WO2022100720A1 - 一种制备增强CNPase活性的化合物或生物药物的方法用于治疗心脏疾病 - Google Patents

一种制备增强CNPase活性的化合物或生物药物的方法用于治疗心脏疾病 Download PDF

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WO2022100720A1
WO2022100720A1 PCT/CN2021/130496 CN2021130496W WO2022100720A1 WO 2022100720 A1 WO2022100720 A1 WO 2022100720A1 CN 2021130496 W CN2021130496 W CN 2021130496W WO 2022100720 A1 WO2022100720 A1 WO 2022100720A1
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cnpase
activity
myocardial
group
expression
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王东方
陈凯欣
卢志强
谭文
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珠海沅芷健康科技有限公司
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Definitions

  • 2',3'-cyclic nucleotide-3'-phosphodiesterase (2',3'-cyclic nucleotide 3'-phosphodiesterase, CNPase) was discovered in the early 1960s, with catalytic 2',3'-cAMP And 2', 3'-cGMP degradation function, with high expression in the central nervous system. The release of extracellular 2',3'-cAMP is associated with injury, and 2',3'-cAMP activates mitochondrial permeability transition pores (mPTPs) leading to apoptosis. At present, 2', 3'-cAMP and CNPase have been found to be related to mitochondrial membrane permeability. Jackson EK et al. reported that CNPase knockout can protect renal function in ischemia/reperfusion (J Am Soc Nephrol. 2016). So far, no one has reported the target and function of CNPase in cardiomyopathy.
  • Heart failure is a severe manifestation or late stage of various heart diseases, with high mortality and rehospitalization rates.
  • Heart failure is a complex clinical syndrome caused by abnormal changes in the structure and/or function of the heart due to various reasons, resulting in dysfunction of ventricular systolic and/or diastolic function, mainly manifested as dyspnea, fatigue, and fluid retention (pulmonary congestion, systemic congestion and peripheral edema).
  • Cardiac remodeling is a change in the size, shape, and function of the heart due to changes in molecular and gene expression upon cardiac injury or in response to hemodynamic stress.
  • Left ventricular hypertrophy is an adaptive response of the heart to pathological conditions such as pressure or volume overload, sarcomeric protein gene mutation, or decreased contractility caused by myocardial infarction.
  • Pathological conditions such as pressure or volume overload, sarcomeric protein gene mutation, or decreased contractility caused by myocardial infarction.
  • Sexual heart disease In the pathological process of these diseases, excessive pressure overload can lead to concentric myocardial hypertrophy, and concentric myocardial hypertrophy is considered to be a compensatory pathological change because it can increase left ventricular contractility, reduce ventricular wall pressure and Myocardial oxygen consumption.
  • left ventricular hypertrophy can also be a risk factor for severe heart failure and malignant arrhythmias. Therefore, effective inhibition of left ventricular hypertrophy without causing circulatory dysfunction is considered to have important clinical significance.
  • abnormal energy metabolism is particularly prominent: under the condition of left ventricular hypertrophy, the energy metabolism of the heart changes from mainly relying on aerobic oxidation of long-chain fatty acids to aerobic oxidation of glucose.
  • the change of energy metabolism substrates can reduce the oxygen consumption required to generate each mole of ATP, that is, it can reduce the generation of intracellular reactive oxygen species (ROS); but on the other hand, this change is unavoidable.
  • ROS reactive oxygen species
  • CNPase is used as a therapeutic target, and overexpression of CNPase in the heart can reverse the course of cardiac hypertrophy.
  • the applicant of this patent has determined the sub-localization of CNPase in mitochondria through immunofluorescence technology, and explored its function of affecting mitochondrial energy supply; through molecular cloning technology, a recombinant AAV packaging plasmid expressing CNPase protein has been constructed, using virus packaging and purification technology, Recombinant AAV2/9-CNPase virus was prepared, and a rat model of myocardial hypertrophy and compensatory cardiac function damage during chronic ischemia was used to determine the protective function and clinical therapeutic potential of overexpressing CNPase on cardiomyopathy. Meanwhile, the present invention discloses that kaurines, such as sodium isosteviol, can enhance the enzymatic activity of CNPase.
  • the purpose of the present invention is to provide a new tool for enhancing the activity or expression of CNPase in the heart, including the use of an adeno-associated virus biological drug and a compound isosteviol sodium that can enhance the activity of CNPase, for the protection of heart failure and myocardial remodeling drug.
  • patients in the preclinical heart failure stage do not have any symptoms or signs of heart failure, but develop structural heart disease, such as left ventricular hypertrophy, asymptomatic valvular heart disease, and the like.
  • kaurines such as isosteviol and its derivatives, can enhance the activity of CNPase, and can be used for the treatment of heart failure and myocardial remodeling.
  • the biotherapy method of the present invention includes recombinant vector construction, recombinant virus packaging, concentration and purification, and corresponding identification.
  • an AAV-CNPase vector was constructed using an AAV overexpression vector combined with molecular cloning technology, and its expression activity was identified by transient transfection and western blot technology.
  • the recombinant AAV2/9-CNPase virus is based on the steps of virus coating, virus particle collection, concentration and purification, and titer identification.
  • the recombinant AAV2/9-CNPase virus of the present invention is used for preventing/or treating cardiac hypertrophy and compensatory cardiac function damage-related diseases in the process of chronic ischemia.
  • AAV2/9-CNPase virus to achieve myocardial-specific CNPase upregulation, targeted prevention and/or treatment of myocardial hypertrophy and compensatory cardiac function damage-related diseases in the process of chronic ischemia in rats.
  • a rat model of chronic myocardial hypertrophy disease was constructed by abdominal aortic ligation. Using ultrasound guidance, each rat was injected with 1 ⁇ 10 12 virus. In the second week, some rats were treated, and the hearts were taken out. Fluorescence quantitative PCR and western blot technology confirmed that the site-specific injection of recombinant AAV2/9-CNPase virus could increase the expression of CNPase mRNA and protein.
  • ultrasonic detection was used, and the detection parameters included ejection fraction, fractional shortening, diastolic blood pressure, systolic blood pressure, LVSP, max pressure, Kinetic parameters such as Max dP/dt.
  • the rats were sacrificed, the hearts were collected, and fixed with electron microscope fixative. After electron microscope lenses were prepared, scanning electron microscope was used for detection. Through the observation of mitochondrial morphology, it was confirmed that CNPase injection can promote mitophagy and alter changes in cardiac energy homeostasis by promoting the clearance of damaged mitochondria.
  • rats were sacrificed, hearts were collected, and total protein was extracted. Western blot was used to detect the changes of TGF- ⁇ 1/2 signaling pathway. CNPase could inhibit the activation of this signaling pathway.
  • coronary artery ligation was used to model myocardial ischemia in rats. Using ultrasound guidance, each rat was injected with 1 ⁇ 10 12 virus. In the fourth week of injection of recombinant virus, ultrasound was used to detect parameters. Including ejection fraction, fractional shortening and other cardiac function indicators. Specifically, the use of CNPase enzyme in the protection medicine of myocardial ischemia injury-related diseases, wherein the myocardial ischemia injury-related diseases are heart failure, arrhythmia, ischemic cardiomyopathy, and cardiac rupture.
  • the present invention reveals for the first time the direct connection between CNPase and cardiac function, and discovers for the first time that CNPase plays a protective role in the compensatory cardiac function in the process of myocardial hypertrophy and chronic ischemia.
  • Up-regulation of CNPase can prevent/protect myocardial hypertrophy and chronic ischemia.
  • the dual effect of compensatory cardiac function in the blood process reveals a new field of CNPase in the treatment of myocardial diseases, and provides a new field for the prevention and treatment of myocardial hypertrophy, myocardial fibrosis and myocardial ischemia-related heart failure, arrhythmia, Ischemic cardiomyopathy, cardiac rupture and other diseases provide new solutions and have broad clinical application prospects.
  • the cases provide experimental methods and results used to support and validate the animal models used in the present invention. Appropriate control experiments and statistical analysis methods were used in the cases involved. The following cases are used to describe rather than limit the application of the present invention. The methods and techniques involved in these cases can be used to prepare chemical or biological drugs that enhance the activity of CNPase. The same method can be used for the evaluation of the therapeutic effect of other formulations of such compounds.
  • mice adult male Sprague Dawley rats, weighing 230g ⁇ 20g, 6-8 weeks old.
  • the rearing environment included constant temperature, humidity, and a strict dark-light cycle, with ad libitum feeding.
  • isosteviol (ent-17-norkaurane-16-oxo-18-oic acid, molecular formula, C 20 H 40 O 3 , molecular weight: 318.5) is obtained from stevioside by acid hydrolysis and crystallization purification.
  • the sodium salt of isosteviol can be obtained by adding NaOH or other sodium-containing bases; the purity of isosteviol sodium salt measured by high performance liquid chromatography is greater than 99%.
  • AAV-CNPase recombinant plasmids SD rats were killed by neck breaking, hearts were taken, and an appropriate weight of cardiac tissue was weighed, and total RNA was extracted by TRIZOL method; 5 ⁇ g RNA was used as a template, and RNA was reverse transcribed using QIAGEN's RNA
  • the kit was used to prepare cDNA; using cDNA as a template, Thermo Scientific TM Phusion TM high-fidelity DNA polymerase was used to amplify the CNPase coding sequence (CDS), and the primers were BamHI and HindIII with enzyme cleavage sites; the PCR product was agar
  • the CNPase CDS product with the sticky end of the restriction site was prepared by gel electrophoresis purification, BamHI and HindIII double digestion and purification, and the pAAV-MCS plasmid was digested and purified with BamHI and HindIII to prepare the cNPase CDS
  • HEK293 cell line is used as a tool for packaging recombinant virus.
  • the specific steps of virus packaging are as follows: MCS-CNPase plasmid, pHELPER plasmid, and pAAV-RC9 plasmid were transfected by liposome transfection; the culture supernatant and cells were collected separately, and the supernatant was precipitated and concentrated with PEG6000, and the virus particles in the cells were subjected to repeated Obtained by freeze-thaw method and concentrated with PEG6000; the concentrated virus was purified by ultracentrifugation and dialysis, subpackaged, and frozen at -80°C for future use; the virus titer was detected by fluorescence quantitative PCR.
  • a 1-0 surgical silk thread was inserted below the abdominal aorta segment between the left and right renal artery branches, and a blunted 7-gauge syringe needle was placed along the direction of the blood vessel.
  • the abdominal aorta and nylon thread were ligated together between the left and right renal artery branches.
  • the left kidney was observed to be white (indicating that the ligation was reliable), and then the needle of the syringe was quickly withdrawn to observe the congestion and reddening of the left kidney, which reduced the rat abdominal aorta cross-sectional area by about 50%. Suture layer by layer after surgery. Postoperative intraperitoneal injection of gentamicin for 3 days to prevent infection.
  • the surgical silk thread was passed through the abdominal aorta after laparotomy, and other operations were the same as in the formal operation group except that the abdominal aorta was not narrowed.
  • all animals were sacrificed and the hearts were removed for further analysis.
  • the rats were anesthetized and electrocardiogram needle electrodes were inserted subcutaneously in the limbs (20% urethane, 12 g/kg, intraperitoneal injection). After the electrocardiogram and body temperature (37°C) were stable, the right common carotid artery was isolated, and the end of the common carotid artery was ligated for arterial cannulation.
  • the pressure sensor is connected with the multi-channel physiological signal acquisition and processing system. Systolic blood pressure (sbp), diastolic blood pressure (dbp) and heart rate (bpm) were recorded 15 minutes after stabilization.
  • the cannula is then inserted into the left ventricle until the ventricular pressure waveform, left ventricular systolic pressure (LVSP) and left ventricular end-diastolic pressure (LVEDP) appear. All (-dp/dtmax) were recorded 15 minutes after stabilization. All variables were monitored by Power Lab software (Power Lab 8/30 AD Instruments, Australia).
  • Rat myocardial tissue was fixed with 4% neutral formalin, embedded in paraffin, cut into 3 mm sections, and then stained with hematoxylin-eosin (H&E) and Masson. Photographs were taken using a Zeiss confocal microscope. Cell morphology and size were detected by H&E staining, and fibrosis was detected by Masson staining. Computer-assisted image analysis (image processing software) was used to determine cell cross-sectional area and interstitial collagen content. The sample size is at least four or five different cardiac tissues.
  • This case mainly illustrates that isosteviol sodium enhances CNPase activity in angiotensin II-induced cardiac hypertrophic cells.
  • the enzyme activity of CNPase in hypertrophic myocardium samples was detected.
  • This case mainly illustrates the effect of overexpression of CNPase on improving TAAC-induced cardiac hypertrophy.
  • HW/BW Heart body weight ratio
  • This case mainly illustrates the role of AAV-CNPase in inhibiting the formation of myocardial fibrosis.

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Abstract

一种制备增强CNPase活性的化合物或生物药物的方法用于治疗心脏疾病。化学方法涉及使用贝壳杉烷类化合物上调CNPase的表达和活性。生物治疗药涉及使用重组腺相关病毒表达CNPase酶的制备及局部介入的疗法。在大鼠心肌肥大和心衰的模型中，上述方法可以有效改善心肌肥大、心肌重构、抑制心肌肥大和纤维化增加，增加心脏功能。

Description

一种制备增强CNPase活性的化合物或生物药物的方法用于治疗心脏疾病 背景技术
2’,3’-环核苷酸-3’-磷酸二酯酶(2',3'-cyclic nucleotide 3'-phosphodiesterase，CNPase)在1960年代早期被发现，具有催化2’，3’-cAMP和2’，3’-cGMP降解的功能，在中枢神经系统具有高量表达。细胞外2’，3’-cAMP的释放与损伤有关，而2’，3’-cAMP可激活线粒体通透性转换孔(mPTPs)导致细胞凋亡。目前发现2’，3’-cAMP及CNPase与线粒体膜通透性相关，Jackson EK等人报道CNPase敲除可保护缺血/再灌注的肾脏功能(J Am Soc Nephrol.2016)。至今，没有人报道CNPase在心肌病的靶点及功能。
心衰是各种心脏疾病的严重表现或晚期阶段，死亡率和再住院率居高不下。心衰是多种原因导致心脏结构和/或功能的异常改变，使心室收缩和/或舒张功能发生障碍，从而引起的一组复杂临床综合征，主要表现为呼吸困难、疲乏和液体潴留(肺淤血、体循环淤血及外周水肿)等。心脏重构是心脏损伤或在血液动力学的应激反应时，由于分子和基因表达的变化，导致心脏的大小、形状和功能发生变化。
左室心肌肥大是心脏对于压力或容量超负荷、肌节蛋白基因突变或心肌梗死导致收缩力下降等病理情况的适应性反应,其伴随着多种心脏疾病,如高血压、瓣膜病及缺血性心脏病。在这些疾病的病理过程中,压力负荷过重会导致向心性心肌肥大,而向心性心肌肥大被认为是一种具有代偿作用的病理改变因其可以增加左室收缩力、降低室壁压力和心肌耗氧量。然而,左室心肌肥大同样可以成为严重心力衰竭以及恶性心律失常的危险因素。因此,在不引起循环功能障碍的前提条件下有效的抑制左室心肌肥大被认为具有重要的临床意义。
在众多心肌肥大所带来的不利因素中,能量代谢异常显得尤为突出：在左室心肌肥大的条件下,心脏的能量代谢方式由主要依靠长链脂肪酸有氧氧化转变为葡萄糖有氧氧化。能量代谢底物的改变一方面可以减少生成每摩尔ATP所需的耗氧量,即能够减少细胞内活性氧簇(Reactive Oxygen Species,ROS)的生成；但另一方面,这一改变不可避免的存在着大量的不利因素,如慢性脂肪酸有氧氧化障碍导致心肌细胞内脂质累积、乳酸堆积、以及糖酵解增多所带来的总ATP水平下降。
本团队采用腹主动脉结扎构建的压力引起的慢性心肌肥大大鼠模型，发现CNPase蛋白表达量没有变化，而酶活下降，这提示CNPase蛋白酶活不足可能是心肌肥大发生的原因，本团队直接以CNPase作为治疗靶点，在心脏过表达CNPase则可以逆转心肌肥大的病程，证明过表达CNPase可以逆转心肌肥大及慢性缺血过程中的代偿性心功能损伤为治疗靶点。
本专利申请人通过免疫荧光技术确定了CNPase在线粒体中的亚定位，探索其影响线粒体能量供应的功能；通过分子克隆技术，构建了表达CNPase蛋白的重组AAV包装质粒，使用病毒包装以及纯化技术，制备了重组AAV2/9-CNPase病毒，并通过了大鼠心肌肥大以及慢性缺血过程中的代偿性心功能损伤模型，确定了过表达CNPase对心肌病的保护功能以及临床上治疗潜力。同时，本发明揭示贝壳杉烷类化合物，如异甜菊醇钠可以增强CNPase的酶活。
发明内容
本发明旨是为提供一种在心脏中增强CNPase活性或表达的新工具，包括使用可以增强CNPase活性的腺相关病毒的生物药物及化合物异甜菊醇钠，用于心衰和心肌重构的保护药物。其中前临床心衰阶段的患者没有任何心衰的症状或体征， 却以发展为结构性心脏病，例如左心室肥厚、无症状瓣膜性心脏病等。本发明揭示贝壳杉烷类化合物，如异甜菊醇及其衍生物，可以增强CNPase的活性，用于治疗心衰和心肌重构。
本发明的生物治疗的方法包括重组载体构建、重组病毒包装、浓缩和纯化以及相应的鉴定。具体地，采用AAV过表达载体，结合分子克隆技术，构建AAV-CNPase载体，其表达活性通过瞬时转染与western blot技术进行鉴定。进一步地，重组AAV2/9-CNPase病毒基于病毒包被、病毒颗粒收集以及浓缩纯化、滴度鉴定等步骤。本发明重组AAV2/9-CNPase病毒用于预防/或治疗心肌肥大及慢性缺血过程中的代偿性心功能损伤相关疾病。进一步地，通过AAV2/9-CNPase病毒实现心肌特异性CNPase上调，针对性的预防和/或治疗大鼠心肌肥大及慢、性缺血过程中的代偿性心功能损伤相关疾病的用途。
具体地，通过腹腔主动脉结扎术构建大鼠的慢性心肌肥大疾病模型，采用超声引导的方式，每只大鼠注射1×10 12病毒，在第二周处理部分大鼠，取心脏，分别采用荧光定量PCR以及western blot技术，证实重组AAV2/9-CNPase病毒的定点注射可以提高CNPase mRNA、蛋白的表达。进一步地，分别在注射病毒之后的第二周、第四周、第六周、第八周，使用超声检测，检测参数包括射血分数、缩短分数、舒张压、收缩压、LVSP、max pressure、Max dP/dt等动力学参数。进一步地，分别在注射病毒之后的第八周，处死大鼠，收集心脏，使用电镜固定液进行固定，经过电镜片子的制备之后，使用扫描电镜进行检测。通过对线粒体形态的观察，确认了CNPase注射可以促进线粒体自噬，通过促进受损线粒体的清除，改变心脏能量稳态的变化。分别在注射病毒之后的第八周，处死大鼠，收集心脏，提取总蛋白，使用western blot检测TGF-β 1/2信号通路的变化，CNPase可以抑制该信号通路的激活。
具体地，采用冠状动脉结扎法对大鼠进行心肌缺血造模，采用超声引导的方式，每只大鼠注射1×10 12病毒，在注射重组病毒的第四周，使用超声检测，检测参数包括射血分数、缩短分数等心功能指标。具体地，CNPase酶在心肌缺血损伤相关疾病的保护药物中的用途，其中所述的心肌缺血损伤相关疾病为心力衰竭，心律失常，缺血性心肌病，心脏破裂。本发明第一次揭示了CNPase与心功能的直接联系，首次发现了CNPase在心肌肥大及慢性缺血过程中的代偿性心功能发挥了保护作用，上调CNPase可以预防/保护心肌肥大及慢性缺血过程中的代偿性心功能的双重功效，揭示了CNPase在心肌疾病治疗中的全新领域，并为预防和治疗心肌肥大、心肌成纤维化及心肌缺血损伤相关的心力衰竭、心律失常、缺血性心肌病、心脏破裂等疾病提供了新的方案，具有广阔的临床应用前景。
以上是本发明整体的介绍。为了更好地说明本发明的方法和技术，以下将给出实施案例，以便可以由本领域技术人员执行。
在以下实施例中详细提供了本发明的方法和实施方式。
具体实施方式
为了进一步说明用于实现本发明目的的技术，下文描述了关于确定和鉴定本发明中化合物的药物和治疗用途的详细方法，技术，流程和特点。案例提供了用于支持及验证本发明所使用的动物模型的实验方法和结果。涉及的案例均使用了适当的对照组实验及统计分析方法。以下的案例均用于描述而非限制本发明的应用。这些案例所涉及的方法及技术可用于制备增强CNPase活性的化学或生物药物的方法。其他此类化合物制剂的治疗效果评价可使用相同的方法。
本发明中列举的案例用于支持本发明的实验方法和结果，并验证本发明中使用的动物模型。本发明的所有实验均采用了适当的对照和统计检验。提供以下实施例来说明而非限制本发明。这些例子说明了一种制备增强CNPase活性的化学或生物药物的方法用于治疗心衰和心肌重构。
实验材料
实验动物：成年雄性Sprague Dawley大鼠，体重230g±20g，6-8周龄。饲养环境包括恒定的温度、湿度以及严格的黑暗光照周期，自由采食。
化学试剂：异甜菊醇(ent-17-norkaurane-16-oxo-18-oic acid，分子式，C 20H 40O 3，分子量：318.5)是由甜菊糖通过酸性水解、结晶纯化而得到。异甜菊醇的钠盐可以通过加入NaOH或其他含钠碱获得；用高效液相色谱法测得异甜菊醇钠盐的纯度大于99％。
AAV-CNPase重组质粒的构建：采用断颈的方法处死SD大鼠，取心脏，称取适当重量的心脏组织，采用TRIZOL的方法提取总RNA；取5μg RNA作为模板，采用QIAGEN公司的RNA逆转录试剂盒制备cDNA；以cDNA作为模板，采用Thermo Scientific TM Phusion TM高保真DNA聚合酶扩增CNPase编码序列(coding sequence，CDS)，其中引物为带有酶切位点BamHI与HindIII；PCR产物采用琼脂糖电泳割胶纯化、BamHI与HindIII双酶切、纯化，制备出带有酶切位点黏性末端的CNPase CDS产物，pAAV-MCS质粒采用采用BamHI与HindIII双酶切、纯化，制备带有黏性末端的线性化质粒；采用T4连接酶，连接酶切质粒与CNPase CDS产物，转入大肠杆菌感受态DH5α细胞中，在LB培养基中37℃孵育一个小时；离心，取沉淀，重悬之后，均匀涂抹到带有氨苄抗生素的琼脂糖固体平板上，37℃培养过夜；提取单克隆，采用菌落PCR的方法筛选阳性克隆，并通过测序的方法确认重组质粒的构建成功。
重组AAV2/9-CNPase病毒的包装、纯化与浓缩以及滴度的鉴定：HEK293细胞系作为包装重组病毒的工具，病毒包装的具体步骤如下：采用质粒大提试剂盒，提取无内毒素的pAAV-MCS-CNPase质粒、pHELPER质粒、pAAV-RC9质粒，采用脂质体转染的方法转染；分别收取培养基上清和细胞，对上清采用PEG6000进行沉淀和浓缩，对细胞中的病毒颗粒采用反复冻融的方法获取，并使用PEG6000进行浓缩；浓缩后的病毒采用超高速离心以及透析的方法进行纯化，分装，冻存在-80℃备用；采用荧光定量PCR的方法检测病毒滴度。
实验方法
腹主动脉狭窄(TAAC)后压力诱导的大鼠肥厚心肌的实验方案
用10％的水合氯醛0.3ml/100g腹腔注射麻醉。麻醉后，将大鼠仰卧，四肢固定。行腹部备皮。沿剑突下腹正中，左肾上方处为手术部位，行2.0～2.5cm纵行切口，逐层打开腹腔。暴露腹后壁及左侧肾脏，找到左、右肾动脉分支，在左肾动脉以上钝性游离腹主动脉。在左、右肾动脉分支之间腹主动脉段下面穿入1-0号手术丝线，沿血管走行方向放置磨钝的7号注射器针头。在左、右肾动脉分支之间将腹主动脉和尼龙线一同结扎。观察左肾发白(表明结扎可靠)，然后迅速抽出注射器针头，观察左肾充血变红，使大鼠腹主动脉横截面积减少50％左右。手术后逐层缝合。术后腹腔注射庆大霉素3日，预防感染。假手术组开腹后将手术丝线穿过腹主动脉，除不缩窄腹主动脉外，其它操作均与正式手术组完全相同。观察期结束，测定体内血流动力学后，将所有动物处死，并把心脏取出做进一步分析。
心脏血流动力学参数测量
造模后的第2周，然后给AAV2/9-CNPase的第6周后，将大鼠麻醉后将心电图针电极插入四肢皮下(20％氨基甲酸乙酯，12g/kg，腹腔注射)。心电图和体 温(37℃)稳定后，分离右侧颈总动脉，结扎颈总动脉末端进行动脉插管。压力传感器与多通道生理信号采集处理系统相连。稳定后15分钟记录收缩压(sbp)、舒张压(dbp)和心率(bpm)。然后，将套管插入左心室，直到出现心室压力波形、左心室收缩压(LVSP)和左心室舒张末期压(LVEDP)。稳定后15分钟记录所有(-dp/dtmax)。所有变量均由Power Lab软件(Power Lab 8/30 AD Instruments，澳大利亚)监测。
组织学分析
利用4％的中性福尔马林固定大鼠心肌组织，进行石蜡包埋，切成3毫米的切片，然后用苏木精-伊红(H&E)及马松染色。采用蔡司共聚焦显微镜拍照。用H&E染色结果检测细胞形态大小，使用马松染色检测纤维化。使用计算机辅助图像分析(图像处理软件)来确定细胞横截面面积和间隙胶原含量。样本量至少为四个或五个不同的心脏组织。
统计分析
依次通过方差分析(单因素方差分析)，Fisher检验比较多组间的差异。所有检验的P值均为双尾，以P<0.05被认为是具有统计学差异。
实施例1
本案例主要说明异甜菊醇钠增强采用在血管紧张素II诱导的心肌肥厚细胞中的CNPase活性。采用小牛肠碱性磷酸酶活性的检测方法，在给予异甜菊醇后,检测肥厚心肌样本中CNPase的酶活。
表1.异甜菊醇钠可以增强采用血管紧张素II诱导的心肌肥厚中的CNPase活性(n＝3)
Figure PCTCN2021130496-appb-000001
实施例2
本案例主要说明过表达CNPase改善TAAC诱导的心肌肥厚的作用。
成年的SD大鼠经过TAAC诱导2周后，分别使AAV-CNPase治疗。心脏体重比(HW/BW)是反映心肌肥大的指数。在8周TAAC模型组中，我们发现TAAC组大鼠的心脏体重比(HW/BW)显著上升；而过表达CNPase可以有效减低心脏体重比。
表2.过表达CNPase对TAAC模型大鼠心重与体重的影响(n＝8-14)
Figure PCTCN2021130496-appb-000002
实施例3
本案例主要说明了AAV-CNPase在抑制心肌纤维化形成中的作用。
为了确定过表达CNPase是否可以减弱TAAC诱导的心肌纤维化，我们使用马松染色检测左心室心肌间质胶原的改变。我们发现TAAC组大鼠心肌纤维化增强及胶原沉积增多；而过表达CNPase可以有效减少心肌纤维化及胶原的沉积。
表3.过表达CNPase抑制TAAC模型大鼠心肌纤维化形成中(n＝8-14)
Figure PCTCN2021130496-appb-000003
实施例4
为了评估CNPase是否也可以改善TAAC大鼠的心功能，我们发现在TAAC引起的心肌肥厚大鼠模型中，过表达CNPase大鼠的左心室射血分数(EF)、分数缩短(FS)和心输出量(CO)均得到了改善。
表4.心脏特异性过表达CNPase有效改善TAAC大鼠的心功能等指标(n＝8-14)
Figure PCTCN2021130496-appb-000004

Claims (10)

  1. 本发明涉及一种使用贝壳杉烷类化合物和生物学方法以提高心肌CNPase的活性或表达的制备方法，用于治疗心脏疾病。
  2. 权力要求1所述的心脏疾病，包括心肌缺血、心肌肥大、心衰及心肌重构。
  3. 根据权利要求1所述的化合物是代表结构式(I)的化合物，其中，R1:氢、羟基或烷氧基；R2：羧基、羧酸盐、酰卤、醛基、羟甲基，和可以生成羧基的酯基、丙烯酰胺基、酰基或醚键基团；R3、R4、R5、R6、R8：氧、羟基、羟甲基和能水解生成羟甲基的酯基或烷氧甲基；R7：甲基、羟基，和能水解生成羟甲基的酯基或烷氧甲基；R9：亚甲基或氧。
    Figure PCTCN2021130496-appb-100001
  4. 根据权利要求3所述的化合物，其特征在于，包括异甜菊醇和甜菊醇及其衍生物。
  5. 根据权利要求1所述的生物学方法，其特征在于通过重组基因方法，使用慢病毒，腺病毒及腺相关病毒方式增强CNPase活性和表达。
  6. 根据权利要求1所述的生物学方法，其特征在于使用非编码RNA方式增加CNPase的活性和表达。
  7. 根据权利要求1所述的提高心肌CNPase的活性或表达，其特征涉及改变相关底物包括2’,3’-cAMP、2’,3’-cGMP、2’,3’-cCMP和2’,3’-cIMP绝对量或浓度的降低。
  8. 根据权利要求1所述的提高活性改变，其特征在于组织中其底物2’,3’-cAMP、2’,3’-cGMP、2’,3’-cCMP和2’,3’-cIMP与相关3’,5’cGMP or 3’,5’cAMP的比值降低。
  9. 根据权利要求6所述的非编码RNA，其特征在于使用rRNA，tRNA，snRNA，snoRNA和microRNA等多种已知功能的RNA和未知功能的RNA增加CNPase的活性的方式。
  10. 根据权利要求1所述的提高心肌CNPase的活性或表达的药物介入方法，其特征在于，以药用赋形剂/载体通过静脉、吸入或局部注射导入心肌的形式，制备方式为针剂、注射剂、水剂、干粉、或者与其他释放剂结合，定点注射到心肌中上调CNPase酶或蛋白表达的功能。
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