【作者】 张旭；

【导师】 于淼； 孙桂波；

【作者基本信息】 哈尔滨商业大学 ， 中药学， 2021， 硕士

【摘要】 心肌缺血再灌注损伤(myocardial ischemia-reperfusion injury,MIRI)是指缺血心脏在一定时间内恢复血液灌注后出现组织损伤加重的病理过程,其中涉及到多种会导致心脏机械活动发生异常的机制,例如氧化应激、细胞自噬、炎症反应、线粒体功能障碍以及钙超载等,目前依然缺乏有效药物用于临床治疗。具有与人类心脏相同遗传背景及生物学特性的人源心肌细胞(hiPSC-CMs)是人体细胞重编程后定向分化而成的心肌细胞,全称人诱导多能干细胞来源的心肌细胞,是研究药物心肌活性的优秀生物细胞模型。通过具有实时、持续、无标记、无损伤监测细胞并记录分析细胞生理活动特点的实时无标记细胞分析系统(real time xCELLigence analysis system,RTCA)测量心肌细胞的收缩-舒张和场电位信号可以得到心肌细胞的生理活动变化,反映出心脏机械活动情况。冠心丹参方是以丹参和三七分别作为君药和臣药配伍而成的治疗冠心病的有效复方,研究表明其具有心肌保护作用,可以治疗MIRI引起的左心室重构。且冠心病症状之一即是冠状动脉的狭窄从而引起的心肌缺血,提示冠心丹参方在治疗MIRI方面可能存在着一定的疗效。此外,课题组先前研究证实丹参中丹酚酸B和三七中人参皂苷Re配伍用药可以通过抗氧化和抗炎机制有效地阻止氧化低密度脂蛋白诱导的内皮细胞凋亡,且60μg/mL的丹酚酸B和12μg/mL的人参皂苷Re具有最佳的协同保护作用。因此本课题对冠心丹参方中丹参的有效成分丹酚酸B和三七的有效成分人参皂苷Re进行了二者单独用药和配伍用药改善MIRI作用的研究,运用优化中药复方研究策略,旨在为研究开发冠心丹参方用于临床治疗MIRI的作用提供理论依据。选用hiPSC-CMs作为细胞模型开展体外实验,通过缺氧6小时复氧24小时建立hiPSC-CMs心肌损伤模型,选取丹酚酸B(7.5、15、30、60、120μg/mL)5个剂量组和人参皂苷Re(7.5、15、30、60、120μg/mL)5个剂量组分别研究二者对hiPSC-CMs及hiPSC-CMs心肌损伤模型阻抗与场电位的影响。综合此部分研究结果和课题组先前研究结果选择丹酚酸B(15、30、60μg/mL)和人参皂苷Re(30、60、120μg/mL)各3剂量进行不同剂量的配伍,共9组,探究丹酚酸B与人参皂苷Re配伍用药对hiPSC-CMs及hiPSC-CMs心肌损伤模型阻抗与场电位的影响。后选择SD大鼠开展动物实验,分为假手术组;心肌缺血再灌注模型组;丹酚酸B低(15mg/kg)、中(30mg/kg)、高(60mg/kg)剂量组;人参皂苷 Re 低(15mg/kg)、中(30mg/kg)、高(60mg/kg)剂量组;丹酚酸B(60mg/kg)与人参皂苷Re(60mg/kg)配伍组以及阳性对照普萘洛尔(2.5mg/kg)组,各给药组大鼠灌胃给药4天后,对模型组及各给药组大鼠开展冠状动脉左前降支结扎手术(缺血30分钟,再灌注24小时)建立大鼠MIRI模型。采用TTC染色法观察大鼠心脏缺血区域面积、HE染色法观察大鼠心肌结构病理形态、全自动生化分析仪测定血清中心肌酶活性探究丹酚酸B、人参皂苷Re及二者配伍用药保护MIRI的作用。使用试剂盒检测丹酚酸B及丹酚酸B配伍人参皂苷Re保护MIRI过程中可能存在的抗氧化机制。向hiPSC-CMs中添加L型钙通道激动剂bay-k-8644 20nM建立心肌细胞内钙超载模型研究人参皂苷Re保护hiPSC-CMs减轻钙超载的作用,并采用Langendorff离体心脏灌流技术研究人参皂苷Re对缺氧再灌注大鼠原代心肌细胞收缩功能与钙瞬变的影响,探讨人参皂苷Re减轻MIRI保护心脏作用中可能存在的调节心肌细胞钙通道机制。为验证实验研究结果利用PCR技术检测基因中钙通道相关蛋白SERCA、RyR2、α1C 的表达。结果显示,丹酚酸B、人参皂苷Re及二者配伍用药均有增强hiPSC-CMs收缩力,减轻心肌损伤,改善场电位功能的作用。丹酚酸B、人参皂苷Re及二者配伍用药可以有效减少大鼠MIRI模型的心肌缺血面积,改善心肌结构损伤,降低心肌酶活性,保护心脏。丹酚酸B及丹酚酸B配伍人参皂苷Re能够降低MIRI大鼠血清中丙二醛(MDA)含量,提升超氧化物歧化酶(SOD)活力和还原型谷胱甘肽(GSH)活性,在改善心肌损伤的过程中发挥着抗氧化作用。人参皂苷Re具有改善缺氧再灌注大鼠原代心肌细胞受损的细胞收缩与钙瞬变功能,通过增加SERCA、降低RyR2和α1C蛋白的表达,发挥着拮抗心肌细胞内钙超载,调节心肌细胞钙通道从而减轻MIRI的作用。综上所述,丹酚酸B减轻MIRI的机制可能是降低MDA水平、提高SOD活力、GSH活性,增强了心肌抗氧化能力从而保护心脏。人参皂苷Re降低MIRI保护心脏的机制可能与调控心肌细胞钙通道,增加SERCA蛋白表达、减少RyR2和α1C蛋白表达,拮抗细胞内钙超载,维持细胞钙稳态有关。丹酚酸B与人参皂苷Re配伍用药保护心脏的机制可能是通过降低MDA水平、提高SOD活力、提升GSH活性减少心肌氧化应激的抗氧化机制减轻MIRI保护心脏。更多还原

【Abstract】 Myocardial ischemia-reperfusion injury(MIRI)refers to the pathological process of tissue damage aggravation after the ischemic heart recovers blood perfusion within a certain period of time.It involves a variety of mechanisms that will lead to abnormal mechanical activity of the heart,such as oxidative stress,autophagy,inflammatory response,mitochondrial dysfunction and calcium overload.Currently,there is still a lack of effective drugs for clinical treatment.Human cardiomyocytes(hiPSC-CMs)with the same genetic background and biological characteristics as those of human heart are cardiomyocytes directionally differentiated after reprogramming from human cells,which refer to cardiomyocytes derived from human induced pluripotent stem cells in full.They are excellent biological cell models for studying the myocardial activity of drugs.The real time xCELLigence analysis system(RTCA)with realtime,continuous,unlabeled and injury-free monitoring cells and recording and analyzing the characteristics of cell physiological activity is used to measure the contraction-relaxation and field potential signals of myocardial cells,so as to obtain the physiological activity changes of myocardial cells and reflect the mechanical activity of the heart.Guanxin Danshen Formulation is an effective compound for the treatment of coronary heart disease with Salviae miltiorrhizae and Notoginseng as the sovereign and ministerial drugs,respectively.Studies have shown that it has a myocardial protection effect,and can treat left ventricular remodeling caused by MIRI.Besides,one of the symptoms of coronary heart disease is myocardial ischemia caused by coronary artery stenosis,suggesting that Guanxin Danshen Formulation may have certain efficacy in the treatment of MIRI.In addition,our research group has confirmed in the previous studies that the combination of salvianolic acid B in Salviae miltiorrhizae and ginsenoside Re in Notoginseng can effectively prevent the apoptosis of endothelial cells induced by oxidized low density lipoprotein through the anti-oxidative and anti-inflammatory mechanisms,and that salvianolic acid B at 60μg/mL and ginsenoside Re at 120μg/mL have the optimal synergistic protection.Therefore,in this study,salvianolic acid B,an active ingredient of Salviae miltiorrhizae in Guanxin Danshen Formulation,and ginsenoside Re,an active ingredient of Notoginseng were used alone or in combination to improve the MIRI effect,using the research strategy of optimizing Chinese herbal compound to provide a theoretical basis for the research and development of the role of Guanxin Danshen Formulation in the clinical treatment of MIRI.HiPSC-CMs was used as the model for in vitro experiments.The myocardial injury model of hiPSC-CMs was established by hypoxia for 6 hours and reoxygenation for 24 hours.Five dosage groups of salvianolic acid B(7.5,15,30,60 and 120μg/mL)and five dosage groups of ginsenoside Re(7.5,15,30,60 and 120μ/mL)were selected to study the effects of the two on the impedance and field potential of hiPSC-CMs and its myocardial injury model.Based on the results of this part of the study and the previous research results of the research group,salvianolic acid B(15,30,and 60μg/mL)and ginsenoside Re(30,60,and 120μg/mL)were combined in three different doses into nine groups to explore the effects of the combination of salvianolic acid B and ginsenoside Re on the impedance and field potential of hiPSC-CMs and its myocardial injury model.Then SD rats were selected for animal experiments and divided into sham operation group.Myocardial ischemia reperfusion model group;Salvianolic acid B low dose group(15mg/kg),medium dose group(30mg/kg),and high dose group(60mg/kg);Ginsenoside Re low dose group(15mg/kg),medium dose group(30mg/kg),and high dose group(60mg/kg);The MIRI models of rats in the salvianolic acid B(60mg/kg)plus ginsenoside Re(60mg/kg)group and the positive control propranolol(2.5mg/kg)group were established by ligation of the left anterior descending coronary artery(ischemia for 30 minutes and reperfusion for 24 hours)in the model group and the rats in the administration groups after four days of intragastric administration.The TTC staining method was used to observe the ischemic area of the rat heart,and HE staining method was used to observe the pathological morphology of the myocardial structure.The activity of serum myocardial enzymes was determined by automatic biochemical analyzer.The effects of salvianolic acid B,ginsenoside Re and their combination on the protection of MIRI were explored.The kit was used to detect the possible antioxidant mechanism during the protection of MIRI by salvianolic acid B and salvianolic acid B combined with ginsenoside Re.The L-type channel agonist bay-k-864420nM was added into hiPSC-CMs to establish an intracellular calcium overload model,to study the effect of ginsenoside Re on protecting hiPSC-CMs against calcium overload,and the Langendorff isolated heart perfusion technique was used to study the effects of ginsenoside Re on the contraction function of primary myocardial cells and calcium transient in rats with hypoxic-reperfusion injury,and to explore the possible mechanism of ginsenoside Re in regulating calcium channels in myocardial cells in order to alleviate the protective effect of MIRI on the heart.In order to verify the experimental results,the expressions of calcium channel related proteins(SERCA,RyR2,and α1C)in the gene were detected by PCR.The results showed that salvianolic acid B,ginsenoside Re and their combination could enhance the contractility of hiPSC-CMs,alleviate myocardial damage and improve field potential function.Salvianolic acid B,ginsenoside Re and their combination could effectively reduce the myocardial ischemic area of the rat MIRI model,improve myocardial structural damage,reduce the activity of myocardial enzymes,and protect the heart.Salvianolic acid B and salvianolic acid B together with ginsenoside Re could reduce the malondialdehyde(MDA)content in serum of MIRI rats,and enhance the superoxide dismutase(SOD)activity and reduced glutathione(GSH)activity,which played an antioxidant role in the process of improving myocardial injury.Ginsenoside Re has the function of improving the damaged cell contraction and calcium transient of primary myocardial cells in rats with hypoxia-reperfusion injury.By increasing SERCA and reducing the expression of RyR2 and α1C proteins,it plays a role in antagonizing calcium overload in myocardial cells and regulating calcium channel in myocardial cells to reduce MIRI.In summary,the mechanism of salvianolic acid B reducing MIRI may be that it reduces MDA level,improves SOD activity and GSH activity,and enhances the myocardial antioxidant capacity to protect the heart.The mechanism of ginsenoside Re in reducing the protection of the heart by MIRI may be related to its efficacy in regulating the calcium channel in myocardial cells,increasing the expression of SERCA protein,reducing the expression of RyR2 and α1C proteins,antagonizing intracellular calcium overload,and maintaining cellular calcium homeostasis.The mechanism of cardioprotection by the combination of salvianolic acid B and ginsenoside Re may be that MIRI protects the heart by reducing the antioxidant mechanism of myocardial oxidative stress,such as MDA level,improving SOD activity and GSH activity.更多还原