基于心肌细胞钙稳态研究益气活血药防治舒张性心功能不全的作用机制

发布时间：2018-01-22 23:47

本文关键词： 钙稳态美托洛尔舒张性心功能不全益气活血药　出处：《北京中医药大学》2017年博士论文　论文类型：学位论文

【摘要】：心力衰竭是21世纪最常见的心血管流行病之一。其中近50%的心衰患者属于舒张性心功能不全,表现为射血分数正常或保留的心衰(heart failure with preserved ejection fraction,HFpEF),其5年生存率仅30%。近20年来,与收缩性心衰防治已经取得的进展相比,舒张性心衰的发病率与病死率没有明显减少,其防治形势十分严峻,已成为心衰研究的难点与热点。心肌细胞钙稳态是钙离子分布及时相变化的动态过程,通过兴奋-收缩耦联机制直接影响心肌细胞的舒缩功能。心肌细胞钙稳态异常贯穿于病理状态下心肌代偿性肥厚至舒张功能障碍,再到舒张性功能不全的全过程。蛋白激酶CaMK Ⅱ介导的肌浆网收缩期钙释放及舒张期钙回摄是调节心肌细胞舒缩功能的关键环节,也是潜在的重要干预靶点,目前临床仍缺乏可有效作用于该靶点的药物。不论HFpEF或收缩性心衰,均可出现心悸、气短、乏力疲倦、胸闷、活动后加重,甚至喘息气促等气虚的证候表现,并可伴有唇甲色暗、舌质紫暗、瘀斑、瘀点等血瘀证征象。基于临证经验及证候规律研究,气虚血瘀被认为是HFpEF的基本病机,贯穿于HFpEF发生发展的始终。基于这一认识开展的临床研究证实益气活血方药在改善HFpEF患者气虚血瘀证的同时,可显著改善心脏舒张功能;我们的前期研究也发现益气活血方药可改善压力负荷大鼠左室舒张功能及肥大心肌细胞的舒缩功能,并对钙转运产生良性调节作用。但益气药、活血药或益气活血药不同配伍对HFpEF是否存在不同影响及其相关机制仍需进一步验证。本研究拟通过复制缩窄腹主动脉致压力负荷HFpEF大鼠模型,运用小动物超声、左心室悬浮导管、激光共聚焦技术、Western blot等技术,从整体、组织、细胞及分子水平验证"益气活血药物可能通过调节心肌细胞钙稳态,改善心肌舒张功能,起到延缓甚至阻断舒张功能不全的作用,并且益气活血药物不同配伍之间存在疗效差异"假说的科学性。方法:1.动物造模:采用改良缩窄腹主动脉术复制压力负荷HFpEF大鼠模型。2.分组及给药:除假手术组外,术后各组大鼠随机分为模型组(给予生理盐水),益气组(给予黄芪+党参),活血组(给予丹参+三七),益气活血1:1组(黄芪+党参vs丹参+三七=1:1),益气活血2:1组(黄芪+党参vs丹参+三七=2:1),美托洛尔组。3.检测方法及指标:(1)术后每周记录大鼠一般情况,包括体重、症状、体征、死亡情况;术后4周、12周小动物超声评价左室心功能,包括舒张期左室前后壁厚度,左室内径,左室射血分数,短轴缩短率,二尖瓣口前向血流速E峰与二尖瓣环运动幅度e峰比值;术后12周大鼠跑台检测运动耐力,左心室悬浮导管测量模型大鼠血流动力学参数以评价左室心功能;术后12周处死动物,采静脉血测量血浆血小板凝集速率、纤维蛋白原含量,并取材计算标化心脏重量及肺重量;(2)术后12周处死动物,急性分离左心室心肌细胞,利用激光共聚焦技术检测心肌细胞舒缩功能及钙释放、钙回摄状态;取心肌组织,Western blot法检测钙转运相关蛋白CaMK Ⅱ、PKA、NCX1、PLB(S16)、PLB(T17)、SERCA2a、RyR2 表达水平。结果:(1)模型评价:与假手术组相比,①术后12周,模型大鼠心肌出现间质纤维化及血管周围纤维化,左室前后壁显著增厚(P0.05),E/e显著增高(平均值大于15)(P0.05),标化心脏重量、肺重量显著增加(P0.05),运动耐力下降(P0.05),舒张期左室压力下降速率-dp/dt降低(P0.05),左室舒张末期压力增高(静息约12mmHg,负荷约16mmHg)(P0.05),颈动脉压力明显增高(P0.05);左室射血分数、短轴缩短率、收缩期左室压力上升速率下降(P0.05),但射血分数均大于50%,收缩功能尚正常;②模型大鼠心肌细胞舒张期下降速率-d1/dt减慢(P0.05),收缩期上升速率+d1/dt下降(P0.05),收缩最大幅度50%时间增加(P0.05);模型大鼠钙瞬变最大幅度50%时间延长(P0.05),钙回落50%时间增加(P0.05),舒张期钙消除时间常数(P0.05)及钙泄漏增大(P0.05),钙储备降低(P0.05),钙转运相关蛋白表达水平异常,表现为CaMK Ⅱ、PKA、NCX1、N/S(NCX1/SERCA2a)表达水平上升(P0.05),PLB(S16)、PLB(T17)、SERCA2a 表达水平下降(P0.05)。此外,模型大鼠出现以运动耐力下降为主的气虚证表现(P0.05)及以纤维蛋白原含量升高为主的血瘀证表现(P0.05)。(2)益气活血药物对压力负荷HFpEF大鼠标化心肺重量、心肌纤维化、证候主要表现干预作用:术后12周①与模型组比较,各干预组标化心肺重量均降低(均P0.05);干预组间比较,益气活血1:1组、益气活血2:1组标化心脏重量改善优于活血组(P0.05),标化肺重量改善益气活血2:1组优于益气组、活血组、美托洛尔组(P0.05),但益气活血2:1组与益气活血1:1间比较标化心肺重量无差异(P0.05);②与模型组相比,仅益气活血2:1组可降低模型大鼠心肌组织纤维化评分(P0.05);③与模型组比较,益气组、益气活血1:1组,益气活血2:1组可改善反映气虚证的力竭距离、力竭时间(P0.05);干预组间比较,益气活血2:1组力竭距离、力竭时间优于益气组、益气活血1:1组(均P0.05);只有活血组、美托洛尔组可降低反映血瘀证的纤维蛋白原含量(P0.05),益气组、益气活血1:1组、益气活血2:1组有降低纤维蛋白原含量的趋势,但无统计学意义(P0.05);仅活血组降低血小板最大聚集率有统计学差异(P0.05),其余各干预组均无统计学差异(PO.05),但显示出下降趋势。(3)益气活血药物对压力负荷HFpEF大鼠左室舒缩功能的干预作用:术后12周,①益气活血2:1组与模型组比较,可显著改善左室射血分数(P0.05);与模型组比较,益气组、美托洛尔组舒张期左室前后壁厚度降低(P0.05),活血组各指标无差异(P0.05),益气活血1:1组、益气活血2:1组不仅可降低舒张期左室前后壁厚度(P0.05),还可降低E/e比值(P0.05),但两组间比较无差异(P0.05);②左心室悬浮导管测定心肌舒张功能—静息状态下,与模型组比较,益气活血2:1组、益气活血1:1组可显著降低左室舒张末期压力(P0.05),益气活血2:1组可显著增大左室舒张期压力下降速率-dp/dt(P0.05),且益气活血2:1组改善左室舒张末期压力优于益气活血1:1组(P0.05);多巴胺负荷状态下,与模型组比较,益气活血2:1组、益气活血1:1组可显著降低左室舒张末期压力、增大-dp/dt(P0.05);益气活血2:1组改善-dp/dt疗效优于益气活血1:1组(P0.05)。(4)益气活血药物对压力负荷HFpEF大鼠心肌细胞舒缩功能、钙稳态及钙转运关键蛋白的干预作用:术后12周,①心肌细胞收缩功能—与模型组比较,益气组、益气活血1:1组、益气活血2:1组可显著缩短心肌细胞收缩最大幅度50%的时间(P0.05);干预组间比较,益气活血1:1组、益气活血2:1组改善心肌细胞收缩最大幅度50%的时间优于益气组(P0.05),益气活血1:1组、2:1组间无统计学差异(P0.0.5);舒张功能—与模型组比较,各干预组均可改善心肌细胞舒张50%的时间(P0.05);干预组间比较,益气活血2:1组改善心肌细胞舒张50%的时间优于益气组(P0.05);益气活血2:1组、益气活血1:1组、美托洛尔组改善心肌细胞舒张50%的时间优于活血组(P0.05);益气活血1:1组、2:1组间比较无差异(P0.05);②心肌细胞钙释放—与模型组比较,活血组、美托洛尔组可降低心肌细胞钙瞬变幅度(P0.05),益气组、益气活血1:1组、益气活血2:1组可缩短钙瞬变最大幅度50%时间(P0.05),三组间比较无差异(P0.05);心肌细胞钙回摄—与模型组相比,益气组、益气活血1:1组、益气活血2:1组对钙回落50%时间、钙消除时间常数Tau值有改善作用(P0.05),三组间比较无差异(P0.05);活血组、美托洛尔组各指标无统计学差异(P0.05));钙泄漏及钙储备:与模型组相比,仅益气活血2:1组可减少心肌细胞钙泄漏(P0.05);各组与模型组比较钙库储备均无差异(P0.05);③钙转运关键蛋白表达水平:与模型组比较,各干预药物均可降低钙转运关键蛋白CaMK Ⅱ、PKA的表达水平及NCX1/SERCA2a(N/S)比值(P0.05),仅益气活血2:1组可降低钙释放相关蛋白RyR2的表达并升高钙回摄相关蛋白SERCA2a的表达(P0.05)),益气组、益气活血1:1组、益气活血2:1组可降低NCX1的表达水平(P0.05),益气组、益气活血2:1组还可升高钙回摄相关蛋白PLB(S16)、PLB(T17)的表达水平(P0.05),美托洛尔组可升高PLB(T17)表达水平(PP0.05);干预组间比较,益气活血2:1组改善CaMK Ⅱ、PKA优于益气组,改善N/S水平优于活血组、美托洛尔组(P0.05)),其余各干预组间未见明显差异(P0.05)。结论:1.益气组在改善运动耐力、左室重构、左室舒张功能、心肌细胞舒缩功能、钙回摄及升高PLB磷酸化位点的表达量方面优于活血组。2.整体上,益气活血配伍组防治舒张性心功能不全疗效优于单纯的益气、活血和美托洛尔组。3.重用益气药物的益气活血配伍组防治舒张性心功能不全疗效优于常规益气活血配伍组。
[Abstract]:Heart failure is one of the most common cardiovascular disease in twenty-first Century. Nearly 50% of patients with heart failure are diastolic dysfunction, manifested as normal ejection fraction or heart failure with preserved (heart failure with preserved ejection fraction, HFpEF), the 5 year survival rate of 30%. for nearly 20 years, compared with the progress and Prevention of systolic heart failure has the incidence and mortality of diastolic heart failure is not significantly reduced, the control situation is very serious, has become the focus and difficulty of heart failure research. Myocardial calcium homeostasis is calcium ion distribution change dynamic process, through the excitation contraction coupling mechanism directly affect the myocardial systolic and diastolic function. Myocardial cell abnormal calcium homeostasis throughout the pathological state of myocardial hypertrophy and diastolic dysfunction, and then to the whole process of diastolic dysfunction. The sarcoplasmic reticulum protein kinase CaMK II mediated charge Systolic and diastolic calcium calcium release back to the camera is a key step in regulating cardiomyocyte contractile function, but also an important potential target for intervention, the clinical effect of the drug is still lack effectively on this target. Whether HFpEF or systolic heart failure may occur, palpitations, shortness of breath, fatigue, chest tightness, aggravated by activities, symptoms even breathing shortness of breath, Qi deficiency, accompanied by a lip color dark, dark purple tongue, ecchymosis petechiae, etc. blood stasis signs. Study on clinical experience and regularity of TCM syndrome based on Qi deficiency and blood stasis is the basic pathogenesis of HFpEF, through the development of HFpEF. The clinical research always understanding of the confirmation of Yiqi Huoxue Prescription on improving HFpEF patients with Qi deficiency and blood stasis based, can significantly improve cardiac diastolic function; our previous studies also found that Yiqi Huoxue Decoction can improve left ventricular pressure overload hypertrophy and diastolic function in rats Myocardial systolic and diastolic function, and have a positive regulatory role on calcium transport. But the Yiqi Huoxue drugs or medicine Yiqi Huoxue Decoction in different combinations on HFpEF if there are different effects and mechanisms still need further verification. This study intends to copy by constriction of abdominal aorta in HFpEF rats model induced by pressure overload, the use of small animal ultrasound. Left ventricular catheter suspension, confocal laser technology, Western blot technology, from the whole, tissue, cellular and molecular level verification of "Yiqi Huoxue drugs may be through the regulation of myocardial cell calcium homeostasis, improve the myocardial diastolic function, to delay or even stop the vasodilation dysfunction, science and efficacy differences between different hypothesis the compatibility of Yiqi Huoxue drugs. Methods: 1. animal model: coarctation of abdominal aorta was made pressure overload rat model of HFpEF.2. by using the modified grouping and administration: except the sham operation group after operation. The rats were randomly divided into model group (saline group), Qi (Huangqi dangshen +), Huoxue group (Salvia miltiorrhiza + 37), 1:1 group (Astragalus Yiqi Huoxue + Salvia miltiorrhiza Codonopsis vs + 37 =1:1), 2:1 group (Astragalus Yiqi Huoxue + Salvia miltiorrhiza Codonopsis vs + 37 =2:1), the United States Metoprolo group.3. detection methods and indicators: (1) a week after operation, the general condition of rats, including body weight, symptoms, signs and death; after 4 weeks, 12 weeks of small animal ultrasound assessment of left ventricular cardiac function, including before and after the left ventricular diastolic wall thickness, left ventricular diameter, left ventricular ejection fraction, short shortening of mitral valve, forward blood flow velocity of mitral annular motion amplitude of peak E and e peak ratio; endurance testing exercise rats 12 weeks after operation, left ventricular catheter suspension measurement model rats with hemodynamic parameters of left ventricular function evaluation; animal were sacrificed 12 weeks after operation, the venous blood platelet plasma measurement agglutination rate, fiber The content of fibrinogen, and were normalized heart weight and lung weight; (2) animal were sacrificed 12 weeks after surgery, acute left ventricular myocytes were isolated myocardial cells were detected in Shu focusing technique and diastolic function and calcium release calcium by laser shooting back state; myocardium Western, blot assay of calcium transport related protein CaMK II, PKA, NCX1, PLB (S16), PLB (T17), SERCA2a, the expression level of RyR2. Results: (1) evaluation model: compared with sham operation group, 12 weeks after operation, the rats model of myocardial fibrosis and around vascular fibrosis, left ventricular wall thickening (P0.05 (E/e), significantly higher mean values greater than 15) (P0.05), the heart weight, lung weight increased significantly (P0.05), decreased exercise tolerance (P0.05), left ventricular diastolic pressure decline rate of -dp/dt decreased (P0.05), left ventricular end diastolic pressure (resting about 12mmHg, about 16mmHg (load) P0.05), carotid artery pressure Ming Xian Zenggao (P0.05); left ventricular ejection fraction, fractional shortening, left ventricular systolic pressure rise rate (P0.05), but decreased ejection fraction was more than 50%, the systolic function is normal; myocardial cells of model rats decreased diastolic rate of -d1/dt decreased (P0.05), systolic rise rate decreased +d1/dt (P0.05) the maximum contraction amplitude, 50% time increased (P0.05); calcium transients in rat model of variable amplitude 50% time (P0.05), calcium (P0.05) fell 50% times increase, diastolic calcium elimination time constant (P0.05) and calcium (P0.05), calcium leakage increases, reduce the reserve (P0.05) calcium transport protein expression abnormal performance for CaMK II, PKA, NCX1, N/S (NCX1/SERCA2a) expression level increased (P0.05), PLB (S16), PLB (T17), the expression level of SERCA2a decreased (P0.05). In addition, rats in exercise performance based performance deficiency (P0.05) and fibrinogen content increased The main manifestation of blood stasis syndrome (P0.05). (2) of heart and lung qi and promoting blood circulation drug on weight, pressure load HFpEF big mouse myocardial fibrosis, effects of syndrome manifestations: 12 weeks of intervention compared with model group after the intervention group, standardized heart lung weight were decreased (P0.05); intervention group comparison of Yiqi Huoxue group 1:1, 2:1 group Yiqihuoxue standardized heart weight is better than blood group (P0.05), standardized lung weight improved Yiqi Huoxue group 2:1 was better than that of Yiqi group, Huoxue group, metoprolol group (P0.05), but 2:1 group and Yiqi Huoxue Yiqi Huoxue 1:1 between standardized heart lung weight (no difference P0.05); compared with the model group, Yiqihuoxue group 2:1 only can reduce the myocardial tissue of model rats with fibrosis score (P0.05); compared with the model group, Yiqi group, Yiqi Huoxue group 1:1 2:1 group, Yiqi Huoxue can improve Qi deficiency reflects the exhaustive distance, exhaustive time (P0.05); the intervention group the comparison of benefits Qi and blood group 2:1 exhaustive distance, exhaustive time is better than that of Yiqi group, Yiqi Huoxue group 1:1 (P0.05); only blood group, metoprolol group can reduce the fibrinogen content reflect the blood stasis syndrome (P0.05), Yiqi group, Yiqi Huoxue Yiqi Huoxue group 1:1, decreased fibrinogen content trend 2:1 group, but no statistical significance (P0.05); only blood group decreased the maximum platelet aggregation rate had significant difference (P0.05), there was no significant difference in the rest of the intervention group (PO.05), but showed a downward trend. (3) the intervention effect of Yiqi Huoxue drugs on negative pressure bearing left ventricular systolic and diastolic function of HFpEF rats after 12 weeks, the 2:1 of Yiqi Huoxue group compared with model group, can significantly improve the left ventricular ejection fraction (P0.05); compared with the model group, Yiqi group, metoprolol group after left ventricular diastolic wall thickness decreased (P0.05), live blood groups had no difference (P0.05), Yiqi Huoxue group 1:1 Yi. 2:1 can not only reduce the blood gas before and after the left ventricular diastolic wall thickness (P0.05), but also can reduce the ratio of E/e (P0.05), but no difference between the two groups (P0.05); the left ventricular diastolic function - Determination of suspended pipe resting state, compared with the model group, Yiqihuoxue group 2:1, 1:1 Yiqihuoxue group can significantly reduce left ventricular end diastolic pressure (P0.05), 2:1 group, Yiqi Huoxue can significantly increase the left ventricular diastolic pressure decline rate of -dp/dt (P0.05), and Yiqihuoxue group 2:1 improved left ventricular end diastolic pressure is better than that of group 1:1 (P0.05) of Supplementing Qi and activating blood circulation; dopamine load condition, compared with the model group, Qi blood group 2:1, Yiqi Huoxue 1:1 group can significantly reduce the left end of the Shi Shuzhang pressure, the increase of -dp/dt (P0.05); Yiqi Huoxue group 2:1 improved -dp/dt curative effect is better than that of group 1:1 (P0.05) Yiqi Huoxue Yiqi Huoxue drugs. (4) the pressure load on contraction of rat myocardial cells HFpEF Schwartz, calcium The intervention effect of steady state and calcium transport key protein: 12 weeks after operation, the myocardial contractile function compared with the model group, Yiqi group and Yiqi Huoxue group 1:1 group 2:1, time of Supplementing Qi and activating blood circulation can significantly shorten the maximum contraction amplitude of 50% myocardial cells (P0.05); intervention groups, Yiqi Huoxue group 1:1 time better than the 2:1 group of Yiqi Huoxue Yiqi group improve myocardial contractile amplitude of 50% (P0.05), Yiqi Huoxue group 1:1, there was no significant difference between the 2:1 group (P0.0.5); diastolic function: compared with the model group, the intervention group can improve the myocardial diastolic cell 50% (P0.05); intervention groups, better than the time Qi group Yiqi Huoxue group 2:1 improved diastolic myocardial cell 50% (P0.05) 2:1; Yiqi Huoxue group, Yiqi Huoxue group 1:1, the time is better than that of metoprolol group and Huoxue group improve the myocardial cell diastolic 50% (P0.05); Yiqi Huoxue group 1:1, there was no difference in 2:1 between groups ( P0.05); myocardial cell calcium release, compared with the model group, Huoxue group, metoprolol group can reduce myocardial cell calcium transient amplitude (P0.05), Yiqi group, Yiqi Huoxue group 1:1 2:1 group, Yiqi Huoxue can shorten the calcium transient amplitude of 50% time (P0.05), there is no difference between the three groups (P0.05); myocardial cell calcium intake back -- compared with the model group, Yiqi group and Yiqi Huoxue group 1:1 2:1 group, Yiqi Huoxue on calcium calcium down 50% time, eliminate time constant Tau value improvement (P0.05), there is no difference between the three groups (P0.05); blood group, dexmedetomidine sotalol group no statistical indexes the difference (P0.05)); calcium leakage and calcium reserves: compared with the model group, Yiqihuoxue group 2:1 only can reduce myocardial calcium leakage (P0.05); each group compared with the model group, calcium reserves were no difference (P0.05); the expression of calcium transport key protein: compared with the model group, each drug intervention can to reduce calcium Transport key protein CaMK II, and the expression level of NCX1/SERCA2a PKA (N/S) ratio (P0.05), only the expression of Yiqi Huoxue group 2:1 can reduce the expression of calcium release related protein RyR2 and increased calcium intake back related protein SERCA2a (P0.05)), Yiqi group, Yiqi Huoxue group 1:1 2:1 group, Yiqi Huoxue can reduce the level of the expression of NCX1 (P0.05), Yiqi group, Yiqi Huoxue group 2:1 can increase calcium shooting back related protein PLB (S16), PLB (T17) expression level (P0.05), metoprolol group can increase the expression level of PLB (T17) (PP0.05); intervention groups, Yiqi Huoxue group 2:1 improved CaMK II PKA, better than Qi group, improve the level of N/S is better than that of blood group, metoprolol group (P0.05)), the rest of the intervention group there was no significant difference between the 1. (P0.05). Conclusion: Yiqi group in improving exercise tolerance, left ventricular remodeling, left ventricular diastolic function and diastolic function of myocardial cells, and increased expression of calcium intake to PLB phosphorylation site The amount of.2. is better than that of the whole blood group, Yiqihuoxue prescription group treatment of diastolic heart failure curative effect is better than pure Yiqi, Huoxue Yiqi Huoxue Prescription group and metoprolol group control.3. Qi drug reuse diastolic dysfunction is more effective than conventional Yiqihuoxue prescription group.

【学位授予单位】：北京中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R541.6

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