平滑肌严重损伤,可能是其NFκB、ADRB2功能降低的原因之一。(2)与热证血液循环障碍关系密切的分子有CD31、Bcl-2、NFκB、IκB、ADRB2,且CD31介导的血管内皮粘附性增高、Bcl-2抑制所致的内皮细胞凋亡增多、NFκB活性增强所致的各种炎症因子产生和释放,可能是热证发生发展的重要因素。(3)寒证中受桃仁影响的分子有：CD31、NFκB、IκB、AT1,其中CD31、NFκB活性增强可能不利于血液循环障碍的恢复,而AT1介导的血管强烈的收缩反应、巨噬细胞活性增强,可能是桃仁改善寒证血管麻痹、促进血流及损伤修复的重要因素。(4)热证中受桃仁影响的分子有：VEGF、Bcl-2、NFκB、IκB,其中VEGF减弱、Bcl-2增强可能是其维持细胞凋亡不变的重要原因；NFκB活性不变但转录增强,则可能与IκB活性降低关系密切,且可能不利于血液循环障碍的恢复。桃仁改善热证血液循环的因素尚待深入研究。更多还原

【Abstract】 The blood circulation disorder (BCD) is a pathophysiological state induced by local or systemic circulatory inadequacy. It can also result in tissue ischemia hypoxia injury, metabolic disorder, organ failure, even life-threatening. Microcirculation disturbance (MCD), which occurs in the local micro-vascular blood circulation, is an important component of BCD. BCD-related disease has been the thorny problem of therapy.Cold stagnation and blood stasis syndrome and heat stagnation and blood stasis syndrome (HS and RS), has been found with characteristics of BCD. Traditional Chinese medicine has achieved ideal effects on these two syndromes. So looking for a safe and effective traditional Chinese medicine to prevent BCD-related disease has great significance, as well as studying on its efficacy characteristics and mechanisms.Taoren, one kind of traditional Chinese medicine, is the seed of heart. It has been found with effects of blood vessel dilation, increasing organ blood flow, inhibiting platelet aggregation, anticoagulation, anti-thrombosis, and so on. Our previous studies found that Taoren act effectively on improving the two syndromes. Therefore, these study committed to find the effects, mechanisms and the differences of Taoren on BCD induced by the two syndromes in rats. The study was divided into three parts:Part I:Study on the Characteristics of Blood Circulation Disorder in HS and RS RatsObjective:To study the different characteristics of BCD in HS and RS rats.Methods:The rats were randomly divided into 4 groups,10 rats in each group:normal control group of HS (HN), model group of HS (HM), normal control group of RS (RN), model group of RS (RM). HM group rats were put into the -18±2℃freezer for 2 hours, two times a day for seven consecutive days. To RM rats, Carrageenan solution was given intraperitoneally for six consecutive days, after that dry yeast solution was given to them subcutaneous ly on the seventh day. On the eighth day, all of the rats were detected the following indexes:(1) the Chinese medicine symptoms, (2) blood flow velocity (Fve) and blood flow score (Fsc), (3) blood viscosity (Vis) and blood fibrinogen (Fib), (4) arteriole and small artery diameter (Adia, s-Adia), venular and small vein diameter (Vdia, s-Vdia), glomerulus area (Gare), thrombosis rate (Trat), organ injury severity score (Isco) (including heart, lung, liver, kidney, spleen) by histopathology analysis (HE, PASM and PTAH staining).Results:(1) The corresponding Chinese medicine symptoms appeared respectively in both HM and RM rats. (2) The decreased blood flow velocity appeared in both HM and RM rats, while the decreased blood flow score appeared just in RM rats. (3) The increased blood viscosity appeared in both HM and RM rats, while the increased blood fibrinogen appeared just in RM rats. (4) The increased small artery diameter appeared just in HM rats. The increased glomerulus area appeared in both HM and RM rats. The arteriole, venular and small vein diameter did not change significantly in both HM and RM rats. The increased thrombosis rate and increased organ injury severity score appeared respectively in both HM and RM rats, in addition to the lung injury severity score in RM rats.Conclusion:There was BCD in rats with cold stagnation and blood stasis syndrome and heat stagnation and blood stasis syndrome, induced by frozen method and Carrageenan respectively. Its systemic manifestations included decreased blood flow velocity and increased blood viscosity. Its local manifestations included changes of vascular diameter, thrombosis, organ injury. The differences between HS and RS included blood flow score, blood fibrinogen, small artery diameter and the range of organ injury.Part II:Effects of Taoren on Blood Circulation Disorder in HS and RS RatsObjective:To study the different effects of Taoren on BCD in HS and RS rats.Methods:The rats were randomly divided into 2 part,4 groups in one part, 10 rats in each group, the 1st 4 groups:normal control group of HS (HN), model group of HS (HM), Taoren treatment group of HS(HT), Chuanxiong control group of HS(HC), the 2nd 4 groups:normal control group of RS(RN), model group of RS (RM), Taoren treatment group of RS(RT), Danshen control group of RS(RD). HM, HT and HC rats were put into the -18±2℃freezer as Part I. The carrageenan and dry yeast solution were given to RM, RT and RD rats as PartⅠ. At the same time, the corresponding traditional Chinese medicines were given to HT, HC, RT and RD rats for sever consecutive days. On the eighth day, all of the rats were detected the following indexes:(1) blood flow velocity (Fve) and blood flow score (Fsc), (2) blood viscosity (Vis) and blood fibrinogen (Fib), (3) small artery diameter (s-Adia), glomerulus area (Gare), thrombosis rate (Trat), organ injury severity score (Isco) (including heart, lung, liver, kidney, spleen) by histopathology analysis (HE, PASM and PTAH staining).Results:The effects of Taoren on BCD included:(1) The increased blood flow velocity appeared in both HT and RT rats, while the blood flow score did not change in both HT and RT rats. (2) The decreased blood viscosity appeared in both HT and RT rats, while the blood fibrinogen did not change in both HT and RT rats. (3) The decreased small artery diameter appeared just in HT rats, while the increased small artery diameter appeared in RT rats. The decreased glomerulus area appeared in RT rats, while the glomerulus area did not change in HT rats. The thrombosis rate did not change in both HT and RT rats. The decreased kidney injury severity score appeared in both HT and RT rats, while the decreased injury severity score of heart, lung and liver appeared just in HT rats. The spleen injury severity score did not change in both HT and RT rats.Conclusion:Taoren improved BCD in rats with HS and RS. The improvements lied in increasing blood flow velocity, decreasing blood viscosity, regulating the local vascular diameter and protecting kidney from injury. The different effects of Taoren on the two syndromes included:decreasing small artery diameter and protecting more organs from injury in HS rats, but increasing small artery diameter and protecting only for kidney from injury in RS rats.PartⅢ:Molecular Mechanisms of Taoren on Blood Circulation Disorder in HS and RS RatsObjective:To study the different molecular expression of HS and RS, the molecular mechanisms of Taoren on BCD in the two syndromes.Methods:The organ specimens were derived from PartⅡ. All of the specimens were detected the molecular expression by histopathology analysis (IHC, TUNEL, ISH). The indexes included:(1) adhesion molecule CD31 and vascular endothelial cell (VEC) growth factor VEGF, (2) VEC apoptosis, apoptotic inhibitor Bcl-2, apoptotic induced protein P53, (3) nucleus factor NFκB p65 and mRNA, inhibitor IκB-a and mRNA, (4) hepatic CD68+ macrophage and Caspase-1 P20 protein, (5) vascular pericyte (VPC) protein a-SM-actin, vascular smooth muscle cell (VSMC) receptor AT1 and ADRB2.Results:Molecular expression in HM and RM rats:(1) Increased CD31 expression appeared just in RM rats, while it did not change in HM rats. VEGF expression did not change in both HM and RM rats. Increased apoptosis appeared in both HM and RM rats. Decreased Bcl-2 expression appeared just in RM rats, while it did not change in HM rats. Decreased P53 expression appeared just in HM rats, while it did not change in RM rats. It was decreased NFκB protein, increased its mRNA and without its nucleus expression in HM rats. While in RM rats, it was decreased NFκB protein, increased its nucleus expression and without its mRNA change. It was increased IκB mRNA, without change on its protein and its nucleus expression in HM rats. While in RM rats, it was increased nucleus expression, without change on its protein and its mRNA. (2) Decreased hepatic CD68+ macrophage appeared just in HM rats, while it did not change in RM rats. Caspase-1 P20 protein did not change in both HM and RM rats. (3) Decreased ADRB2 expression appeared in both HM and RM rats, while expression of a-SM-actin and AT1 did not change in the two models.The changes of molecular expression in HT and RT:(1) Increased CD31 expression appeared just in HT rats, while it did not change in RT rats. Decreased VEGF expression appeared just in RT rats, while it did not change in HT rats. Apoptosis did not change in both HT and RT rats. Increased Bcl-2 expression appeared just in RT rats, while it did not change in HT rats. P53 expression did not change in both HT and RT rats. It was increased NFκB nucleus expression, without change on its protein and its mRNA in HT rats. While in RT rats, it was increased NFκB protein and its mRNA, without change on its nucleus expression. It was decreased IκB protein, without its nucleus expression and its mRNA change in HT rats. While in RT rats, it was decreased nucleus expression, without change on its protein and its mRNA.Conclusion:(1) Molecules closely related to HS included P53, NFκB, IκB and ADRB2. The promoting factors of HS might be the increased P53-induced apoptosis of endothelial cells and the decreased repair capacity of macrophages. The serious injury of endothelial cells and smooth muscle cells, caused by repeated freezing and rewarming, might be the reason of decreased activity of NFκB and ADRB2.(2) Molecules closely related to RS included CD31, Bcl-2, NFκB, IκB and ADRB2. The promoting factors of RS might be the increased CD31-induced adhesion and Bcl-2-related apoptosis of endothelial cells, besides of the increased inflammatory factor induced by activated NFκB.(3) Molecules affected by Taoren in HS included CD31, NFκB, IκB and AT1. The inhibited factors of circulation recovery might be increased CD31 and activated NFκB. The acceleration of circulation recovery might be the vascular contractile response induced by AT1 and the increased repair capacity of macrophages.(4) Molecules affected by Taoren in RS included VEGF, Bcl-2, NFκB and IκB. The persistent factors of apoptosis might be the decreased VEGF and increased Bcl-2 of endothelial cells. The persistent activity and increased transcription of NFκB might be closely related to the decreased activity of IκB. These might not be conductive to the recovery of BCD, and remained to be in-depth study.更多还原