冠心病心血瘀阻证模型大鼠的建立及方药评价

Establishment of Rat Model of Coronary Heart Disease with Xinxueyuzu(心血瘀阻)Syndrome and Evaluation of Prescription Efficacy

目的:研究冠状动脉左前降支结扎模型的病理特点及血府逐瘀汤的干预效应,以“方证对应”理论探讨冠心病心血瘀阻证模型的建立方法。方法:通过结扎冠状动脉左前降支法制备冠心病模型大鼠,随机分为正常对照组、假手术对照组、模型对照组、血府逐瘀汤14 g/kg组、曲美他嗪5.4 mg/kg组,连续灌胃给药14 d。观察舌象色度测定R(Red,红色)、G(Green,绿色)、B(Blue,蓝色)分量值;心电图检测心率及ST段的变化;超声心动图检测心功能指标,包括左室射血分数(LVEF)、短轴缩短率(LVFS)、左室收缩末内径(LVIDs)、左室舒张末内径(LVIDd);血液流变学检测全血黏度、卡松粘度和红细胞聚集指数;HE染色观察心肌组织病理学变化;Masson染色观察心肌组织纤维化情况,使用Image J软件计算胶原容积分数(CVF)、透射电镜观察心肌细胞超微结构变化;ELISA法检测血清血栓素B2(TXB2)、6-酮-前列腺素1α(6-keto-PGF1α)、纤溶酶原激活物抑制剂-1(PAI-1)、组织型纤溶酶原激活物(t-PA)含量;免疫组化(IHC)法检测心肌组织细胞间粘附分子-1(ICAM-1)、血管内皮细胞粘附分子-1(VCAM-1)蛋白阳性表达情况。结果:与正常对照组、假手术对照组比较,模型对照组大鼠舌象R、G、B值均显著降低(P<0.01);心电图心率、ST段抬高幅度显著升高(P<0.01);LVEF、LVFS值显著下降,LVIDd、LVIDs值显著升高(P<0.01);心肌组织病理形态学显示心肌细胞排列紊乱、有大量炎性细胞浸润、胶原纤维增多、CVF显著升高(P<0.01),线粒体膜、嵴结构出现肿胀、断裂、溶解,可见空泡化改变;全血粘度(低、中、高切)、卡松粘度、红细胞聚集指数显著升高(P<0.01);TXB2、PAI-1含量显著升高(P<0.01),6-keto-PGF1α、t-PA含量显著降低(P<0.01);黏附因子ICAM-1、VCAM-1蛋白阳性表达显著升高(P<0.01)。与模型对照组比较,血府逐瘀汤14 g/kg组可显著升高大鼠舌象R、G、B值,心电图心率、ST段抬高幅度显著降低(P<0.01);LVIDd、LVIDs值显著降低,LVEF、LVFS值显著升高(P<0.01),心肌组织病理形态学显示心肌细胞排列相对整齐、心肌间隙明显减小、有少量炎性细胞浸润、胶原纤维减少、CVF显著降低(P<0.01),线粒体膜、嵴结构的肿胀、断裂、溶解、空泡化程度减轻;全血粘度(低、中、高切)、卡松粘度、红细胞聚集指数显著下降(P<0.01),TXB2、PAI-1含量显著降低(P<0.01),6-keto-PGF1α、t-PA含量显著升高(P<0.01);黏附因子ICAM-1、VCAM-1蛋白阳性表达显著降低(P<0.01)。结论:冠状动脉左前降支结扎模型出现心功能障碍、心肌结构损伤、血液“黏稠凝聚”、舌色紫暗、血管内皮损伤、纤溶系统紊乱、黏附因子沉积、等病理特征,活血化瘀代表方血府逐瘀汤可有效调节上述指标,基于“正向评价+逆向反证”印证模型可较好地模拟冠心病心血瘀阻证病理过程。

Objective:To investigate the pathological features of the model established by left anterior descending coronary artery ligation and the intervention effect of Xuefuzhuyu(血府逐瘀)Decoction,and explore the establishment method of Xinxueyuzu(心血瘀阻)Syndrome based on the theory of prescription corresponding to syndrome.Methods:A rat model of coronary heart disease was established by left anterior descending coronary artery ligation.Rats were randomized into normal control,sham surgery,model control,Xuefuzhuyu Decoction(14 g/kg),and trimetazidine(5.4 mg/kg)groups.All the rats were administered with corresponding drugs by gavage for 14 days.A tongue color analyzer was used to determine the R(red),G(green),and B(blue)values of the tongue.The heart rate and ST segment changes were observed by electrocardiogram(ECG)measurement.Echocardiography was employed to determine the left ventricular ejection fraction(LVEF),left ventricular fractional shortening(LVFS),left ventricular end-systolic diameter(LVIDs),and left ventricular end-diastolic diameter(LVIDd).The whole blood viscosity,Casson viscosity,and red blood cell aggregation index were determined.Hematoxylin-eosin(HE)staining and Masson’s staining were performed to observe histopathological changes and myocardial fibrosis,respectively.Image J was used to calculate the collagen volume fraction(CVF).Transmission electron microscopy was employed to observe the ultrastructural changes in myocardial cells.Enzyme-linked immunosorbent assay(ELISA)was employed to measure the serum levels of thromboxane B2(TXB2),6-ketoprostaglandin F1α(6-keto-PGF1α),plasminogen activator inhibitor-1(PAI-1),and tissue-type plasminogen activator(t-PA).Immunohistochemistry(IHC)was employed to assess the positive expression of intercellular adhesion molecule-1(ICAM-1)and vascular cell adhesion molecule-1(VCAM-1)in the myocardial tissue.Results:Compared with the normal control group and the sham surgery group,the model control group showed decreases in tongue R,G,and B values(P<0.01),increases in heart rate and ST segment elevation(P<0.01),lowered LVEF and LVFS(P<0.01),and elevated LVIDd and LVIDs(P<0.01).The histopathological examination of the model control group revealed disarrangement of myocardial cells,extensive infiltration of inflammatory cells,increased collagen fibers,and elevated CVF(P<0.01).The mitochondrial membrane and ridge structures exhibited swelling,fragmentation,dissolution,and visible vacuolar changes.Moreover,the model control group showed increased whole blood viscosity(low,medium,and high shear),Casson viscosity,and red blood cell aggregation index(P<0.01),elevated levels of TXB2 and PAI-1(P<0.01),lowered levels of 6-keto-PGF1αand t-PA(P<0.01),and increased positive expression of ICAM-1 and VCAM-1 in the myocardial tissue(P<0.01).Compared with the model control group,the Xuefuzhuyu Decoction group showed increased tongue R,G,and B values(P<0.01),decreased heart rate and ST segment elevation(P<0.01),lowered LVIDd and LVIDs(P<0.01),and elevated LVEF and LVFS(P<0.01).The histopathological examination revealed regular arrangement of myocardial cells,significant reduction in myocardial gap,mild infiltration of inflammatory cells,decreased collagen fibers,and reduced CVF(P<0.01).The degree of swelling,fragmentation,dissolution,and vacuolar changes in mitochondrial membrane and ridge structures was alleviated.Furthermore,the Xuefuzhuyu Decoction group showed decreased whole blood viscosity(low,medium,and high shear),Casson viscosity,and red blood cell aggregation index(P<0.01),lowered levels of TXB2 and PAI-1(P<0.01),elevated levels of 6-keto-PGF1αand t-PA(P<0.01),and inhibited positive expression of ICAM-1 and VCAM-1(P<0.01).Conclusion:The modeling by the left anterior descending coronary artery ligation leads to the pathological features such as cardiac dysfunction,myocardial structural damage,sticky blood coagulation,purple tongue,vascular endothelial injury,fibrinolysis system disorder,and adhesion factor deposition.The above changes can be adjusted by Xuefuzhuyu Decoction,a representative prescription with the Huoxuehuayu(活血化瘀)effect.The combination of evaluation and disproof can well simulate the pathological process of Xinxueyuzu syndrome in coronary heart disease.