黄芪水提取物拮抗高血压及其所致心肌纤维化的作用研究

发布时间：2018-04-21 05:01

本文选题：黄芪水提取物 + 心肌纤维化　；参考：《辽宁中医药大学》2016年博士论文

【摘要】：目的:心肌纤维化是一种在临床常见的慢性心血管疾病,其与动脉粥样硬化、心肌梗死、心律失常、高血压等众多疾病和病理过程有着密切的关系。高血压所致的心肌纤维化,是一种比较常见的病理表现,现阶段研究已发展到分子水平。高血压诱发心肌纤维化的原因可能与炎症、缺氧、血流动力学变化等息息相关。心肌纤维化在中医理论中有着对应的描述。心肌纤维化贯穿于多种疾病的病理过程中,比如高血压、冠心病、糖尿病、心肌炎等多种病症。上述这些病症,在中医理论中可以归为“胸痹”、“心悸”、“眩晕”、“痰饮”、“水肿”等范畴。心肌纤维化的中医病机,可以概括为本虚标实。因多属久病劳损,病入络脉所致,阴阳亏虚所致的本虚,表现为痰浊瘀阻、水湿横生、痞块热毒等标实现象。黄芪则是我国传统中医学中常用的一种药材,经过药理学研究,已经证实含有多种有效成分,如黄芪皂苷、黄芪多糖、黄酮类和氨基酸类等。黄芪具有增强机体免疫功能、保护肝脏功能、利尿、抗衰老氧化、抗应激作用,广谱抗菌作用,并且能够降低血压。传统医学的验方中,使用黄芪配伍其他药物,治疗相关心血管疾病的比比皆是,但其中起效的具体成分仍不清楚。黄芪水提取物是通过水提取法从黄芪的根部中提取的主要活性成分之一。本实验室对于黄芪水提取物的以往研究表明,黄芪水提取物具有抗炎活性,可降低巨噬细胞迁移和血管内皮细胞的粘附,通过核转录因子(NF)-κB信号通路减轻局部炎症,抗动脉粥样硬化。但是黄芪水提取物对于高血压相关作用,对心肌纤维化的相关影响及其机制并未见诸于报道。因此,本研究旨在通过文献研究对高血压及心肌纤维化的证型、使用药物等作出分析,同时对本实验室近期所研究的药物黄芪水提取物是否对于高血压及其所致的心肌纤维化具有拮抗作用加以研究。材料与方法:1.文献研究收集高血压相关的研究共计3663篇,经过筛选后入选文献共527篇,通过对上述文献进行归纳,整理出高血压相关的证型与用药的初步总结。然后再次收集心肌纤维化相关的文献490篇,通过文献研究心肌纤维化的临床治疗用药习惯来探讨高血压治疗与心肌纤维化治疗的共通之处,从而在临床治疗上能够有效的对这种疾病进行治疗。2.相关实验研究实验一中的文献研究显示,高血压比较常见的证型表现为肝阳上亢、痰瘀互结、肝肾阴虚等。心肌纤维化比较常见的证型包括气虚血瘀、阳虚水泛、阴虚热盛等证型。中药黄芪的具有的作用非常多,包括益气固表,利尿托毒,排脓敛疮等,对于气虚、阴阳两虚等症状表现的治疗作用全面,在临床治疗心肌纤维化的应用中应该是较为常见的一种药材。而现阶段,随着药理学与临床医学的发展,中药提取物的使用逐渐成为医学研究的后续热潮,黄芪的提取物研究也在逐步展开,曾经有相关报道提示,黄芪甲苷通过对内皮的保护等,对于拮抗高血压具有一定的作用。同时,黄芪甲苷对于心肌纤维化也有一定的抑制作用。而黄芪水提取物是另一种较为常见的黄芪提取物,本实验室既往研究便是围绕黄芪水提取物对内皮因子作用进行研究。本次的后续实验试图研究,黄芪水提取物这种中药提取物是否也一样对于高血压性心肌纤维化具有相关作用。2.1.试剂黄芪水提取物由中国科学院大连化学物理研究所提供,在蒸馏水中溶解并稀释(3克/公斤/天)。辛伐他汀(simvastatin)购自sigma-aldrich(saintlouis,mo,usa)。血管紧张素Ⅱ购自美仑生物制药(大连)。皮下埋入植入式胶囊渗透压泵(usa/alzet生产)。2.2.小鼠雄性昆明小鼠(n=24),年龄8周,18~20克,来源于swiss小鼠的背景,购自沈阳军区总医院医学动物科。所有的小鼠全部分笼分组喂养,以常规平价鼠粮进行适应性,常规性的喂养一周后,将小鼠按照盲法随机分为空白对照组、血管紧张素Ⅱ模型组、血管紧张素Ⅱ加辛伐他汀组、血管紧张素Ⅱ加黄芪水提取物组(每组n=6)。除空白对照组外,其他三组全部麻醉后皮下埋入植入式胶囊渗透压泵,内容物为血管紧张素Ⅱ(1.3毫克/公斤/天),血管紧张素Ⅱ加辛伐他汀组小鼠每日服用0.4%辛伐他汀溶液灌胃(30毫克/公斤/天),血管紧张素Ⅱ加黄芪水提取物组小鼠给予口服剂量黄芪水提取物溶液(3克/公斤/天)。在皮下埋泵28日时收集组织标本,进一步准备进行数据分析。按照相关要求,所有涉及动物实验程序的相关实验,都已经获得伦理委员会的批准。2.3he染色实验收集到达标点时间的小鼠心脏标本,清洗干净后投入预先配制准备的固定液(一般为4%多聚甲醛)中,固定脱水后浸蜡、包块、切片(心脏横切,切片厚度大约5-8微米)后,将切片放入45℃恒温箱进行烘干步骤,通过浸泡在酒精和二甲苯中进行脱蜡,按照步骤要求将切片投入regaud(或weigert)苏木素溶液中染色5-10分钟,然后投入1%盐酸乙醇与氨水中分化数秒,使用流水冲洗大约20分钟以上等待细胞核反蓝,下一步骤,使用蒸馏水反复冲洗后,将切片投入0.5%的伊红溶液中染色约1-3分钟,分别投入80%-100%酒精中按顺序脱水后,投入二甲苯中依次序换缸,采用中性树脂进行封片固定,切片标本制备后即可采用显微镜下观察。2.4masson染色实验将小鼠的心脏标本切片准备后,投入已经配置准备的固定液(4%多聚甲醛)中,固定脱水后浸蜡、包块、切片(心脏横切,一般厚度5-8微米)后,放入45℃恒温箱内烘干,石蜡切片使用酒精进行脱蜡、脱水,然后使用自来水和蒸馏水分别冲洗。用regaud苏木素溶液或weigert苏木素溶液5-10分钟,充分进行水洗后,观察效果如果认为切片过染,可以使用盐酸酒精进行分化,分化后再次使用蒸馏水冲洗。使用苏木素染核结束后,使用masson丽春红酸性复红液浸染,大概时间为5-10分钟,然后切片以2%冰醋酸水溶液侵染片刻,再使用1%的磷钼酸水溶液分化进行约3-5分钟。不必经过流水重新,直接用苯胺蓝溶液进行染色5分钟,染色结束后再用0.2%冰醋酸水溶液浸洗片刻,使用80%、90%、95%酒精、无水酒精进行脱水后,再用二甲苯进行透明、中性树脂封固,就可以对切片进行镜下观察。2.5免疫组化技术检验将手术后采集的小鼠心脏标本清洗后,投入预先配好的固定液(4%多聚甲醛)中,固定脱水后浸蜡、包块、切片(心脏横切,一般厚度5-8微米)后,放入45℃恒温箱内烘干,石蜡切片在酒精、二甲苯中进行脱蜡脱水,然后将切片在清水中反复冲洗一段时间,置入3%过氧化氢溶液中浸泡10分钟,(这一步骤可以除去内源性的过氧化氢酶)然后倒掉过氧化氢溶液,将切片放入清水中反复清洗后,取出滴加抗原修复液(本实验室使用为柠檬酸缓冲液),密闭放入沸水中煮40分钟后捞出冷却至室温。pbs溶液冲洗后,动物血清封闭,去除动物血清添加一抗,一抗滴加完毕后,将切片盒于4℃冰箱内过夜,第二日取出在室温下复温30分钟,使用pbs溶液冲洗后在切片上添加二抗,滴加适量的辣根酶或碱性磷酸酶标记的链霉卵白素工作液后,将切片用pbs溶液反复冲洗,冲洗后进行dab染色观察,待dab显色达到理想程度后,清水冲洗切片以终止显色反应,然后使用苏木素复染染色细胞核,梯度酒精进行脱水,二甲苯溶液透明后采用中性树胶对切片封片固定,标本即可观察使用。2.6免疫印迹分析(westernblot)提取各组小鼠心脏组织,测定不同组别间的心脏组织胶原含量。本实验中采用westernblot检测小鼠心脏组织的collagen-3。使用材料为100微升裂解液(50毫摩尔/升的tris,10毫摩尔/升的氯化镁,0.5摩尔/升氯化钠,1%tritonx-100)用来提取心肌总蛋白。心脏组织保持在冰上裂解及收取。在离心机使用14,000转速度离心10分钟后,将分离出来的上清收集。按照相关试剂盒上的实验方法提取蛋白,离心后上清保存。用bca法测定上清液中的蛋白质水平。等分30微克蛋白质样品进行聚丙烯酰胺凝胶电泳,将其转移到聚偏二氟乙烯膜。转膜结束后,采用5%脱脂奶粉将聚偏二氟乙烯膜封闭在tris中缓冲生理盐水,然后在膜上添加需要的一抗,置于在4°c冰箱中,按照孵育条件过夜,次日取出后采用pbs洗涤3次,冲洗后使用1:2,000稀释的辣根过氧化物酶(hrp)标记的羊抗兔,羊抗鼠,兔抗山羊二抗在室温下培养2小时。洗涤三次后,添加发光剂检测活性,暗室曝光。2.7统计分析实验数据采用平均值±sd表示。所有实验中的数据均采用spss13.0统软件统计结果。两组之间的差异统计采用unpairedstudent’st检验。三个相关实验组之间的差异采用方差分析(anova)。p0.05(双尾)具有统计学意义。结论:1.高血压病的证型分布以肝阳上亢型为最多、肝肾阴虚、瘀血阻络、肝火亢盛、痰湿中阻、痰瘀互结、气虚血瘀、阴阳两虚、肾气亏虚等都是较为常见的证型,此外还有多种证型存在,临床治疗要根据具体证候进行论治。2.高血压病的证素中以阳亢、阴虚、气滞为最主要,临床中出现频率最高,其次为血瘀、气虚、痰浊、火热等。3.高血压病的常用药物以补虚药、镇肝熄风药、活血化瘀药、清热药、理气化痰药为主,其他各种不同药物根据证型加以配伍改变。4.心肌纤维化的临床治疗以活血化瘀、平肝潜阳、降浊消积、温阳利水、宽胸散结法最为常见。现阶段临床治疗中以活血化瘀药、平肝熄风药、温里药、补虚药、利水渗湿药为主,通常选用复方汤剂或者其他复方制剂,中药提取物也逐渐开始广泛应用于临床。5.补虚类药物在高血压病与心肌纤维化重都有着大量的应用。6.黄芪水提取物能够拮抗血管紧张素Ⅱ刺激产生的血压升高与心室重构,从而抑制心肌纤维化的形成。
[Abstract]:Objective: myocardial fibrosis is a common chronic cardiovascular disease, which has a close relationship with many diseases and pathological processes, such as atherosclerosis, myocardial infarction, arrhythmia, hypertension, and so on. Myocardial fibrosis caused by hypertension is a common pathological manifestation. At present, the study has developed to molecular level. The causes of myocardial fibrosis induced by blood pressure may be closely related to inflammation, hypoxia and hemodynamic changes. Myocardial fibrosis has a corresponding description in traditional Chinese medicine theory. Myocardial fibrosis runs through the pathological process of various diseases, such as hypertension, coronary heart disease, diabetes, myocarditis and many other diseases. These diseases are in Chinese medicine. The theory can be classified as "chest Bi", "palpitation", "dizziness", "phlegm" and "edema". The TCM pathogenesis of myocardial fibrosis can be summed up as a virtual standard. Because of many chronic diseases, the disease entered the collateral veins, the deficiency of yin and Yang caused by the deficiency of phlegm and blood stasis, the water wet cross, the heat poison of the lump, and so on. A kind of medicinal herbs commonly used in traditional Chinese medicine has proved to contain a variety of effective components, such as Astragalus saponins, astragalus polysaccharides, flavonoids and amino acids. Astragalus membranaceus can enhance the immune function of the body, protect liver function, diuresis, anti aging oxidation, anti stress effect, broad-spectrum antibacterial effect, and can reduce blood pressure. In traditional medicine, the use of Astragalus and other drugs to treat related cardiovascular diseases is abound, but the specific components of the effect are still unclear. The water extract of Astragalus is one of the main active ingredients extracted from the root of Astragalus membranaceus through water extraction. The extract of Astragalus membranaceus has anti-inflammatory activity, which can reduce the migration of macrophages and the adhesion of vascular endothelial cells, alleviate local inflammation and anti atherosclerosis through the nuclear factor (NF) - kappa B signaling pathway, but the effect of Astragalus membranaceus water extract on hypertension and the related effects on myocardial fibrosis and its mechanism are not reported. The purpose of this study is to analyze the evidence of hypertension and myocardial fibrosis by literature, and to analyze the antagonistic effects of Astragalus membranaceus water extract on hypertension and myocardial fibrosis in recent years. Materials and methods: 1. literature studies are related to the collection of hypertension. A total of 3663 articles were selected and 527 articles were selected after screening. Through the induction of the above literature, a preliminary summary of hypertension related syndromes and drugs was sorted out. Then 490 articles related to myocardial fibrosis were collected again. The treatment and heart of hypertension in the treatment and heart of hypertension were studied through literature Study on the habit of treating myocardial fibrosis in the treatment of bed. The common place of the treatment of muscle fibrosis, so as to be effective in the treatment of the disease in clinical treatment, the literature study in the experimental study of.2. related experiments shows that the common syndromes of hypertension are liver yang hyperactivity, phlegm and blood stasis, deficiency of liver and kidney, etc. the common syndromes of myocardial fibrosis include Qi deficiency, blood stasis and yang deficiency. The effect of traditional Chinese medicine Astragalus membranaceus is very much, including Qi Qi and fixation, diuresis, purulent and collecting sores, etc., in the treatment of qi deficiency, yin and yang two deficiency symptoms and other symptoms, in the clinical treatment of myocardial fibrosis should be a more common medicine. At the present stage, with pharmacology and clinical medicine The use of Chinese medicine extracts has gradually become a subsequent upsurge of medical research. The study on the extract of Astragalus has also been carried out gradually. It has been reported that astragaloside has a certain effect on antagonizing hypertension through the protection of endothelium and so on. At the same time, astragaloside also has certain inhibitory effect on myocardial fibrosis. The Astragalus membranaceus extract is a more common extract of Astragalus membranaceus. The previous study in our laboratory was to study the effect of Huang Qishui extract on the endothelial factor. This follow-up experiment tried to study whether the extract of Astragalus membranaceus water extract was also the same as the.2.1. test for high blood pressure myocardial fibrosis. The aqueous extract of Astragalus membranaceus was provided by the Dalian Institute of Chemical Physics, Chinese Academy of Sciences, dissolved and diluted in distilled water (3 grams per kilogram / day). Simvastatin was purchased from Sigma-Aldrich (saintlouis, Mo, USA). Angiotensin II was purchased from MLUN biopharmaceutical (Dalian). Subcutaneous embedded capsule osmotic pressure pump (usa/alzet production).2.2. The male mouse Kunming mice (n=24), aged 8 weeks, 18~20 gram, derived from the background of Swiss mice, were purchased from the Medical Animal Department of the General Hospital of Shenyang military region. All the mice were fed in all the cage groups and were fed with conventional parity rats. After a week of routine feeding, the rats were randomly divided into blank control groups according to the blind method, angiotensin II model. Group, angiotensin II plus simvastatin group, angiotensin II Plus Astragalus water extract group (each group of n=6). Except for the blank control group, all the other three groups were subcutaneously embedded into the implanted capsule osmotic pressure pump, the contents were angiotensin II (1.3 mg / kg / day), and angiotensin II plus simvastatin group took 0.4% daily. Simvastatin solution (30 mg / kg / day), angiotensin II and Astragalus membranaceus water extract group were given oral dose of Astragalus membranaceus water extract solution (3 g / kg / day). Tissue specimens were collected at 28 days of subcutaneous embedded pump to further prepare for data analysis. All related experiments involving animal experiment procedures were conducted according to the relevant requirements. It has been approved by the ethics committee's approval.2.3he staining experiment to collect the heart specimens of the mice that arrive at the punctuation time and put into the pre prepared fixed liquid (usually 4% polyformaldehyde) after cleaning. After dehydration, paraffin, lumps, slices (heart crosscutting, thickness about 5-8 microns), the slices are put into a thermostat at 45 degrees centigrade to be baked. Dry steps, dewaxing by soaking in alcohol and dimethylbenzene, dyeing the slice into regaud (or Weigert) soubin solution for 5-10 minutes according to the steps, and then putting into 1% hydrochloric acid ethanol and ammonia water for several seconds, and using water for more than 20 minutes to wait for the nucleus anti blue for more than 20 minutes, and the next step, using distilled water to rinse again and again. After putting the slice into 0.5% eosin solution for about 1-3 minutes, then putting into 80%-100% alcohol in order to dehydrate in sequence, then put into the dimethylbenzene in order to change the cylinder in order, and use the neutral resin to fix the seal. After the preparation of the slice specimen, the specimen of the heart of the mice can be observed under the microscope and then the specimens of the mice are prepared and put into the section. After the fixed solution (4% polyformaldehyde), after the fixed dehydration, the wax is dipped, the block, the slice (the heart crosses, the general thickness is 5-8 microns), then dried in the constant temperature box at 45 C, and the paraffin section is dewaxed and dehydrated using alcohol. Then, the water and the distilled water are used respectively. 5-10 points are used with regaud hematoxylin solution or Weigert hematoxylin solution. When the clock is washed fully, the observation effect, if it is considered that the slice is dyed, can be differentiated by hydrochloric acid and then flushed with distilled water after differentiation. After the use of hematoxylin to dye the nucleus, the Masson Li Chun Hong acid reddish liquid is used for 5-10 minutes, and then the slice is infected by 2% glacial acetic acid solution for a moment, and then use 1%. The solution of Phospho molybdate water is divided for about 3-5 minutes. Without water, it will be dyed for 5 minutes directly with aniline blue solution. After the dyeing, it is soaking for a moment with 0.2% glacial acetic acid solution, using 80%, 90%, 95% alcohol, and anhydrous alcohol to dehydrate, then dimethylbenzene is transparent, and the neutral resin is sealed. After cleaning the specimens of the mice after the operation, after the.2.5 immunohistochemical technique, they were put into the pre matched fixed solution (4% polyformaldehyde). After the dehydration was fixed, the wax was impregnated, the paraffin block and the slice (the heart crosscutting, the general thickness of 5-8 microns) were dried in a constant temperature box at 45 C, and paraffin section was dewaxed and dehydrated in alcohol and xylene. Then, the paraffin section was dewaxed and dehydrated. Then, the paraffin section was dewaxed and dehydrated. The slice was rinsed in clear water for a period of time and was soaked in 3% hydrogen peroxide solution for 10 minutes. (this step could remove the endogenous catalase) and then pour out the hydrogen peroxide solution. After rinsing the slice into the clean water, the antigen repair solution was extracted (the laboratory was used as a citric acid buffer) and closed into the boiling water. After 40 minutes of water boiled in the water and cooled to room temperature.Pbs solution, the animal serum was closed and the animal serum was added to add a resistance. After the first anti drop, the sliced box was taken overnight in the refrigerator at 4, and the temperature was reheated at room temperature for second days for 30 minutes. After washing with the PBS solution, the slices were added to the slice and added a proper amount of horseradish or alkaline phosphoric acid. After the enzyme labelled streptomycin working fluid, the slice was rinsed with PBS solution repeatedly and followed by DAB staining. After the DAB coloration reached the ideal degree, the clear water was flushed to terminate the color reaction. Then the cell nucleus was stained with hematoxylin and the gradient alcohol was used to dehydrated, and the dimethylbenzene solution was transparent with neutral gum after the slice. .2.6 immunoblotting analysis (Westernblot) was used to observe the cardiac tissue of each group and determine the collagen content of cardiac tissue in different groups. In this experiment, the use of Westernblot to detect collagen-3. in the cardiac tissue of mice was 100 microlitre solution (50 mole / liter Tris, 10 mole / liter chlorine). Magnesium, 0.5 mole / liter of sodium chloride, 1%tritonx-100) used to extract total myocardial protein. Cardiac tissue remains frozen and collected on ice. The isolated supernatant is collected after 14000 rotation centrifugation in centrifuge for 10 minutes. The protein is extracted according to the experimental method on the related kit and preserved in the supernatant after centrifugation. The supernatant is determined in the supernatant by BCA method. Protein level. 30 microgram protein samples were divided into polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (pvvdf) membrane. After the film was finished, 5% skimmed milk powder was used to close the polyvinylidene fluoride membrane in Tris Physiological Buffered Saline, and then the required first resistance was added to the membrane. It was placed in the 4 degree C refrigerator and spent the night according to the incubation conditions. After taking out the next day, 3 times were washed with PBS. After rinsing, the Sheep anti rabbit, Sheep anti rat and Rabbit anti goat two anti goat were cultured with 1:2000 diluted horseradish peroxidase (HRP) for 2 hours at room temperature. After three times washing, the activity was detected by adding luminescent agent. The statistical analysis data of dark room exposure were expressed by mean value of + SD. All the data in the experiment The statistical results of SPSS13.0 software were used. The difference between the two groups was measured by unpairedstudent 'st test. The difference between the three related experimental groups was statistically significant by using variance analysis (ANOVA).P0.05 (double tail). Conclusion: the syndrome type distribution of 1. hypertension is the most in liver yang hyperactivity, liver kidney yin deficiency, blood stasis obstructing collaterals, liver fire hyperactivity, The obstruction of phlegm dampness, phlegm and blood stasis, Qi deficiency and blood stasis, yin and yang two deficiency and kidney qi deficiency are common syndrome types. In addition, there are many kinds of syndrome types. The clinical treatment should be based on specific syndromes to treat.2. hypertension with yang hyperactivity, yin deficiency and qi stagnation as the most important, the second is blood stasis, Qi deficiency, phlegm turbidity, and heat. The commonly used drugs for.3. hypertension are to fill in the deficiency drugs, the antipyretic drug of the liver, the medicine of activating blood and removing stasis, the antipyretic medicine and the medicine of Qi and chemical phlegm, and the other different drugs are combined with the syndrome type to change the clinical treatment of.4. myocardial fibrosis with the treatment of activating blood and removing stasis, smoothing the liver and Yang, reducing turbidity and eliminating the accumulation, warming Yangli water, and the broad chest disintegration. In the treatment, the drugs of promoting blood circulation and removing blood stasis, the medicine of extinguishing the liver, the medicine of warm water, the medicine of filling the deficiency, the medicine of the water and the dampness, usually use the compound decoction or other compound preparation. The extract of the Chinese medicine has gradually started to be widely used in the clinical.5. supplement deficiency drugs in the hypertension and the myocardial fibrosis, and the.6. Astragalus water extract can antagonize the blood. Angiotensin II stimulates increased blood pressure and ventricular remodeling, thereby inhibiting the formation of myocardial fibrosis.

【学位授予单位】：辽宁中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R285.5

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