大蒜素对寒凝血瘀心肌缺血气体信号分子调控及微循环影响的研究

【作者】 谷万里；

【导师】 史载祥；

【作者基本信息】 北京中医药大学 ， 中西医结合临床， 2007， 博士

【摘要】 背景:冠心病心绞痛、急性心肌梗死(AMI)是临床最常见的心肌缺血性疾病,均属于中医血瘀证范畴。寒凝血瘀是缺血性心脏病临床常见的中医证型,前期临床研究表明,大蒜素能够作用于冠心病心绞痛的多个发病环节,具有中药多靶点起效的特性,对缺血心肌有良好的保护作用,尤其对寒证的治疗作用明显优于热证。大蒜素对寒凝血瘀证的治疗作用与历代文献记载的大蒜具有温通活血作用颇为一致,提示大蒜素也可能具有温通活血的作用,发挥对寒凝血瘀心肌缺血的治疗作用。目的:探讨大蒜素对寒凝血瘀心肌缺血的作用机制,从大蒜素对气体信号分子调控和对微循环的改善,揭示大蒜素温通活血治疗寒凝血瘀心肌缺血的病理生理及药理机制,并对血瘀证和寒凝血瘀心肌缺血患者的微循环进行观测。方法:本研究分为理论探讨、实验研究和临床研究三大部分。(1)理论探讨:围绕研究目的,从寒凝血瘀心肌缺血的证治研究、气体信号分子与活血化瘀研究、冠脉微循环障碍的中西医结合研究三方面进行了理论探讨。(2)实验研究:①首先采用间断反复冷应激和垂体后叶素(Pit)联合造模的方法,建立了大鼠寒凝血瘀心肌缺血病证结合模型:将大鼠分别装入笼内,置于-5℃±1℃环境中,连续3h,使大鼠全身暴露于低温环境。然后将大鼠取出,恢复20℃±1℃室温,并给予Pit20U/kg/d腹腔注射,连续3天。造模第3天,大鼠寒凝血瘀心肌缺血模型造模成功。同时,从大鼠外观状况、体温、舌耳微循环血氧饱和度(StO2)、心电图、血液生化指标、心肌病理、染色等方面对模型进行了综合评价。②在大鼠寒凝血瘀心肌缺血动物模型的基础上,观察了大蒜素对寒凝血瘀心肌缺血的一般生理指标(证候、体温、心电图)、生化指标(FT3、FT4、TSH、cTnT、MDA、SOD)、血管舒缩活性肽(CGRP、ET)、心肌病理的作用。③从大蒜素对气体信号分子NO、NOS、H2S和对心肌eNOS、ET-1mRNA表达的影响,研究了大蒜素对寒凝血瘀心肌缺血的气体信号分子调控机制。④从可视光微循环StO2、微血管内皮细胞Caveolin-1、CD34、心肌毛细血管超微结构,研究了大蒜素对寒凝血瘀心肌缺血微循环的影响。(3)临床研究:①通过对健康成人筛查,取得舌、面部可视光StO2正常值,并测定血瘀证患者活血化瘀治疗前后的舌、面部可视光StO2,对血瘀证的微循环功能进行了临床研究。②通过对急诊PCI及溶栓治疗后的急性期AMI患者进行超声心动图和MCE检查,评价心肌微循环,观测寒凝血瘀心肌缺血的心肌微循环特点。结果:(1)大鼠外观状况观察评价,符合中医寒凝血瘀证候表现:耳温和肛温降低分别反映了寒冷因素对外周和内部体温的影响;耳、舌StO2降低表明外周微循环的功能减退;心电图J点的变化可判断心肌缺血模型的成立;心脏StO2降低表明心肌微循环功能受损;心率的减慢、血清FT3、FT4、TSH的降低反映了寒的征象;血浆ET的增高反映了血管痉挛、血瘀的情况;心肌组织病理切片、染色证实了模型心肌缺血、坏死的存在。应用具有热性药属性的抗心肌缺血药硝酸甘油治疗进行反证,显著改善了各项指标,验证了模型的寒凝血瘀心肌缺血诊断。综合上述各项指标,可以确定采用间断反复冷应激和Pit联合造模的方法建立的大鼠寒凝血瘀心肌缺血病证结合模型成立,符合中医理论。(2)大蒜素对寒凝血瘀心肌缺血的一般指标、生化指标、血管舒缩活性肽调节、心肌病理均有改善作用:低剂量大蒜素可明显减少寒凝血瘀心肌缺血大鼠心肌中MDA含量,增加SOD活力。(3)在对气体信号分子调控机制方面,大蒜素低剂量明显能上调eNOSmRNA表达,促进eNOS的合成,显著提高大鼠心肌组织NOS、NO含量;中剂量能显著提高血浆H2S含量,降低心肌组织H2S合酶含量。(4)在微循环方面,大蒜素中、低剂量能显著提高寒凝血瘀心肌缺血大鼠的耳、舌、心脏StO2,大蒜素中剂量能显著降低Caveolin-1的表达,增加CD34的表达,减轻心肌超微结构病变程度,有改善微循环的作用。(5)临床研究表明,可视光StO2面部最低,口唇最高;舌各部位的StO2值以舌面最高,活血化瘀治疗后各部位StO2明显增高。(6)超声心动检查寒凝血瘀证患者对负荷试验的反映更明显,MCE心尖k值显著低于非寒凝血瘀证。结论:(1)间断反复冷应激和Pit联合造模的方法建立的大鼠寒凝血瘀心肌缺血模型符合中医证候特点。(2)大蒜素具有多重作用机制,能针对寒凝血瘀心肌缺血的多个发病环节,通过调控气体信号分子和改善微循环,多层面发挥对缺血心肌的保护作用,以中、低剂量作用最佳。①大蒜素能提高心肌组织抗氧化能力,保护缺血心肌;通过调控血管活性肽CGRP、ET的作用,拮抗由于造模因素所致的冠脉痉挛收缩,舒张血管,改善心电图J点变化,降低cTnT,减轻心肌缺血损伤。②大蒜素对寒凝血瘀心肌缺血的气体信号分子调控机制是:通过上调eNOSmRNA表达,下调ET-1mRNA表达,升高NOS,促进NO的释放,同时下调H2S,多方面发挥对缺血心肌的保护作用。③大蒜素对寒凝血瘀心肌缺血的心肌微循环具有改善作用:能增加心肌组织毛细血管数量,作用于微血管内皮细胞的Caveolin-1,升高外周耳、舌和心肌组织的StO2,维持心肌氧供和氧利用,改善微循环功能。(3)可视光StO2测定是一种新的定量评价组织微循环功能的指标,对血瘀证舌、面部望诊的客观化研究有重要价值。(4)AMI患者介入治疗后MCE检测,寒凝血瘀者较非寒凝血瘀者心肌微循环受损有加重趋势。(5)应用中药大蒜的单体化合物有效成分大蒜素,也要辨证用药,才能发挥其最大功效。更多还原

【Abstract】 Background: Coronary heart disease(CHD) angina pectoris and acute myocardial infarction(AMI) are the most frequent myocardial ischemia disease in clinic.They all belong to the syndrome of blood stasis category in TCM. Blood stasis due to cold coagulation is the frequent type of syndrome in TCM. The prophase research indicate that garlicin can interfere in CHD angina pectoris in several morbility component element and has the character of the traditional Chinese drug effects on multitarget point. It has satisfactory protection on ischemia myocardium especially in the therapeutic action on cold syndrome surpass pyretic syndrome. This concordance to successive dynasties record and indicate that garlicin possess the function of warming and activating meridian to promote blood flow.Garlicin has the therapeutic action of treating Myocardial ischemia of blood stasis due to cold coagulation (MIBSCC).Aim: Disscuss the garlicin functions to regulate gaseous signaling molecule and to improve microcirculation. Reveal therapeutic mechanism of garlicin to MIBSCC in patho-physiology and pharmacology mechanism by warming and activating meridian to promote blood flow. Survey the microcirculation of patients with syndrome of blood stasis and patients with MIBSCC.Methods: This study consist of three portions: theoretical discuss, expetimental research and clinical research.(1)Theoretical discuss: According to the study purpose, theory approach was carried out from three aspects: study on the pattern and treatment of MIBSCC; study on gaseous signaling molecule and promoting blood circulation to removing blood stasis; and study on integrated traditional and western medicine of coronary artery microcirculation disturbance.(2)Empirical research:①First,we established integrated disease and syndrome rat model of MIBSCC adopting interrupt cold stress repeatedly and Pit. Synthesis evaluation was done from the following aspects to determine the animal model’s establishment: the state of rat, body temperature, microcirculation evaluated by tongue and ear StO2, ECG, biochemical indicator of the blood and myocardium pathology.②On the bases of these, garlicin’s role on MIBSCC in common physiological index (syndrome, body temperature and ECG), biochemical indicator (FT3,FT4,TSH,cTnT,MDA and SOD), vasomotion bioactive peptide (CGRP and ET) and myocardium pathology were observed.③The regulating mechanism of garlicin on MIBSCC by gaseous signaling molecule were researched from the influence of garlicin to gaseous signaling molecule NO, NOS and H2S as well as myocardium eNOS and ET-1 mRNA expression.④The influence of garlicin on microcirculation of MIBSCC were researched from visible light StO2, vascular endothelial cell Caveolin-1, CD34 and heart muscle capillary ultrastructure.(3)Clinical research:①Through the healthy adult screening, the normal value of visual light StO2 in tongue and face were obtained. The visual light StO2 in tongue and face of ptients with syndrome of blood stasis were detected pretherapy and post-treatment. By this way, clinical research on microcirculation function was done.②To patients in acute stage of AMI after emergency PCI or thrombolysis treatment to evaluating myocardial microcirculation and surveying the myocardial microcirculation characteristic on MIBSCC by ultrasoundcardiogram and MCE examination.Results:(1)Each model evaluation index above-mentioned support the establishment of rat MIBSCC model.The model is correspond to the TCM theory.(2)Garlicin can improve common index, biochemical indicator, vasomotion bioactive peptide regulation and myocardial pathology of MIBSCC.(3)In the aspect of gaseous signaling molecule regulation mechanism, low dose garlicin can up-regulate eNOS mRNA expression obviously, promote the synthesis of eNOS, elevate the contents of NOS and NO in rat myocardium tissue significantlly, degrade the H2S synthase contents in myocardium tissue.(4)In microcirculation aspects, middle and low dose garlicin can significantlly improve the MIBSCC rat’s StO2 of ear,tongue and heart, middle dose garlicin can lower the Caveolin-1 and raise CD34 expression notablely, lessen the pathological changes degree of myocardium ultrastructure and improve microcirculation.(5)Clinical research indicate that visible light StO2 lowest in the face and highest in oral lips. Lingual surface StO2 is the highest in each regional of the tongue. StO2 in each part above-mentioned increased obviously after treated by activating blood circulation to dissipate blood stasis medicine.(6)MIBSCC patients response more obvious to echocardiography stress test and the k value in ischemic region significantlly less than the other syndrome.Conclusions:(1)The method of MIBSCC animal model by interrupting and repeatedly cold stress connected with Pit correspond to TCM syndrome character. (2)Garlicin can aim directly at MIBSCC multitude morbidity link, and protect ischemia myocardium in many ways through regulating gaseous signaling molecule and improve microcirculation.The middle and low dose of garlicin produce the best effect.(3)The determination of StO2 with visual light is a new index quantitative assessment tissue microcirculation function.(4)The research of AMI patients detected by MCE indicate that the myocardial microcirculation of MIBSCC patients had aggravate tendency compared with non MIBSCC patients.(5) According to differentiation of symptoms and signs, the utilization of garlicin can get its best effect.更多还原