基于肌浆网钙转运研究黄芪、丹参对肥大心肌细胞舒缩功能干预机制

【作者】 孙娅楠；

【导师】 林谦；

【作者基本信息】 北京中医药大学 ， 中西医结合临床， 2013， 博士

【摘要】 心力衰竭是高血压、缺血性心脏病、心脏瓣膜病等各种心血管系统疾病发展的终末阶段。心衰一旦发生,预后极差,其死亡率占心血管疾病总死亡率的40%。心衰发生发展的基本机制是心肌重构,其病理生理核心在于心肌细胞钙稳态平衡紊乱,进而导致心肌舒缩功能受损。其早期主要表现为舒张功能障碍,心肌顺应性降低,进而影响收缩功能,最终导致心功能不全。兴奋-收缩偶联障碍则是导致其舒缩功能不全的重要环节。心肌胞浆的Ca2+浓度周期性变化主要来源于肌浆网,肌浆网通过兰尼丁受体2(Ryanodine receptor, RyR2)快速释放Ca2+,引起胞浆Ca2+浓度迅速升高,引发心肌收缩,随后又由肌浆网钙泵(sarcoendoplastic reticulum Ca2+-ATPase, SERCA2a)在受磷蛋白(phospholamban, PLB)的调节下主动将Ca2+转运回肌浆网中,使胞浆Ca2+浓度回落而引起心肌细胞舒张,由肌浆网释放-摄取Ca2+引起的钙瞬变,将心肌中的电活动与机械活动偶联,与心肌收缩-舒张功能直接相关。以上钙转运关键因子都受到钙调素依赖性蛋白激酶Ⅱ(Calmodulin kinase, CaMKⅡ)的调控。肌浆网钙转运是心衰早期潜在的干预靶点。运用抑制剂或基因敲除方法抑制CaMKⅡ的过度激活,解除PLB对SERCA2a的抑制可以延缓心衰的进展,但如何推广到临床应用,尚需要大量试验研究。因此目前尚缺乏能被临床试验证实有效的药物可供使用。中医辨证论治在心衰早期干预方面具有一定的优势,临床研究认为气虚、血瘀是引起心衰发生、发展的基本病机。益气药黄芪、活血药丹参是治疗心衰的常用药,具有确切的临床疗效,在改善气虚血瘀证同时,可以改善心肌肥厚、延缓心衰的进展。本课题在大量文献整理、分析及既往研究工作的基础上,从心肌细胞肌浆网钙转运机制着手,阐释黄芪、丹参治疗心肌肥厚、预防心衰发生发展的药理学基础。方法1.心肌细胞特性观察实验：体外原代培养乳鼠心肌细胞,分为对照组(Control)与模型组(Model)。于给药后24、48、72h后运用倒置相差显微镜观察心肌细胞一般特性。HE染色及图像分析法记录细胞核／细胞质面积与直径等检测肥大心肌细胞模型的建立；相差显微镜下记录细胞搏动频率；活细胞工作站记录细胞搏动幅度。2.肥大心肌细胞钙转运的变化及黄芪、丹参对其调节作用：体外原代培养乳鼠心肌细胞,分为对照组(Control、模型组(Model).黄芪组(HQ)、丹参组(DS)、芪丹组(QD)、氯沙坦组(Losartan)。用钙荧光探针Fluo-4/AM进行负载,然后在波宽0.5ms,强度为20V的电刺激起搏下,于给药后24、48、72h,用单细胞收缩与离子测量系统动态记录单个心肌细胞的钙瞬变,提取特征参数：荧光强度变化率(△F)、基线荧光强度(Fo)、钙瞬变幅度(Ft)、峰回落时间(RTso)、衰减时间常数(τ)、-dp/dt max.观察不同时点AngⅡ对心肌细胞内钙瞬变的影响。同时观察丹参注射液和黄芪注射液的干预作用。3.肥大心肌细胞钙转运相关蛋白变化及黄芪、丹参对其干预作用：采用免疫荧光法及Western blot去在24、48、72h,观察不同时点AngⅡ对钙转运调控蛋白CaMKⅡ、SERCA2a、PLB荧光强度与蛋白含量及SERCA2a/PLB的影响。同时观察丹参注射液和黄芪注射液的干预作用。4.KN-93阻断CaMKⅡ后黄芪、丹参对钙瞬变的影响：采用KN-93阻断CaMKⅡ后应用激光共聚焦显微镜,观察48h黄芪注射液和丹参注射液对钙瞬变的干预作用。结果1.对心肌细胞特性的影响10-7mol/L AngⅡ刺激乳鼠心肌细胞24、48、72h后,乳鼠心肌细胞核／细胞质面积与直径显著增加；48h、72h后心肌细胞总蛋白含量与对照组比较显著增加。给予AngⅡ后24h模型组搏动频率较对照组下降,48h后显著减慢,随着AngⅡ作用时间延长,心肌细胞搏动频率不断减慢,至72h搏动频率达到最低。2.黄芪、丹参对肥大心肌细胞钙瞬变及其收缩功能的影响(1)给予Ang11后24h模型组与对照组比较,心肌细胞RTso与T值显著增加,-dp/dt max显著降低(P<0.05)；各中药组可使RTso与τ值降低至正常水平(P<0.05)(2)48h, AngⅡ对心肌细胞RTso、τ值与-dp/dt max的作用持续(P<0.05),并显著降低Fo、△F、Ft(P<0.05),各中药组可改善AngⅡ引起的上述变化使RT50、T值、-dp/dt max、F0、△F、Ft恢复至正常水平,氯沙坦组可降低F0、RT50与τ值(P<0.05)。(3)72h,模型组心肌细胞RT50、T值与.-dp/dt max的持续增加(P<0.05),F0、△F、Ft持续降低(P<0.05),各中药组可改善AngⅡ引起的上述变化,芪丹组可显著降低τ值且优于丹参组,氯沙坦组可显著增加△F、Ft与-dp/dt max (P<0.05).3.黄芪、丹参对肥大心肌细胞蛋白含量的影响免疫荧光检测：(1)24h模型组CaMK Ⅱ荧光强度较对照组显著增加(P<0.05),SERCA2a与PLB荧光强度虽略有下降,但无统计学意义。(2)48h模型组CaMK Ⅱ荧光强度增加的同时SERCA2a与PLB荧光强度显著下降(P<0.05)；各给药组可显著降低CaMK Ⅱ荧光强度,并使SERCA2a与PLB荧光强度显著增加。(3)模型组对CaMK Ⅱ. SERCA2a与PLB荧光强度的影响仍然持续；各给药组虽仍可抑制AngⅡ的上述作用,但仅对SERCA2a荧光强度的增加有统计学意义(P<0.05),对于PLB荧光强度的影响,只有芪丹组与氯沙坦组有显著差异(P<0.05)。Western blot检测：(1)24h,模型组和对照组相比CaMK Ⅱ蛋白含量显著升高(P<0.05),SERCA2a蛋白含量下降(P<0.05)；PLB蛋白含量与SERCA2a/PLB比率下降,但无统计学意义；黄芪组可降低CaMKⅡ蛋白含量,增加SERCA2a蛋白含量,至正常水平,同时可大幅度升高SERCA2a/PLB比例；丹参组与模型组相比可降低CaMKⅡ蛋白含量,略升高SERCA2a与SERCA2a/PLB比例,但没有统计学意义；芪丹组显著降低CaMKⅡ蛋白含量,增加SERCA2a与PLB蛋白含量,且优于单药使用。(2)48h与对照组相比模型组CaMKⅡ蛋白含量持续增加,SERCA2a、PLB蛋白含量有仍呈下降趋势,SERCA2a/PLB比率大幅度下降(P<0.05)；黄芪组与丹参组可显著降低CaMKⅡ蛋白含量,增加SERCA2a、PLB蛋白含量和SERCA2a/PLB比率；联合用药组可使CaMKⅡ蛋白含量显著下降,使SERCA2a、PLB蛋白含量和SERCA2a/PLB比率上升,且均优于单药组。4.黄芪、丹参对Ca2+/CaMKⅡ通路的影响48h,模型组和对照组相比Fo及RT50上升,△F下降(P<0.05)；黄芪组、丹参组与模型组相比可降低F0及RT5o,回升△F(P<0.05)；芪丹组与模型组比较使RT5o及Fo显著降低,并大幅度回升△F,且与单药组相比有显著差异(P<0.05)；氯沙坦组可降低RT50并增加△F(P<0.05)。给予模型组及各治疗组KN-93后检测心肌细胞钙瞬变。模型组心肌细胞△F、F0、Ft无显著性差异,提示肥大心肌细胞收缩功能得以改善,但RT50与对照组相比显著增加(P<0.05),表明心肌细胞舒张功能仍存在异常。黄芪注射液、丹参注射液、两药联合使用及氯沙坦在此基础上可以进一步RT50(P<0.05)。结论AngⅡ导致心肌细胞肥大-衰竭的过程中可引起心肌细胞核/细胞质面积与心肌细胞核/细胞质直径显著增加,搏动频率下降。给予AngⅡ后24h钙瞬变峰回落时间增加-舒张功能异常,至48h发生钙瞬变荧光强度变化率降低-收缩功能异常。提示在心肌细胞肥大-衰竭过程中,舒张功能障碍发生先于收缩功能。在早期即可引起心肌细胞钙转运相关蛋白CaMKⅡ、PLB、SERCA2a蛋白表达水平的变化,影响心肌细胞内钙离子浓度。黄芪、丹参对钙离子的调节作用是通过干预钙转运调控蛋白CaMKⅡ、PLB、 SERCA2a而实现,从而改善了心肌细胞的舒缩功能。黄芪在心肌细胞肥大早期既可改善钙调控蛋白含量与钙瞬变峰回落时间异常,衰竭初期黄芪,丹参可以调整心肌细胞钙瞬变与钙调控蛋白含量异常,从而改善心肌细胞兴奋-收缩偶联,两药联合应用在早期并未优于黄芪单独应用,但在衰竭期选择联合用药可以更好的改善肌浆网对钙离子的调节作用。两药联合的长程干预不但可以保护心肌细胞的舒缩特性,也可以拮抗心衰时心肌细胞钙瞬变及其调控蛋白的异常变化。氯沙坦在心肌细胞肥大早期并未体现组对与钙瞬变的调节作用,在衰竭初期可通过调节心肌细胞峰回落时间与T值改善舒张功能,在衰竭期则可显著改善钙瞬变幅度及其荧光变化率,影响心肌细胞收缩功能。此种变化显示了其对心肌细胞舒缩功能改善的不均一性,常常是舍弃其中一方而保全另一方,中药则不仅可以改善心肌细胞收缩功能,并且对舒张功能的作用也非常显著,体现了中药调节的整体性。黄芪、丹参改善肥大心肌细胞收缩功能的机制可能部分是通过抑制钙调蛋白激酶途径中CaMKⅡδ的表达而实现的,但对心肌细胞舒张功能的改善可能是另有其他途径,需要进一步研究。更多还原

【Abstract】 Heart failure is the terminal stage of a variety of cardiovascular diseases, such as hypertension, coronary heart disease, valvular heart disease. Once heart failure occurs, the prognosis is extremely poor, and accounted for40%of the total mortality of cardiovascular disease mortality. The basic mechanism of heart failure is myocardial remodeling, and the pathphysiology is myocardial calcium homeostasis balance disorders, leading to impaired myocardial function.The early manifestation of heart failure is diastolic dysfunction and reduced myocardial compliance, thereby affecting systolic function, eventually leading to heart dysfunction. Excitation-contraction coupling (ECC) barriers are an important part of the heart systolic and diastolic dysfunction. In normal excitation-contraction coupling (ECC), firstly, small amounts of calcium ions enter the cell through L-type calcium channel during depolarization, secondly, a large of calcium release from sarcoendoplasmic reticulium (SR) by ryanodine receptors. This is called the calcium-induced calcium release. The result is a rapid rise of cytosolic calcium concentration that causes myocardium contraction. Then during repolarization, most of the cytosolic calcium is uptaken into sarcoendoplasmic reticulum by its Ca+-ATPase (SERCA2) under the regulation of phospholamban. So the concentration of calcium in cytosol decreases and the myocardium relaxes. This change of intracellular calcium ions during excitation-contraction coupling is called calcium transient, and it is the molecular basis of myocardial contraction. The above of calcium transport protein is regulated by calmodulin-dependent protein kinase Ⅱ (CaMK Ⅱ). Calcium transport has become to be considered is one of the most mechanisms during the development of heart failure, and may be the target for intervention in the future. Use of inhibitors or gene knockout method to inhibit excessive activation of CaMK Ⅱ can delay heart failure progress, but how to promote clinical applications, still need a lot of tests. Therefore no corresponding drugs can be used for clinical application.Traditional Chinese medicine has certain advantages in early intervention in heart failure. During the clinical studies, the researcher suggests that deficiency qi and blood stasis is the cause of heart failure occurred, and a key factor for heart failure. Previous experiments also suggested that traditional Chinese medicine has different regulation for sarcoplasmic reticulum calcium transport. Salvia and Astragali is effective to heart failure which is commonly used drugs, with the exact clinical efficacy. There can improve cardiac hypertrophy, but also can delay the progress of heart failure.Our research is based on sorting out and analyzing a large of reference data and previous research. Use cardiac sarcoplasmic reticulum calcium transport mechanism to explain Astragalus, Sal via treatment of cardiac hypertrophy and pharmacological basis for the prevention of heart development.Methods1. Cardiomyocytes characteristics observed experiment:The cultured of neonatal rat cardiomyocytes were divided into control group, Model group, Salvia group (DS), Astragali Group (HQ), Salvia and Astragali group (DQ), Losartan group. At24th hour,48th hour,72nd hour, use inverted phase contrast microscope to observe the characteristics of cardiomyocytes. HE staining and image analysis record the model of the nucleus/cytoplasm area and diameter of cardiomyocytes; Use phase contrast microscope to record beating frequency of cardiomyocytes; The cell area before and after cell contraction was measured by the living cells workstation.2. Effects of Salvia and Astragali on the Calcium Transients:The cardiomyocytes were loaded with Fluo-4/AM. The Salvia and Astragali on the contractility, diastolic [Ca2+] and calcium transients were assessed by the Fluorescence Measurement and Cell Dimensioning Systems. Observe the changes of Salvia and Astragali effect on calcium transients at24th hour,48th hour,72nd hour.3. Effects of Salvia and Astragali on the expression of calcium handing proteins:Using immunofluorescence and Western bolt method observed Salvia and Astragali on the fluorescence intensity and protein content of calcium handing protein:CaMK Ⅱ, SERCA2a and PLB at different time points (24h,48h,72h).4. Effects of Salvia and Astragali on the Calcium transients after KN-93blocked CaMK Ⅱ:The Salvia and Astragali on calcium transients were assessed by the Fluorescence Measurement and Cell Dimensioning Systems and Laser Scanning Confocal Microscopy (LSCM). Using KN-93to block CaMK Ⅱ, observe the change of Salvia and Astragali effect on calcium transients in24th hour,48th hour, and72nd hour. Results1. The nuclear/cytoplasm area and diameter of myocardial are significantly increased at24th hour,48th hour, and72nd hour by10-7mol/L angiotensin Ⅱ. The total protein content of cardiomyocytes increased significantly; the beating rate decreased significantly with time.2.24th hour, compare to the control group, the relaxation time to50%baseline (RT50) and τ of model group significantly increased (P<0.05). In herbs group, RT50and τ decreased.48th hour, the F0, τ and RT50of model group increased, the△F and F decreased (P<0.05). The herbs group not only reduced RT50, τ and Fo, but also recovered the△F and F to normal levels. The Losartan group reduce RT50, τ and Fo.72nd hour, the F0,△and RT50of model group still increased, the△E and F were significantly lower (P<0.01). HQ and DS can improve the change by Ang Ⅱ, and the two drugs used in combination are better than their individual use to change τThe Losartan group increased△F, F and-dp/dt max.3. Effects of Salvia and Astragali on the mean IOD of calcium handing proteins.(1)24th hour, the CaMK11IOD of model group compared with control group increased significantly(P<0.05), SERCA2a mean IOD and PLB mean IOD decreased slightly, but not statistically significant. Each dose group can be varying degrees of improvement.(2)48th hour, model group CaMK Ⅱ mean IOD increased, while SERCA2a mean IOD and PLB mean IOD was significantly decreased(P<0.05). Each dose group can significantly reduce the CaMK Ⅱ mean IOD, and SERCA2a mean IOD and PLB mean IOD increased.(3)72nd hour, the model group mean IOD of CaMK Ⅱ, SERCA2a and PLB is still continuing to change(F <0.05). Each dose group increased SERCA2a mean IOD. Only the combined group and Losartan group increased PLB mean IOD. Effects of Salvia and Astragali on the expression of calcium handing proteins.(1)24th hour, compared to the model group and the control group CaMK Ⅱ expression level was higher(P <0.05) and the SERCA2a expression level decreased(P<0.05). The HQ group reduced the expression level of CaMK Ⅱ, and increased SERCA2a protein content to normal levels, while significantly elevated SERCA2a/PLB ratio. The DS group compared with the model group can be reduced CaMK Ⅱ protein expression.(2)48th hour, the model group continued to increase CaMK Ⅱ expression level and the expression of SERCA2a, PLB contimued to drop; SERCA2a/PLB ratio dropped significantly. HQ group and DS group can significantly reduce the CaMK Ⅱ protein expression, recovered SERCA2a and PLB protein expression, and SERCA2a/PLB ratio. The therapeutic effect of the QD group was better than.single-agent group.4.48th hour, the F0and RT50of model group increased, the△F decreased (P<0.05). HQ/DS can reduce RT50and FO, and recovery the△F to normal levels. The Losartan group can reduce RT50and recovery the△F. We study the calcium transients of KN-93blocked CaMK Ⅱ at48h. Compared of the model group and the control group F0and△F were no significant difference, but RT50increased significantly(P<0.05). The result indicated that the systolic function of cardiomyocytes was improved and the diastolic function exist abnomal. Each dose group can be further reduced on RT50.ConclusionAng Ⅱ causes the myocardial cell dynamic changes of survival ratio during the process of hypertrophy to heart failure, and causing myocardial nuclear/cytoplasmic area and diameter increased significantly. The diastolic function changed at24th hour, and the systolic dysfunction at48th hour by using Ang Ⅱ. Ang Ⅱ cause CaMKⅡ, PLB and SERCA2a dynamic changes of protein expression levels. Those affect the intracellular calcium ion concentration, thus affecting the changer of normal calcium transient.Astragalus, Salvia regulated calcium ions through the intervention of calcium transports regulatory proteins CaMK Ⅱ, PLB, SERCA2a, which resulting in improved myocardial systolic and diastolic function of the cell. Astragali can improve abnormal calcium transient, and regulate protein expression in early cardiomyocyte hypertrophy. Note to Ang Ⅱ could induce the changes of transcription and expression of calcium handing protein resulting in sarcoplasmic reticulum calcium transport barriers. This progress can be inhibitited by Salvia and Astragali in failure of myocardial cell. They improve cardiacmyocyte excitation-contraction coupling and slow the progress of heart failure. The combination of two drugs used in the early was not superior to Astragli, but in the failure stage the combination therapy was better. The two drugs combination not only protect the heart’s overall function, but also has a protective effect in a single cardiac cell contraction, during the long course of treatment in heart failure. They also can antagonize the abnormal changes of calcium transient and calcium handing protein in heart failure. Losartan improve either systolic function or diastolic function, but the Chinese medicine is different from western medicine. The herbs improve not only the systolic function but also the diastolic function of myocardial cells, which reflects the integrity of regulation.The effect of Salvia and Astragali on regulating systolic dysfunction, partly by regulating the changes of CaMK Ⅱ δ pathway, but the improvement of diastolic dysfunction may be by another pathway. Further research is needed.更多还原