益气活血中药通过外膜干预血管衰老及血管衰老的中医证型特点研究
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摘要
随着经济社会的发展,医疗水平的提高,我国人口平均寿命不断提高,老龄人口的比例逐年增加,针对这一庞大的特殊人群,研究血管衰老,即随增龄心血管系统发生的结构和功能的特征性改变,已成为刻不容缓的问题。研究发现,血管衰老与各种心血管疾病相互作用,影响了疾病的发生的阈值、严重程度和预后,有望成为临床防治心血管病的新靶点。血管衰老的重要特点之一为血管壁弹性的下降和僵硬度的增加,而血管外膜及外膜成纤维细胞(adventitial fibroblasts, AF)具有重要的免疫及内分泌功能,在分泌胶原等细胞外基质,维持血管弹性等方面发挥着关键的作用,可能成为血管衰老研究的新的突破口。
课题组前期已经对血管内皮细胞、平滑肌细胞的衰老规律及益气活血中药的作用机制进行了探讨。本研究在此基础上,以血管外膜为切入点,以血管紧张素Ⅱ(angiotensin Ⅱ, Ang Ⅱ)、烟酰胺腺嘌呤二核苷酸(nicotinamide-adenine dinucleotide phosphate, NADPH)氧化酶p47phox、活性氧(reactive oxygen species,ROS)通路为主线,从动物、细胞、分子水平探讨血管衰老的机制,观察益气活血中药(人参三七川芎提取物)延缓血管衰老的作用,进一步探讨其作用机制。同时,通过临床调查,初步探讨血管衰老的中医证型特点。以期通过本研究,为血管衰老的中医药研究奠定基础,为临床运用中医药延缓血管衰老提供科学依据。
本研究分为三个部分：文献综述、基础研究和临床研究。
1.文献综述通过对血管外膜及AF相关文献的归纳,综述了血管外膜和AF在血管疾病发生发展过程中的作用。
2.基础研究由以下三个基础实验构成：
实验一：益气活血中药延缓自然衰老大鼠血管外膜重构的实验研究
目的：观察自然衰老大鼠血管外膜的病理改变并探讨其机制；观察益气活血中药(人参三七川芎提取物)对衰老血管外膜病变的干预作用,并探讨其作用机制。
方法：建立自然衰老大鼠模型,分为衰老模型组(蒸馏水)、中药高剂量组(人参、三七、川芎醇提物1493.4mg/kg/d)、中药中剂量组(人参、三七、川芎醇提物746.7mg/kg/d),中药低剂量组(人参、三七、川芎醇提物373.4mg/kg/d)、氯沙坦对照组(10mg/kg/d),另设一青年对照组(蒸馏水)。给予相应干预15周后,采用Morris水迷宫自主活动法观察动物的学习能力、记忆获取能力和记忆保存能力,HE染色法观察胸主动脉壁形态,苦味酸天狼猩红染色法观测血管壁胶原种类、分布和含量,扫描电子显微镜观察外膜超微结构,放射免疫分析法检测血浆肾素活性(plasma renin activity, PRA)、血浆Ang Ⅱ浓度及外膜组织Ang Ⅱ含量,生物化学分析法检测外膜组织羟脯氨酸、抗超氧阴离子自由基(superoxide anion, O2)活力单位含量,酶联免疫吸附法(enzyme linked immunosorbent assay, ELISA)检测外膜组织1型基质金属蛋白酶(matrix metalloproteinases type1, MMP-1)及其抑制剂(tissue inhibitor of metalloproteinase type1, TIMP-1)含量,RT-PCR法检测外膜组织血管紧张素Ⅱ1型受体(angiotensin Ⅱ receptor1, AT1R)、血管紧张素Ⅱ2型受体(angiotensin Ⅱ receptor2, AT2R)和NADPH氧化酶p47phox的mRNA含量,western bloting法检测外膜组织AT1R、AT2R和NADPH氧化酶p47phox蛋白表达。
结果：与青年组比较,衰老模型组血管外膜外膜增厚,AF增生,外膜厚度/管径增高,胶原分布致密,胶原纤维堆积,粗大,排列紊乱,外膜组织Ⅰ型胶原增加,Ⅲ型胶原减少,Ⅲ型胶原/Ⅰ型胶原降低,羟脯氨酸含量增高,MMP-1含量降低,TIMP-1含量增高,MMP-1/TIMP-1降低,02-合成增加,Ang Ⅱ含量增高,AT1R的mRNA及蛋白表达下调,AT2R及NADPH氧化酶p47phox的mRNA及蛋白表达上调,动物的学习能力、记忆获取能力和记忆保存能力下降。人参三七川芎提取物能够改善衰老血管形态学改变,抑制AF过度增殖,改善胶原比例紊乱,降低外膜组织中Ang Ⅱ的水平,抑制02合成,调节MMP-1/TIMP-1平衡,从mRNA及蛋白水平上调AT1R及AT2R的表达,下调NADPH氧化酶p47phox的表达,改善动物学习能力、记忆获取能力和记忆保存能力。
结论：衰老血管出现外膜重构,表现为外膜增厚,AF过度增殖,MMP-1/TIMP-1平衡失调,胶原堆积,Ⅰ型、Ⅲ型胶原比例紊乱等,其机制可能与AngⅡ、NADPH氧化酶p47phox、活性氧(ROS)通路有关；人参三七川芎提取物可能通过对外膜局部Ang Ⅱ、AT1R等多靶位的干预,改善外膜重塑,增加血管弹性,进而延缓血管衰老,提高整个机体的状态。
实验二：益气活血中药延缓AF复制性衰老的实验研究
目的：观察AF复制性衰老的病理变化,观察益气活备中药(人参三七川芎提取物)延缓AF复制性衰老的作用,并探讨其机制。
方法：采用原代培养的方法,通过传代建立复制性衰老AF模型,分为衰老模型组(DMEM完全培养液),中药高剂量组(100m/L人参三七川芎提取液+DMEM完全培养液),中药低剂量组(50mg/L人参三七川芎提取液+DMEM完全培养液),VitE组(100mg/LVitE+DMEM完全培养液),另设一5代空白组(DMEM完全培养液)。采用细胞衰老特异性β半乳糖苷酶(senescence-associated-β-galactosidase, SA-β-gal)染色法观察细胞衰老改变,噻唑兰(MTT)比色法分析细胞增殖情况,流式细胞仪分析细胞周期,免疫细胞化学染色法检测细胞内MMP-1及TIMP-1含量,荧光探针法检测细胞内活性氧(ROS)含量,生物化学法检测培养液内抗O2-活力单位含量,ELISA法检测培养液内Ⅰ型胶原、Ⅲ型胶原含量,RT-PCR法检测细胞AT1R、AT2R和NADPH氧化酶p47phox的mRNA含量,western bloting法检测细胞AT1R、AT2R和NADPH氧化酶p47phox蛋白表达。
结果：衰老AF SA-β-gal染色阳性率增加,G1期细胞减少,MMP-1表达降低,TIMP-1表达增加,细胞内ROS荧光强度增加,O2-合成增加,Ⅰ胶原浓度升高,Ⅲ型胶原浓度降低,AT1R mRNA及蛋白表达下调,AT2R、NADPH氧化酶P47phox的mRNA及蛋白表达上调。人参三七川芎提取物干预后,AF的SA-p-gal染色阳性率降低,G1期细胞增加,MMP-1表达增加,TIMP-1表达降低,细胞内ROS荧光强度减弱,O2-合成减少,Ⅰ胶原浓度降低,Ⅲ型胶原浓度升高,AT1R和AT2R的mRNA及蛋白表达上调,NADPH氧化酶P47phoxmRNA及蛋白表达下调。
结论：衰老AF SA-β-gal染色阳性表达增加,MMP-1/TIMP-1合成失衡,胶原分泌紊乱,病变特点与衰老动物一致,可能是衰老血管外膜重构病理过程的参与者；人参三七川芎提取物可能从mRNA及蛋白水平下调NADPH氧化酶p47phox的表达,减少ROS合成,从而降低SA-β-gal染色阳性表达,调节MMP-1/TIMP-1平衡,调节胶原代谢,进而改善血管重构,延缓血管衰老。
实验三：益气活血中药对Ang Ⅱ诱导的AF增殖及胶原生成等相关因素的影响
目的：观察Ang Ⅱ对AF的作用,并探讨其机制；观察益气活血中药(人参三七川芎提取物)对Ang Ⅱ诱导的AF病理变化的影响,并探讨其作用机制。
方法：采用原代培养的方法,建立Ang Ⅱ诱导的AF模型,分为空白对照组(DMEM完全培养液)、Ang Ⅱ诱导模型组(10-6mol/L Ang Ⅱ+DMEM完全培养液)、中药高剂量组(10-6mol/L Ang Ⅱ+100mg/L人参三七川芎提取液+DMEM完全培养液)、中药低剂量组(10-6mol/L Ang Ⅱ+50mg/L人参三七川芎提取液+DMEM完全培养液)、Valsartan组(10-6mol/L Ang Ⅱ+10-6mol/LValsartan+DMEM完全培养液)。采用SA-β-gal染色法观察细胞衰老改变,噻唑兰(MTT)比色法分析增殖情况,流式细胞仪分析细胞周期,免疫细胞化学染色法检测细胞内MMP-1及TIMP-1含量,荧光探针法检测细胞内ROS含量,生物化学法检测培养液内抗O2-活力单位含量,ELISA法检测培养液内Ⅰ型胶原、Ⅲ型胶原含量,RT-PCR法检测细胞AT1R、AT2R和NADPH氧化酶p47phox的mRNA含量,western bloting法检测细胞AT1R、AT2R和NADPH氧化酶p47phox蛋白表达。
结果：经Ang Ⅱ诱导后,AF的SA-β-gal阳性染色率增加,S期细胞增多,MMP-1表达降低,TIMP-1表达增加,ROS荧光强度增强,02-合成增加,Ⅰ胶原浓度升高,Ⅲ型胶原浓度降低,AT1R mRNA及蛋白表达下调,AT2R和NADPH氧化酶p47phox mRNA及蛋白表达上调。人参三七川芎提取物干预后,AF的SA-β-gal阳性染色率降低,G1期细胞增多,MMP-1表达增加,TIMP-1表达降低,ROS荧光强度减弱,02-合成下降,Ⅰ胶原浓度降低,Ⅲ型胶原浓度升高,AT1R和AT2RmRNA及蛋白表达上调,NADPH氧化酶p47phox mRNA及蛋白表达下调。
结论：Ang Ⅱ对AF具有促进增殖和SA-β-gal染色阳性表达,诱导胶原分泌紊乱,破坏MMP-1/TIMP-1合成平衡等作用,人参三七川芎提取物可能经由AT1R从mRNA及蛋白水平下调NADPH氧化酶p47phox的表达,减少ROS合成,从而拮抗Ang Ⅱ对AF的不良刺激,抑制细胞增殖,调节MMP-1/TIMP-1平衡,调节胶原代谢,进而改善血管重构,延缓血管衰老。
3.临床研究：血管衰老的中医证型特征研究
目的：观察血管衰老的中医证型特点。
方法：选择231例原发性高血压患者作为研究对象,利用聚类分析和主成分分析的统计学方法,分析其血管衰老相关指标及中医四诊信息。
结果：高血庄合并血管衰老的主要证型为血.瘀证、气虚证、阴虚证,可兼见痰浊及肝阳上亢证候；高血压血管衰老病变发展至动脉粥样硬化时,其主要证型为痰浊证、血瘀证、气虚证,可兼见阴虚及阴阳两虚证候。
结论：血瘀证和气虚证贯穿高血压血管衰老发生发展始终,益气活血延缓血管衰老有据可循。
本研究创新点
(1)本研究从血管研究的新热点——血管外膜入手,以AngⅡ、NADPH氧化酶p47phox、ROS通路为主线,研究益气活血中药延缓衰老作用及机制,是该领域的独创性研究。
(2)本研究建立相关的AF模型,首次从成纤维细胞角度进行血管衰老研究。
(3)本研究采用聚类分析、主成分分析的统计学方法分析血管衰老相关指标和中医四诊信息,从而阐明血管衰老的中医证型特点,经文献检索未见相关报道。
With the developing of society as well as the medicine technology, the expectation of life of Chinese is increasing day by day and the percentage of the aged is increasing accordingly, which means that we are stepping into an aging society. Therefore, the study of vascular aging, the structural and/or functional changes of cardiovascular system with aging, is of great importance. It has been found out that there is an interaction between vascular aging and cardiovascular diseases, which has a harmful effect on the pathogenesis. Besides, adventitia as well as adventitial fibroblasts (AF) is becoming a new breakthrough point of the study of vascular aging for its role in flexibility keeping by secreting extracellular matrix such as collagen.
In the preparatory works, the mechanism of delaying senility with traditional Chinese medicine of tonifying qi and activating blood has been studied from endothelial cell and smooth muscle cell aspect. Based on those, this study researched vascular aging and the treatment with traditional Chinese medicine from the adventitial aspect following the way of angiotensin Ⅱ (Ang Ⅱ), nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase component as p47phox and reactive oxygen species(ROS).Besides, an investigation was made to identify the patterns of syndrome of vascular aging. It is expected that this study could provide a scientific basis for the subject of vascular aging delaying and the corresponding treatment with traditional Chinese medicine.
This study is divided into three parts:literature review, basic research and clinical research.
1. Literature review
By sorting and summing up the literatures of adventitia and AF, the effects of adventitia and AF in the vascular diseases were summarized.
2. Basic research This part includes three basic experiments.
Experiment Ⅰ:Experimental Study on Traditional Chinese Medicine of Tonifying Qi and Activating Blood Delaying Vascular Aging of Senescent Rat
Objective:To explore the structural and functional changes of vascular in senescent rat as well as the therapeutic effect of traditional Chinese medicine(extracts from Radix Ginseng, Radix Notoginseng and Rhizoma Chanxiong)
Methods:The model of natural aging was built, and all rats were divided into6groups as aging-model group, young control group, traditional Chinese medicine high-dose group, traditional Chinese medicine medium-dose, traditional Chinese medicine low-dose group, and Losartan control group. After15-week intervention the following indexes were observed with corresponding methods:memory acquisition and preservation ability by Morris water maze, morphological changes of aorta by HE and picrosirius red stainings, adventitial ultra-structure by scanning electron microscopy, the plasma renin activity and the content of rennin AngⅡ by radio immunoassay, the content of hydroxyproline by biochemical analysis, the levels of anti-endogenous superoxide anion (O2-) by biochemical analysis, the content of MMP-1and TIMP-1with enzyme linked immunosorbent assay(ELISA), mRNA and protein expression of NADPH oxidase p47phox, type1and2receptor (AT1R, AT2R)of angiotensin by real-time polymerase chain reaction(RT-PCR) and Western Bloting.
Results:Compared with young control group, the aging model showed characteristics as following:increased thickness of adventitia, hyperplasia of AF, abnormality of collagen, abnormality of MMP-1/TIMP-1, increased content of AngⅡ, hydroxyproline and O2-of adventitia, decreased expression of mRNA and protein of AT1R, increased expression of mRNA and protein of AT2R and p47phox, declined ability of memory acquisition and preservation. And the Traditional Chinese Medicine could improve the adventitial morphology changes, inhibit hyperplasia of AF, improve the abnormality of collagen as well as MMP-1/TIMP-1,decrease content of AngⅡ, hydroxyproline and O2-of adventitia, increase the expression of mRNA and protein of AT1R and AT2R, decrease the expression of mRNA and protein of p47phox, improve the ability of memory acquisition and preservation of rats.
Conclusion:Remodeling was showed in adventitia of aged vascular, which may happen following the way of AngⅡ, NADPH oxidase p47phox and ROS; The extracts from Radix Ginseng, Radix Notoginseng and Rhizoma Chanxiong could delaying vascular aging possibly by its interference with multitarget, such as AngⅡ and AT1R of adventitia.
Experiment Ⅱ:Experimental Study on Traditional Chinese Medicine of Tonifying Qi and Activating Blood Delaying AF Replicative Senescence
Objective:To observe the patholological changes of replicative senescence in AF, as well as the therapeutic effect of traditional Chinese medicine(extracts from Radix Ginseng, Radix Notoginseng and Rhizoma Chanxiong)
Methods:AFs were induced to aging by generation culvitation in order to make models. The AFs in vitro were divided into5groups:the control group, the cell-senescence untreated group, traditional Chinese medicine high-dose group, and traditional Chinese medicine low-dose group, Vitamin E control group. After intervention, the following indexes were observed with the corresponding methods: changes of aging by senescence-associated-β-galactosidase(SA-β-gal) staining method, status of hyperplasy by MTT (3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2H tetrazolium bromide) assay, mitotic cycle by flow cytometer, the content of MMP-1and TIMP-1by immunocytochemical method, the levels of O2-by biochemical analysis, the content of type Ⅰ collagen and type Ⅲcollagen by ELISA, mRNA and protein expression of NADPH oxidase p47phox, type1and2receptor (AT1R, AT2R)of angiotensin by real-time polymerase chain reaction(RT-PCR) and Western Bloting.
Results:Ageing AF showed characteristics as following:the increased expression of SA-β-gal staining, increase the percentage of AF in S Phase, increased content of type Ⅰ collagen while decreased type Ⅲ collagen, increased expression of TIMP-1while decreased of MMP-1, increased fluorescence intensity of ROS in cells, increased production of O2-, increased expression of mRNA and protein of p47phox and AT2R, decreased expression of mRNA and protein of AT1R. The traditional Chinese medicine could decrease the positive expression of SA-β-gal staining, increase the percentage of AF in G1Phase, improve the abnormality of collagen, increase the expression of type Ⅰ collagen and decrease type Ⅲcollagen, decrease fluorescence intensity of ROS in cells, decrease production of O2-increase the expression of mRNA and protein of AT1R and AT2R, decrease the expression of mRNA and protein of p47phox
Conclusion:The extracts from Radix Ginseng, Radix Notoginseng and Rhizoma Chanxiong could improve AF replicative senescence possibly by the way of NADPH oxidase p47phox and ROS.
Experiment Ⅲ:Experimental Study on Traditional Chinese Medicine of Tonifying Qi and Activating Blood improving proliferation, collagenation abnormality, et al of AF induced by Ang Ⅱ
Objective:To observe the patholological changes of AF induced by Ang Ⅱ, as well as the therapeutic effect of traditional Chinese medicine(extracts from Radix Ginseng, Radix Notoginseng and Rhizoma Chanxiong)
Methods:AFs were induced by AngⅡ in order to make models. The cultured AFs in vitro were divided into5groups:the control group, Ang Ⅱ induced group, traditional Chinese medicine high-dose group, traditional Chinese medicine low-dose group, and valsartan control group. After intervention, the following indexes were observed with the corresponding methods:changes of aging by SA-β-gal staining method, status of hyperplasy by MTT (3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2H tetrazolium bromide) assay, mitotic cycle by flow cytometer, the content of MMP-1and TIMP-1by immunocytochemical method, the levels of O2-by biochemical analysis, the content of type Ⅰ collagen and type Ⅲcollagen by ELISA, mRNA and protein expression of NADPH oxidase p47phox, type1and2receptor (AT1R, AT2R)of angiotensin by real-time polymerase chain reaction(RT-PCR) and Western Bloting.
Results:The AF induced by Ang Ⅱ showed characteristics as following:the increased expression of SA-β-gal staining, increase the percentage of AF in S Phase, increased content of type I collagen while decreased type Ⅲ collagen, increased expression of TIMP-1while decreased of MMP-1, increased fluorescence intensity of ROS in cells, increased production of O2-, increased expression of mRNA and protein of p47phox and AT2R, decreased expression of mRNA and protein of AT1R. The traditional Chinese medicine could decrease the positive expression of SA-β-gal staining, increase the percentage of AF in G1Phase, improve the abnormality of collagen, increase the expression of type Ⅰ collagen and decrease type Ⅲ collagen, decrease fluorescence intensity of ROS in cells, decrease production of O2-, increase the expression of mRNA and protein of AT1R and AT2R, decrease the expression of mRNA and protein of p47phox.
Conclusion:The extracts from Radix Ginseng, Radix Notoginseng and Rhizoma Chanxiong could improve AF induced by AngⅡ possibly by the way of AngⅡ, NADPH oxidase p47phox and ROS.
3. Clinical Research:Study on syndrome types of vascular aging
Objective:To investigate the patterns of syndrome in the population with vascular aging
Methods:231patients with essential hypertension were selected as research object. After physical examinations of both modern medicine and traditional Chinese medicine, the results relating vascular aging were analyzed by the statistics methods of cluster analysis and principal component analysis.
Results:The main patterns of syndrome are the syndrome of blood stasis, the syndrome of deficiency of qi, and the syndrome of yin asthenia of the patients with essential hypertension as well as vascular aging; The main patterns of syndrome are the syndrome of phlegm, the syndrome of blood stasis, and the syndrome of deficiency of qi of the patients with essential hypertension, vascular aging and atherosclerosis.
Conclusion:The syndrome of blood stasis and the syndrome of deficiency of qi, are the main patterns of syndrome of vascular aging.
课题组前期已经对血管内皮细胞、平滑肌细胞的衰老规律及益气活血中药的作用机制进行了探讨。本研究在此基础上,以血管外膜为切入点,以血管紧张素Ⅱ(angiotensin Ⅱ, Ang Ⅱ)、烟酰胺腺嘌呤二核苷酸(nicotinamide-adenine dinucleotide phosphate, NADPH)氧化酶p47phox、活性氧(reactive oxygen species,ROS)通路为主线,从动物、细胞、分子水平探讨血管衰老的机制,观察益气活血中药(人参三七川芎提取物)延缓血管衰老的作用,进一步探讨其作用机制。同时,通过临床调查,初步探讨血管衰老的中医证型特点。以期通过本研究,为血管衰老的中医药研究奠定基础,为临床运用中医药延缓血管衰老提供科学依据。
本研究分为三个部分：文献综述、基础研究和临床研究。
1.文献综述通过对血管外膜及AF相关文献的归纳,综述了血管外膜和AF在血管疾病发生发展过程中的作用。
2.基础研究由以下三个基础实验构成：
实验一：益气活血中药延缓自然衰老大鼠血管外膜重构的实验研究
目的：观察自然衰老大鼠血管外膜的病理改变并探讨其机制；观察益气活血中药(人参三七川芎提取物)对衰老血管外膜病变的干预作用,并探讨其作用机制。
方法：建立自然衰老大鼠模型,分为衰老模型组(蒸馏水)、中药高剂量组(人参、三七、川芎醇提物1493.4mg/kg/d)、中药中剂量组(人参、三七、川芎醇提物746.7mg/kg/d),中药低剂量组(人参、三七、川芎醇提物373.4mg/kg/d)、氯沙坦对照组(10mg/kg/d),另设一青年对照组(蒸馏水)。给予相应干预15周后,采用Morris水迷宫自主活动法观察动物的学习能力、记忆获取能力和记忆保存能力,HE染色法观察胸主动脉壁形态,苦味酸天狼猩红染色法观测血管壁胶原种类、分布和含量,扫描电子显微镜观察外膜超微结构,放射免疫分析法检测血浆肾素活性(plasma renin activity, PRA)、血浆Ang Ⅱ浓度及外膜组织Ang Ⅱ含量,生物化学分析法检测外膜组织羟脯氨酸、抗超氧阴离子自由基(superoxide anion, O2)活力单位含量,酶联免疫吸附法(enzyme linked immunosorbent assay, ELISA)检测外膜组织1型基质金属蛋白酶(matrix metalloproteinases type1, MMP-1)及其抑制剂(tissue inhibitor of metalloproteinase type1, TIMP-1)含量,RT-PCR法检测外膜组织血管紧张素Ⅱ1型受体(angiotensin Ⅱ receptor1, AT1R)、血管紧张素Ⅱ2型受体(angiotensin Ⅱ receptor2, AT2R)和NADPH氧化酶p47phox的mRNA含量,western bloting法检测外膜组织AT1R、AT2R和NADPH氧化酶p47phox蛋白表达。
结果：与青年组比较,衰老模型组血管外膜外膜增厚,AF增生,外膜厚度/管径增高,胶原分布致密,胶原纤维堆积,粗大,排列紊乱,外膜组织Ⅰ型胶原增加,Ⅲ型胶原减少,Ⅲ型胶原/Ⅰ型胶原降低,羟脯氨酸含量增高,MMP-1含量降低,TIMP-1含量增高,MMP-1/TIMP-1降低,02-合成增加,Ang Ⅱ含量增高,AT1R的mRNA及蛋白表达下调,AT2R及NADPH氧化酶p47phox的mRNA及蛋白表达上调,动物的学习能力、记忆获取能力和记忆保存能力下降。人参三七川芎提取物能够改善衰老血管形态学改变,抑制AF过度增殖,改善胶原比例紊乱,降低外膜组织中Ang Ⅱ的水平,抑制02合成,调节MMP-1/TIMP-1平衡,从mRNA及蛋白水平上调AT1R及AT2R的表达,下调NADPH氧化酶p47phox的表达,改善动物学习能力、记忆获取能力和记忆保存能力。
结论：衰老血管出现外膜重构,表现为外膜增厚,AF过度增殖,MMP-1/TIMP-1平衡失调,胶原堆积,Ⅰ型、Ⅲ型胶原比例紊乱等,其机制可能与AngⅡ、NADPH氧化酶p47phox、活性氧(ROS)通路有关；人参三七川芎提取物可能通过对外膜局部Ang Ⅱ、AT1R等多靶位的干预,改善外膜重塑,增加血管弹性,进而延缓血管衰老,提高整个机体的状态。
实验二：益气活血中药延缓AF复制性衰老的实验研究
目的：观察AF复制性衰老的病理变化,观察益气活备中药(人参三七川芎提取物)延缓AF复制性衰老的作用,并探讨其机制。
方法：采用原代培养的方法,通过传代建立复制性衰老AF模型,分为衰老模型组(DMEM完全培养液),中药高剂量组(100m/L人参三七川芎提取液+DMEM完全培养液),中药低剂量组(50mg/L人参三七川芎提取液+DMEM完全培养液),VitE组(100mg/LVitE+DMEM完全培养液),另设一5代空白组(DMEM完全培养液)。采用细胞衰老特异性β半乳糖苷酶(senescence-associated-β-galactosidase, SA-β-gal)染色法观察细胞衰老改变,噻唑兰(MTT)比色法分析细胞增殖情况,流式细胞仪分析细胞周期,免疫细胞化学染色法检测细胞内MMP-1及TIMP-1含量,荧光探针法检测细胞内活性氧(ROS)含量,生物化学法检测培养液内抗O2-活力单位含量,ELISA法检测培养液内Ⅰ型胶原、Ⅲ型胶原含量,RT-PCR法检测细胞AT1R、AT2R和NADPH氧化酶p47phox的mRNA含量,western bloting法检测细胞AT1R、AT2R和NADPH氧化酶p47phox蛋白表达。
结果：衰老AF SA-β-gal染色阳性率增加,G1期细胞减少,MMP-1表达降低,TIMP-1表达增加,细胞内ROS荧光强度增加,O2-合成增加,Ⅰ胶原浓度升高,Ⅲ型胶原浓度降低,AT1R mRNA及蛋白表达下调,AT2R、NADPH氧化酶P47phox的mRNA及蛋白表达上调。人参三七川芎提取物干预后,AF的SA-p-gal染色阳性率降低,G1期细胞增加,MMP-1表达增加,TIMP-1表达降低,细胞内ROS荧光强度减弱,O2-合成减少,Ⅰ胶原浓度降低,Ⅲ型胶原浓度升高,AT1R和AT2R的mRNA及蛋白表达上调,NADPH氧化酶P47phoxmRNA及蛋白表达下调。
结论：衰老AF SA-β-gal染色阳性表达增加,MMP-1/TIMP-1合成失衡,胶原分泌紊乱,病变特点与衰老动物一致,可能是衰老血管外膜重构病理过程的参与者；人参三七川芎提取物可能从mRNA及蛋白水平下调NADPH氧化酶p47phox的表达,减少ROS合成,从而降低SA-β-gal染色阳性表达,调节MMP-1/TIMP-1平衡,调节胶原代谢,进而改善血管重构,延缓血管衰老。
实验三：益气活血中药对Ang Ⅱ诱导的AF增殖及胶原生成等相关因素的影响
目的：观察Ang Ⅱ对AF的作用,并探讨其机制；观察益气活血中药(人参三七川芎提取物)对Ang Ⅱ诱导的AF病理变化的影响,并探讨其作用机制。
方法：采用原代培养的方法,建立Ang Ⅱ诱导的AF模型,分为空白对照组(DMEM完全培养液)、Ang Ⅱ诱导模型组(10-6mol/L Ang Ⅱ+DMEM完全培养液)、中药高剂量组(10-6mol/L Ang Ⅱ+100mg/L人参三七川芎提取液+DMEM完全培养液)、中药低剂量组(10-6mol/L Ang Ⅱ+50mg/L人参三七川芎提取液+DMEM完全培养液)、Valsartan组(10-6mol/L Ang Ⅱ+10-6mol/LValsartan+DMEM完全培养液)。采用SA-β-gal染色法观察细胞衰老改变,噻唑兰(MTT)比色法分析增殖情况,流式细胞仪分析细胞周期,免疫细胞化学染色法检测细胞内MMP-1及TIMP-1含量,荧光探针法检测细胞内ROS含量,生物化学法检测培养液内抗O2-活力单位含量,ELISA法检测培养液内Ⅰ型胶原、Ⅲ型胶原含量,RT-PCR法检测细胞AT1R、AT2R和NADPH氧化酶p47phox的mRNA含量,western bloting法检测细胞AT1R、AT2R和NADPH氧化酶p47phox蛋白表达。
结果：经Ang Ⅱ诱导后,AF的SA-β-gal阳性染色率增加,S期细胞增多,MMP-1表达降低,TIMP-1表达增加,ROS荧光强度增强,02-合成增加,Ⅰ胶原浓度升高,Ⅲ型胶原浓度降低,AT1R mRNA及蛋白表达下调,AT2R和NADPH氧化酶p47phox mRNA及蛋白表达上调。人参三七川芎提取物干预后,AF的SA-β-gal阳性染色率降低,G1期细胞增多,MMP-1表达增加,TIMP-1表达降低,ROS荧光强度减弱,02-合成下降,Ⅰ胶原浓度降低,Ⅲ型胶原浓度升高,AT1R和AT2RmRNA及蛋白表达上调,NADPH氧化酶p47phox mRNA及蛋白表达下调。
结论：Ang Ⅱ对AF具有促进增殖和SA-β-gal染色阳性表达,诱导胶原分泌紊乱,破坏MMP-1/TIMP-1合成平衡等作用,人参三七川芎提取物可能经由AT1R从mRNA及蛋白水平下调NADPH氧化酶p47phox的表达,减少ROS合成,从而拮抗Ang Ⅱ对AF的不良刺激,抑制细胞增殖,调节MMP-1/TIMP-1平衡,调节胶原代谢,进而改善血管重构,延缓血管衰老。
3.临床研究：血管衰老的中医证型特征研究
目的：观察血管衰老的中医证型特点。
方法：选择231例原发性高血压患者作为研究对象,利用聚类分析和主成分分析的统计学方法,分析其血管衰老相关指标及中医四诊信息。
结果：高血庄合并血管衰老的主要证型为血.瘀证、气虚证、阴虚证,可兼见痰浊及肝阳上亢证候；高血压血管衰老病变发展至动脉粥样硬化时,其主要证型为痰浊证、血瘀证、气虚证,可兼见阴虚及阴阳两虚证候。
结论：血瘀证和气虚证贯穿高血压血管衰老发生发展始终,益气活血延缓血管衰老有据可循。
本研究创新点
(1)本研究从血管研究的新热点——血管外膜入手,以AngⅡ、NADPH氧化酶p47phox、ROS通路为主线,研究益气活血中药延缓衰老作用及机制,是该领域的独创性研究。
(2)本研究建立相关的AF模型,首次从成纤维细胞角度进行血管衰老研究。
(3)本研究采用聚类分析、主成分分析的统计学方法分析血管衰老相关指标和中医四诊信息,从而阐明血管衰老的中医证型特点,经文献检索未见相关报道。
With the developing of society as well as the medicine technology, the expectation of life of Chinese is increasing day by day and the percentage of the aged is increasing accordingly, which means that we are stepping into an aging society. Therefore, the study of vascular aging, the structural and/or functional changes of cardiovascular system with aging, is of great importance. It has been found out that there is an interaction between vascular aging and cardiovascular diseases, which has a harmful effect on the pathogenesis. Besides, adventitia as well as adventitial fibroblasts (AF) is becoming a new breakthrough point of the study of vascular aging for its role in flexibility keeping by secreting extracellular matrix such as collagen.
In the preparatory works, the mechanism of delaying senility with traditional Chinese medicine of tonifying qi and activating blood has been studied from endothelial cell and smooth muscle cell aspect. Based on those, this study researched vascular aging and the treatment with traditional Chinese medicine from the adventitial aspect following the way of angiotensin Ⅱ (Ang Ⅱ), nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase component as p47phox and reactive oxygen species(ROS).Besides, an investigation was made to identify the patterns of syndrome of vascular aging. It is expected that this study could provide a scientific basis for the subject of vascular aging delaying and the corresponding treatment with traditional Chinese medicine.
This study is divided into three parts:literature review, basic research and clinical research.
1. Literature review
By sorting and summing up the literatures of adventitia and AF, the effects of adventitia and AF in the vascular diseases were summarized.
2. Basic research This part includes three basic experiments.
Experiment Ⅰ:Experimental Study on Traditional Chinese Medicine of Tonifying Qi and Activating Blood Delaying Vascular Aging of Senescent Rat
Objective:To explore the structural and functional changes of vascular in senescent rat as well as the therapeutic effect of traditional Chinese medicine(extracts from Radix Ginseng, Radix Notoginseng and Rhizoma Chanxiong)
Methods:The model of natural aging was built, and all rats were divided into6groups as aging-model group, young control group, traditional Chinese medicine high-dose group, traditional Chinese medicine medium-dose, traditional Chinese medicine low-dose group, and Losartan control group. After15-week intervention the following indexes were observed with corresponding methods:memory acquisition and preservation ability by Morris water maze, morphological changes of aorta by HE and picrosirius red stainings, adventitial ultra-structure by scanning electron microscopy, the plasma renin activity and the content of rennin AngⅡ by radio immunoassay, the content of hydroxyproline by biochemical analysis, the levels of anti-endogenous superoxide anion (O2-) by biochemical analysis, the content of MMP-1and TIMP-1with enzyme linked immunosorbent assay(ELISA), mRNA and protein expression of NADPH oxidase p47phox, type1and2receptor (AT1R, AT2R)of angiotensin by real-time polymerase chain reaction(RT-PCR) and Western Bloting.
Results:Compared with young control group, the aging model showed characteristics as following:increased thickness of adventitia, hyperplasia of AF, abnormality of collagen, abnormality of MMP-1/TIMP-1, increased content of AngⅡ, hydroxyproline and O2-of adventitia, decreased expression of mRNA and protein of AT1R, increased expression of mRNA and protein of AT2R and p47phox, declined ability of memory acquisition and preservation. And the Traditional Chinese Medicine could improve the adventitial morphology changes, inhibit hyperplasia of AF, improve the abnormality of collagen as well as MMP-1/TIMP-1,decrease content of AngⅡ, hydroxyproline and O2-of adventitia, increase the expression of mRNA and protein of AT1R and AT2R, decrease the expression of mRNA and protein of p47phox, improve the ability of memory acquisition and preservation of rats.
Conclusion:Remodeling was showed in adventitia of aged vascular, which may happen following the way of AngⅡ, NADPH oxidase p47phox and ROS; The extracts from Radix Ginseng, Radix Notoginseng and Rhizoma Chanxiong could delaying vascular aging possibly by its interference with multitarget, such as AngⅡ and AT1R of adventitia.
Experiment Ⅱ:Experimental Study on Traditional Chinese Medicine of Tonifying Qi and Activating Blood Delaying AF Replicative Senescence
Objective:To observe the patholological changes of replicative senescence in AF, as well as the therapeutic effect of traditional Chinese medicine(extracts from Radix Ginseng, Radix Notoginseng and Rhizoma Chanxiong)
Methods:AFs were induced to aging by generation culvitation in order to make models. The AFs in vitro were divided into5groups:the control group, the cell-senescence untreated group, traditional Chinese medicine high-dose group, and traditional Chinese medicine low-dose group, Vitamin E control group. After intervention, the following indexes were observed with the corresponding methods: changes of aging by senescence-associated-β-galactosidase(SA-β-gal) staining method, status of hyperplasy by MTT (3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2H tetrazolium bromide) assay, mitotic cycle by flow cytometer, the content of MMP-1and TIMP-1by immunocytochemical method, the levels of O2-by biochemical analysis, the content of type Ⅰ collagen and type Ⅲcollagen by ELISA, mRNA and protein expression of NADPH oxidase p47phox, type1and2receptor (AT1R, AT2R)of angiotensin by real-time polymerase chain reaction(RT-PCR) and Western Bloting.
Results:Ageing AF showed characteristics as following:the increased expression of SA-β-gal staining, increase the percentage of AF in S Phase, increased content of type Ⅰ collagen while decreased type Ⅲ collagen, increased expression of TIMP-1while decreased of MMP-1, increased fluorescence intensity of ROS in cells, increased production of O2-, increased expression of mRNA and protein of p47phox and AT2R, decreased expression of mRNA and protein of AT1R. The traditional Chinese medicine could decrease the positive expression of SA-β-gal staining, increase the percentage of AF in G1Phase, improve the abnormality of collagen, increase the expression of type Ⅰ collagen and decrease type Ⅲcollagen, decrease fluorescence intensity of ROS in cells, decrease production of O2-increase the expression of mRNA and protein of AT1R and AT2R, decrease the expression of mRNA and protein of p47phox
Conclusion:The extracts from Radix Ginseng, Radix Notoginseng and Rhizoma Chanxiong could improve AF replicative senescence possibly by the way of NADPH oxidase p47phox and ROS.
Experiment Ⅲ:Experimental Study on Traditional Chinese Medicine of Tonifying Qi and Activating Blood improving proliferation, collagenation abnormality, et al of AF induced by Ang Ⅱ
Objective:To observe the patholological changes of AF induced by Ang Ⅱ, as well as the therapeutic effect of traditional Chinese medicine(extracts from Radix Ginseng, Radix Notoginseng and Rhizoma Chanxiong)
Methods:AFs were induced by AngⅡ in order to make models. The cultured AFs in vitro were divided into5groups:the control group, Ang Ⅱ induced group, traditional Chinese medicine high-dose group, traditional Chinese medicine low-dose group, and valsartan control group. After intervention, the following indexes were observed with the corresponding methods:changes of aging by SA-β-gal staining method, status of hyperplasy by MTT (3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2H tetrazolium bromide) assay, mitotic cycle by flow cytometer, the content of MMP-1and TIMP-1by immunocytochemical method, the levels of O2-by biochemical analysis, the content of type Ⅰ collagen and type Ⅲcollagen by ELISA, mRNA and protein expression of NADPH oxidase p47phox, type1and2receptor (AT1R, AT2R)of angiotensin by real-time polymerase chain reaction(RT-PCR) and Western Bloting.
Results:The AF induced by Ang Ⅱ showed characteristics as following:the increased expression of SA-β-gal staining, increase the percentage of AF in S Phase, increased content of type I collagen while decreased type Ⅲ collagen, increased expression of TIMP-1while decreased of MMP-1, increased fluorescence intensity of ROS in cells, increased production of O2-, increased expression of mRNA and protein of p47phox and AT2R, decreased expression of mRNA and protein of AT1R. The traditional Chinese medicine could decrease the positive expression of SA-β-gal staining, increase the percentage of AF in G1Phase, improve the abnormality of collagen, increase the expression of type Ⅰ collagen and decrease type Ⅲ collagen, decrease fluorescence intensity of ROS in cells, decrease production of O2-, increase the expression of mRNA and protein of AT1R and AT2R, decrease the expression of mRNA and protein of p47phox.
Conclusion:The extracts from Radix Ginseng, Radix Notoginseng and Rhizoma Chanxiong could improve AF induced by AngⅡ possibly by the way of AngⅡ, NADPH oxidase p47phox and ROS.
3. Clinical Research:Study on syndrome types of vascular aging
Objective:To investigate the patterns of syndrome in the population with vascular aging
Methods:231patients with essential hypertension were selected as research object. After physical examinations of both modern medicine and traditional Chinese medicine, the results relating vascular aging were analyzed by the statistics methods of cluster analysis and principal component analysis.
Results:The main patterns of syndrome are the syndrome of blood stasis, the syndrome of deficiency of qi, and the syndrome of yin asthenia of the patients with essential hypertension as well as vascular aging; The main patterns of syndrome are the syndrome of phlegm, the syndrome of blood stasis, and the syndrome of deficiency of qi of the patients with essential hypertension, vascular aging and atherosclerosis.
Conclusion:The syndrome of blood stasis and the syndrome of deficiency of qi, are the main patterns of syndrome of vascular aging.
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