芩丹胶囊抑制血管外膜重构的实验研究
|
摘要
研究背景
原发性高血压是临床常见病和多发病,是引发心、脑血管和肾病变的一个重要的危险因素。血管重构(Vascular remodeling, VR)是高血压靶器官损害的病理基础,发掘和研究能对抗和逆转VR的抗高血压药物具有重大意义。
血管外膜是最早发生病理变化的血管结构,是VR的重要参与者,近年来发现其对高血压的反应较血管内膜和中膜更为敏感。血管外膜的主要细胞类型是外膜成纤维细胞(Adventitial fibroblast, AF),在系统调节机制作用下,成为参与VR的重要细胞。在损伤作用下,AF可发生表型改变、增殖和迁移活性提高,释放细胞外基质(Extracellular matrix, ECM),促进新生外膜形成,导致血管外膜重构。血管外膜在高血压血管重构中的作用越来越受到重视,针对干预血管外膜重构的药物研究具有理论依据和应用价值。
胶原是ECM的重要成分,Ⅰ型和Ⅲ胶原蛋白是血管壁的主要胶原蛋白类型。胶原的合成多于降解,胶原蛋白异常表达是高血压血管病变的重要病理变化。血管壁AF也是合成胶原蛋白的主要细胞类型,转化生长因子β1(Transforming growth factor β1, TGF-β1)是刺激AF合成和胶原蛋白沉积的强大始动因子,可在明显促进胶原蛋白基因转录的同时,抑制新合成胶原蛋白的降解。TGF-β1的经典下游信号通路是Smad信号通路。Smad3是受体调节型Smad(Receptor activated Smad, R-Smad)蛋白的重要成员,在TGF-β1细胞内信号转导通路中发挥着核心的作用。我们的前期体外研究,发现Smad3在TGF-β1诱导的AF转化和胶原沉积中起重要作用。
芩丹胶囊(Qindan Capsule, QC)是导师的临床经验方,用于原发性高血压病肝热血瘀证的治疗。多年来,针对QC的疗效和药理作用机制进行了系列研究。以往的临床实验研究,发现QC能够在有效降压的同时,降低原发性高血压患者血浆中的TGF-β1水平,改善其肝热血瘀证的临床症状和血栓前状态；在体动物实验研究,发现QC能够有效降低自发性高血压大鼠(Spontaneous Hypertensive Rat, SHR)的血压和血浆中的血管紧张素Ⅱ(Angiotonin Ⅱ, AngⅡ)水平,抑制骨桥蛋白(Osteopondin, OPN)表达和血管平滑肌细胞(Vascular Smooth Muscle Cell, VSMC)转化,下调动脉中膜的胶原容积比例,提高动脉形态学指标,起到逆转SHR动脉中膜重构的作用；近期的体外细胞研究,发现QC在降低TGF-β1刺激引起AF的胶原蛋白高表达的同时,抑制了Smad3的活性。本研究在已往研究的基础上,通过对SHR动脉外膜病理变化的观察,及Ⅰ、Ⅲ型胶原和TGF-β1、Smad3在外膜表达的检测,进一步研究和探讨QC对高血压血管外膜重构的作用和机制,为其临床应用提供科学依据。
目的
1.观察芩丹胶囊对SHR大鼠血压和胸主动脉外膜形态学的改变。
2.检测芩丹胶囊对SHR大鼠胸主动脉外膜中Ⅰ、Ⅲ型胶原和TGF-β1、 Smad3表达的改变。
方法
1.研究对象：
(1)实验动物：24只(重250-290g)的14周龄雄性SHR大鼠和8只(重240-280g)的14周龄京都(Wistar-Kyoto, WKY)大鼠。
(2)实验分组：24只SHR大鼠随机平均分为3组：阳性对照组、QC治疗组、氯沙坦治疗组；8只WKY大鼠设为阴性对照组。
(3)实验给药：QC治疗组给予芩丹胶囊750mg/kg/d,氯沙坦治疗组给予氯沙坦30mg/kg/d,阳性对照组和阴性对照组分别给予等量生理盐水。所有大鼠均每日1次同时灌胃,连续12周。
(4)实验取材：大鼠末次灌胃后禁食24h,10%水合氯醛腹腔注射,麻醉后取胸主动脉血管。
2.研究内容：
(1) tail-cuff plethysmographic (TCP)测压法测定大鼠收缩压
(2)Van Gieson (V-G)染色法观察大鼠胸主动脉外膜的组织形态。
(3)逆转录聚合酶链式反应(Reverse transcription PCR, RT-PCR)法检测Ⅰ、Ⅲ型胶原和TGF-β1、Smad3的mRNA表达。
(4)免疫组化法检测Ⅰ、Ⅲ型胶原和TGF-β1、Smad3的蛋白表达。
3.统计学处理：
实验数据均用x±s表示,应用统计软件SPSS20.0进行分析,P＜0.05为差异有统计学意义。t检验用于两组间的比较,双因素方差分析用于多组间的比较。
结果
1.各实验组大鼠血压的比较
在用药前,与阴性对照组中血压正常的WKY大鼠(14周龄)比较,阳性对照组、QC治疗组、氯沙坦治疗组中所有SHR大鼠(14周龄)血压已经明显升高(P＜0.01),阳性对照组和QC治疗组、氯沙坦治疗组相比差异无显著性(P均＞0.05)。在用药过程中,阳性对照组SHR大鼠血压呈升高趋势,QC治疗组、氯沙坦治疗组中所有SHR大鼠血压持续下降,阴性对照组中WKY大鼠血压稳定在正常范围。用药结束,与阴性对照组中血压正常的WKY大鼠(26周龄)相比,阳性对照组SHR大鼠(26周龄)血压显著降低(P＜0.01)。从给药第3周起,QC治疗组、氯沙坦治疗组大鼠和阳性对照组大鼠相比,血压均有下降(P＜0.05或P＜0.01)。给药5周后至给药结束,QC治疗组、氯沙坦治疗组大鼠和阳性对照组大鼠相比,血压有明显下降(P均＜0.01)。给药期间,QC治疗组、氯沙坦治疗组大鼠血压下降的比较,差异无统计学意义(P＞0.05)。
2.各实验组大鼠用药后胸主动脉外膜组织形态比较
V-G染色显示,阴性对照组WKY大鼠血管壁外膜排列整齐,胶原组织分布均匀,着色浅淡；阳性对照组SHR大鼠管壁外膜明显增厚,僵硬变直甚至断裂剥脱,胶原面积明显增加,着色深染；与阳性对照组相比,QC治疗组、氯沙坦治疗组血管壁外膜明显变薄,排列较规整,胶原面积明显减少,着色变浅。QC治疗组和氯沙坦治疗组间的比较无明显差别。
3.各实验组大鼠用药后胸主动脉外膜Ⅰ、Ⅲ型胶原和TGF-β1、Smad3的mRNA表达比较
与阴性对照组26周龄血压正常的WKY大鼠相比,阳性对照组26周龄的SHR大鼠胸主动脉外膜组织中Ⅰ、Ⅲ型胶原和TGF-β1、Smad3的mRNA表达显著升高(P＜0.01)；而同周龄QC治疗组、氯沙坦治疗组SHR大鼠外膜组织中Ⅰ、Ⅲ型胶原和TGF-β1、Smad3的mRNA表达,较阳性对照组SHR大鼠均下降(P＜0.05或P＜0.01)；QC治疗组和氯沙坦治疗组间的比较,差异无统计学意义(P＞0.05)。
4.各实验组大鼠用药后胸主动脉外膜Ⅰ、Ⅲ型胶原和TGF-β1、Smad3的蛋白表达比较
与阴性对照组26周龄血压正常的WKY大鼠相比,阳性对照组26周龄的SHR大鼠胸主动脉外膜组织中Ⅰ、Ⅲ型胶原和TGF-β1、Smad3的蛋白表达显著升高(P＜0.01)；而QC治疗组、氯沙坦治疗组中同周龄SHR大鼠外膜组织中Ⅰ、Ⅲ型胶原和TGF-β1、Smad3的蛋白表达,较阳性对照组SHR大鼠均下降(P＜0.05或P＜0.01)；QC治疗组和氯沙坦治疗组间的比较,差异无统计学意义(P＞0.05)。
结论
1.芩丹胶囊对SHR大鼠有明显的的降压作用,降压机制与降低SHR大鼠血管外膜TGF-β1和Smad3的表达有关。
2.芩丹胶囊能够降低SHR大鼠血管外膜中胶原的表达,改善外膜形态学,对血管外膜重构有抑制或逆转作用,机制与抑制TGF-β1/Smad3信号转导通路有关。
研究背景
血管重构(Vascular remodeling, VR)是心血管疾病发病的病理基础,也是高血压靶器官损害的病理基础,发掘和研究能对抗和逆转VR的抗高血压药物具有重大意义。
血管外膜对血压的改变最为敏感,成纤维细胞是血管外膜中的主要细胞成分,其生物学活性异常可导致血管损伤后不良重构和血管再狭窄的发生。高血压可促使血管外膜成纤维细胞(Adventitial fibroblast, AF)变为肌成纤维细胞(Myofibroblast, MF), MF迁移至中膜或内膜,同时可促进细胞外基质(Extracellular matrix, ECM)合成,发生纤维化,缩窄血管管腔,导致VR发生
转化生长因子β1(Transforming growth factor β1, TGF-β1)与VR的发生和发展关系最为密切,是刺激AF发生表型转换、增殖和迁移、合成ECM最重要和最直接的信号因子。Smad信号通路也是近年发现的,与众多组织纤维化密切相关的TGF-β1下游信号通路。我们以前的研究已证实Smad2和Smad3介导了AF对TGF-β1的反应。而TGF-β1的生物学功能也受丝裂素活化蛋白激酶(Mitogen-activated protein kinase, MAPK)的调控,该作用能被特异性MAPK抑制剂阻断。MAPK信号途径(主要包括ERK、JNK及p38)是细胞内最重要的网络信号之一,参与介导细胞增殖、迁移、分化等多种病理生理过程,与纤维化性疾病的发生密切相关。由受体酪氨酸激酶激活的MAPK通过磷酸化Smads作用可以改变TGF-β1信号。研究具体信号通路TGF-β1/ERK在高血压血管重构中的作用和机制具有重要意义。
血管重构的过程是一个伴随着ECM大量合成与降解的过程。基质金属蛋白酶(Matrix metalloproteinase, MMP)是机体内降解ECM的主要酶系,MMP2和MMP9是其主要成分,基质金属蛋白酶合成(Matrix metalloproteinase synthesis,MMPs)在高血压的血管重构过程中发挥了重要作用。
结缔组织生长因子(Connective tissue growth factor, CTGF)是一种可刺激成纤维细胞增殖和分泌胶原蛋白的生长因子,能够促进细胞有丝分裂、参与凋亡调节及血管形成,与动脉粥样硬化和器官纤维化疾病的发生密切相关。CTGF基因属即刻早期基因,启动子中含有TGF-β1的应答元件,能够被TGF-β1选择性地诱导后激活,并且作为其下游介质,促进ECM的合成。目前,CTGF与血管功能和不良重构的研究成为研究心血管病防治的重要靶点。
高血压病属于中医学“眩晕”范畴,肝热血瘀证是临床常见证型。芩丹胶囊(Qindan capsule, QC)是导师应用多年治疗肝热血瘀型高血压病的临床经验方,以往的研究表明,QC通过影响TGF-β1/Smad信号通路,抑制AF增殖和胶原合成,在逆转老年自发性高血压大鼠(Spontaneous hypertensive rat, SHR)血管外膜重构中发挥重要作用,但是其对AF的生物活性、MMP2和MMP9、CTGF的合成及TGF-β1/ERK信号通路的影响尚不明了。本研究在已往研究的基础上继续探讨QC对TGF-β1诱导的AF增殖和迁移活性、周期和凋亡调控及MMPs和CTGF合成的影响,以及在此过程中TGF-β1/ERK信号转导通路的作用,以期为该临床验方的应用和开发提供更多科学依据。
目的
1.观察QC含药血清对TGF-β1诱导的AF增殖、迁移、周期和凋亡调控的变化,证实QC对TGF-β1诱导的AF生物活性的影响。
2.观察QC含药血清对TGF-β1诱导的AF中细胞外信号调节激酶1/2(Extracellular signal-regulated kinase1/2, ERK1/2)、磷酸化的细胞外信号调节激酶1/2(phospho-ERK1/2, p-ERK1/2)、MMP2、MMP9和CTGF表达的变化,证实QC对AF中基质金属蛋白酶和结缔组织生长因子合成的影响,探讨TGF-β1/ERK信号转导通路在血管外膜重构中的作用。
方法
1.制备芩丹胶囊含药血清：90只(重240-280g)14周龄雄性京都(Wistar-Kyoto, WKY)大鼠随机分成3组：芩丹胶囊大剂量组(QCH组)给予芩丹胶囊750mg/kg/d,芩丹胶囊小剂量组(QCL组)给予芩丹胶囊150mg/kg/d,氯沙坦组(Losartan组)给予氯沙坦30mg/kg/d。各实验组大鼠均予以灌胃给药,每日2次,连续4天。大鼠末次给药后禁食24h,麻醉1.5h后,经腹主动脉取血,分离血清,同一组混匀,滤菌,56℃灭活30min,在低温真空干燥机中制成血清冰冻干粉,-20℃保存备用。
2.外膜成纤维细胞获取和干预：(1)剥脱WKY大鼠胸主动脉血管外膜,组织贴块法原代培养纤维母细胞,冻存、复苏、传代培养成纤维细胞。(2)含药血清预刺激：传代培养的外膜成纤维细胞换无血清培养基培养4h后,在无血清培养基中加入等容的血清冻干粉培养48h。阴性对照组不加任何处理因素。(3)TGF-β1诱导：用含有TGF-β1(20ng/ml)的普通培养液培养24h。
3.实验分组：分为5组：(1)阴性对照(negative control, NC)组(2)阳性对照(positive control, PC)组(3)芩丹胶囊大剂量(QCH)组(4)芩丹胶囊小剂量(QCL)组(5)氯沙坦(Losartan)组
4.研究内容：(1)MTT比色法(MTT colorimetry assay)检测细胞增殖率。(2)划痕技术(Transwell assay)测定细胞迁移能力。(3)流式细胞学检测和分析细胞周期和凋亡调控。(4)反转录酶聚合酶链锁反应(Reverse Transcription-Polymerase Chain Reaction, RT-PCR)检测CTGF.MMP2和MMP9的mRNA表达。(5)蛋白质印迹(Western blot)检测ERK1/2、p-ERK1/2、CTGF、MMP2和MMP9的蛋白表达。
5.统计学处理：
实验数据均用x±s表示,应用统计软件SPSS20.0进行分析,t检验用于两组间的比较,方差分析用于多组间的比较,P<0.05为差异有统计学意义。
结果
1.各实验组AF增殖活性比较
与NC组相比,PC组AF增殖活性明显增强(P＜0.01)；经药物处理后,QCH组、QCL组和Losartan组增殖活性均有减弱,与PC组相比,有统计学差异(P＜0.05或P＜0.01)；QCH组和Losartan组间的比较,无统计学意义(P>0.05)。
2.各实验组AF迁移数量比较
与NC组相比,PC组AF迁移数量明显增多(P＜0.01)；经药物处理后,QCH组、QCL组和Losartan组迁移数量均有减少,与PC组相比,均有统计学差异(P＜0.05)；QCH组和Losartan组间的比较,无统计学意义P＞0.05)。
3.各实验组AF细胞周期比较
与NC组相比,PC组AF的G0/G1和G2/M的细胞百分比明显增大(P＜0.01和P＜0.05)；经药物处理后,QCH组、QCL组和Losartan组AF的G0/G1和G2/M细胞百分比均有减少,与PC组相比,均有统计学差异(P＜0.05)；QCH组和Losartan组间的比较,无统计学意义(P＞0.05)。
4.各实验组AF细胞凋亡比较
与NC组相比,PC组AF的早期凋亡细胞百分比明显减少(P＜0.01)；经药物处理后,QCH组、QCL组和Losartan组早期凋亡细胞百分比均有增大,与PC组相比,均有统计学差异(P＜0.01或P＜0.05)；QCH组和Losartan组间的比较,无统计学意义(P＞0.05)。
5.各实验组AF中CTGF、MMP2和MMP9的mRNA表达比较
与NC组相比,PC组AF的CTGF、MMP2和MMP9的mRNA表达明显增强(P＜0.01)；经药物处理后,QCH组、QCL组和Losartan组CTGF、MMP2和MMP9的mRNA表达明显减弱,与PC组相比,均有统计学差异(P＜0.05)；QCH组和Losartan组间的比较,无统计学意义(P＞0.05)。
6.各实验组AF中ERK1/2、p-ERK1/2、CTGF、MMP2和MMP9的蛋白表达比较
与NC组相比,PC组的p-ERK1/2、CTGF、MMP2和MMP9的蛋白表达明显增强(P＜0.01)；经药物处理后,QCH组、QCL组和Losartan组AF的p-ERK1/2、CTGF、MMP2和MMP9的蛋白表达明显减弱,与PC组相比,均有统计学差异(P＜0.05)；QCH组和Losartan组间的比较,无统计学意义(P＞0.05)。ERK1/2蛋白的表达在所有实验组间的比较,无统计学意义(P＞0.05)。
结论
1.芩丹胶囊能够通过影响TGF-β1诱导的AF的生物活性、基质金属蛋白酶和结缔组织生长因子合成,具有抑制或逆转血管外膜重构的作用。
2.芩丹胶囊能够抑制TGF-β1/ERK信号转导通路,可能与抑制或逆转血管外膜重构的机制有关。
Background
As a clinical common disease and frequently occurring disease, essential hypertension is an important risk factor to vascular diseases of heart, brain and kidney. Vascular remodeling (VR) is the pathological basis of target organ damage during high blood pressure. It is of be great significance to discover and study the antihypertensive drugs that can fight and reverse VR.
Vascular adventitia is an important participant in VR. Recent studies have shown that the adventitia is the most sensitive layer to respond to blood pressure, and is the earliest vascular structure in which pathological changes occur. Adventitial fibroblasts (AFs), the main adventitial cells, play a key role in controlling VR through regulatory systems. Under the action of damage, AFs can change phenotype, increase activity of proliferation and migration, release the extracellular matrix, promote new adventitia formation, and participate in the occurrence of vascular adventitia remodeling. The role of vascular adventitia in the vascular remodeling during hypertension is increasingly arousing attention in clinically. Therefore, the research on medicine to intervene VR has theoretical basis and application value.
The main components of extracellular matrix (ECM) is collagen, with type I and III as the main types in vascular wall. The abnormal synthesis and expression in collagen is regarded as the important pathological changes of vascular lesions in hypertension. Transforming growth factor β1(TGF β1) is powerful initiating factor to stimulate the synthesis and deposition of collagen in AFs. The classical signal transduction pathway of the TGF-P family is based on Smad signal pathway. Smad3belongs to receptor activated Smad (R-Smad) and plays a core role in the TGF-β1intracellular signal transduction pathways. Our previous study showed that Smad3can respond to collagen deposition in AFs induced by TGF-β1.
Qindan capsule (QC), a prepared compound used in traditional Chinese medicine, has been used as an anti-hypertensive agent in clinical settings for years to treat patients with essential hypertension manifests as "liver fire and blood stagnation" symptoms. Our previous clinical study showed that QC can decrease blood pressure and plasma TGF-β1levels so as to help in clearing up clinical symptoms and the prothrombotic state of of this kind of patients.
Additionally, our previous animal studies showed that QC has the actions in decreasing blood pressure and angiotensin-Ⅱ level, inhibiting or reversing aorta damage by improving the morphological index of the artery, down-regulating the collagen volume fraction in the media and inhibiting the expression of osteopontin and the transformation of smooth muscle cells. The latest in vitro research has confirmed that QC can reduce the high expression of collagen and inhibit the activity of Smad3excited by TGF-β1in AFs.
This research is based on the past studies, further study and discuss the effect and mechanism of QC on vascular remodeling in hypertension by observing the pathological changes and detecting Ⅰ,Ⅲcollagen and TGF-β1and Smad3expression in adventitia of spontaneously hypertensive rats (SHRs). Our study will provide scientific reason for the clinical application of QC.
Aims
1. To investigate the effect of QC on blood pressure and vascular adventitial morphology change in SHRs.
2. To investigate the effect of QC on collagen synthesis and the mechanism underlying the process in SHRs.
Methods
1. Object of study
(1) Experimental rats:Twenty-four male SHRs aged14weeks and weighing 250-290g and8male WKY rats aged14weeks and weighing240-280g were used in the study.
(2) Experimental groups:Twenty-four SHRs were divided into three groups: the positive control (PC) group, the QC treatment (QD) group, and the losartan treatment (LSD) group. Eight male WKY rats were included in the negative control (NC) group.
(3) Experimental treatment:Rats in the QD group were administered QC at a dose of750mg/kg/day through irrigation of the stomach. Rats in the LSD group were administered losartan at a dose of30mg/kg/day through irrigation of the stomach. Rats in the PC group and the NC group were administered an equal volume of distilled water for three months.
(4) Obtain adventitia:Rats were fast for24h at the end of medical administration of whole-day drugs and anesthetized with10%chloral hydrate by intraperitoneal injection. The thoracic aorta adventitia of the experimental rats was segregated.
2. Contents of study
(1) The systolic blood pressure (SBP) of the rats was monitored by tail-cuff plethysmographic (TCP) method.
(2) The thoracic aorta adventitia was observed by Van Gieson (V-G) histological staining method.
(3) The mRNA expression of TGF-β1, Smad3, and collagens Ⅰ and Ⅲ was measured by reverse transcription PCR method.
(4) The protein expression of TGF-β1, Smad3, and collagens Ⅰ and Ⅲ was measured by immunohistochemical staining method.
3. Statistical analysis
Data are expressed as mean±SD. All of the experiments data were analyzed by SPSS20.0software. An independent-sample t-test was applied when only two groups were compared, whereas comparisons among groups were analyzed by two-way ANOVA. Differences were considered to be statistically significant when the P values were less than0.05.
Results
1. Comparison of SBP before and after experimental treatments.
Before experimental treatment, compared with WKY rats (aged14weeks) in NC group, the SBP of SHR rats (aged14weeks) in PC, QD and LSD groups were significantly increased, the difference was statistically significant (p<0.01), the compares among PC, QD and LSD groups were no statistically significance (p>0.05). During experimental treatment, the SBP of SHR rats in PC group was keeping high level, the SBP of SHR rats in QD and LSD groups was significantly continue decreased, and the SBP of WKY rats in PC group was keeping normal level. By the end of experimental treatment, compared with WKY rats (aged26weeks) in NC group, the SBP of SHR rats (aged26weeks) in QD and LSD groups were significantly decreased, the difference was statistically significant (p<0.01). From the third week after the treatments, the compares between PC, QD and LSD groups have statistical difference (p<0.05or p<0.01). From the fifth week after the treatments to the end of treatments, the compares between PC, QD and LSD groups both had significantly statistical difference (p<0.01) and the compare between QD and LSD groups was no statistically significance (p>0.05)
2. Morphological observation of thoracic aorta adventitia histological after experimental treatments.
The Van Gieson stain showed that the vascular adventitia in the PC group was thicker than that in the NC group, and QC-and losartan-treated rats showed lower adventitial thicknesses than the control rats in the PC group. Compared with WKY rats, the collagen fibers of the adventitia in SHRs showed significant hyperplasia and were intensely dyed. However, a significant reverse effect was observed in the QD group and the LSD group at the end of treatment. The QD group was not significantly different from the LSD group.
3. Comparison of the mRNA expression of TGF-β1, Smad3, and collagens Ⅰ and Ⅲ after experimental treatments.
The expression of TGF-β1, Smad3, and collagens Ⅰ and Ⅲ in the PC group was significantly increased compared with that in the NC group (p<0.01). However, the expression was significantly decreased in the QD group and the LSD group compared with the PC group (p<0.05or p<0.01). There was no significant difference in TGF-β1, Smad3, or collagen Ⅰ and Ⅲ mRNA expression between the QD group and the LSD group (p>0.05).
4. Comparison of the protein expression of TGF-β1, Smad3, and collagens Ⅰ and Ⅲ after experimental treatments.
TGF-β1, Smad3, and collagen Ⅰ and Ⅲ expression in the PC group was significantly increased compared with that in the NC group (p<0.01). However, the expression of TGF-β1, Smad3, and collagens Ⅰ and Ⅲ was significantly decreased in the QD group and the LSD group compared with the PC group (p<0.05or p<0.01). There was no significant difference in TGF-β1, Smad3, or collagen Ⅰ and Ⅲ protein expression between the QD group and the LSD group (p>0.05).
Conclusions
1. The significantly antihypertensive effect of QC on SHR rats was verified again, our study suggested that its antihypertensive mechanism through down-regulating the expression of TGF-β1, Smad3, and collagen Ⅰ and Ⅲ in thoracic aorta adventitia.
2. QC could reduce collagen content of SHR rats in vascular adventitia, improve the adventitia morphology of vascular and reverse the vascular remodeling. The possible mechanism was related to inhibit the TGF-β1/Smad3signal pathway.
Background
Harmful vascular remodeling (VR) caused by vascular hyperplastic lesions is the common pathological basis on a variety of cardiovascular diseases. Fibroblasts are the most abundant cell type in the adventitia, which lead to vascular harmful reconstruction and restenosis after damage. Hypertention could prompt the adventitial fibroblasts (AFs) turn into myofibroblasts (MFs), MFs migrate to the tunica media or the intima, and prompt the composition or fibrosis of extracellular matrix (ECM) at the same time, and lead to coarctation or VR. More and more evidence to support the AFs is "the central part" of vascular hyperplastic lesions, and become a new target to the prevention and control of harmful VR.
The studies have found that the TGF-β1is the most important and the most direct cell factor to promote AFs to proliferation, migration, phenotypic switching and synthetic function, and has most close relations to vascular adventitia reconstruction. Smad signal path is a TGF-β1downstream signaling pathways found in recent years and closely related with many tissue fibrosis. Our previous studies have confirmed that Smad2and Smad3mediated the AFs to TGF-β1reaction.
And the biological function of TGF-β1is also controlled by the mitogen activated protein kinase (MAPK). This effect can be blocked by specific MAPK inhibitor. MAPK signaling pathways (mainly including ERK, JNK and p38) is one of the most important intracellular signals in the network. It is involved in many kinds of pathophysiological process such as mediating cell proliferation, differentiation and so on, and closely related with the happening of fibrosis diseases. The receptor tyrosine kinase activated MAPK may be participate in cross-talk with the Smad pathway by phosphorylation and change the TGF-β1signal. But there has little research for specific signal path TGF-β1/ERK on vascular remodeling in hypertension so far.
The process of VR is along with synthesis and degradation of ECM. Matrix metalloproteinase (MMP) is in the main enzyme system in degradation of ECM, MMP2and MMP9are the main composition. Matrix metalloproteinase synthesis (MMPs) participated in the process of hypertensive heart and vascular reconstruction.
Connective tissue growth factor (CTGF) is a kind of ecretory polypeptide with rich cysteine. The main biological activities are to promote cell mitosis, chemotactic cytokines, inducing adhesion, apoptosis regulation and the formation of blood vessels, it also can promote cell proliferation and ECM synthesis. CTGF can be secreted by fibroblast under the induction of TGF-β1. The studies found that the CTGF excessive expression is closely related with the happening of some hyperplastic or fibrosis disease s. However, the relation between CTGF and vascular function has been less reported.
Hypertension disease belongs to the "vertigo" category in traditional Chinese medicine,"live fire and blood stagnation"s the common symptoms. Qindan capsule (QC) is a clinical empirical formula by the tutor to treat hypertension patients with "liver fire and blood stagnation" symptoms. Our previous clinical study showed that QC could influent TGF-β1/Smad signaling pathways, restrain AFs proliferation and collagen synthesis and play an important role in reversing vascular adventitia remodeling of SHRs.
But the influence on the AFs biological activity. MMP2, MMP9, CTGF, and TGF-β1/ERK signaling pathway is still unknown. This research is based on our previous studies to continue to explore the influence of QC on AFs proliferation, migration, cycle and apoptosis regulation and MMP2and MMP9, CTGF synthesis induced by TGF-β1, and the role of ERK signal transduction pathway during the process. Our study would provide more scientific basis for the clinical application of QC
Aims
1. To investigate the effect of QC medicated serum on the biological activity of AFs induced by TGF-(31by observing the proliferation, migration, cycle and apoptosis regulation.
2. To investigate the role of TGF-β1/ERK signaling pathway by observing the expression of extracellular signal-regulated kinase1/2(ERK1/2)、phospho-ERK1/2(p-ERK1/2)、CTGF、MMP2and MMP9in AFs induced by TGF-β1and affected by QC medicated serum.
Methods
1. Preparation of QC-containing serum
Ninety male WKY rats (weight:240-280g) were divided into three groups randomly:the high dosage QC group (QCH,750mg/kg/d), the low dosage QC group (QCL,150mg/kg/d) and Losartan group (30mg/kg/d)). Medicines were given2times a day and four consecutive days. Rats were starved for24h and were anaesthetised for1.5h after the final medicine treatment. Blood was drawn from the abdominal aorta. Serum was separated and mixed in the same group, filtered bacteria, at56℃inactivated for30min. Serum freeze dry powder was made in low temperature vacuum drying machine and was stored at-20℃for use.
2. Acquisition and intervention of AFs
(1) Vascular adventitia was stripped from thoracic aorta of WKY rat. Paste tissue pieces method was used to cultivate original generation fibroblasts. AFs were frozen storage, carried anabiosis and subculture.
(2) Medicated serum preliminary stimulus:The subcultured AFs were continue to be cul tured in serum free medium for4h, then isovolumetric serum freeze-dried powder was added and continue to be cultured for48h. And the negative control group was not given any processing factors.
(3) TGF-β1induced AFs:AFs were cultured by common nutrient solution with TGF-β1(20ng/ml) for24h.
3. Experimental groups
The AFs were divided into5groups as follows:
(1) The negative control group (NC).
(2) The positive control group (PC).
(3) The QC high dose group (QCH).
(4) The QC low dose group (QCL).
(5) The Losartan group (Losartan).
4. Contents of study
(1) The cell proliferation rate was measured by MTT colorimetry assay.
(2) The cell migration ability was measured by transwell assay.
(3) The cell cycle and apoptosis regulation was measured by flow cytometry analysis.
(4) The mRNA expression of MMP2, MMP9and CTGF was measured by reverse transcription-polymerase chain reaction (RT-PCR).
(5) The protein expression of MMP2, MMP9and ERK1/2, p-ERK1/2and CTGF was measured by western blot.
5. Statistical analysis
Data are expressed as mean±SD. All of the experiments data were analyzed by SPSS20.0software. An independent-sample t-test was applied when only two groups were compared, whereas comparisons among groups were analyzed by ANOVA. Differences were considered to be statistically significant when the P values were less than0.05.
Results
1. Comparison of proliferation activity of AFs among experimental groups
The proliferation activity of AFs in the PC group was significantly increased compared with that in NC group (P<0.01). After treated by medicine, the proliferation activity of AFs in QCH, QCL and Losartan group was decreased compared with that in PC group (P<0.05or P<0.01). The compare between QCH and Losartan groups has no statistically significance (P>0.05).
2. Comparison of migration number of AFs among experimental groups
The migration number of AFs in the PC group was significantly increased compared with that in NC group (P<0.01). After treated by medicine, the cell migration number of AFs in QCH, QCL and Losartan group was decreased compared with that in PC group (P<0.05). The compare between QCH and Losartan groups has no statistically significance (P>0.05).
3. Comparison of cell cycle of AFs among experimental groups
The cell percentage of G0/G1and G2/M in the PC group was increased compared with that in NC group (.P<0.05or P<0.01). After treated by medicine, the cell percentage of G0/G1and G2/M in QCH, QCL and Losartan group was decreased compared with that in PC group (P<0.05). The compare between QCH and Losartan groups has no statistically significance (P>0.05).
4. Comparison of cell apoptisis of AFs among experimental groups
The cell percentage of early apoptosis in the PC group was significantly decreased compared with that in NC group (P<0.01). After treated by medicine, the cell percentage of early apoptosis in QCH, QCL and Losartan group was increased compared with that in PC group (P<0.05). The compare between QCH and Losartan groups has no statistically significance (P>0.05).
5. Comparison of the mRNA expression of MMP2, MMP9and CTGF in AFs among experimental groups
The mRNA expression of MMP2, MMP9and CTGF in the PC group was significantly increased compared with that in NC group (P<0.01). After treated by medicine, the mRNA expression of MMP2, MMP9and CTGF in QCH, QCL and Losartan group was decreased compared with that in PC group (P<0.05). The compare between QCH and Losartan groups has no statistically significance (P>0.05).
6. Comparison of protein expression of MMP2, MMP9and ERK1/2, p-ERK1/2and CTGF in AFs among experimental groups
The protein expression of MMP2, MMP9and p-ERK1/2and CTGF in the PC group was significantly increased compared with that in NC group (P<0.01). After treated by medicine, the protein expression of MMP2, MMP9and p-ERK1/2and CTGF in QCH, QCL and Losartan group was decreased compared with that in PC group (P<0.05). The compare between QCH and Losartan groups has no statistically significance (P>0.05).
The protein expression of ERK1/2among five experimental groups has no statistically significance (P>0.05).
Conclusion
1. QC could inhibit or reverse vascular adventitia remodeling by inhibiting the biological activity of AFs and the synthesis of MMP2, MMP9and CTGF induced by TGF-β1.
2. The mechanism of QC to inhibit or reverse vascular adventitia remodeling maybe related to the inhibiting effect of TGF-β1/ERK signaling pathway.
原发性高血压是临床常见病和多发病,是引发心、脑血管和肾病变的一个重要的危险因素。血管重构(Vascular remodeling, VR)是高血压靶器官损害的病理基础,发掘和研究能对抗和逆转VR的抗高血压药物具有重大意义。
血管外膜是最早发生病理变化的血管结构,是VR的重要参与者,近年来发现其对高血压的反应较血管内膜和中膜更为敏感。血管外膜的主要细胞类型是外膜成纤维细胞(Adventitial fibroblast, AF),在系统调节机制作用下,成为参与VR的重要细胞。在损伤作用下,AF可发生表型改变、增殖和迁移活性提高,释放细胞外基质(Extracellular matrix, ECM),促进新生外膜形成,导致血管外膜重构。血管外膜在高血压血管重构中的作用越来越受到重视,针对干预血管外膜重构的药物研究具有理论依据和应用价值。
胶原是ECM的重要成分,Ⅰ型和Ⅲ胶原蛋白是血管壁的主要胶原蛋白类型。胶原的合成多于降解,胶原蛋白异常表达是高血压血管病变的重要病理变化。血管壁AF也是合成胶原蛋白的主要细胞类型,转化生长因子β1(Transforming growth factor β1, TGF-β1)是刺激AF合成和胶原蛋白沉积的强大始动因子,可在明显促进胶原蛋白基因转录的同时,抑制新合成胶原蛋白的降解。TGF-β1的经典下游信号通路是Smad信号通路。Smad3是受体调节型Smad(Receptor activated Smad, R-Smad)蛋白的重要成员,在TGF-β1细胞内信号转导通路中发挥着核心的作用。我们的前期体外研究,发现Smad3在TGF-β1诱导的AF转化和胶原沉积中起重要作用。
芩丹胶囊(Qindan Capsule, QC)是导师的临床经验方,用于原发性高血压病肝热血瘀证的治疗。多年来,针对QC的疗效和药理作用机制进行了系列研究。以往的临床实验研究,发现QC能够在有效降压的同时,降低原发性高血压患者血浆中的TGF-β1水平,改善其肝热血瘀证的临床症状和血栓前状态；在体动物实验研究,发现QC能够有效降低自发性高血压大鼠(Spontaneous Hypertensive Rat, SHR)的血压和血浆中的血管紧张素Ⅱ(Angiotonin Ⅱ, AngⅡ)水平,抑制骨桥蛋白(Osteopondin, OPN)表达和血管平滑肌细胞(Vascular Smooth Muscle Cell, VSMC)转化,下调动脉中膜的胶原容积比例,提高动脉形态学指标,起到逆转SHR动脉中膜重构的作用；近期的体外细胞研究,发现QC在降低TGF-β1刺激引起AF的胶原蛋白高表达的同时,抑制了Smad3的活性。本研究在已往研究的基础上,通过对SHR动脉外膜病理变化的观察,及Ⅰ、Ⅲ型胶原和TGF-β1、Smad3在外膜表达的检测,进一步研究和探讨QC对高血压血管外膜重构的作用和机制,为其临床应用提供科学依据。
目的
1.观察芩丹胶囊对SHR大鼠血压和胸主动脉外膜形态学的改变。
2.检测芩丹胶囊对SHR大鼠胸主动脉外膜中Ⅰ、Ⅲ型胶原和TGF-β1、 Smad3表达的改变。
方法
1.研究对象：
(1)实验动物：24只(重250-290g)的14周龄雄性SHR大鼠和8只(重240-280g)的14周龄京都(Wistar-Kyoto, WKY)大鼠。
(2)实验分组：24只SHR大鼠随机平均分为3组：阳性对照组、QC治疗组、氯沙坦治疗组；8只WKY大鼠设为阴性对照组。
(3)实验给药：QC治疗组给予芩丹胶囊750mg/kg/d,氯沙坦治疗组给予氯沙坦30mg/kg/d,阳性对照组和阴性对照组分别给予等量生理盐水。所有大鼠均每日1次同时灌胃,连续12周。
(4)实验取材：大鼠末次灌胃后禁食24h,10%水合氯醛腹腔注射,麻醉后取胸主动脉血管。
2.研究内容：
(1) tail-cuff plethysmographic (TCP)测压法测定大鼠收缩压
(2)Van Gieson (V-G)染色法观察大鼠胸主动脉外膜的组织形态。
(3)逆转录聚合酶链式反应(Reverse transcription PCR, RT-PCR)法检测Ⅰ、Ⅲ型胶原和TGF-β1、Smad3的mRNA表达。
(4)免疫组化法检测Ⅰ、Ⅲ型胶原和TGF-β1、Smad3的蛋白表达。
3.统计学处理：
实验数据均用x±s表示,应用统计软件SPSS20.0进行分析,P＜0.05为差异有统计学意义。t检验用于两组间的比较,双因素方差分析用于多组间的比较。
结果
1.各实验组大鼠血压的比较
在用药前,与阴性对照组中血压正常的WKY大鼠(14周龄)比较,阳性对照组、QC治疗组、氯沙坦治疗组中所有SHR大鼠(14周龄)血压已经明显升高(P＜0.01),阳性对照组和QC治疗组、氯沙坦治疗组相比差异无显著性(P均＞0.05)。在用药过程中,阳性对照组SHR大鼠血压呈升高趋势,QC治疗组、氯沙坦治疗组中所有SHR大鼠血压持续下降,阴性对照组中WKY大鼠血压稳定在正常范围。用药结束,与阴性对照组中血压正常的WKY大鼠(26周龄)相比,阳性对照组SHR大鼠(26周龄)血压显著降低(P＜0.01)。从给药第3周起,QC治疗组、氯沙坦治疗组大鼠和阳性对照组大鼠相比,血压均有下降(P＜0.05或P＜0.01)。给药5周后至给药结束,QC治疗组、氯沙坦治疗组大鼠和阳性对照组大鼠相比,血压有明显下降(P均＜0.01)。给药期间,QC治疗组、氯沙坦治疗组大鼠血压下降的比较,差异无统计学意义(P＞0.05)。
2.各实验组大鼠用药后胸主动脉外膜组织形态比较
V-G染色显示,阴性对照组WKY大鼠血管壁外膜排列整齐,胶原组织分布均匀,着色浅淡；阳性对照组SHR大鼠管壁外膜明显增厚,僵硬变直甚至断裂剥脱,胶原面积明显增加,着色深染；与阳性对照组相比,QC治疗组、氯沙坦治疗组血管壁外膜明显变薄,排列较规整,胶原面积明显减少,着色变浅。QC治疗组和氯沙坦治疗组间的比较无明显差别。
3.各实验组大鼠用药后胸主动脉外膜Ⅰ、Ⅲ型胶原和TGF-β1、Smad3的mRNA表达比较
与阴性对照组26周龄血压正常的WKY大鼠相比,阳性对照组26周龄的SHR大鼠胸主动脉外膜组织中Ⅰ、Ⅲ型胶原和TGF-β1、Smad3的mRNA表达显著升高(P＜0.01)；而同周龄QC治疗组、氯沙坦治疗组SHR大鼠外膜组织中Ⅰ、Ⅲ型胶原和TGF-β1、Smad3的mRNA表达,较阳性对照组SHR大鼠均下降(P＜0.05或P＜0.01)；QC治疗组和氯沙坦治疗组间的比较,差异无统计学意义(P＞0.05)。
4.各实验组大鼠用药后胸主动脉外膜Ⅰ、Ⅲ型胶原和TGF-β1、Smad3的蛋白表达比较
与阴性对照组26周龄血压正常的WKY大鼠相比,阳性对照组26周龄的SHR大鼠胸主动脉外膜组织中Ⅰ、Ⅲ型胶原和TGF-β1、Smad3的蛋白表达显著升高(P＜0.01)；而QC治疗组、氯沙坦治疗组中同周龄SHR大鼠外膜组织中Ⅰ、Ⅲ型胶原和TGF-β1、Smad3的蛋白表达,较阳性对照组SHR大鼠均下降(P＜0.05或P＜0.01)；QC治疗组和氯沙坦治疗组间的比较,差异无统计学意义(P＞0.05)。
结论
1.芩丹胶囊对SHR大鼠有明显的的降压作用,降压机制与降低SHR大鼠血管外膜TGF-β1和Smad3的表达有关。
2.芩丹胶囊能够降低SHR大鼠血管外膜中胶原的表达,改善外膜形态学,对血管外膜重构有抑制或逆转作用,机制与抑制TGF-β1/Smad3信号转导通路有关。
研究背景
血管重构(Vascular remodeling, VR)是心血管疾病发病的病理基础,也是高血压靶器官损害的病理基础,发掘和研究能对抗和逆转VR的抗高血压药物具有重大意义。
血管外膜对血压的改变最为敏感,成纤维细胞是血管外膜中的主要细胞成分,其生物学活性异常可导致血管损伤后不良重构和血管再狭窄的发生。高血压可促使血管外膜成纤维细胞(Adventitial fibroblast, AF)变为肌成纤维细胞(Myofibroblast, MF), MF迁移至中膜或内膜,同时可促进细胞外基质(Extracellular matrix, ECM)合成,发生纤维化,缩窄血管管腔,导致VR发生
转化生长因子β1(Transforming growth factor β1, TGF-β1)与VR的发生和发展关系最为密切,是刺激AF发生表型转换、增殖和迁移、合成ECM最重要和最直接的信号因子。Smad信号通路也是近年发现的,与众多组织纤维化密切相关的TGF-β1下游信号通路。我们以前的研究已证实Smad2和Smad3介导了AF对TGF-β1的反应。而TGF-β1的生物学功能也受丝裂素活化蛋白激酶(Mitogen-activated protein kinase, MAPK)的调控,该作用能被特异性MAPK抑制剂阻断。MAPK信号途径(主要包括ERK、JNK及p38)是细胞内最重要的网络信号之一,参与介导细胞增殖、迁移、分化等多种病理生理过程,与纤维化性疾病的发生密切相关。由受体酪氨酸激酶激活的MAPK通过磷酸化Smads作用可以改变TGF-β1信号。研究具体信号通路TGF-β1/ERK在高血压血管重构中的作用和机制具有重要意义。
血管重构的过程是一个伴随着ECM大量合成与降解的过程。基质金属蛋白酶(Matrix metalloproteinase, MMP)是机体内降解ECM的主要酶系,MMP2和MMP9是其主要成分,基质金属蛋白酶合成(Matrix metalloproteinase synthesis,MMPs)在高血压的血管重构过程中发挥了重要作用。
结缔组织生长因子(Connective tissue growth factor, CTGF)是一种可刺激成纤维细胞增殖和分泌胶原蛋白的生长因子,能够促进细胞有丝分裂、参与凋亡调节及血管形成,与动脉粥样硬化和器官纤维化疾病的发生密切相关。CTGF基因属即刻早期基因,启动子中含有TGF-β1的应答元件,能够被TGF-β1选择性地诱导后激活,并且作为其下游介质,促进ECM的合成。目前,CTGF与血管功能和不良重构的研究成为研究心血管病防治的重要靶点。
高血压病属于中医学“眩晕”范畴,肝热血瘀证是临床常见证型。芩丹胶囊(Qindan capsule, QC)是导师应用多年治疗肝热血瘀型高血压病的临床经验方,以往的研究表明,QC通过影响TGF-β1/Smad信号通路,抑制AF增殖和胶原合成,在逆转老年自发性高血压大鼠(Spontaneous hypertensive rat, SHR)血管外膜重构中发挥重要作用,但是其对AF的生物活性、MMP2和MMP9、CTGF的合成及TGF-β1/ERK信号通路的影响尚不明了。本研究在已往研究的基础上继续探讨QC对TGF-β1诱导的AF增殖和迁移活性、周期和凋亡调控及MMPs和CTGF合成的影响,以及在此过程中TGF-β1/ERK信号转导通路的作用,以期为该临床验方的应用和开发提供更多科学依据。
目的
1.观察QC含药血清对TGF-β1诱导的AF增殖、迁移、周期和凋亡调控的变化,证实QC对TGF-β1诱导的AF生物活性的影响。
2.观察QC含药血清对TGF-β1诱导的AF中细胞外信号调节激酶1/2(Extracellular signal-regulated kinase1/2, ERK1/2)、磷酸化的细胞外信号调节激酶1/2(phospho-ERK1/2, p-ERK1/2)、MMP2、MMP9和CTGF表达的变化,证实QC对AF中基质金属蛋白酶和结缔组织生长因子合成的影响,探讨TGF-β1/ERK信号转导通路在血管外膜重构中的作用。
方法
1.制备芩丹胶囊含药血清：90只(重240-280g)14周龄雄性京都(Wistar-Kyoto, WKY)大鼠随机分成3组：芩丹胶囊大剂量组(QCH组)给予芩丹胶囊750mg/kg/d,芩丹胶囊小剂量组(QCL组)给予芩丹胶囊150mg/kg/d,氯沙坦组(Losartan组)给予氯沙坦30mg/kg/d。各实验组大鼠均予以灌胃给药,每日2次,连续4天。大鼠末次给药后禁食24h,麻醉1.5h后,经腹主动脉取血,分离血清,同一组混匀,滤菌,56℃灭活30min,在低温真空干燥机中制成血清冰冻干粉,-20℃保存备用。
2.外膜成纤维细胞获取和干预：(1)剥脱WKY大鼠胸主动脉血管外膜,组织贴块法原代培养纤维母细胞,冻存、复苏、传代培养成纤维细胞。(2)含药血清预刺激：传代培养的外膜成纤维细胞换无血清培养基培养4h后,在无血清培养基中加入等容的血清冻干粉培养48h。阴性对照组不加任何处理因素。(3)TGF-β1诱导：用含有TGF-β1(20ng/ml)的普通培养液培养24h。
3.实验分组：分为5组：(1)阴性对照(negative control, NC)组(2)阳性对照(positive control, PC)组(3)芩丹胶囊大剂量(QCH)组(4)芩丹胶囊小剂量(QCL)组(5)氯沙坦(Losartan)组
4.研究内容：(1)MTT比色法(MTT colorimetry assay)检测细胞增殖率。(2)划痕技术(Transwell assay)测定细胞迁移能力。(3)流式细胞学检测和分析细胞周期和凋亡调控。(4)反转录酶聚合酶链锁反应(Reverse Transcription-Polymerase Chain Reaction, RT-PCR)检测CTGF.MMP2和MMP9的mRNA表达。(5)蛋白质印迹(Western blot)检测ERK1/2、p-ERK1/2、CTGF、MMP2和MMP9的蛋白表达。
5.统计学处理：
实验数据均用x±s表示,应用统计软件SPSS20.0进行分析,t检验用于两组间的比较,方差分析用于多组间的比较,P<0.05为差异有统计学意义。
结果
1.各实验组AF增殖活性比较
与NC组相比,PC组AF增殖活性明显增强(P＜0.01)；经药物处理后,QCH组、QCL组和Losartan组增殖活性均有减弱,与PC组相比,有统计学差异(P＜0.05或P＜0.01)；QCH组和Losartan组间的比较,无统计学意义(P>0.05)。
2.各实验组AF迁移数量比较
与NC组相比,PC组AF迁移数量明显增多(P＜0.01)；经药物处理后,QCH组、QCL组和Losartan组迁移数量均有减少,与PC组相比,均有统计学差异(P＜0.05)；QCH组和Losartan组间的比较,无统计学意义P＞0.05)。
3.各实验组AF细胞周期比较
与NC组相比,PC组AF的G0/G1和G2/M的细胞百分比明显增大(P＜0.01和P＜0.05)；经药物处理后,QCH组、QCL组和Losartan组AF的G0/G1和G2/M细胞百分比均有减少,与PC组相比,均有统计学差异(P＜0.05)；QCH组和Losartan组间的比较,无统计学意义(P＞0.05)。
4.各实验组AF细胞凋亡比较
与NC组相比,PC组AF的早期凋亡细胞百分比明显减少(P＜0.01)；经药物处理后,QCH组、QCL组和Losartan组早期凋亡细胞百分比均有增大,与PC组相比,均有统计学差异(P＜0.01或P＜0.05)；QCH组和Losartan组间的比较,无统计学意义(P＞0.05)。
5.各实验组AF中CTGF、MMP2和MMP9的mRNA表达比较
与NC组相比,PC组AF的CTGF、MMP2和MMP9的mRNA表达明显增强(P＜0.01)；经药物处理后,QCH组、QCL组和Losartan组CTGF、MMP2和MMP9的mRNA表达明显减弱,与PC组相比,均有统计学差异(P＜0.05)；QCH组和Losartan组间的比较,无统计学意义(P＞0.05)。
6.各实验组AF中ERK1/2、p-ERK1/2、CTGF、MMP2和MMP9的蛋白表达比较
与NC组相比,PC组的p-ERK1/2、CTGF、MMP2和MMP9的蛋白表达明显增强(P＜0.01)；经药物处理后,QCH组、QCL组和Losartan组AF的p-ERK1/2、CTGF、MMP2和MMP9的蛋白表达明显减弱,与PC组相比,均有统计学差异(P＜0.05)；QCH组和Losartan组间的比较,无统计学意义(P＞0.05)。ERK1/2蛋白的表达在所有实验组间的比较,无统计学意义(P＞0.05)。
结论
1.芩丹胶囊能够通过影响TGF-β1诱导的AF的生物活性、基质金属蛋白酶和结缔组织生长因子合成,具有抑制或逆转血管外膜重构的作用。
2.芩丹胶囊能够抑制TGF-β1/ERK信号转导通路,可能与抑制或逆转血管外膜重构的机制有关。
Background
As a clinical common disease and frequently occurring disease, essential hypertension is an important risk factor to vascular diseases of heart, brain and kidney. Vascular remodeling (VR) is the pathological basis of target organ damage during high blood pressure. It is of be great significance to discover and study the antihypertensive drugs that can fight and reverse VR.
Vascular adventitia is an important participant in VR. Recent studies have shown that the adventitia is the most sensitive layer to respond to blood pressure, and is the earliest vascular structure in which pathological changes occur. Adventitial fibroblasts (AFs), the main adventitial cells, play a key role in controlling VR through regulatory systems. Under the action of damage, AFs can change phenotype, increase activity of proliferation and migration, release the extracellular matrix, promote new adventitia formation, and participate in the occurrence of vascular adventitia remodeling. The role of vascular adventitia in the vascular remodeling during hypertension is increasingly arousing attention in clinically. Therefore, the research on medicine to intervene VR has theoretical basis and application value.
The main components of extracellular matrix (ECM) is collagen, with type I and III as the main types in vascular wall. The abnormal synthesis and expression in collagen is regarded as the important pathological changes of vascular lesions in hypertension. Transforming growth factor β1(TGF β1) is powerful initiating factor to stimulate the synthesis and deposition of collagen in AFs. The classical signal transduction pathway of the TGF-P family is based on Smad signal pathway. Smad3belongs to receptor activated Smad (R-Smad) and plays a core role in the TGF-β1intracellular signal transduction pathways. Our previous study showed that Smad3can respond to collagen deposition in AFs induced by TGF-β1.
Qindan capsule (QC), a prepared compound used in traditional Chinese medicine, has been used as an anti-hypertensive agent in clinical settings for years to treat patients with essential hypertension manifests as "liver fire and blood stagnation" symptoms. Our previous clinical study showed that QC can decrease blood pressure and plasma TGF-β1levels so as to help in clearing up clinical symptoms and the prothrombotic state of of this kind of patients.
Additionally, our previous animal studies showed that QC has the actions in decreasing blood pressure and angiotensin-Ⅱ level, inhibiting or reversing aorta damage by improving the morphological index of the artery, down-regulating the collagen volume fraction in the media and inhibiting the expression of osteopontin and the transformation of smooth muscle cells. The latest in vitro research has confirmed that QC can reduce the high expression of collagen and inhibit the activity of Smad3excited by TGF-β1in AFs.
This research is based on the past studies, further study and discuss the effect and mechanism of QC on vascular remodeling in hypertension by observing the pathological changes and detecting Ⅰ,Ⅲcollagen and TGF-β1and Smad3expression in adventitia of spontaneously hypertensive rats (SHRs). Our study will provide scientific reason for the clinical application of QC.
Aims
1. To investigate the effect of QC on blood pressure and vascular adventitial morphology change in SHRs.
2. To investigate the effect of QC on collagen synthesis and the mechanism underlying the process in SHRs.
Methods
1. Object of study
(1) Experimental rats:Twenty-four male SHRs aged14weeks and weighing 250-290g and8male WKY rats aged14weeks and weighing240-280g were used in the study.
(2) Experimental groups:Twenty-four SHRs were divided into three groups: the positive control (PC) group, the QC treatment (QD) group, and the losartan treatment (LSD) group. Eight male WKY rats were included in the negative control (NC) group.
(3) Experimental treatment:Rats in the QD group were administered QC at a dose of750mg/kg/day through irrigation of the stomach. Rats in the LSD group were administered losartan at a dose of30mg/kg/day through irrigation of the stomach. Rats in the PC group and the NC group were administered an equal volume of distilled water for three months.
(4) Obtain adventitia:Rats were fast for24h at the end of medical administration of whole-day drugs and anesthetized with10%chloral hydrate by intraperitoneal injection. The thoracic aorta adventitia of the experimental rats was segregated.
2. Contents of study
(1) The systolic blood pressure (SBP) of the rats was monitored by tail-cuff plethysmographic (TCP) method.
(2) The thoracic aorta adventitia was observed by Van Gieson (V-G) histological staining method.
(3) The mRNA expression of TGF-β1, Smad3, and collagens Ⅰ and Ⅲ was measured by reverse transcription PCR method.
(4) The protein expression of TGF-β1, Smad3, and collagens Ⅰ and Ⅲ was measured by immunohistochemical staining method.
3. Statistical analysis
Data are expressed as mean±SD. All of the experiments data were analyzed by SPSS20.0software. An independent-sample t-test was applied when only two groups were compared, whereas comparisons among groups were analyzed by two-way ANOVA. Differences were considered to be statistically significant when the P values were less than0.05.
Results
1. Comparison of SBP before and after experimental treatments.
Before experimental treatment, compared with WKY rats (aged14weeks) in NC group, the SBP of SHR rats (aged14weeks) in PC, QD and LSD groups were significantly increased, the difference was statistically significant (p<0.01), the compares among PC, QD and LSD groups were no statistically significance (p>0.05). During experimental treatment, the SBP of SHR rats in PC group was keeping high level, the SBP of SHR rats in QD and LSD groups was significantly continue decreased, and the SBP of WKY rats in PC group was keeping normal level. By the end of experimental treatment, compared with WKY rats (aged26weeks) in NC group, the SBP of SHR rats (aged26weeks) in QD and LSD groups were significantly decreased, the difference was statistically significant (p<0.01). From the third week after the treatments, the compares between PC, QD and LSD groups have statistical difference (p<0.05or p<0.01). From the fifth week after the treatments to the end of treatments, the compares between PC, QD and LSD groups both had significantly statistical difference (p<0.01) and the compare between QD and LSD groups was no statistically significance (p>0.05)
2. Morphological observation of thoracic aorta adventitia histological after experimental treatments.
The Van Gieson stain showed that the vascular adventitia in the PC group was thicker than that in the NC group, and QC-and losartan-treated rats showed lower adventitial thicknesses than the control rats in the PC group. Compared with WKY rats, the collagen fibers of the adventitia in SHRs showed significant hyperplasia and were intensely dyed. However, a significant reverse effect was observed in the QD group and the LSD group at the end of treatment. The QD group was not significantly different from the LSD group.
3. Comparison of the mRNA expression of TGF-β1, Smad3, and collagens Ⅰ and Ⅲ after experimental treatments.
The expression of TGF-β1, Smad3, and collagens Ⅰ and Ⅲ in the PC group was significantly increased compared with that in the NC group (p<0.01). However, the expression was significantly decreased in the QD group and the LSD group compared with the PC group (p<0.05or p<0.01). There was no significant difference in TGF-β1, Smad3, or collagen Ⅰ and Ⅲ mRNA expression between the QD group and the LSD group (p>0.05).
4. Comparison of the protein expression of TGF-β1, Smad3, and collagens Ⅰ and Ⅲ after experimental treatments.
TGF-β1, Smad3, and collagen Ⅰ and Ⅲ expression in the PC group was significantly increased compared with that in the NC group (p<0.01). However, the expression of TGF-β1, Smad3, and collagens Ⅰ and Ⅲ was significantly decreased in the QD group and the LSD group compared with the PC group (p<0.05or p<0.01). There was no significant difference in TGF-β1, Smad3, or collagen Ⅰ and Ⅲ protein expression between the QD group and the LSD group (p>0.05).
Conclusions
1. The significantly antihypertensive effect of QC on SHR rats was verified again, our study suggested that its antihypertensive mechanism through down-regulating the expression of TGF-β1, Smad3, and collagen Ⅰ and Ⅲ in thoracic aorta adventitia.
2. QC could reduce collagen content of SHR rats in vascular adventitia, improve the adventitia morphology of vascular and reverse the vascular remodeling. The possible mechanism was related to inhibit the TGF-β1/Smad3signal pathway.
Background
Harmful vascular remodeling (VR) caused by vascular hyperplastic lesions is the common pathological basis on a variety of cardiovascular diseases. Fibroblasts are the most abundant cell type in the adventitia, which lead to vascular harmful reconstruction and restenosis after damage. Hypertention could prompt the adventitial fibroblasts (AFs) turn into myofibroblasts (MFs), MFs migrate to the tunica media or the intima, and prompt the composition or fibrosis of extracellular matrix (ECM) at the same time, and lead to coarctation or VR. More and more evidence to support the AFs is "the central part" of vascular hyperplastic lesions, and become a new target to the prevention and control of harmful VR.
The studies have found that the TGF-β1is the most important and the most direct cell factor to promote AFs to proliferation, migration, phenotypic switching and synthetic function, and has most close relations to vascular adventitia reconstruction. Smad signal path is a TGF-β1downstream signaling pathways found in recent years and closely related with many tissue fibrosis. Our previous studies have confirmed that Smad2and Smad3mediated the AFs to TGF-β1reaction.
And the biological function of TGF-β1is also controlled by the mitogen activated protein kinase (MAPK). This effect can be blocked by specific MAPK inhibitor. MAPK signaling pathways (mainly including ERK, JNK and p38) is one of the most important intracellular signals in the network. It is involved in many kinds of pathophysiological process such as mediating cell proliferation, differentiation and so on, and closely related with the happening of fibrosis diseases. The receptor tyrosine kinase activated MAPK may be participate in cross-talk with the Smad pathway by phosphorylation and change the TGF-β1signal. But there has little research for specific signal path TGF-β1/ERK on vascular remodeling in hypertension so far.
The process of VR is along with synthesis and degradation of ECM. Matrix metalloproteinase (MMP) is in the main enzyme system in degradation of ECM, MMP2and MMP9are the main composition. Matrix metalloproteinase synthesis (MMPs) participated in the process of hypertensive heart and vascular reconstruction.
Connective tissue growth factor (CTGF) is a kind of ecretory polypeptide with rich cysteine. The main biological activities are to promote cell mitosis, chemotactic cytokines, inducing adhesion, apoptosis regulation and the formation of blood vessels, it also can promote cell proliferation and ECM synthesis. CTGF can be secreted by fibroblast under the induction of TGF-β1. The studies found that the CTGF excessive expression is closely related with the happening of some hyperplastic or fibrosis disease s. However, the relation between CTGF and vascular function has been less reported.
Hypertension disease belongs to the "vertigo" category in traditional Chinese medicine,"live fire and blood stagnation"s the common symptoms. Qindan capsule (QC) is a clinical empirical formula by the tutor to treat hypertension patients with "liver fire and blood stagnation" symptoms. Our previous clinical study showed that QC could influent TGF-β1/Smad signaling pathways, restrain AFs proliferation and collagen synthesis and play an important role in reversing vascular adventitia remodeling of SHRs.
But the influence on the AFs biological activity. MMP2, MMP9, CTGF, and TGF-β1/ERK signaling pathway is still unknown. This research is based on our previous studies to continue to explore the influence of QC on AFs proliferation, migration, cycle and apoptosis regulation and MMP2and MMP9, CTGF synthesis induced by TGF-β1, and the role of ERK signal transduction pathway during the process. Our study would provide more scientific basis for the clinical application of QC
Aims
1. To investigate the effect of QC medicated serum on the biological activity of AFs induced by TGF-(31by observing the proliferation, migration, cycle and apoptosis regulation.
2. To investigate the role of TGF-β1/ERK signaling pathway by observing the expression of extracellular signal-regulated kinase1/2(ERK1/2)、phospho-ERK1/2(p-ERK1/2)、CTGF、MMP2and MMP9in AFs induced by TGF-β1and affected by QC medicated serum.
Methods
1. Preparation of QC-containing serum
Ninety male WKY rats (weight:240-280g) were divided into three groups randomly:the high dosage QC group (QCH,750mg/kg/d), the low dosage QC group (QCL,150mg/kg/d) and Losartan group (30mg/kg/d)). Medicines were given2times a day and four consecutive days. Rats were starved for24h and were anaesthetised for1.5h after the final medicine treatment. Blood was drawn from the abdominal aorta. Serum was separated and mixed in the same group, filtered bacteria, at56℃inactivated for30min. Serum freeze dry powder was made in low temperature vacuum drying machine and was stored at-20℃for use.
2. Acquisition and intervention of AFs
(1) Vascular adventitia was stripped from thoracic aorta of WKY rat. Paste tissue pieces method was used to cultivate original generation fibroblasts. AFs were frozen storage, carried anabiosis and subculture.
(2) Medicated serum preliminary stimulus:The subcultured AFs were continue to be cul tured in serum free medium for4h, then isovolumetric serum freeze-dried powder was added and continue to be cultured for48h. And the negative control group was not given any processing factors.
(3) TGF-β1induced AFs:AFs were cultured by common nutrient solution with TGF-β1(20ng/ml) for24h.
3. Experimental groups
The AFs were divided into5groups as follows:
(1) The negative control group (NC).
(2) The positive control group (PC).
(3) The QC high dose group (QCH).
(4) The QC low dose group (QCL).
(5) The Losartan group (Losartan).
4. Contents of study
(1) The cell proliferation rate was measured by MTT colorimetry assay.
(2) The cell migration ability was measured by transwell assay.
(3) The cell cycle and apoptosis regulation was measured by flow cytometry analysis.
(4) The mRNA expression of MMP2, MMP9and CTGF was measured by reverse transcription-polymerase chain reaction (RT-PCR).
(5) The protein expression of MMP2, MMP9and ERK1/2, p-ERK1/2and CTGF was measured by western blot.
5. Statistical analysis
Data are expressed as mean±SD. All of the experiments data were analyzed by SPSS20.0software. An independent-sample t-test was applied when only two groups were compared, whereas comparisons among groups were analyzed by ANOVA. Differences were considered to be statistically significant when the P values were less than0.05.
Results
1. Comparison of proliferation activity of AFs among experimental groups
The proliferation activity of AFs in the PC group was significantly increased compared with that in NC group (P<0.01). After treated by medicine, the proliferation activity of AFs in QCH, QCL and Losartan group was decreased compared with that in PC group (P<0.05or P<0.01). The compare between QCH and Losartan groups has no statistically significance (P>0.05).
2. Comparison of migration number of AFs among experimental groups
The migration number of AFs in the PC group was significantly increased compared with that in NC group (P<0.01). After treated by medicine, the cell migration number of AFs in QCH, QCL and Losartan group was decreased compared with that in PC group (P<0.05). The compare between QCH and Losartan groups has no statistically significance (P>0.05).
3. Comparison of cell cycle of AFs among experimental groups
The cell percentage of G0/G1and G2/M in the PC group was increased compared with that in NC group (.P<0.05or P<0.01). After treated by medicine, the cell percentage of G0/G1and G2/M in QCH, QCL and Losartan group was decreased compared with that in PC group (P<0.05). The compare between QCH and Losartan groups has no statistically significance (P>0.05).
4. Comparison of cell apoptisis of AFs among experimental groups
The cell percentage of early apoptosis in the PC group was significantly decreased compared with that in NC group (P<0.01). After treated by medicine, the cell percentage of early apoptosis in QCH, QCL and Losartan group was increased compared with that in PC group (P<0.05). The compare between QCH and Losartan groups has no statistically significance (P>0.05).
5. Comparison of the mRNA expression of MMP2, MMP9and CTGF in AFs among experimental groups
The mRNA expression of MMP2, MMP9and CTGF in the PC group was significantly increased compared with that in NC group (P<0.01). After treated by medicine, the mRNA expression of MMP2, MMP9and CTGF in QCH, QCL and Losartan group was decreased compared with that in PC group (P<0.05). The compare between QCH and Losartan groups has no statistically significance (P>0.05).
6. Comparison of protein expression of MMP2, MMP9and ERK1/2, p-ERK1/2and CTGF in AFs among experimental groups
The protein expression of MMP2, MMP9and p-ERK1/2and CTGF in the PC group was significantly increased compared with that in NC group (P<0.01). After treated by medicine, the protein expression of MMP2, MMP9and p-ERK1/2and CTGF in QCH, QCL and Losartan group was decreased compared with that in PC group (P<0.05). The compare between QCH and Losartan groups has no statistically significance (P>0.05).
The protein expression of ERK1/2among five experimental groups has no statistically significance (P>0.05).
Conclusion
1. QC could inhibit or reverse vascular adventitia remodeling by inhibiting the biological activity of AFs and the synthesis of MMP2, MMP9and CTGF induced by TGF-β1.
2. The mechanism of QC to inhibit or reverse vascular adventitia remodeling maybe related to the inhibiting effect of TGF-β1/ERK signaling pathway.
引文
1.Risler NR, Cruzado MC, Miatello RM. Vascular remodeling in experimental hypertension【J]. ScientificWorldJournal,2005,12(5):959-971.
2.Toblli JE, Grana D, Cao G, Milei J. Effects of ACE inhibition and beta-blockade on plasminogen activator inhibitor-1 and transforming growth factor-betal in carotid glomus and autonomic ganglia in hypertensive rats[J]. Am J Hypertens,2007, 20(3):326-334.
3.Zheng L, Xu CC, Chen WD, et al. MicroRNA-155 regulates angiotensin Ⅱ type 1 receptor expression and phenotypic differentiation in vascular adventitial fibroblasts [J]. Biochem Biophys Res Commun,2010,400(4):483-488.
4.Stenmark KR, Frid MG, Yeager M, et al.Targeting the adventitial microenvironment in pulmonary hypertension:A potential approach to therapy that considers epigenetic change[J]. Pulm Circ,2012,2(1):3-14.
5.Schreier D, Hacker T, Song G, et al. The role of collagen synthesis in ventricular and vascular adaptation to hypoxic pulmonary hypertension [J]. J Biomech Eng, 2013,135(2):021018.
6.Massague J, Blain SW, Lo RS. TGF beta signaling in growth control, cancer, and heritable disorders[J]. Cell,2000,103(2):295-309.
7.Ren M, Wang B, Zhang J, et al.Smad2 and Smad3 as mediators of the response of adventitial fibroblasts induced bytransforming growth factor (31 [J]. Mol Med Rep,2011,4(3):561-567.
8.Liang S, Zhang J. Effect of Qindan Capsule on Prothrombotic State in Essential Hypertension Patientsof Yang Hyperactivity and Blood Stagnation[J]. Shanghai Journal of Traditional Chinese,2006,40(10):21-23.
9.Wang B, Zhang JD, Feng JB, et al. Effect of traditional Chinese medicine Qin-Dan-Jiang-Ya-Tang on remodeled vascular phenotype and osteopontin in spontaneous hypertensive rats[J]. J Ethnopharmacol,2007.110:176-182.
10.Ren M, Zhang J, Wang B, et al. Qindan-capsule inhibits proliferation of adventitial fibroblasts and collagen synthesis [J]. J Ethnopharmacol,2010,129:53-58.
11.Ren M, Wang B, Zhang J. Study on quality standard of Qindan Capsule[J]. Chinese Journal of Integrative Medicine on Cardio-/Cerebrovascular Disease, 2009,7:597-98.
12.Yagil Y, Yagil C. Genetic models of hypertension in experimental animals [J]. Exp Nephrol,2001,9(1):1-9.
13. Siragy, HM, Carey RM. Angiotensin type 2 receptors:potential importance in the regulation of blood pressure [J]. Curr Opin Nephrol Hypertens,2001,10(1):99-103.
14.Rouet-Benzineb P, Gontero B, Dreyfus P, et al. Angiotensin Ⅱ induces nuclear factor-kappa B activation in cultured neonatal rat cardiomyocytes through protein kinase C signaling pathway [J]. J Mol Cell Cardiol,2000,32(10):1767-1778.
15.Matsubara H. Pathophysiological role of angiotensin Ⅱ type 2 receptor in cardiovascular and renal diseases[J]. Circ Res,1998,83(12):1182-1191.
16.Tamarat R, Silvestre JS, Durie M, et al. Angiotensin Ⅱ angiogenic effect in vivo involves vascular endothelial growth factor-and inflammation-related pathways [J]. Lab Invest,2002,82 (6),747-756
17.陈洁,孙建平.氯沙坦和百令胶囊对马兜铃酸肾病小鼠肾间质纤维化过程的干预研究[J].中国中西医结合肾病杂志,2008,9(5)：440-442.
18.王博,张继东,王世华.芩丹胶囊降压作用及其机制的实验研究[J].山东大学学报(医学版),2006,44(10)：1024-1027.
19.Staessen JA, Wang J, Bianchi G, et al. Essential hypertension [J]. Lancet,2003,361(9369):1629-1641.
20.Derhaschnig U, Shehata M, Herkner H, et al. Increased levels of transforming growth factor-betal in essential hypertension [J]. Am J Hyperten,2002,15(3):207-211.
21.Dahly AJ, Hoagland KM, Flash AK, et al. Antihypertensive effects of chronic anti-TGF-beta antibody therapy in Dahl S rats [J]. Am J Physiol Regul Integr Comp Physiol,2002,283(3):R757-767.
22.Peterson MC. Circulating transforming growth factor beta-1:a partial molecular explanation for associations between hypertension, diabetes, obesity, smoking and human disease involving fibrosis[J]. Med Sci Monit,2005,11(7):RA229-232.
23.闫宏,孙刚.高血压血管重塑的研究进展[J].中华高血压杂志,2009,17(2)：107-109.
24.August P, Leventhal B, Suthanthiran M. Hypertension-induced organ damage in African Americans:transforming growth factor-beta(1) excess as a mechanism for increased prevalence[J]. Curr Hypertens Rep,2000,2(2):184-191.
25.Chrysant SG. Vascular remodeling:the role of angiotensin-converting enzyme inhibitors[J]. Am Heart J,1998,135(2 Pt 2):S21-30.
26.Williams B. Angiotensin Ⅱ and the pathophysiology of cardiovascular remodeling[J]. Am J Cardiol,2001,87(8A):10C-17C.
27.Asano Y. Future treatments in systemic sclerosis[J]. J Dermatol,2010, 37(1):54-70.
28. Hara K. Molecular mechanism and therapeutic strategy for cerebral small vessel disease[J]. Rinsho Shinkeigaku,2010,50(11):852-854.
29.Ikushima H, Miyazono K. Biology of transforming growth factor-β signaling[J]. Curr Pharm Biotechnol,2011,12(12):2099-2107.
30.Lan HY, Chung AC.Transforming growth factor-β and Smads[J].Contrib Nephrol, 2011,170:75-82.
31.Heldin CH, Moustakas A. Role of Smads in TGFβ signaling[J].Cell Tissue Res,2012,347(1):21-36.
32.Zi Z, Chapnick DA, Liu X. Dynamics of TGF-β/Smad signaling[J].FEBS Lett, 2012,586(14):1921-1928.
33.Biernacka A, Dobaczewski M, Frangogiannis NG. TGF-β signaling in fibrosis[J]. Growth Factors,2011,29(5):196-202.
34.Lan HY. Diverse roles of TGF-β/Smads in renal fibrosis and inflammation[J]. Int J Biol Sci,2011,7(7):1056-1067.
35.Lan HY, Chung AC.TGF-β/Smad signaling in kidney disease[J]. Semin Nephrol, 2012,32(3):236-243.
36.Calvieri C, Rubattu S, Volpe M. Molecular mechanisms underlying cardiac antihypertrophic and antifibrotic effects of natriuretic peptides[J]. J Mol Med (Berl),2012,90(1):5-13.
37.Toma I, McCaffrey TA. Transforming growth factor-(3 and atherosclerosis: interwoven atherogenic and atheroprotective aspects[J].Cell Tissue Res,2012, 347(1):155-175.
38.Doetschman T, Barnett JV, Runyan RB, et al. Transforming growth factor beta signaling in adult cardiovascular diseases and repair[J].Cell Tissue Res,2012, 347(1):203-223.
39.牟华明,祝之明.外膜对血管重塑和功能的调控[J].国外医学·心血管疾病分册,2004,31(5)：267-269.
40.Huang XR, Chung AC, Yang F, et al. Smad3 mediates cardiac inflammation and fibrosis in angiotensin β-induced hypertensive cardiac remodeling[J]. Hypertension,2010,55(5):1165-1171.
41.邱志兵,陈鑫,万松.血管外膜和胶原分布对内膜增生及血管重塑的影响[J].中国胸心血管外科临床杂志,2008,15(4)：272-276.
42.Bhogal RK, Stoica CM, McGaha TL, et al. Molecular aspects of regulation of collagen gene expression in fibrosis[J]. J Clin Immunol,2005,25(6):592-603.
43.Laurent S, Boutouyrie P, Lacolley P.Structural and genetic bases of arterial stiffness[J]. Hypertension,2005,45(6):1050-1055.
44.葛汝青,张继东,黄山英,等.芩丹胶囊对老年自发性高血压大鼠血管外膜重构作用机制的研究[J].上海中医药杂志,2011,45(3),62-66.
45.Verrecchia F, Mauviel A. Transforming growth factor-beta and fibrosis[J]. World J Gastroenterol,2007,13(22):3056-3062.
46.Verrecchia F, Chu ML. Mauviel A. Identification of novel TGF-beta/Smad gene targets in dermal fibroblasts using a combined cDNA microarray/promoter transactivation approach[J]. J Biol Chem,2001,276(20):17058-17062.
47.Verrecchia F, Laboureau J, Verola O, et al. Skin involvement in scleroderma--where histological and clinical scores meet[J]. Rheumatology (Oxford),2007,46(5):833-841.
48.覃国辉,黄俊,马业新TGF-β1及其信号蛋白Smad2/3在大鼠心肌细胞肥大中的作用[J].现代医学,2003,31(5)：301-304.
49.邓长柏,杨作成.阻断Smad3表达对转化生长因子β1诱导肌成纤维细胞增殖的影响[J].中国动脉硬化杂志,2008,16(4)：281-283.
50.Leivonen SK, Hakkinen L, Liu D, et al. Smad3 and extracellular signal-regulated kinase 1/2 coordinately mediate transforming growth factor-beta-induced expression of connective tissue growth factor in human fibroblasts[J]. J Invest Dermatol,2005, 124(6):1162-1169.
51.Liu P, Zhang C, Feng JB, et al. Cross talk among Smad, MAPK, and integrin signaling pathways enhances adventitial fibroblast functions activated by transforming growth factor-betal and inhibited by Gax[J]. Arterioscler Thromb Vasc Biol,2008,28(4):725-731.
52.Hu B, Wu Z, Phan SH. Smad3 mediates transforming growth factor-beta-induced alpha-smooth muscle actin expression[J]. Am J Respir Cell Mol Biol,2003,29(3 pt 1):397-404.
53.葛汝青,张继东,黄山英,等.芩丹胶囊对高血压血管外膜重构及TGF-β1/Smad信号转导通路的影响.山东大学学报(医学版),2011,49(4)：7-12.
54.高学敏,张廷模,张俊荣,等.中药学[M].中国中医药出版社,2002,9(1)：51-500.
55.赵华英主编.生药学[M].山东大学出版社,2005,139-140.
56.史雪靖.黄芩药理作用研究进展[J].中医药信息,2010,27(4)：128-130.
57.赵红艳,张蟠,范书铎,等.黄芩甙对发热大鼠下丘脑PGE2和cAMP含量的影响[J].中国应用生理学志,2002,18(2)：139.
58.侯艳宁,朱秀嫒,程桂芳,等.黄芩甙的抗炎机理[J].药学学报,2000,35(3)：161.
59.Chen ZY, Su YL, Lau CW, et al. Endothelium-dependent contraction and direct relaxation induced by baicalein in rat mesenteric artery[J]. Eur J Pharmacol,1999, 374(1):41-47.
60.黑爱莲,孙颂三,王泽生.黄芩甙对培养的大鼠主动脉平滑肌细胞内游离钙浓度的影响[J].中药药理与临床,1998,14(4)：6.
61.杨学青,黄力.中药治疗高血压研究进展[J].中日友好医院学报,2002,16(5)：328-331.
62.Kimura Y, Yokoi K, Matsushita N, et al. Effects of flavonoids isolated from scutellariae radix on the production of tissue-type plasminogen activator and plasminogen activator inhibitor-1 induced by thrombin and thrombin receptor agonist peptide in cultured human umbilical vein endothelial cells[J]. J Pharm Pharmacol,1997,49(8):816-822.
63.Liu JJ, Huang TS, Cheng WF, et al. Baicalein and baicalin are potent inhibitors of angiogenesis:Inhibition of endothelial cell proliferation migration and differentiation [J]. Int J Cancer,2003,106 (4):559-565.
64.陈锋,熊伟,何庆.黄芩对皮肤瘢痕形成过程中转化生长因子-β1及胶原蛋白Ⅲ表达的影响[J].实用医院临床杂志,2007,4(5)：32-33.
65.李林,曾耀英,黄秀艳,等.黄芩苷对小鼠T淋巴细胞体外增殖和细胞周期的影响[J].细胞与分子免疫学杂志,2009,25(1)：75-78.
66.章沐曦,李立.黄芩苷对兔tenon囊成纤维细胞增殖及TGF-β1表达的影响[J].激光杂志,2010,31(3)：86-89.
67.Dong LH, Wen JK, Liu G, et al. Blockade of the Ras-extracellular signal-regulated kinase 1/2 pathway is involved in smooth muscle 22 alpha-mediated suppression of vascular smooth muscle cell proliferation and neointima hyperplasia[J]. Arterioscler Thromb Vasc Biol,2010,30(4):683-691
68.高学敏,张廷模,张俊荣,等.中药学[M].中国中医药出版社,,2002,9(1)：51-500.
69.刘诚进,张绍杰.丹参的药理及在临床的合理应用[J].中华临床医学研究杂志,2007,13(10)：1399-1400.
70.刘晓梅,李树青.丹参对自发性高血压大鼠转化生长因子β、Ⅳ型胶原的影响[J].中国心血管病研究杂志,2005,3(2)：146-147.
71.刘虹彬,温进坤.丹参对血管平滑肌细胞基质金属蛋白酶和骨桥蛋白基因表达及细胞增殖的影响[J].中国中西医结合杂志,2002,22(10)：764-766.
72.张淑涛.丹参对原发性高血压患者hs-CRP、sICAM-1及内皮功能的影响[J].中国实用医药,2010,5(17)：3-5.
73.张知新,高福云,李克明,等.丹参酚酸B镁对自由基损伤人主动脉内皮细胞的影响[J].中国中西医结合杂志,2004,24(6)：521-524.
74.Au-Yeung KK, Zhu DY, Siow YL,et al. Inhibition of stress-activated protein kinase in the ischemic/reperfused heart:role of magnesium tanshinoate B in preventing apoptosis[J]. Biochem Pharmacol,2001,62(4):483-493.
75.蔡辉,赵智明,修春英,等.丹参酮ⅡA对溶血磷脂酸致新生大鼠心脏成纤维细胞 增殖及分泌转化生长因子β1的影响[J].医学研究生学报,2008,21(2)：126-128.
76.张冬梅,秦英,牛福玲,等.丹参酮ⅡA对AngⅡ诱导的心肌成纤维细胞增殖及Ⅰ型胶原合成的影响[J].辽宁中医杂志,2008,35(12)：1934-1936.
77.谢辉,郑智,龚丽娅.丹参酮ⅡA对自发性高血压大鼠左室肥厚的影响及机制[J].实用医学杂志,2004,20(3)：252-254.
78.邹昌群,占成业,白祥军,等.丹参酮ⅡA抗心肌纤维化作用机制实验研究[J].内科急危重症杂志,2008,14(5)：242-245.
79.Lau CW, Yao XQ, Chen ZY, et al. Cardiovascular actions of berberine[J]. Cardiovasc Drug Rev,2001,19(3):234.
80.张明发.黄连素的抗高血压作用[J].医院用药评价与分析,2001,1(5)：299-301.
81.陈惠卿.黄连素的心血管作用药理及其临床应用[J].佛山科学技术学院学报(自然科学版),2008,26(2)：75-77.
82.郝钰,徐泊文,郑宏,等.小檗碱对人脐静脉内皮细胞增殖与凋亡的作用[J].中国病理生理杂志,2005,21(6)：1124-1127.
83.娄金丽,邱全瑛,郝钰,等.小檗碱抗肿瘤新生血管形成作用机制的研究[J].中国免疫学杂志,2006,22(3)：235-236,243.
84.李凝,陆付耳,徐丽君,等.小檗碱对大鼠糖尿病早期肾脏转化生长因子β1的影响[J].中国康复,2008,23(1)：11-13.
85.解欣然,洪缨,董世芬.小檗碱抑制血管紧张素Ⅱ诱导的心肌成纤维细胞增殖和胶原合成的作用及机制研究[J].北京中医药大学学报,2008,31(11)：748-751.
86.吴红金,吕俊萍,马增春,等.川芎嗪对培养人脐静脉内皮细胞基因表达谱的影响[J].中国实验方剂学杂志,2005,11(3)：40.
87.熊石龙,文志斌,王前,等.川芎嗪对肿瘤坏死因子致血管内皮细胞组织因子表达的影响[J].中国现代医学,2007,17(19)：2330-2333.
88.高雪,杨洁,元英进.盐酸川芎嗪对血管紧张素Ⅱ损伤内皮细胞的保护作用[J].细胞生物学杂志,2007,29：895-900.
89.张冬梅,秦英,杨君,等.川芎嗪对血管紧张素Ⅱ诱导的人α1(Ⅰ)胶原基因启动子活性的影响[J].中国中医药信息杂志,2005,12(9)：21.
90.郑红花,李映红,罗德生.川芎嗪对血管紧张素诱导的血管平滑肌细胞增殖的抑制作用及机制[J].中国动脉硬化杂志,2006,14(4)：301.
91.李国凡,张延斌,陈清枝.川芎嗪对凝血酶诱导的血管平滑肌细胞增殖的影响[J]心脏杂志,2007,19(6)：646-648.
92.张海啸,史载祥,贾海忠,等.川芎嗪对成纤维细胞及TGF-β/Smads信号传导的影响[J].北京中医药大学学报,2007,30(40)：252-255.
93.宋纯清,樊懿,黄伟晖,等.钩藤中不同成分降压作用的差异[J].中草药,2000,31(10)：762-764.
94.余文海.钩藤对自发性高血压大鼠内皮的影响[J].国外医学·中医中药分册,2000,22(6)：351-352.
95.怡悦.钩藤对自发性高血压大鼠血管内皮功能的影响[J].国外医学·中医中药分册,2000,22(1)：28-29.
96.刘建斌,任江华.钩藤对自发性高血压大鼠心肌重构及原癌基因C-fos表达的影响[J].中国中医基础医学杂志,2000,6(5)：40-44.
97.李艳菊,孙安盛,余丽梅,等.钩藤碱对Ang Ⅱ诱导大鼠血管平滑肌细胞增殖的抑制作用[J].中国药学杂志,2008,43(21)：1624-1624.
98.丁伯平,熊莺,徐朝阳,等.益母草碱对急性血瘀证大鼠血液流变学的影响[J].中国中医药科技,2004,11(1)：36-37
99.Pang S, Tsuchiya S, Horie S, et al. Enhancement of phenyle-phedrine- induced contraction in the isolated rat aorta with endothelium by H2O- extract from an oriental medical plant leonuri herba[J]. Jpn J Pharmacol,2001,86(2):215-220.
100.陈少如,郑鸿翱,陈韩秋,等.益母草制剂治疗心肌缺血及其机制研究[J].中国危重病急救医学,2002,14(1)：19-22.
101.阮奕满,林进国,刘甘泉.益母草对兔心肌缺血再灌注损伤氧自由基的影响[J].卫生职业教育,2007,12(25)：123.
102.杨得坡,苏薇薇.参环毛蚓脂类挥发性成分分析[J].中药材,2000,23(1)：31-33.
103.葛勤,张恩娟,刘同华,等.地龙的化学成分及制剂质量研究概况[J].综述报告,2003,13(3)：69-71.
104.徐宗佩,王益明,张吉正,等.地龙提取物对健康小鼠红细胞变形性的影响[J].天津中医,2000,17(3)：37.
105.殷出梅,储益平,吴鹏.地龙活性提取物的主要药效学实验[J].中草药,2002,33(10)：926-928.
106.陈洪,陆亚琴,刘顺英,等.地龙2号对肝纤维化模型大鼠肝星状细胞活化及 TGF-β1蛋白表达的影响[J].江苏中医药,2005,26(1)：50-52.
107.陈洪,陆亚琴,刘顺英,等.地龙2号对大鼠肝纤维化α-SMA、TGF-β1、MMP-13及TIMP-1蛋白表达的影响[J].胃肠病学和肝病学杂志,2005,14(2)：156-159.
108.翟宝伟,康白,毛淑梅,等.地龙提取物对自发性高血压大鼠左室肥厚的影响[J].现代中西医结合杂志,2007,16(29)：4272-4274.
109.李承德,康白,毛淑梅,等.地龙降压蛋白对自发性高血压大鼠降压作用及其机制的影响[J].中华中医药杂志,2008,23(5)：450-452.
110.陈乐生.桑寄生药理研究[J].陕西中医,2000,21(11)：520-521.
111.叶立新,王继红,黄华利.桑寄生对肾性高血压大鼠血浆p-内啡肽浓度影响的量效作用[J].中国临床康复,2005,9(27)：84-85.
112.张朝阳.降压中药的有关研究进展[J].内蒙古中医药,2008,6：66-69.
113.刘丽娟.桑寄生现代临床应用研究进展[J].检验医学与临床,2009,6(12)：1001-1002.
1.Zhu DL, Herembert T, Marche P. Increased proliferation of ad vent it ial fibroblasts from spontaneously hypertensive rat aorta[J]. J Hypertens,1991,9(12):1161-1168.
2.Zhu DL, Herembert T, Caruelle D, et al. Signaling mechanisms of basic fibroblast growth factor in arterial cells from genetically hypertensive rat[J]. Am J Hypertens, 1994,7(4 Pt 1):351-356.
3.Shi Y, OBrien JE, Fard A, et al. Adventitial myofibroblasts contribute to neointimal formation in injured porcine coronary arteries [J]. Circulation,1996,4:1655-1664.
4.Hoshino R, Tanimura S, Watanabe K, et al. Blockade of the extracellular signal-regulated kinase pathway induces marked G1 cell cycle arrest and apoptosis in tumor cell in which the pathway is constitutively activated:up-regulation of p27 (Kip1) [J]. J Biol Chem,2001,276:2686-2692.
5.Gibbons GH, Dzau VJ. The emerging concept of vascular remodeling[J]. The New England Journal of Medicine,1994,330(20):1431-1438.
6.F.E. Rey, R.J. Pagano. The Reactive Adventitia:Fibroblast Oxidase in vascular function. Arterioscler[J]. Thromb Vasc. Biol,2002,22:1962-1971.
7.Clakr DA, Coker R. Transforming growth factor-beta (TGF-beta)[J].In J Biochem Cell Biol,1998,30:293-298.
8.Anderson KM, Brawer ME, Nichols AJ. Transforming growth factor-beta in restenosis [A]:In Anderson KM. Coronary restenosis:form genetics to therapeutics 1sted [M]. New York Marcel Dekker,2000,99-127.
9. Camoretti-Mercado B'Solway J. Transforming growth factor-betal and disorders of the lung[J].Cell Biochem Biophys,2005,43(1):131-148.
10.Saika S, Yamanaka O, Okada Y, et al. TGF beta in fibroproliferative diseases in the eye[J]. Front Biosci (Schol Ed),2009,1:376-390.
11.Ryna ST, Koetlinansky VE, Gowtals PJ, et al. Transforming growth factor-beta-dependent events in vascular remodeling following arterial injury[J]. J Vase Res,2003, 40:37-46.
12. Jiang YL, Dai AG, Li QF, et al. Transforming growth factor-betal induces transdifferentiation of fibroblasts into myofibroblasts in hypoxic pulmonary vascular remodeling [J]. Acta Biochim Biophys Sin,2006,38:29-36.
13.Toma I, McCaffrey TA. Transforming growth factor-β and atherosclerosis: interwoven atherogenic and atheroprotective aspects [J]. Cell Tissue Res,2012 347(1):155-175.
14.Lan HY, Chung AC.Transforming growth factor-β and Smads [J]. Contrib Nephrol,2011,170:75-82.
15.Lan HY.Transforming growth factor-β/Smad signalling in diabetic nephropathy [J]. Clin Exp Pharmacol Physiol,2012,39 (8):731-738.
16.Inagaki Y, Higashiyama R, Higashi K. Novel anti-fibrotic modalities for liver fibrosis:molecular targeting and regenerative medicine in fibrosis therapy [J]. J Gastroenterol Hepatol,2012, Suppl2:85-88.
17.Mulder M. Role of Ras and Mapks in TGF beta signaling [J]. Cytokine Growth Factor Rev,2000,11 (1-2):23-35.
18.Bobick BE, Kulyk WM. Regulation of cartilage formation and maturation by mitogen-activated protein kinase signaling[J]. Birth Defects Res C Embryo Today, 2008,84(2):131-154.
19.Mu Y, Gudey SK, Landstrom M. Non-Smad signaling pathways[J]. Cell Tissue Res,2012,347(1):11-20.
20.Samarakoon R, Higgins PJ. Integration of non-SMAD and SMAD signaling in TGF-betal-induced plasminogen activator inhibitor type-1 gene expression in vascular smooth muscle cells [J]. Thromb Haemost,2008,100 (6):976-983.
21.Guo X, Wang XF. Signaling cross-talk between TGF-beta/BMP and other pathways[J]. Cell Res,2009,19(1):71-88.
22.Biondi RM, Nebreda AR. Signalling specificity of Ser/Thr protein kinases through docking-site-mediated interactions[J]. Biochem J,2003,372(Pt 1):1-13.
23.Bobik A, Tkachuk V. Metalloproteinases and plasminogen activators in vessel remodeling[J]. Curr Hypertens Rep,2003,5(6):466-472.
24.Janicki JS, Brower GL, Gardner JD, et al. Cardiac mast cell regulation of matrix metalloproteinase-related ventricular remodeling in chronic pressure or volume overload[J]. Cardiovasc Res,2006,69(3):657-665.
25.Steed MM, Tyagi SC.Mechanisms of cardiovascular remodeling in hyperhomocysteinemia[J]. Antioxid Redox Signal,2011,15(7):1927-1943.
26.Raffetto JD, Khalil RA. Matrix metalloproteinases and their inhibitors in vascular remodeling and vascular disease[J]. Biochem Pharmacol,2008, 75(2):346-359.
27.Lim CS, Shalhoub J, Gohel MS, et al. Matrix metalloproteinases in vascular disease--a potential therapeutic target[J]? Curr Vasc Pharmacol,2010, 8(1):75-85.
28.Chelladurai P, Seeger W, Pullamsetti SS. Matrix metalloproteinases and their inhibitors in pulmonary hypertension[J]. Eur Respir J,2012,40(3):766-782.
29.Moussad EE, Brigstock DR.Connective tissue growth factor:what's in a name[J]? Mol Genet Metab,2000,71 (1-2):276-292.
30.Rachfal AW, Brigstock DR. Structural and functional properties of CCN proteins[J]. Vitam Horm,2005,70:69-103.
31.Luft FC. CCN2, the connective tissue growth factor[J]. J Mol Med (Berl),2008, 86(1):1-3.
32.Grotendorst GR.Connective tissue growth factor:a mediator of TGF-beta action on fibroblasts[J]. Cytokine Growth Factor Rev,1997,8 (3):171-179.
33.Tabibzadeh S.Homeostasis of extracellular matrix by TGF-beta and lefty[J]. Front Biosci,2002,7:1231-1246.
34.Burgess JK. Connective tissue growth factor:a role in airway remodelling in asthma[J]? Clin Exp Pharmacol Physiol,2005,32(11):988-994.
35.Leask A, Holmes A, Abraham DJ. Connective tissue growth factor:a new and important player in the pathogenesis of fibrosis[J]. Curr Rheumatol Rep,2002, 4(2):136-142.
36.Leask A, Abraham DJ. The role of connective tissue growth factor, a multifunctional matricellular protein, in fibroblast biology [J]. Biochem Cell Biol.2003,81(6):355-363.
37.Leask A, Abraham DJ. TGF-beta signaling and the fibrotic response[J]. FASEB J,2004,18(7):816-827.
38.Ruiz-Ortega M, Rodriguez-Vita J, Sanchez-Lopez E, Carvajal G, Egido J.TGF-beta signaling in vascular fibrosis[J].Cardiovasc Res,2007,74 (2):196-206.
39.Chen XM, Qi W, Pollock CA. CTGF and chronic kidney fibrosis[J]. Front Biosci (Schol Ed),2009,1:132-141.
40.Arnott JA, Lambi AG, Mundy C, Hendesi H, Pixley RA, Owen TA, Safadi FF, Popoff SN. The role of connective tissue growth factor (CTGF/CCN2) in skeletogenesis[J]. Crit Rev Eukaryot Gene Expr,2011,21(1):43-69.
41.Ng K, Butlin M, Avolio AP. Persistent effect of early, brief angiotensin-converting enzyme inhibition on segmental pressure dependency of aortic stiffness in spontaneously hypertensive rats[J]. J Hypertens,2012,30(9):1782-1790.
42.Soro-Paavonen A, Gordin D, Forsblom C, et al. FinnDiane Study Group. Circulating ACE2 activity is increased in patients with type ldiabetes and vascular complications [J]. J Hypertens,2012,30(2):375-383.
43.Ren M, Zhang J, Wang B, et al. Qindan-capsule inhibits proliferation of adventitial fibroblasts and collagen synthesis[J]. J Ethnopharmacol,2010,129:53-58.
44.Ren M, Wang B, Zhang J. Study on quality standard of Qindan Capsule[J]. Chinese Journal of Integrative Medicine on Cardio-/Cerebrovascular Disease, 2009,7:597-98.
45.Iwama H, Amagaya S, Ogihara Y. Effect of shosaikoto, a Japanese and Chinese traditional herbal medicinal mixture, on the mitogenic activity of lipopolysaccharide: a new pharmacological testing method[J]. J Ethnopharmacol,1987,21(1):45-53.
46.He YZ. Research survey of serum pharmacology and serum medicinal Chemistry in Japanese Chinese herbs[J]. Int J Chin Med,1998,20(5):3-7.
47.王喜军.中药血清药物化学的研究动态及发展趋势[J].中国中药杂志,2006,31(10)：789.
48.任敏,张继东,王博,等.芩丹胶囊对血管外膜成纤维细胞的作用机制.山东大学 学报(医学版)[J].2010,04：27-32.
49.Itoh H, Nakao K. Antagonism between the vascular renin-angiotensin and natriuretic peptide systems in vascular remodelling[J]. Blood Press Suppl,1994, 5:49-53.
50.Teng J, Fukuda N, Suzuki R, et al. Inhibitory effect of a novel angiotensin Ⅱ type 1 receptor antagonist RNH-6270 on growth of vascular smooth muscle cells from spontaneously hypertensive rats:different anti-proliferative effect to angiotensin-converting enzyme inhibitor[J]. J Cardiovasc Pharmacol,2002,39(2):161-171.
51.Chen K, Mehta JL, Li D, et al. Transforming growth factor beta receptor endoglin is expressed in cardiac fibroblasts and modulates profibrogenic actions of angiotensin Ⅱ [J]. Circ Res,2004,10(12):1167-1173.
52.Kawano H, Do YS, Kawano Y, et al. Angiotensin Ⅱ has multiple prebrothic effects in human cardiac fibroblasts [J]. Circulation,2000,1(10):1130-1137.
53.刘平,张运,胡维成.血管外膜及其成纤维细胞在血管增殖性病变中的作用[J].中华老年医学杂志,2005,24(12)：936-938.
54.Orton EC, LaRue SM, Ensley B, et al. Bromodeoxyuridine labeling and DNA content of pulmonary arterial medial cells from hypoxia-exposed and nonexposed healthy calves [J]. Am J Vet Res,1992,53(10):1925-1930.
55.Scott NA, Cipolla GD, Ross CE, et al. Identification of a potential role for the adventitia in vascular lesion formation after balloon overstretch injury of porcine coronary arteries[J]. Circulation,1996,93(12):2178-2187.
56.Shi Y, Niculescu R, Wang D, et al. Increased NAD(P)H oxidase and reactive oxygen species in coronary arteries after balloon injury[J]. Arterioscler Thromb Vasc Biol,2001,21(5):739-745.
57.Krick S, Hanze J, Eul B, et al. Hypoxia-driven proliferation of human pulmonary artery fibroblasts:cross-talk between HIF-1 alpha and an autocfine angiotensin system[J]. FASEB J,2005,19(7):857-859.
58.Stenmark KR, Gerasimovskaya E, Nemenoff RA, et al. Hypoxic activation of adventitial fibroblasts:role in vascular remodeling[J]. Chest,2002,122(6):326-334.
59.Ryan ST, Koteliansky VE, Gotwals PJ, et al. Transforming growth factor-beta-dependent events in vascular remodeling following arterial injury[J]. J Vasc Res, 2003,40(1):37-46.
60.Lijnen PJ, Ptrov VV, Fagard RH. Association between transforming growth factor-β1 and hypertension[J]. Am J Hypertens,2003,16(7):604-611.
61.梁粟,张继东.芩丹胶囊对阳亢血瘀型高血压患者血栓前状态的影响[J].上海中医药杂志2006,40(10)：21-23.
62.Li G, Chen SJ, Oparil S, et al. Direct in vivo evidence demonstrating neointimal migration of adventitial fibroblasts after balloon injury of rat carotid arteries[J]. Circulation,2000,101(12):1362-1365.
63.Rey FE, Pagano PJ. The reactive adventitia:fibroblast oxidase in vascular function[J]. Arterioscler Thromb Vasc Biol,2002,22(12):1962-1971.
64.王永利,贺能树,张家兴,等.血管成形术后外膜细胞表型转化和迁移的实验研究[J].介入放射学杂志,2006,15(7)：424-429.
65.孙爱军,高平进,刘建军,等.骨桥蛋白增强自发性高血压大鼠血管外膜成纤维细胞的迁移活性[J].生理学报,2004,56(1)：21-24.
66.Wang B, Zhang JD, Feng JB, et al. Effect of traditional Chinese medicine Qin-Dan-Jiang-Ya-Tang on remodeled vascular phenotype and osteopontin in spontaneous hypertensive rats[J]. J Ethnopharmacol 2007,110:176-182.
67.Chatelain RE, Dardik BN. Increased DNA replication in the arterial adventitia after aortic ligation[J]. Hypertension,1988,11(suppl Ⅰ):Ⅰ-130-Ⅰ-134.
68.裴端卿.基质金属蛋白酶家族介绍[J].中国生物化学与分子生物学报.2001,17(1)：17-20.
69.Van den Steen PE, Dubois B, Nelissen I,et al. Biochemistry and molecular biology of gelatinase B or matrix metalloproteinase-9 (MMP-9)[J]. Crit Rev Biochem Mol Biol,2002,37(6):375-536.
70.Newby AC. Metalloproteinase expression in monocytes and macrophages and its relationship to atherosclerotic plaque instability [J]. Arterioscler Thromb Vasc Biol, 2008,28(12):2108.
71.Berry E, Bosonea AM, Wang X, et al. Insights into the activity, differential expression, mutual regulation, and functions of matrix metalloproteinases and a disintegrin and metalloproteinases in hypertension and cardiac disease[J]. Crit Rev Biochem Mol Biol,2002,37(6):375-536.
72.Sankaralingam S, Arenas IA, Lalu MM, et al. Preeclampsia:current understanding of the molecular basis of vascular dysfunction[J]. Expert Rev Mol Med,2006,8:1-20.
73.Benjamin MM, Khalil RA. Matrix metalloproteinase inhibitors as investigative tools in the pathogenesis and management of vascular disease[J]. EXS,2012,103: 209-279.
74.Jackson C, Nguyen M, Arkell J, et al. Selective matrix metalloproteinase (MMP) inhibition in rheumatoid arthritis targetting gelatinase A activation[J]. Inflamm Res, 2001,50(4):183-186.
75.Tayebjee MH, Nadar SK, Blann AD, et al. Matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 in hypertension and their relationship to cardiovascular risk and treatment:a substudy of the Anglo-Scandinavian Cardiac Outcomes Trial (ASCOT) [J]. Am J Hypertens,2004,17:764-769.
76.Derosa G, D'Angelo A, Ciccarelli L, et al. Matrix metalloproteinase-2,-9,and tissue inhibitor of metalloproteinase-1 in patients with hypertension[J]. Endothelium 2006,13:227-231.
77.Roberts AB, Sporn MB, Assoian RK, et al. Transforming growth factor-β:rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro[J]. Proc Natl Acad Sci USA,1986,83:4167-4171.
78.Axisa B, Loftus IM, Naylor AR, et al. Prospective, randomized, double-blind trial investigating the effect of doxycycline on matrix metalloproteinase expression within atherosclerotic carotid plaques[J]. Stroke,2002,33:2858-2864.
79.Beaudeux JL, Giral P, Bruckert E, et al. Matrix metalloproteinases, inflammation and atherosclerosis:therapeutic perspectives [J]. Clin Chem Lab Med,2004, 42:121-131.
80.Bremer C, Tung CH, Weissleder R. In vivo molecular target assessment of matrix metalloproteinase inhibition[J]. Nat Med,2001,7:743-748.
81.Brown S, Meroueh SO, Fridman R, et al. Quest for selectivity in inhibition of matrix metalloproteinases [J]. Curr Top Med Chem,2004,4:1227-1238.
82.Cook GR, Manivannan E, Underdahl T, et al. Synthesis and evaluation of novel oxazoline MMP inhibitors [J]. Bioorg Med Chem Lett,2004,14:4935-4939.
83.Agrawal A, Romero-Perez D, Jacobsen JA, et al. Zinc-binding groups modulate selective inhibition of MMPs [J].Chem Med Chem,2008,3:812-820.
84.Bradham DM, Igarashi A, Potter RL, et al.Connective tissue growth factor:a cysteine-rich mitogen secreted by human vascular endothelial cells is related to the SRC-induced immediate early gene product CEF-10[J]. J Cell Biol,1991, 114(6):1285-1294.
85.Igarashi A, Bradham DM, Okochi H, et al. Connective tissue growth factor[J]. J Dermatol,1992,19(11):642-643.
86.Frazier K, Williams S, Kothapalli D, et al. Stimulation of fibroblast cell growth, matrix production, and granulation tissue formation by connective tissue growth factor[J]. J Invest Dermatol,1996,107:404-411.
87.Chen MM, et al. CTGF expression is induced by TGF- beta in cardiac fibroblasts and cardiac myocytes:a potential role in heart fibrosis[J]. J Mol Cell Cardiol,2000, 32(10):1805-1819.
88.Ruperez M, et al. Connective tissue growth factor is a mediator of angiotensin Ⅱ-induced fibrosis. Circulation[J].2003,108(12):1499-1505.
89.Sato S, et al. Serum levels of connective tissue growth factor are elevated in patients with systemic sclerosis:association with extent of skin sclerosis and severity of pulmonary fibrosis. J Rheumatol[J].2000,27(1):149-154.
90.Gressner AM, et al. Connective tissue growth factor in serum as a new candidate test for assessment of hepatic fibrosis[J]. Clin Chem,2006,52(9):1815-1817.
91.Lee YS, Byun J, Kim JA, et al. Monocrotaline-induced pulmonary hypertension correlates with upregulation of connective tissue growth factor expression in the lung[J]. Exp Mol Med,2005,37(1):27-35.
92.田锋,徐永健,张珍祥,等.结缔组织生长因子在烟雾暴露大鼠肺血管重塑中的作用[J].中华结核和呼吸杂志,2007,30：921-925.
93.田锋,徐永健,张珍祥,等CTGF在烟雾暴露大鼠肺血管中的表达及其与肺血管重建的关系[J].中国病理生理杂志,2008,24：7-11.
94.王再,徐永健,刘先胜,曾大雄,向敏.结缔组织生长因子和周期蛋白D1在烟雾暴露大鼠肺血管重构中的表达变化[J].中华结核和呼吸杂志,2010,33(9)：679-683.
95. Wang R, Xu YJ, Liu XS, et al. Knockdown of connective tissue growth factor by plasmid-based short hairpin RNA prevented pulmonary vascular remodeling in cigarette smoke-exposed rats. Arch Biochem Biophys[J].2011,508(1):93-100.
96.Massague J. The transforming growth factor-beta family[J]. Anna Rev Cell Biol, 1990,6:597-641.
97.Heldin CH, Miyazono K, ten Dijke P. TGF-beta signalling from cell membrane to nucleus through SMAD proteins[J]. Nature,1997,390(6659):465-471.
98.Moustakas A, Souchelnytskyi S, Heldin CH. Smad regulation in TGF-beta signal transduction[J]. J Cell Sci,2001,114(24):4359-4369.
99.陈琛,叶冬梅.转化生长因子-β/Smad信号通路研究进展[J].医学分子生物学杂志,2006,3(2)：126-128.
100.Massague J. How cells read TGF-beta signals[J]. Nat Rev Mol Cell Biol,2000, 1(3):169-178.
101.Ren M, Wang B, Zhang J, et al.Smad2 and Smad3 as mediators of the response of adventitial fibroblasts induced by transforming growth factor (31 [J]. Mol Med Rep,2011,4(3):561-567.
102.Yu L, Hebert MC, Zhang YE. TGF-beta receptor-activated p38 MAP kinase mediates Smad-independent TGF-beta responses [J]. EMBO J,2002, 21(14):3749-3759.
103.Imamichi Y, Waidmann O, Hein R, et al. TGF beta-induced focal complex formation in epithelial cells is mediated by activated ERK and JNK MAP kinases and is independent of Smad4[J]. Biol Chem,2005,86(3):225-236.
104.Muscogiuri G, Chavez AO, Gastaldelli A, et al. The crosstalk between insulin and renin-angiotensin-aldosterone signaling systems and its effect on glucose metabolism and diabetes prevention [J]. Curr Vase Pharmacol,2008,6(4):301-312.
105.Jing L, Zhang J, Sun J, et al. Inhibition of extracellular signal-regulated kinases ameliorates hypertension-induced renal vascular remodeling in rat models[J]. Int J Mol Sci,2011,12(12):8333-8346.
106.Takayama N, Kai H, Kudo H, et al. Simvastatin prevents large blood pressure variability induced aggravation of cardiac hypertrophy in hypertensive rats by inhibiting RhoA/Ras-ERK pathways[J]. Hypertens Res,2011,34(3):341-347.
107.Bonacasa B, Sanchez ML, Rodriguez F, et al.2-Methoxyestradiol attenuates hypertension and coronary vascular remodeling in spontaneously hypertensive rats [J]. Maturitas,2008,61(4):310-316.
108.Yan JT, Wang T, Wang DW. Recombinant adeno-associated virus-mediated human kallikrein gene therapy protects against hypertensive target organ injuries through inhibiting cell apoptosis[J]. Acta Pharmacol Sin,2009,30(9):1253-1261.
109.Redondo A, Estrella N, Lorenzo AG, et al. Quercetin and catechin synergistically inhibit angiotensin Ⅱ-induced redox-dependent signalling pathways in vascular smooth muscle cells from hypertensive rats[J]. Free Radic Res,2012, 46(5):619-627.
110.Jing L, Zhang JZ, Zhao L, et al. High-expression of transforming growth factor betal and phosphorylation of extracellular signal-regulated protein kinase in vascular smooth muscle cells from aorta and renal arterioles of spontaneous hypertensionrats[J]. Clin Exp Hypertens,2007,29(2):107-117.
111.Rezaei HB, Kamato D, Ansari G, et al. Cell biology of Smad2/3 linker region phosphorylation in vascular smooth muscle[J]. Clin Exp Pharmacol Physiol,2012, 39(8):661-667.
112.Krause C, Kloen P. Concurrent inhibition of TGF-β and mitogen driven signaling cascades in Dupuytren's disease-non-surgical treatment strategies from a signaling point of view[J]. Med Hypotheses,2012,78(3):385-388.
113.Huang XY, Chen CX. Effect of oxymatrine, the active component from Radix Sophorae flavescentis (Kushen) on ventricular remodeling in spontaneously hypertensive rats[J]. Phytomedicine,2013,20(3-4):202-212.
2.Toblli JE, Grana D, Cao G, Milei J. Effects of ACE inhibition and beta-blockade on plasminogen activator inhibitor-1 and transforming growth factor-betal in carotid glomus and autonomic ganglia in hypertensive rats[J]. Am J Hypertens,2007, 20(3):326-334.
3.Zheng L, Xu CC, Chen WD, et al. MicroRNA-155 regulates angiotensin Ⅱ type 1 receptor expression and phenotypic differentiation in vascular adventitial fibroblasts [J]. Biochem Biophys Res Commun,2010,400(4):483-488.
4.Stenmark KR, Frid MG, Yeager M, et al.Targeting the adventitial microenvironment in pulmonary hypertension:A potential approach to therapy that considers epigenetic change[J]. Pulm Circ,2012,2(1):3-14.
5.Schreier D, Hacker T, Song G, et al. The role of collagen synthesis in ventricular and vascular adaptation to hypoxic pulmonary hypertension [J]. J Biomech Eng, 2013,135(2):021018.
6.Massague J, Blain SW, Lo RS. TGF beta signaling in growth control, cancer, and heritable disorders[J]. Cell,2000,103(2):295-309.
7.Ren M, Wang B, Zhang J, et al.Smad2 and Smad3 as mediators of the response of adventitial fibroblasts induced bytransforming growth factor (31 [J]. Mol Med Rep,2011,4(3):561-567.
8.Liang S, Zhang J. Effect of Qindan Capsule on Prothrombotic State in Essential Hypertension Patientsof Yang Hyperactivity and Blood Stagnation[J]. Shanghai Journal of Traditional Chinese,2006,40(10):21-23.
9.Wang B, Zhang JD, Feng JB, et al. Effect of traditional Chinese medicine Qin-Dan-Jiang-Ya-Tang on remodeled vascular phenotype and osteopontin in spontaneous hypertensive rats[J]. J Ethnopharmacol,2007.110:176-182.
10.Ren M, Zhang J, Wang B, et al. Qindan-capsule inhibits proliferation of adventitial fibroblasts and collagen synthesis [J]. J Ethnopharmacol,2010,129:53-58.
11.Ren M, Wang B, Zhang J. Study on quality standard of Qindan Capsule[J]. Chinese Journal of Integrative Medicine on Cardio-/Cerebrovascular Disease, 2009,7:597-98.
12.Yagil Y, Yagil C. Genetic models of hypertension in experimental animals [J]. Exp Nephrol,2001,9(1):1-9.
13. Siragy, HM, Carey RM. Angiotensin type 2 receptors:potential importance in the regulation of blood pressure [J]. Curr Opin Nephrol Hypertens,2001,10(1):99-103.
14.Rouet-Benzineb P, Gontero B, Dreyfus P, et al. Angiotensin Ⅱ induces nuclear factor-kappa B activation in cultured neonatal rat cardiomyocytes through protein kinase C signaling pathway [J]. J Mol Cell Cardiol,2000,32(10):1767-1778.
15.Matsubara H. Pathophysiological role of angiotensin Ⅱ type 2 receptor in cardiovascular and renal diseases[J]. Circ Res,1998,83(12):1182-1191.
16.Tamarat R, Silvestre JS, Durie M, et al. Angiotensin Ⅱ angiogenic effect in vivo involves vascular endothelial growth factor-and inflammation-related pathways [J]. Lab Invest,2002,82 (6),747-756
17.陈洁,孙建平.氯沙坦和百令胶囊对马兜铃酸肾病小鼠肾间质纤维化过程的干预研究[J].中国中西医结合肾病杂志,2008,9(5)：440-442.
18.王博,张继东,王世华.芩丹胶囊降压作用及其机制的实验研究[J].山东大学学报(医学版),2006,44(10)：1024-1027.
19.Staessen JA, Wang J, Bianchi G, et al. Essential hypertension [J]. Lancet,2003,361(9369):1629-1641.
20.Derhaschnig U, Shehata M, Herkner H, et al. Increased levels of transforming growth factor-betal in essential hypertension [J]. Am J Hyperten,2002,15(3):207-211.
21.Dahly AJ, Hoagland KM, Flash AK, et al. Antihypertensive effects of chronic anti-TGF-beta antibody therapy in Dahl S rats [J]. Am J Physiol Regul Integr Comp Physiol,2002,283(3):R757-767.
22.Peterson MC. Circulating transforming growth factor beta-1:a partial molecular explanation for associations between hypertension, diabetes, obesity, smoking and human disease involving fibrosis[J]. Med Sci Monit,2005,11(7):RA229-232.
23.闫宏,孙刚.高血压血管重塑的研究进展[J].中华高血压杂志,2009,17(2)：107-109.
24.August P, Leventhal B, Suthanthiran M. Hypertension-induced organ damage in African Americans:transforming growth factor-beta(1) excess as a mechanism for increased prevalence[J]. Curr Hypertens Rep,2000,2(2):184-191.
25.Chrysant SG. Vascular remodeling:the role of angiotensin-converting enzyme inhibitors[J]. Am Heart J,1998,135(2 Pt 2):S21-30.
26.Williams B. Angiotensin Ⅱ and the pathophysiology of cardiovascular remodeling[J]. Am J Cardiol,2001,87(8A):10C-17C.
27.Asano Y. Future treatments in systemic sclerosis[J]. J Dermatol,2010, 37(1):54-70.
28. Hara K. Molecular mechanism and therapeutic strategy for cerebral small vessel disease[J]. Rinsho Shinkeigaku,2010,50(11):852-854.
29.Ikushima H, Miyazono K. Biology of transforming growth factor-β signaling[J]. Curr Pharm Biotechnol,2011,12(12):2099-2107.
30.Lan HY, Chung AC.Transforming growth factor-β and Smads[J].Contrib Nephrol, 2011,170:75-82.
31.Heldin CH, Moustakas A. Role of Smads in TGFβ signaling[J].Cell Tissue Res,2012,347(1):21-36.
32.Zi Z, Chapnick DA, Liu X. Dynamics of TGF-β/Smad signaling[J].FEBS Lett, 2012,586(14):1921-1928.
33.Biernacka A, Dobaczewski M, Frangogiannis NG. TGF-β signaling in fibrosis[J]. Growth Factors,2011,29(5):196-202.
34.Lan HY. Diverse roles of TGF-β/Smads in renal fibrosis and inflammation[J]. Int J Biol Sci,2011,7(7):1056-1067.
35.Lan HY, Chung AC.TGF-β/Smad signaling in kidney disease[J]. Semin Nephrol, 2012,32(3):236-243.
36.Calvieri C, Rubattu S, Volpe M. Molecular mechanisms underlying cardiac antihypertrophic and antifibrotic effects of natriuretic peptides[J]. J Mol Med (Berl),2012,90(1):5-13.
37.Toma I, McCaffrey TA. Transforming growth factor-(3 and atherosclerosis: interwoven atherogenic and atheroprotective aspects[J].Cell Tissue Res,2012, 347(1):155-175.
38.Doetschman T, Barnett JV, Runyan RB, et al. Transforming growth factor beta signaling in adult cardiovascular diseases and repair[J].Cell Tissue Res,2012, 347(1):203-223.
39.牟华明,祝之明.外膜对血管重塑和功能的调控[J].国外医学·心血管疾病分册,2004,31(5)：267-269.
40.Huang XR, Chung AC, Yang F, et al. Smad3 mediates cardiac inflammation and fibrosis in angiotensin β-induced hypertensive cardiac remodeling[J]. Hypertension,2010,55(5):1165-1171.
41.邱志兵,陈鑫,万松.血管外膜和胶原分布对内膜增生及血管重塑的影响[J].中国胸心血管外科临床杂志,2008,15(4)：272-276.
42.Bhogal RK, Stoica CM, McGaha TL, et al. Molecular aspects of regulation of collagen gene expression in fibrosis[J]. J Clin Immunol,2005,25(6):592-603.
43.Laurent S, Boutouyrie P, Lacolley P.Structural and genetic bases of arterial stiffness[J]. Hypertension,2005,45(6):1050-1055.
44.葛汝青,张继东,黄山英,等.芩丹胶囊对老年自发性高血压大鼠血管外膜重构作用机制的研究[J].上海中医药杂志,2011,45(3),62-66.
45.Verrecchia F, Mauviel A. Transforming growth factor-beta and fibrosis[J]. World J Gastroenterol,2007,13(22):3056-3062.
46.Verrecchia F, Chu ML. Mauviel A. Identification of novel TGF-beta/Smad gene targets in dermal fibroblasts using a combined cDNA microarray/promoter transactivation approach[J]. J Biol Chem,2001,276(20):17058-17062.
47.Verrecchia F, Laboureau J, Verola O, et al. Skin involvement in scleroderma--where histological and clinical scores meet[J]. Rheumatology (Oxford),2007,46(5):833-841.
48.覃国辉,黄俊,马业新TGF-β1及其信号蛋白Smad2/3在大鼠心肌细胞肥大中的作用[J].现代医学,2003,31(5)：301-304.
49.邓长柏,杨作成.阻断Smad3表达对转化生长因子β1诱导肌成纤维细胞增殖的影响[J].中国动脉硬化杂志,2008,16(4)：281-283.
50.Leivonen SK, Hakkinen L, Liu D, et al. Smad3 and extracellular signal-regulated kinase 1/2 coordinately mediate transforming growth factor-beta-induced expression of connective tissue growth factor in human fibroblasts[J]. J Invest Dermatol,2005, 124(6):1162-1169.
51.Liu P, Zhang C, Feng JB, et al. Cross talk among Smad, MAPK, and integrin signaling pathways enhances adventitial fibroblast functions activated by transforming growth factor-betal and inhibited by Gax[J]. Arterioscler Thromb Vasc Biol,2008,28(4):725-731.
52.Hu B, Wu Z, Phan SH. Smad3 mediates transforming growth factor-beta-induced alpha-smooth muscle actin expression[J]. Am J Respir Cell Mol Biol,2003,29(3 pt 1):397-404.
53.葛汝青,张继东,黄山英,等.芩丹胶囊对高血压血管外膜重构及TGF-β1/Smad信号转导通路的影响.山东大学学报(医学版),2011,49(4)：7-12.
54.高学敏,张廷模,张俊荣,等.中药学[M].中国中医药出版社,2002,9(1)：51-500.
55.赵华英主编.生药学[M].山东大学出版社,2005,139-140.
56.史雪靖.黄芩药理作用研究进展[J].中医药信息,2010,27(4)：128-130.
57.赵红艳,张蟠,范书铎,等.黄芩甙对发热大鼠下丘脑PGE2和cAMP含量的影响[J].中国应用生理学志,2002,18(2)：139.
58.侯艳宁,朱秀嫒,程桂芳,等.黄芩甙的抗炎机理[J].药学学报,2000,35(3)：161.
59.Chen ZY, Su YL, Lau CW, et al. Endothelium-dependent contraction and direct relaxation induced by baicalein in rat mesenteric artery[J]. Eur J Pharmacol,1999, 374(1):41-47.
60.黑爱莲,孙颂三,王泽生.黄芩甙对培养的大鼠主动脉平滑肌细胞内游离钙浓度的影响[J].中药药理与临床,1998,14(4)：6.
61.杨学青,黄力.中药治疗高血压研究进展[J].中日友好医院学报,2002,16(5)：328-331.
62.Kimura Y, Yokoi K, Matsushita N, et al. Effects of flavonoids isolated from scutellariae radix on the production of tissue-type plasminogen activator and plasminogen activator inhibitor-1 induced by thrombin and thrombin receptor agonist peptide in cultured human umbilical vein endothelial cells[J]. J Pharm Pharmacol,1997,49(8):816-822.
63.Liu JJ, Huang TS, Cheng WF, et al. Baicalein and baicalin are potent inhibitors of angiogenesis:Inhibition of endothelial cell proliferation migration and differentiation [J]. Int J Cancer,2003,106 (4):559-565.
64.陈锋,熊伟,何庆.黄芩对皮肤瘢痕形成过程中转化生长因子-β1及胶原蛋白Ⅲ表达的影响[J].实用医院临床杂志,2007,4(5)：32-33.
65.李林,曾耀英,黄秀艳,等.黄芩苷对小鼠T淋巴细胞体外增殖和细胞周期的影响[J].细胞与分子免疫学杂志,2009,25(1)：75-78.
66.章沐曦,李立.黄芩苷对兔tenon囊成纤维细胞增殖及TGF-β1表达的影响[J].激光杂志,2010,31(3)：86-89.
67.Dong LH, Wen JK, Liu G, et al. Blockade of the Ras-extracellular signal-regulated kinase 1/2 pathway is involved in smooth muscle 22 alpha-mediated suppression of vascular smooth muscle cell proliferation and neointima hyperplasia[J]. Arterioscler Thromb Vasc Biol,2010,30(4):683-691
68.高学敏,张廷模,张俊荣,等.中药学[M].中国中医药出版社,,2002,9(1)：51-500.
69.刘诚进,张绍杰.丹参的药理及在临床的合理应用[J].中华临床医学研究杂志,2007,13(10)：1399-1400.
70.刘晓梅,李树青.丹参对自发性高血压大鼠转化生长因子β、Ⅳ型胶原的影响[J].中国心血管病研究杂志,2005,3(2)：146-147.
71.刘虹彬,温进坤.丹参对血管平滑肌细胞基质金属蛋白酶和骨桥蛋白基因表达及细胞增殖的影响[J].中国中西医结合杂志,2002,22(10)：764-766.
72.张淑涛.丹参对原发性高血压患者hs-CRP、sICAM-1及内皮功能的影响[J].中国实用医药,2010,5(17)：3-5.
73.张知新,高福云,李克明,等.丹参酚酸B镁对自由基损伤人主动脉内皮细胞的影响[J].中国中西医结合杂志,2004,24(6)：521-524.
74.Au-Yeung KK, Zhu DY, Siow YL,et al. Inhibition of stress-activated protein kinase in the ischemic/reperfused heart:role of magnesium tanshinoate B in preventing apoptosis[J]. Biochem Pharmacol,2001,62(4):483-493.
75.蔡辉,赵智明,修春英,等.丹参酮ⅡA对溶血磷脂酸致新生大鼠心脏成纤维细胞 增殖及分泌转化生长因子β1的影响[J].医学研究生学报,2008,21(2)：126-128.
76.张冬梅,秦英,牛福玲,等.丹参酮ⅡA对AngⅡ诱导的心肌成纤维细胞增殖及Ⅰ型胶原合成的影响[J].辽宁中医杂志,2008,35(12)：1934-1936.
77.谢辉,郑智,龚丽娅.丹参酮ⅡA对自发性高血压大鼠左室肥厚的影响及机制[J].实用医学杂志,2004,20(3)：252-254.
78.邹昌群,占成业,白祥军,等.丹参酮ⅡA抗心肌纤维化作用机制实验研究[J].内科急危重症杂志,2008,14(5)：242-245.
79.Lau CW, Yao XQ, Chen ZY, et al. Cardiovascular actions of berberine[J]. Cardiovasc Drug Rev,2001,19(3):234.
80.张明发.黄连素的抗高血压作用[J].医院用药评价与分析,2001,1(5)：299-301.
81.陈惠卿.黄连素的心血管作用药理及其临床应用[J].佛山科学技术学院学报(自然科学版),2008,26(2)：75-77.
82.郝钰,徐泊文,郑宏,等.小檗碱对人脐静脉内皮细胞增殖与凋亡的作用[J].中国病理生理杂志,2005,21(6)：1124-1127.
83.娄金丽,邱全瑛,郝钰,等.小檗碱抗肿瘤新生血管形成作用机制的研究[J].中国免疫学杂志,2006,22(3)：235-236,243.
84.李凝,陆付耳,徐丽君,等.小檗碱对大鼠糖尿病早期肾脏转化生长因子β1的影响[J].中国康复,2008,23(1)：11-13.
85.解欣然,洪缨,董世芬.小檗碱抑制血管紧张素Ⅱ诱导的心肌成纤维细胞增殖和胶原合成的作用及机制研究[J].北京中医药大学学报,2008,31(11)：748-751.
86.吴红金,吕俊萍,马增春,等.川芎嗪对培养人脐静脉内皮细胞基因表达谱的影响[J].中国实验方剂学杂志,2005,11(3)：40.
87.熊石龙,文志斌,王前,等.川芎嗪对肿瘤坏死因子致血管内皮细胞组织因子表达的影响[J].中国现代医学,2007,17(19)：2330-2333.
88.高雪,杨洁,元英进.盐酸川芎嗪对血管紧张素Ⅱ损伤内皮细胞的保护作用[J].细胞生物学杂志,2007,29：895-900.
89.张冬梅,秦英,杨君,等.川芎嗪对血管紧张素Ⅱ诱导的人α1(Ⅰ)胶原基因启动子活性的影响[J].中国中医药信息杂志,2005,12(9)：21.
90.郑红花,李映红,罗德生.川芎嗪对血管紧张素诱导的血管平滑肌细胞增殖的抑制作用及机制[J].中国动脉硬化杂志,2006,14(4)：301.
91.李国凡,张延斌,陈清枝.川芎嗪对凝血酶诱导的血管平滑肌细胞增殖的影响[J]心脏杂志,2007,19(6)：646-648.
92.张海啸,史载祥,贾海忠,等.川芎嗪对成纤维细胞及TGF-β/Smads信号传导的影响[J].北京中医药大学学报,2007,30(40)：252-255.
93.宋纯清,樊懿,黄伟晖,等.钩藤中不同成分降压作用的差异[J].中草药,2000,31(10)：762-764.
94.余文海.钩藤对自发性高血压大鼠内皮的影响[J].国外医学·中医中药分册,2000,22(6)：351-352.
95.怡悦.钩藤对自发性高血压大鼠血管内皮功能的影响[J].国外医学·中医中药分册,2000,22(1)：28-29.
96.刘建斌,任江华.钩藤对自发性高血压大鼠心肌重构及原癌基因C-fos表达的影响[J].中国中医基础医学杂志,2000,6(5)：40-44.
97.李艳菊,孙安盛,余丽梅,等.钩藤碱对Ang Ⅱ诱导大鼠血管平滑肌细胞增殖的抑制作用[J].中国药学杂志,2008,43(21)：1624-1624.
98.丁伯平,熊莺,徐朝阳,等.益母草碱对急性血瘀证大鼠血液流变学的影响[J].中国中医药科技,2004,11(1)：36-37
99.Pang S, Tsuchiya S, Horie S, et al. Enhancement of phenyle-phedrine- induced contraction in the isolated rat aorta with endothelium by H2O- extract from an oriental medical plant leonuri herba[J]. Jpn J Pharmacol,2001,86(2):215-220.
100.陈少如,郑鸿翱,陈韩秋,等.益母草制剂治疗心肌缺血及其机制研究[J].中国危重病急救医学,2002,14(1)：19-22.
101.阮奕满,林进国,刘甘泉.益母草对兔心肌缺血再灌注损伤氧自由基的影响[J].卫生职业教育,2007,12(25)：123.
102.杨得坡,苏薇薇.参环毛蚓脂类挥发性成分分析[J].中药材,2000,23(1)：31-33.
103.葛勤,张恩娟,刘同华,等.地龙的化学成分及制剂质量研究概况[J].综述报告,2003,13(3)：69-71.
104.徐宗佩,王益明,张吉正,等.地龙提取物对健康小鼠红细胞变形性的影响[J].天津中医,2000,17(3)：37.
105.殷出梅,储益平,吴鹏.地龙活性提取物的主要药效学实验[J].中草药,2002,33(10)：926-928.
106.陈洪,陆亚琴,刘顺英,等.地龙2号对肝纤维化模型大鼠肝星状细胞活化及 TGF-β1蛋白表达的影响[J].江苏中医药,2005,26(1)：50-52.
107.陈洪,陆亚琴,刘顺英,等.地龙2号对大鼠肝纤维化α-SMA、TGF-β1、MMP-13及TIMP-1蛋白表达的影响[J].胃肠病学和肝病学杂志,2005,14(2)：156-159.
108.翟宝伟,康白,毛淑梅,等.地龙提取物对自发性高血压大鼠左室肥厚的影响[J].现代中西医结合杂志,2007,16(29)：4272-4274.
109.李承德,康白,毛淑梅,等.地龙降压蛋白对自发性高血压大鼠降压作用及其机制的影响[J].中华中医药杂志,2008,23(5)：450-452.
110.陈乐生.桑寄生药理研究[J].陕西中医,2000,21(11)：520-521.
111.叶立新,王继红,黄华利.桑寄生对肾性高血压大鼠血浆p-内啡肽浓度影响的量效作用[J].中国临床康复,2005,9(27)：84-85.
112.张朝阳.降压中药的有关研究进展[J].内蒙古中医药,2008,6：66-69.
113.刘丽娟.桑寄生现代临床应用研究进展[J].检验医学与临床,2009,6(12)：1001-1002.
1.Zhu DL, Herembert T, Marche P. Increased proliferation of ad vent it ial fibroblasts from spontaneously hypertensive rat aorta[J]. J Hypertens,1991,9(12):1161-1168.
2.Zhu DL, Herembert T, Caruelle D, et al. Signaling mechanisms of basic fibroblast growth factor in arterial cells from genetically hypertensive rat[J]. Am J Hypertens, 1994,7(4 Pt 1):351-356.
3.Shi Y, OBrien JE, Fard A, et al. Adventitial myofibroblasts contribute to neointimal formation in injured porcine coronary arteries [J]. Circulation,1996,4:1655-1664.
4.Hoshino R, Tanimura S, Watanabe K, et al. Blockade of the extracellular signal-regulated kinase pathway induces marked G1 cell cycle arrest and apoptosis in tumor cell in which the pathway is constitutively activated:up-regulation of p27 (Kip1) [J]. J Biol Chem,2001,276:2686-2692.
5.Gibbons GH, Dzau VJ. The emerging concept of vascular remodeling[J]. The New England Journal of Medicine,1994,330(20):1431-1438.
6.F.E. Rey, R.J. Pagano. The Reactive Adventitia:Fibroblast Oxidase in vascular function. Arterioscler[J]. Thromb Vasc. Biol,2002,22:1962-1971.
7.Clakr DA, Coker R. Transforming growth factor-beta (TGF-beta)[J].In J Biochem Cell Biol,1998,30:293-298.
8.Anderson KM, Brawer ME, Nichols AJ. Transforming growth factor-beta in restenosis [A]:In Anderson KM. Coronary restenosis:form genetics to therapeutics 1sted [M]. New York Marcel Dekker,2000,99-127.
9. Camoretti-Mercado B'Solway J. Transforming growth factor-betal and disorders of the lung[J].Cell Biochem Biophys,2005,43(1):131-148.
10.Saika S, Yamanaka O, Okada Y, et al. TGF beta in fibroproliferative diseases in the eye[J]. Front Biosci (Schol Ed),2009,1:376-390.
11.Ryna ST, Koetlinansky VE, Gowtals PJ, et al. Transforming growth factor-beta-dependent events in vascular remodeling following arterial injury[J]. J Vase Res,2003, 40:37-46.
12. Jiang YL, Dai AG, Li QF, et al. Transforming growth factor-betal induces transdifferentiation of fibroblasts into myofibroblasts in hypoxic pulmonary vascular remodeling [J]. Acta Biochim Biophys Sin,2006,38:29-36.
13.Toma I, McCaffrey TA. Transforming growth factor-β and atherosclerosis: interwoven atherogenic and atheroprotective aspects [J]. Cell Tissue Res,2012 347(1):155-175.
14.Lan HY, Chung AC.Transforming growth factor-β and Smads [J]. Contrib Nephrol,2011,170:75-82.
15.Lan HY.Transforming growth factor-β/Smad signalling in diabetic nephropathy [J]. Clin Exp Pharmacol Physiol,2012,39 (8):731-738.
16.Inagaki Y, Higashiyama R, Higashi K. Novel anti-fibrotic modalities for liver fibrosis:molecular targeting and regenerative medicine in fibrosis therapy [J]. J Gastroenterol Hepatol,2012, Suppl2:85-88.
17.Mulder M. Role of Ras and Mapks in TGF beta signaling [J]. Cytokine Growth Factor Rev,2000,11 (1-2):23-35.
18.Bobick BE, Kulyk WM. Regulation of cartilage formation and maturation by mitogen-activated protein kinase signaling[J]. Birth Defects Res C Embryo Today, 2008,84(2):131-154.
19.Mu Y, Gudey SK, Landstrom M. Non-Smad signaling pathways[J]. Cell Tissue Res,2012,347(1):11-20.
20.Samarakoon R, Higgins PJ. Integration of non-SMAD and SMAD signaling in TGF-betal-induced plasminogen activator inhibitor type-1 gene expression in vascular smooth muscle cells [J]. Thromb Haemost,2008,100 (6):976-983.
21.Guo X, Wang XF. Signaling cross-talk between TGF-beta/BMP and other pathways[J]. Cell Res,2009,19(1):71-88.
22.Biondi RM, Nebreda AR. Signalling specificity of Ser/Thr protein kinases through docking-site-mediated interactions[J]. Biochem J,2003,372(Pt 1):1-13.
23.Bobik A, Tkachuk V. Metalloproteinases and plasminogen activators in vessel remodeling[J]. Curr Hypertens Rep,2003,5(6):466-472.
24.Janicki JS, Brower GL, Gardner JD, et al. Cardiac mast cell regulation of matrix metalloproteinase-related ventricular remodeling in chronic pressure or volume overload[J]. Cardiovasc Res,2006,69(3):657-665.
25.Steed MM, Tyagi SC.Mechanisms of cardiovascular remodeling in hyperhomocysteinemia[J]. Antioxid Redox Signal,2011,15(7):1927-1943.
26.Raffetto JD, Khalil RA. Matrix metalloproteinases and their inhibitors in vascular remodeling and vascular disease[J]. Biochem Pharmacol,2008, 75(2):346-359.
27.Lim CS, Shalhoub J, Gohel MS, et al. Matrix metalloproteinases in vascular disease--a potential therapeutic target[J]? Curr Vasc Pharmacol,2010, 8(1):75-85.
28.Chelladurai P, Seeger W, Pullamsetti SS. Matrix metalloproteinases and their inhibitors in pulmonary hypertension[J]. Eur Respir J,2012,40(3):766-782.
29.Moussad EE, Brigstock DR.Connective tissue growth factor:what's in a name[J]? Mol Genet Metab,2000,71 (1-2):276-292.
30.Rachfal AW, Brigstock DR. Structural and functional properties of CCN proteins[J]. Vitam Horm,2005,70:69-103.
31.Luft FC. CCN2, the connective tissue growth factor[J]. J Mol Med (Berl),2008, 86(1):1-3.
32.Grotendorst GR.Connective tissue growth factor:a mediator of TGF-beta action on fibroblasts[J]. Cytokine Growth Factor Rev,1997,8 (3):171-179.
33.Tabibzadeh S.Homeostasis of extracellular matrix by TGF-beta and lefty[J]. Front Biosci,2002,7:1231-1246.
34.Burgess JK. Connective tissue growth factor:a role in airway remodelling in asthma[J]? Clin Exp Pharmacol Physiol,2005,32(11):988-994.
35.Leask A, Holmes A, Abraham DJ. Connective tissue growth factor:a new and important player in the pathogenesis of fibrosis[J]. Curr Rheumatol Rep,2002, 4(2):136-142.
36.Leask A, Abraham DJ. The role of connective tissue growth factor, a multifunctional matricellular protein, in fibroblast biology [J]. Biochem Cell Biol.2003,81(6):355-363.
37.Leask A, Abraham DJ. TGF-beta signaling and the fibrotic response[J]. FASEB J,2004,18(7):816-827.
38.Ruiz-Ortega M, Rodriguez-Vita J, Sanchez-Lopez E, Carvajal G, Egido J.TGF-beta signaling in vascular fibrosis[J].Cardiovasc Res,2007,74 (2):196-206.
39.Chen XM, Qi W, Pollock CA. CTGF and chronic kidney fibrosis[J]. Front Biosci (Schol Ed),2009,1:132-141.
40.Arnott JA, Lambi AG, Mundy C, Hendesi H, Pixley RA, Owen TA, Safadi FF, Popoff SN. The role of connective tissue growth factor (CTGF/CCN2) in skeletogenesis[J]. Crit Rev Eukaryot Gene Expr,2011,21(1):43-69.
41.Ng K, Butlin M, Avolio AP. Persistent effect of early, brief angiotensin-converting enzyme inhibition on segmental pressure dependency of aortic stiffness in spontaneously hypertensive rats[J]. J Hypertens,2012,30(9):1782-1790.
42.Soro-Paavonen A, Gordin D, Forsblom C, et al. FinnDiane Study Group. Circulating ACE2 activity is increased in patients with type ldiabetes and vascular complications [J]. J Hypertens,2012,30(2):375-383.
43.Ren M, Zhang J, Wang B, et al. Qindan-capsule inhibits proliferation of adventitial fibroblasts and collagen synthesis[J]. J Ethnopharmacol,2010,129:53-58.
44.Ren M, Wang B, Zhang J. Study on quality standard of Qindan Capsule[J]. Chinese Journal of Integrative Medicine on Cardio-/Cerebrovascular Disease, 2009,7:597-98.
45.Iwama H, Amagaya S, Ogihara Y. Effect of shosaikoto, a Japanese and Chinese traditional herbal medicinal mixture, on the mitogenic activity of lipopolysaccharide: a new pharmacological testing method[J]. J Ethnopharmacol,1987,21(1):45-53.
46.He YZ. Research survey of serum pharmacology and serum medicinal Chemistry in Japanese Chinese herbs[J]. Int J Chin Med,1998,20(5):3-7.
47.王喜军.中药血清药物化学的研究动态及发展趋势[J].中国中药杂志,2006,31(10)：789.
48.任敏,张继东,王博,等.芩丹胶囊对血管外膜成纤维细胞的作用机制.山东大学 学报(医学版)[J].2010,04：27-32.
49.Itoh H, Nakao K. Antagonism between the vascular renin-angiotensin and natriuretic peptide systems in vascular remodelling[J]. Blood Press Suppl,1994, 5:49-53.
50.Teng J, Fukuda N, Suzuki R, et al. Inhibitory effect of a novel angiotensin Ⅱ type 1 receptor antagonist RNH-6270 on growth of vascular smooth muscle cells from spontaneously hypertensive rats:different anti-proliferative effect to angiotensin-converting enzyme inhibitor[J]. J Cardiovasc Pharmacol,2002,39(2):161-171.
51.Chen K, Mehta JL, Li D, et al. Transforming growth factor beta receptor endoglin is expressed in cardiac fibroblasts and modulates profibrogenic actions of angiotensin Ⅱ [J]. Circ Res,2004,10(12):1167-1173.
52.Kawano H, Do YS, Kawano Y, et al. Angiotensin Ⅱ has multiple prebrothic effects in human cardiac fibroblasts [J]. Circulation,2000,1(10):1130-1137.
53.刘平,张运,胡维成.血管外膜及其成纤维细胞在血管增殖性病变中的作用[J].中华老年医学杂志,2005,24(12)：936-938.
54.Orton EC, LaRue SM, Ensley B, et al. Bromodeoxyuridine labeling and DNA content of pulmonary arterial medial cells from hypoxia-exposed and nonexposed healthy calves [J]. Am J Vet Res,1992,53(10):1925-1930.
55.Scott NA, Cipolla GD, Ross CE, et al. Identification of a potential role for the adventitia in vascular lesion formation after balloon overstretch injury of porcine coronary arteries[J]. Circulation,1996,93(12):2178-2187.
56.Shi Y, Niculescu R, Wang D, et al. Increased NAD(P)H oxidase and reactive oxygen species in coronary arteries after balloon injury[J]. Arterioscler Thromb Vasc Biol,2001,21(5):739-745.
57.Krick S, Hanze J, Eul B, et al. Hypoxia-driven proliferation of human pulmonary artery fibroblasts:cross-talk between HIF-1 alpha and an autocfine angiotensin system[J]. FASEB J,2005,19(7):857-859.
58.Stenmark KR, Gerasimovskaya E, Nemenoff RA, et al. Hypoxic activation of adventitial fibroblasts:role in vascular remodeling[J]. Chest,2002,122(6):326-334.
59.Ryan ST, Koteliansky VE, Gotwals PJ, et al. Transforming growth factor-beta-dependent events in vascular remodeling following arterial injury[J]. J Vasc Res, 2003,40(1):37-46.
60.Lijnen PJ, Ptrov VV, Fagard RH. Association between transforming growth factor-β1 and hypertension[J]. Am J Hypertens,2003,16(7):604-611.
61.梁粟,张继东.芩丹胶囊对阳亢血瘀型高血压患者血栓前状态的影响[J].上海中医药杂志2006,40(10)：21-23.
62.Li G, Chen SJ, Oparil S, et al. Direct in vivo evidence demonstrating neointimal migration of adventitial fibroblasts after balloon injury of rat carotid arteries[J]. Circulation,2000,101(12):1362-1365.
63.Rey FE, Pagano PJ. The reactive adventitia:fibroblast oxidase in vascular function[J]. Arterioscler Thromb Vasc Biol,2002,22(12):1962-1971.
64.王永利,贺能树,张家兴,等.血管成形术后外膜细胞表型转化和迁移的实验研究[J].介入放射学杂志,2006,15(7)：424-429.
65.孙爱军,高平进,刘建军,等.骨桥蛋白增强自发性高血压大鼠血管外膜成纤维细胞的迁移活性[J].生理学报,2004,56(1)：21-24.
66.Wang B, Zhang JD, Feng JB, et al. Effect of traditional Chinese medicine Qin-Dan-Jiang-Ya-Tang on remodeled vascular phenotype and osteopontin in spontaneous hypertensive rats[J]. J Ethnopharmacol 2007,110:176-182.
67.Chatelain RE, Dardik BN. Increased DNA replication in the arterial adventitia after aortic ligation[J]. Hypertension,1988,11(suppl Ⅰ):Ⅰ-130-Ⅰ-134.
68.裴端卿.基质金属蛋白酶家族介绍[J].中国生物化学与分子生物学报.2001,17(1)：17-20.
69.Van den Steen PE, Dubois B, Nelissen I,et al. Biochemistry and molecular biology of gelatinase B or matrix metalloproteinase-9 (MMP-9)[J]. Crit Rev Biochem Mol Biol,2002,37(6):375-536.
70.Newby AC. Metalloproteinase expression in monocytes and macrophages and its relationship to atherosclerotic plaque instability [J]. Arterioscler Thromb Vasc Biol, 2008,28(12):2108.
71.Berry E, Bosonea AM, Wang X, et al. Insights into the activity, differential expression, mutual regulation, and functions of matrix metalloproteinases and a disintegrin and metalloproteinases in hypertension and cardiac disease[J]. Crit Rev Biochem Mol Biol,2002,37(6):375-536.
72.Sankaralingam S, Arenas IA, Lalu MM, et al. Preeclampsia:current understanding of the molecular basis of vascular dysfunction[J]. Expert Rev Mol Med,2006,8:1-20.
73.Benjamin MM, Khalil RA. Matrix metalloproteinase inhibitors as investigative tools in the pathogenesis and management of vascular disease[J]. EXS,2012,103: 209-279.
74.Jackson C, Nguyen M, Arkell J, et al. Selective matrix metalloproteinase (MMP) inhibition in rheumatoid arthritis targetting gelatinase A activation[J]. Inflamm Res, 2001,50(4):183-186.
75.Tayebjee MH, Nadar SK, Blann AD, et al. Matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 in hypertension and their relationship to cardiovascular risk and treatment:a substudy of the Anglo-Scandinavian Cardiac Outcomes Trial (ASCOT) [J]. Am J Hypertens,2004,17:764-769.
76.Derosa G, D'Angelo A, Ciccarelli L, et al. Matrix metalloproteinase-2,-9,and tissue inhibitor of metalloproteinase-1 in patients with hypertension[J]. Endothelium 2006,13:227-231.
77.Roberts AB, Sporn MB, Assoian RK, et al. Transforming growth factor-β:rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro[J]. Proc Natl Acad Sci USA,1986,83:4167-4171.
78.Axisa B, Loftus IM, Naylor AR, et al. Prospective, randomized, double-blind trial investigating the effect of doxycycline on matrix metalloproteinase expression within atherosclerotic carotid plaques[J]. Stroke,2002,33:2858-2864.
79.Beaudeux JL, Giral P, Bruckert E, et al. Matrix metalloproteinases, inflammation and atherosclerosis:therapeutic perspectives [J]. Clin Chem Lab Med,2004, 42:121-131.
80.Bremer C, Tung CH, Weissleder R. In vivo molecular target assessment of matrix metalloproteinase inhibition[J]. Nat Med,2001,7:743-748.
81.Brown S, Meroueh SO, Fridman R, et al. Quest for selectivity in inhibition of matrix metalloproteinases [J]. Curr Top Med Chem,2004,4:1227-1238.
82.Cook GR, Manivannan E, Underdahl T, et al. Synthesis and evaluation of novel oxazoline MMP inhibitors [J]. Bioorg Med Chem Lett,2004,14:4935-4939.
83.Agrawal A, Romero-Perez D, Jacobsen JA, et al. Zinc-binding groups modulate selective inhibition of MMPs [J].Chem Med Chem,2008,3:812-820.
84.Bradham DM, Igarashi A, Potter RL, et al.Connective tissue growth factor:a cysteine-rich mitogen secreted by human vascular endothelial cells is related to the SRC-induced immediate early gene product CEF-10[J]. J Cell Biol,1991, 114(6):1285-1294.
85.Igarashi A, Bradham DM, Okochi H, et al. Connective tissue growth factor[J]. J Dermatol,1992,19(11):642-643.
86.Frazier K, Williams S, Kothapalli D, et al. Stimulation of fibroblast cell growth, matrix production, and granulation tissue formation by connective tissue growth factor[J]. J Invest Dermatol,1996,107:404-411.
87.Chen MM, et al. CTGF expression is induced by TGF- beta in cardiac fibroblasts and cardiac myocytes:a potential role in heart fibrosis[J]. J Mol Cell Cardiol,2000, 32(10):1805-1819.
88.Ruperez M, et al. Connective tissue growth factor is a mediator of angiotensin Ⅱ-induced fibrosis. Circulation[J].2003,108(12):1499-1505.
89.Sato S, et al. Serum levels of connective tissue growth factor are elevated in patients with systemic sclerosis:association with extent of skin sclerosis and severity of pulmonary fibrosis. J Rheumatol[J].2000,27(1):149-154.
90.Gressner AM, et al. Connective tissue growth factor in serum as a new candidate test for assessment of hepatic fibrosis[J]. Clin Chem,2006,52(9):1815-1817.
91.Lee YS, Byun J, Kim JA, et al. Monocrotaline-induced pulmonary hypertension correlates with upregulation of connective tissue growth factor expression in the lung[J]. Exp Mol Med,2005,37(1):27-35.
92.田锋,徐永健,张珍祥,等.结缔组织生长因子在烟雾暴露大鼠肺血管重塑中的作用[J].中华结核和呼吸杂志,2007,30：921-925.
93.田锋,徐永健,张珍祥,等CTGF在烟雾暴露大鼠肺血管中的表达及其与肺血管重建的关系[J].中国病理生理杂志,2008,24：7-11.
94.王再,徐永健,刘先胜,曾大雄,向敏.结缔组织生长因子和周期蛋白D1在烟雾暴露大鼠肺血管重构中的表达变化[J].中华结核和呼吸杂志,2010,33(9)：679-683.
95. Wang R, Xu YJ, Liu XS, et al. Knockdown of connective tissue growth factor by plasmid-based short hairpin RNA prevented pulmonary vascular remodeling in cigarette smoke-exposed rats. Arch Biochem Biophys[J].2011,508(1):93-100.
96.Massague J. The transforming growth factor-beta family[J]. Anna Rev Cell Biol, 1990,6:597-641.
97.Heldin CH, Miyazono K, ten Dijke P. TGF-beta signalling from cell membrane to nucleus through SMAD proteins[J]. Nature,1997,390(6659):465-471.
98.Moustakas A, Souchelnytskyi S, Heldin CH. Smad regulation in TGF-beta signal transduction[J]. J Cell Sci,2001,114(24):4359-4369.
99.陈琛,叶冬梅.转化生长因子-β/Smad信号通路研究进展[J].医学分子生物学杂志,2006,3(2)：126-128.
100.Massague J. How cells read TGF-beta signals[J]. Nat Rev Mol Cell Biol,2000, 1(3):169-178.
101.Ren M, Wang B, Zhang J, et al.Smad2 and Smad3 as mediators of the response of adventitial fibroblasts induced by transforming growth factor (31 [J]. Mol Med Rep,2011,4(3):561-567.
102.Yu L, Hebert MC, Zhang YE. TGF-beta receptor-activated p38 MAP kinase mediates Smad-independent TGF-beta responses [J]. EMBO J,2002, 21(14):3749-3759.
103.Imamichi Y, Waidmann O, Hein R, et al. TGF beta-induced focal complex formation in epithelial cells is mediated by activated ERK and JNK MAP kinases and is independent of Smad4[J]. Biol Chem,2005,86(3):225-236.
104.Muscogiuri G, Chavez AO, Gastaldelli A, et al. The crosstalk between insulin and renin-angiotensin-aldosterone signaling systems and its effect on glucose metabolism and diabetes prevention [J]. Curr Vase Pharmacol,2008,6(4):301-312.
105.Jing L, Zhang J, Sun J, et al. Inhibition of extracellular signal-regulated kinases ameliorates hypertension-induced renal vascular remodeling in rat models[J]. Int J Mol Sci,2011,12(12):8333-8346.
106.Takayama N, Kai H, Kudo H, et al. Simvastatin prevents large blood pressure variability induced aggravation of cardiac hypertrophy in hypertensive rats by inhibiting RhoA/Ras-ERK pathways[J]. Hypertens Res,2011,34(3):341-347.
107.Bonacasa B, Sanchez ML, Rodriguez F, et al.2-Methoxyestradiol attenuates hypertension and coronary vascular remodeling in spontaneously hypertensive rats [J]. Maturitas,2008,61(4):310-316.
108.Yan JT, Wang T, Wang DW. Recombinant adeno-associated virus-mediated human kallikrein gene therapy protects against hypertensive target organ injuries through inhibiting cell apoptosis[J]. Acta Pharmacol Sin,2009,30(9):1253-1261.
109.Redondo A, Estrella N, Lorenzo AG, et al. Quercetin and catechin synergistically inhibit angiotensin Ⅱ-induced redox-dependent signalling pathways in vascular smooth muscle cells from hypertensive rats[J]. Free Radic Res,2012, 46(5):619-627.
110.Jing L, Zhang JZ, Zhao L, et al. High-expression of transforming growth factor betal and phosphorylation of extracellular signal-regulated protein kinase in vascular smooth muscle cells from aorta and renal arterioles of spontaneous hypertensionrats[J]. Clin Exp Hypertens,2007,29(2):107-117.
111.Rezaei HB, Kamato D, Ansari G, et al. Cell biology of Smad2/3 linker region phosphorylation in vascular smooth muscle[J]. Clin Exp Pharmacol Physiol,2012, 39(8):661-667.
112.Krause C, Kloen P. Concurrent inhibition of TGF-β and mitogen driven signaling cascades in Dupuytren's disease-non-surgical treatment strategies from a signaling point of view[J]. Med Hypotheses,2012,78(3):385-388.
113.Huang XY, Chen CX. Effect of oxymatrine, the active component from Radix Sophorae flavescentis (Kushen) on ventricular remodeling in spontaneously hypertensive rats[J]. Phytomedicine,2013,20(3-4):202-212.