【作者】 张大武；

【导师】 陈可冀； 王承龙；

【作者基本信息】 中国中医科学院 ， 中西医结合临床， 2010， 博士

【摘要】 1研究目的1.1研究急性冠脉综合征(acute coronary syndromes, ACS)患者介入治疗前中医证候要素的分布规律,探索中医证候要素与ACS危险分层的相关性,为中医药早期预防和干预ACS患者的不良预后提供临床依据。1.2观察ACS患者介入治疗后中医证候要素演变的规律,以及益气活血中药对中医证候要素演变的影响,为中医药临床治疗提供依据。1.3从炎症信号通路Toll样受体(toll-like receptors, TLRs) 2、4和下游促炎性细胞因子方面探讨益气活血中药联合缺血后适应干预大鼠急性心肌梗死再灌注损伤的作用机制,为中医药防治急性心肌梗死血运重建后再灌注损伤提供实验依据。2方法2.1临床证候要素规律研究本研究是一个前瞻性、随机、对照的多中心临床试验,将6家三级医院收集合格的241例ACS介入患者,随机分为益气活血干预组及对照组,对照组给予西医常规标准化治疗,益气活血干预组在西医常规标准化治疗基础上给予益气活血中药,分别对ACS介入患者入组前及入组后第3、6、12个月进行随访,具体方法包括：(1)通过横断面研究方法,对入组的241例ACS患者进行一般资料的频数分析,并对ACS患者进行全球急性冠状动脉事件注册(global registry of acute coronary event, GRACE)危险评分,基于GRACE危险评分的结果,采用多因子降维法和Logistic回归分析评价ACS高危患者的中医证候要素分布。(2)通过前瞻性调查研究方法,提取241例ACS患者入组后第3、6、12个月不同时间点的四诊信息,总结各个时间点的主要中医证候要素,观察ACS患者介入治疗后中医证候要素演变的规律,以及益气活血中药对中医证候要素演变的影响。2.2动物实验研究建立大鼠急性心肌梗死再灌注损伤模型,将SD大鼠随机分成假手术组、缺血再灌注组、缺血后适应组、益气活血+缺血后适应组以及福辛普利钠+缺血后适应组。通过血清心肌损伤标志物、心肌梗死面积、大体病理和超微结构的变化,观察益气活血中药联合缺血后适应辅助性治疗对再灌注损伤大鼠心肌的干预作用,并从TLR2.4及其下游促炎性细胞因子白细胞介素1β(interleukin-1β, IL-1β)、白细胞介素6(interleukin-6, IL-6)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)和单核细胞趋化蛋白-1(monocyte chemotactic protein-1, MCP-1)方面探讨其作用机理。3结果3.1临床证候要素规律研究3.1.1横断面研究分析的结果3.1.1.1241例ACS患者主要中医证候要素和证候要素组合241例ACS患者7个证候要素出现的比例由高到低依次为：血瘀(88.4%)>气虚(62.2%)>阴虚(32.8%)>痰浊偏寒(20.3%)>痰浊偏热(12.0%)>阳虚(8.7%)>气滞(0.8%),结果显示血瘀和气虚是ACS患者的主要证候要素；在具体中医证候分布中,2证候要素组合气虚+血瘀比例最高,占32%,其次是3证候要素组合气虚+阴虚+血瘀占11.6%,两者占241例ACS患者的43.6%。以本虚和标实证候要素组合的患者,所占比例为86.4%,表明ACS患者呈现的是“本虚标实,虚实夹杂”的特点。3.1.1.2 ACS高危患者中医证候要素分布对241例ACS患者进行GRACE危险评分,其中高危患者21人,中低危患者220人,基于GRACE评分结果,应用多因子降维法和Logistic回归分析ACS高危患者的中医证候要素分布,结果表明同时表现为血瘀、痰浊偏寒和阳虚证候要素的患者与不出现这复合中医证候要素的ACS患者相比,发生不良预后危险的比数比例为7.17,可以认为出现阳虚+血瘀+痰浊偏寒的ACS患者较不出现的患者6个月内可能发生全因死亡事件的百分比要高。3.1.2前瞻性研究分析的结果3.1.2.1 ACS介入后患者中医证候要素及其组合自然演变规律对照组患者西医常规治疗,血瘀、痰浊偏热在介入后显著降低,而气虚和阳虚逐渐增加,入组后12个月气虚和阳虚比例显著增加(共占63.8%)；血瘀比例虽有所下降,但仍然占有较大比例,表明ACS介入后期中医证候以本虚为主,标实为辅的特征。中医证候要素组合在介入治疗前后也出现了显著的变化,介入治疗前中医证候要素组合形式以复合证候要素为主,2证候要素组合以上的比例占86%,随血运重建治疗后时间的延长,逐渐由较多证候组合类型向较少证候组合类型演变,到入组后第12月,单证候要素组合以下的比例占有54.2%,其中单证候要素中又以气虚和阳虚为主,表现出本虚的证候特征。3.1.2.2 ACS介入后患者中医证候要素及其组合在中药干预下的演变规律在西医常规治疗基础上,益气活血干预组的患者血瘀、气虚、阴虚和阳虚证候要素的百分比在四个时间点均有不同程度的下降,尤以血瘀和阴虚的降幅最大,痰浊偏寒在入组后第3月也有下降趋势,但在第6、12月出现波动未见明显下降,痰浊偏热整个病程变化不大,表明益气活血中药对痰浊的干预作用微弱。中医证候要素组合方面,2证候要素组合和3证候要素组合均在入组后第3个月显著减少,而无证候要素和单证候要素在入组后第3个月显著升高,至第12个月,有49例患者症状和舌脉好转,占益气活血干预组患者45.8%,单证候要素和2证候要素组合次之,分别占31.8%和16.8%,3证候要素组合和4证候要素组合少见。以上结果表明益气活血中药干预下,证候要素从复杂组合向简单组合的演变过程较对照组明显加快,ACS介入患者有向良好预后发展的趋势。3.1.2.3益气活血干预组和对照组患者中医证候要素各时间点的比较入组后第3、6、12个月各时间点的对照组和益气活血干预组进行单一证候要素的比较,结果显示益气活血干预组患者入组后第3、6个月时间点血瘀、气虚和阳虚显著下降,与对照组比较,差异有统计学意义(P<0.05,P<0.01),但是益气活血干预组中的痰浊与对照组比较未有显著差异；另外在第12个月的时间点,益气活血中药组患者血瘀和阳虚比例较对照组显著下降,差异有统计学意义(P<0.01),但气虚证候要素未见显著差异,这与益气活血中药仅干预ACS介入患者到入组后第6个月有一定的关系。另外,241例ACS患者共有16例患者发生心血管事件,其中12例是对照组,4例是益气活血干预组,两组进行卡方检验,χ2=4.137,P<0.05,差异有统计学意义。3.2动物实验研究3.2.1益气活血中药联合缺血后适应对再灌注损伤大鼠心肌的保护作用益气活血中药预处理联合缺血后适应能进一步减小缺血后适应心肌梗死面积,减少血清肌酸激酶同功酶(creatine kinase-MB, CK-MB)的含量(P<0.01)；同时血清肌钙蛋白T(cardiac troponin T, cTnT)水平也较缺血后适应有所降低,但差异无统计学意义。大体病理和透射电镜观察结果也显示给予益气活血中药预处理联合缺血后适应后,心肌细胞内未见明显水肿,心肌肌原纤维排列尚规则,线粒体结构完整,无肿胀和空泡样变性,细胞核和核仁显示良好,未见明显胞核空洞样变及核固缩现象。3.2.2益气活血中药对缺血后适应心肌TLR2、4及下游促炎性细胞因子的影响与缺血后适应比较,益气活血中药预处理联合缺血后适应能进一步降低TLR2、4的表达(P<0.01)；同时,缺血后适应基础上,应用益气活血中药能进一步降低缺血后适应心肌组织IL-1β、IL-6、TNF-α和MCP-1水平(P<0.05,P<0.01)。4结论4.1血瘀、气虚是ACS的主要证候要素,气虚血瘀是主要证候表现,高危ACS患者中医证候要素组合特点多表现为阳虚+血瘀+痰浊偏寒；4.2血瘀、气虚贯穿于ACS介入术后1年的整个病程,由实转虚,由繁化简是ACS介入术后中医证候要素的演变特点；4.3本研究进一步验证中医益气活血法是ACS介入术后患者的主要治法；4.4益气活血中药可加强缺血后适应对急性心肌梗死后再灌注损伤心肌的保护作用,其机制与抑制TLRs及其下游促炎性细胞因子有关。更多还原

【Abstract】 1 Objectives1.1 To study distribution of TCM syndrome elements in patients with acute coronary syndromes (ACS) before percutaneous coronary intervention (PCI), and to explore the correlation of ACS risk stratification and TCM syndrome elements, so as to provide the clinical basis for early prevention and intervention of Chinese medicine from adverse outcome of ACS patients.1.2 To observe dynamic changes of TCM syndrome elements in ACS patients after PCI, and effect of Chinese herbs for nourishing qi and activating blood circulation (NQABC) on evolution of TCM syndrome element, so as to provide scientific basis for treatment of Chinese medicine.1.3 From aspects of toll-like receptor (TLR)2,4 and downstream pro-inflammatory cytokines, to observe mechanisms of Chinese herbs of NQABC combined with ischemic postconditioning (IPoC) on rat model of reperfusion injury after acute myocardial infarction, so as to provide experimental evidence for early prevention and treatment of Chinese medicine from reperfusion injury of acute myocardial infarction after revascularization.2 Methods2.1 Clinical investigation of syndrome element regularityThis study is a prospective, randomized, controlled multicenter clinical trial, the 241 qualified ACS patients after PCI from six tertiary hospitals were randomly divided into NQABC intervention group and control group. The control group was treated by western medicine standardized therapy, and NQABC intervention group was treated by Chinese herbs of NQABC on the basis of western medicine standardized therapy, and then to follow up these ACS patients after PCI at four time points, which were respectively before enrolled into group and the 3rd,6th,12th month after enrolled into group. Detailed methods as follows:(1) By cross-sectional research method, to analyze general information of 241 ACS patients through frequency analysis, and to assess the prognosis of these patients through global registry of acute coronary event (GRACE) risk score. Based on the result of GRACE risk score, we assessed the distribution of TCM syndrome elements in ACS high-risk patients through multifactor dimensionality reduction (MDR) and Logistic regression analysis method.(2) By prospective research method, to investigate the four diagnostic information of ACS patients at different time points of 3rd,6th,12th after PCI, and to summarize the main TCM syndrome elements at each time point. At the same time, to observe the dynamic changes of TCM syndrome elements in ACS patients after PCI, and effect of Chinese herbs of NQABC on evolution of TCM syndrome elements. 2.2 Animal experiment researchTo established rat model of acute myocardial infarction reperfusion injury, SD rats were randomly divided into sham group, ischemic reperfusion group, IPoC group, NQABC+IPoC group and fosinopril sodium+IPoC group. By the changes of serum myocardial injury markers, myocardial infarction size, macropathology and ultrastructure changes, to observe the influence of Chinese herbs of NQABC combined with IPoC on rat myocardium with reperfusion injury, and to explore its possible mechanism from aspects of TLR2,4 and its downstream pro-inflammatory cytokines interleukin-1β(IL-1β), interleukin-6 (IL-6), tumor necrosis factor-a (TNF-a) and monocyte chemotactic protein-1 (MCP-1).3 Results3.1 Clinical investigation of syndrome element regularity3.1.1 Cross-sectional analytic results3.1.1.1 The main TCM syndrome elements and combination of syndrome elements in ACS patientsThe proportion of 7 TCM syndrome elements in ACS patients in order from high to low are blood stasis (88.4%)>qi deficiency (62.2%)>yin deficiency (32.8%)> turbid phlegm tending toward cold (20.3%)>turbid phlegm tending toward heat (12.0%)>yang deficiency (8.7%)>qi stagnation (0.8%), the results showed that blood stasis and qi deficiency are main syndrome elements of ACS patients. In the specific TCM syndromes, the proportion of combination of 2 syndrome elements blood stasis+qi deficiency was the highest, accounting for 32.0%, and the second is combination of 3 syndrome elements qi deficiency+yin deficiency+blood stasis, accounted for 11.6%, the two combinations accounted for 43.6% in 241 ACS patients. The proportion of combinations of syndrome elements which include root vacuity and tip repletion accounted for 86.4%, which demonstrated that the syndrome features of ACS patients are root vacuity and tip repletion and deficiency complicated with excess.3.1.1.2 The distribution of TCM syndrome elements in ACS high-risk patients241 ACS patients were evaluated by GRACE risk score, and 21 patients were high-risk,220 patients were low-risk and mediate-risk. Based on the results of GRACE risk score, the analytic results of MDR and Logistic regression analysis method demonstrated that the adverse outcomes of the patients with blood stasis, turbid phlegm tending toward cold and yang deficiency were 7.17 times as much as that of the patients without blood stasis, turbid phlegm tending toward cold and yang deficiency, so probability of all-cause death in the ACS patients with blood stasis, turbid phlegm tending toward cold and yang deficiency during six months were higher than that of the patients without blood stasis, turbid phlegm tending toward cold and yang deficiency.3.1.2 Prospective analytic results3.1.2.1 The natural dynamic changes of TCM syndrome elements and their combinations in ACS patients after PCIACS patients in the control group were treated by western medicine standardized therapy method, blood stasis and turbid phlegm tending toward heat reduced significantly after PCI, qi deficiency and yang deficiency increased gradually, the proportion of qi deficiency and yang deficiency increased significantly at the 12th month, accounted for 63.8%, at the same time blood stasis decreased somewhat, but still accounted for a large proportion, which demonstrated that the feature of ACS patients in the later period after PCI was that deficiency syndrome is primary and repletion pattern is subordinate.The combinations of TCM syndrome elements before and after PCI also had a significant change, the main combinations of TCM syndrome elements before PCI was compound syndrome elements, the proportion of combination of 2 syndrome elements or more accounted for 86%. With the extension of revascularization time, the type of more combinations of syndrome elements turned to the type of less combinations of syndrome elements, the proportion of 1 syndrome element or less accounted for 54.2% at the 12th month, blood stasis and yang deficiency were main syndrome elements in the 1 syndrome element, which showed the feature of root deficiency syndrome.3.1.2.2 The dynamic changes of TCM syndrome elements and their combinations in ACS patients taking Chinese herbs after PCIOn the basis of western medicine stantardized therapy, the proportion of the TCM syndrome elements blood stasis, qi deficiency, yin deficiency and yang deficiency in NQABC intervention group had different downward trends in four time points, particularly downward trends of blood stasis and yin deficiency were larger, the proportion of turbid phlegm tending toward heat also had a downward trend at the 3rd after enrolled into group, but had not significant downward trend at the 6th and the 12th. The proportion of turbid phlegm tending toward heat in the whole course of disease had not significant change, which demonstrated that the effect of Chinese herbs of NQABC on turbid phlegm is ineffective.About the combinations of TCM syndrome elements, both the combination of 2 syndrome elements and the combination of 3 syndrome elements reduced significantly at the 3rd month after enrolled into group, while 0 syndrome element and 1 syndrome element increased significantly at the 3rd month after enrolled into group. At 12th month, symptoms as well as tongue and pulse presentations in 49 patient were improved, accounted for 45.8% in NQABC intervention group, the 1 syndrome element and the combination of 2 syndrome elements accounted for 31.8% and 16.8% respectively, the combination of 3 syndrome elements and the combination of 4 syndrome elements were rare. These results demonstrated that the evolutional time from more combinations of syndrome elements to less combinations of syndrome elements in NQABC intervention group is shorter than that of control group, and the ACS patients taking Chinese herbs of NQABC has the trend to good prognosis.3.1.2.3 The comparison of TCM syndrome elements in NQABC intervention group and control group at different time pointsEach syndrome element between two groups was compared at the 3rd,6th,12th month. The results show that the proportions of blood stasis, qi deficiency and yang deficiency were lower significantly in the NQABC intervention group than that of control group at the 3rd,6th month, the difference had statistical significance (P<0.05, P<0.01), but the turbid phlegm between two groups had not different significantly. At the 12th month, the proportions of blood stasis and yang deficiency were lower significantly in the NQABC intervention group than that of control group, the difference had statistical significance (P<0.01), but qi deficiency between two groups had not different significantly, which related to the intervention time of NQABC Chinese herbs. In addition,16 cases in 241 ACS patients had cardiovascular events, and there were 12 cases in the control group and 4 cases in the NQABC intervention group, the result of chi square test showed that the difference between two groups has statistical significance (χ2=4.137, P<0.05).3.2 Animal experiment research3.2.1 The effects of Chinese herbs of NQABC combined with IPoC protecting rat myocardium with reperfusion injury.Compared with IPoC group, NQABC plus IPoC further reduced infarction size and release of myocardial enzyme CK-MB (P<0.01), the content of serum cTNT was also lower in the NQABC+IPoC group than that of IPoC group, but the difference between two groups had not statistical significance. The results of macropathology and transmission electron microscope observations in the NQABC+IPoC group showed that there were not significant intracellular edema, myocardial fiber arrangement was still regular, mitochondrial structure was integrity, no swelling and vacuolation, nucleus and nucleolus were normal, no obvious nucleus cavity-like degeneration and nuclear pycnosis phenomenon.3.2.2 The effects Chinese herbs of NQABC combined with IPoC on TLR2.4 and downstream pro-inflammatory cytokines of rat myocardium with reperfusion injury.Compared with IPoC group, NQABC plus IPoC further decreased the expression of TLR2,4 (P<0.01). At the same time, Chinese herbs of NQABC on the basis of IPoC could further decrease the contents of IL-1β, IL-6, TNF-αand MCP-1 in rat myocardium with IPoC (P<0.05,P<0.01).4 Conclusions4.1 Blood stasis and qi deficiency were main syndrome elements of ACS patients, qi deficiency and blood stasis was main syndrome performance, the feature of TCM syndrome elements combinations in ACS high-risk patients mainly manifested yang deficiency+blood stasis+turbid phlegm tending toward cold.4.2 Blood stasis and qi deficiency run through the whole course of disease of ACS during one year after PCI, the evolutional features of TCM syndrome elements of ACS after PCI were from excess to deficiency and from complex to simple.4.3 This study further validated that NQABC might be the main therapy method of ACS after PCI. 4.4 The pretreatment with Chinese herbs of NQABC enhanced the protective effect of IPoC on rat myocardial I/R injury, and its mechanism might be related to the inhibition of TLRs expression and the expression of the downstream proinflammatory cytokines.更多还原