大蒜素对左室重构和心肌纤维化的影响及机理研究
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摘要
高血压病(Hypertension,HT)是全球性常见病和多发病,严重损害人们的身心健康,心脏是高血压最常损害的靶器官之一。长期慢性压力超负荷必将引起心脏结构和功能的适应性改变即心脏重塑,主要表现为左室肥厚(LVH)。虽然左室肥厚初期是对心脏负荷增加的适应性改变,以平衡心肌应激的增加,但长期的负荷应激终至适应不良性肥厚。现在认为左室重构不仅是心肌细胞实质成分的变化,还有心肌间质成分和血管成分的变化。心肌间质成分相对于实质成分的不成比例增生,构成心肌结构的异质性,即心肌纤维化,是高血压性心脏病(hypertension heart disease, HHD)心肌重塑的主要病理特征之一,是出现心律失常、心力衰竭、心脏性猝死等并发症的决定因素。
心肌发生纤维化时最主要的病理改变为细胞外基质(ECM)的大量积聚。在心脏中,ECM含量最多的是胶原蛋白,其不成比例的堆积或纤维化对组织硬度具有决定作用。心脏成纤维细胞在局部神经体液因子的作用下过度激活,是胶原分泌增多的主要原因,但其发生机制尚未完全阐明。
本研究致力于探讨左室重构过程中心肌纤维化的发生机制并寻求有效的药物干预手段。全文共分为以下五部分:
第一部分大蒜素对压力负荷大鼠左室重构的影响
目的:观察压力负荷大鼠左室重构模型情况及大蒜素对其的影响。
方法:将90只SD雄性大鼠随机分为6组:假手术组(Sham)、模型组(Control)、大蒜素注射液组( Garlicin 1 )、大蒜素口服组( Garlicin 2 )、川芎嗪组(Tetramethylpyrazine,TMP)、氯沙坦组( Losartan ),每组15只。以腹主动脉缩窄方法制备压力负荷性左室重构模型。于术后3天开始给药,给药30天。给药结束后行心脏超声检查;其后采血样用于后续的生化检测,取心脏作组织形态学检查。
结果:腹主动脉缩窄30天造成的压力超负荷大鼠左室重构模型经超声心动检测证实腹主动脉缩窄引起的心肌肥厚属于向心性肥厚,室壁增厚而室腔容积减少,心室的收缩功能尚处于代偿阶段。各给药组同模型组比较室壁有不同程度的减轻(室壁厚度:Control组>Garlicin 2组>Garlicin 1组/TMP组>Losartan组>sham组)但是除losartan组收缩期左室后壁厚度外(P<0.05),均未出现明显统计学差异,增大样本量有可能出现。模型组心脏体重指数高于假手术组(P<0.05),氯沙坦组明显低于模型组(P<0.05),两个大蒜素组和川芎嗪组较模型组有降低趋势,但没有出现统计学差异。从病理形态来看,光镜下假手术组心肌纤维排列整齐,心肌细胞大小正常,模型组心肌纤维排列紊乱,细胞横径稍大,各药物治疗组有不同程度的改善。电镜下见假手术组细胞为正常超微结构,模型组的细胞超微结构的改变主要为:心肌细胞变大、线粒体变性、肌原纤维变性、细胞核不规则等,各治疗组也有所改善。
结论:大蒜素注射液与川芎嗪注射液有相似的抑制左室重构作用,但其效果不如氯沙坦。大蒜素口服组的疗效不如大蒜素注射液。
第二部分大蒜素对压力负荷大鼠心肌纤维化的影响
目的:观察压力负荷大鼠左室重构过程中的心肌纤维化及大蒜素对其的影响。
方法:检测左室心肌羟脯氨酸(HOP)、血清胶原代谢物Ⅰ型前胶原羧基端肽(PⅠCP)及Ⅲ型前胶原氨基端肽(PⅢNP)、血清透明质酸(HA)及层粘蛋白(LN)。对心肌组织进行苦味酸天狼猩红染色,计算左室心肌胶原容积分数(CVF,%)和血管周围胶原面积(PVCA,%);制备电镜标本观察心肌胶原纤维和超微结构。
结果:模型组心肌羟脯氨酸明显增多,说明胶原的合成增加;同时,通过天狼猩红染色观察到心肌纤维被胶原纤维栅栏状包绕,CVF和PVCA较假手术组明显增高,呈间质纤维化和血管周围纤维化现象,属反应性纤维化阶段。血清中PⅠCP、PⅢNP、LN、HA较假手术组均明显增高,,也说明腹主动脉缩窄造成的压力负荷大鼠存在心肌纤维化现象。阳性药物氯沙坦与模型组相比,可抑制心肌羟脯氨酸,减少心肌胶原(P<0.05),并降低血清PⅠCP、PⅢNP、LN(P<0.001或P<0.05),降低CVF和PVCA(P<0.05),减少心肌胶原的排列紊乱。大蒜素注射液和川芎嗪注射液均可降低血清PⅠCP、PⅢNP和HA的增多(P<0.05),降低PVCA(P<0.05),可改善血管周围胶原的增生和排列紊乱。大蒜素口服仅能改善PⅠCP、PⅢNP(同模型组相比P<0.05)。
结论:大蒜素注射液对腹主动脉缩窄导致的心肌反应性纤维化有一定的抑制作用。大蒜素口服后对心肌纤维化也有一定的作用。
第三部分大蒜素对压力负荷大鼠AngⅡ和SOD、MDA的影响
目的:观察大蒜素对压力负荷大鼠AngⅡ和SOD、MDA的影响。
方法:通过制备腹主动脉缩窄造成的压力负荷大鼠动物模型,检测大蒜素对血浆和心肌中AngⅡ,血清中SOD活性和MDA含量。
结果:腹主动脉缩窄后,模型组比假手术组的血浆和心肌AngⅡ含量升高(P<0.05),其均值可达假手术组的2倍以上。氯沙坦治疗后血浆中AngⅡ均值水平较模型组升高;心肌组织中的AngⅡ却出现降低趋势(P>0.05)。川芎嗪组、Garlicin 1组的血浆中AngⅡ水平较模型组明显降低(P<0.05),余未出现统计学差异。SOD活力在模型组中有降低趋势(Control组:Sham组,P=0.06);各药物治疗后均有不同程度的提高,其中,Losartan组和TMP组较模型组的总SOD活力明显提高(P<0.05),Garlicin 1组也有较为明显的提高,但是未出现统计学差异(P=0.07)。MDA含量的变化在模型组较假手术组增高(P<0.05),四个治疗组与模型组相比出现明显的降低(P<0.05)。
结论:压力负荷使大鼠AngⅡ增高,导致RAAS的激活。大蒜素可降低压力负荷导致的循环和心肌局部的AngⅡ异常增高,而口服大蒜素使AngⅡ降低的程度不大。大蒜素能够通过抑制血清MDA、提高SOD活力,减轻左室重构过程中的过氧化损伤。
第四部分大蒜素对成纤维细胞增殖和胶原分泌的影响
目的:观察大蒜素对成纤维细胞增殖和胶原分泌的影响,探讨抗纤维化作用机理。
方法:以NIH3T3细胞为模型,通过H3-thymidine DNA掺入量测定大蒜素对NIH3T3细胞DNA合成的影响;DNA ladder检测细胞凋亡情况;碱消化法检测细胞培养液中羟脯氨酸含量;荧光免疫染色方法检测NIH3T3细胞Ⅰ型胶原蛋白的表达。
结果:大蒜素作用于NIH3T3细胞24h,在0.2-5ug/ml范围内剂量依赖的抑制NIH3T3细胞的DNA合成;但没有观察到DNA ladder。大蒜素可使细胞培养液中的羟脯氨酸含量减少,Ⅰ型胶原蛋白的表达减少。
结论:大蒜素对NIH3T3细胞生长增殖有抑制作用,减少细胞胶原的分泌,减少Ⅰ型胶原的表达,可能是大蒜素抗纤维化的作用机理之一。
第五部分大蒜素对TGF-β1及其信号传导系统的影响
目的:探讨大蒜素对心肌纤维化过程中TGF-β1及其信号传导的影响。
方法:采用ELISA方法检测压力负荷大鼠模型血清TGF-β1含量,通过免疫组化方法检测心肌TGF-β1表达,并通过图像分析计算阳性表达的面积(Area)、平均光密度(Density)和IOD值。采用Real-time PCR方法检测TGF-β1下游信号心肌Smad2、Smad7 mRNA表达的变化。同时通过体外实验Mv1Lu-(CAGA)12-Luc(稳定转染TGF-β1信号响应基因片段的细胞系)荧光酶活性的变化进一步验证大蒜素对TGF-β1信号传导系统的影响。
结果:模型组同假手术组比较血清TGF-β1水平明显升高,氯沙坦组同模型组相比TGF-β1水平明显下降, Garlicin 1、Garlicin 2和TMP组的血清同模型组相比TGF-β1水平明显下降,同假手术组比较仍存在明显的升高。免疫组化观察TGF-β1蛋白阳性表达为棕黄色沉积,主要在大鼠心肌间质中表达。经图像分析模型组同假手术组比较存在统计学意义上的升高。氯沙坦组、大蒜素注射液组、川芎嗪组同模型组相比Area和IOD两个方面都出现了明显的降低,而大蒜素口服组仅Area值表现出统计学意义上的降低。
Real-time PCR结果显示模型组的Smad2表达量比假手术组上调了1.18倍,而Smad7下调了0.55倍。氯沙坦组与模型组相比Smad2下调至0.63倍,而Smad7仍下调至0.88倍;川芎嗪组与模型组相比Smad2下调至0.49倍, Smad7上调至1.40倍;大蒜素注射液组与模型组相比Smad2下调至0.75倍,Smad7上调至1.27倍;大蒜素口服组与模型组相比Smad2下调至0.71倍,而Smad7却下调至0.72倍。体外实验Mv1Lu-(CAGA)12-Luc细胞荧光酶活性在2ng/mL TGF-β刺激的情况下,大蒜素的中剂量(1ug/mL)组表现出对荧光素酶活性的明显抑制,其抑制率达20%左右,增大剂量至5ug/mL时此作用减弱。大蒜素药物血清在大中小三个剂量组与药物直接作用表现出相同的趋势,大剂量组甚至有增强荧光素酶活性的趋势。
结论:压力负荷可使大鼠TGF-β1信号传导通路活化,心肌TGF-β1蛋白、Smad2 mRNA的表达上调,而Smad7 mRNA表达下调;大蒜素可在一定程度上缓解该信号传导通路的异常。大蒜素可调节细胞TGF-β1报告系统的荧光素酶活性,提示大蒜素可调节TGF-β1信号传导通路,在一定剂量时可抑制TGF-β1信号传导通路的活化。
Hypertension is one kind of common, frequently-occurring disease, which seriously impairs people's healthy in mind and body. The heart is one of impaired target organ in hypertension-related complications. Lasting pressure overload leads to adaptive change of heart structure and function, that is heart remodeling which mainly character is left ventricular hypertrophy (LVH). The adaptational process at initial stages of LVH is arm to balance increased stress on cardiac muscle, but extended overload stress leads to maladjust.
Scholars and researchers consider LVH not only changes on cardiac myocytes, but also on cardiac interstitium and blood vessels. Inproportional proliferation of Cardiac interstitium constituent relative to substance constructs myocardium structural hetertogneity, that is myocardial fibrosis. Myocardial fibrosis is fibrous tissue accumulation or collagen contents alteration in heart tissue, and is the characteristic pathological change in LVH and hypertension heart disease(HHD). Excess cardiac fibrosis is a major determinant that contributes to the progression of arrhythmia, heart failure and cardiac sudden death in HHD. However, mechanisms of which are not well addressed.
The present study was carried out to investigate the mechanisms responsible for myocardial fibrosis in left ventricular remodeling (LVR) and seek for effective pharmaceutical intervention. The whole study included five parts as follows:
PartⅠ: Effects of Garlicin on Cardiac Hypertrophy of Pressure Overload Rat
Objective: To establish animal models of cardiac hypertrophy of pressure overload rats and observe effects of garlicin treated.
Methods: Pressure overloaded myocardial hypertrophy was produced by banding of aorta abdominalis in male SD rats.The 90 rats randomized to 6 groups,they were groups of Sham, Control, Garlicin 1, Garlicin 2, Tetramethylpyrazine(TMP), Losartan, respectively. The rats were administered with drugs 3d after operation, They were examined by echocardiography on after 30 days treatment, then were sacrificed. Portions of heart were processed for histological and morphological examination,blood samples were obtained to conduct subsequent biochemical analysis.
Results:LVH and dysfunction were verified by echocardiography. systolic function of left ventricular is on compensation phase, myocardial hypertrophy of this model belongs to concentric hypertrophy, the thickness of ventricular wall increased and volume decreased. Garlicin treatment could lead to certain lessening. Heart weight/body weight lowered in comparison with controls, LVH was seen in hearts of rats on 30 days after operation, Garlicin treated could ameliorate this pathological change.
Conclusion:It showed that Garlicin can prevent cardiac remodeling of hypertrophy cardium induced by pressure over-load including myocardial hypertrophy.。
PartⅡ:Effects of Garlicin on Myocardial Fibrosis of Pressure Overload Rats
Objective: To observ effects of garlicin on myocardial fibrosis of pressure overload rats.
Methods: We detected hydroxyproline(HOP) of left ventricular and carboxy terminal propeptide of procollagen type I (PⅠCP), amino terminal propeptide of procollagen type III(PⅢNP), hyaluronic acid (HA), laminin(LN) of serum. hearts were processed for histological and morphological examination.
Results: Histological and serological findings showed that was reparative fibrosis, promotion of synthesis and lightened degradation of cardiac collagens were presented in heart tissue of model rats. Garlicin treatment could reduce HOP of left ventricular, PⅠCP, PⅢNP and HA of serum in comparison with controls. Garlicin could also ameliorate collagen surrounding blood vessel in heart histologically.
Conclusion: It showed that Garlicin can prevent myocardial fibrosis of hypertrophy cardium induced by pressure over-load.
PartⅢ: Decreasing AngⅡand Ameliorating Peroxidative Stress of Pressure Overload Rats by Garlicin treatment
Objective: To observ effects of Garlicin on AngⅡand SOD, MDA of pressure overload rats.
Methods:we detected AngⅡo f blood plasma and left ventricular and superoxide anion (SOD) and malondialdehyde (MDA) of serum.
Results: Pressure overload could induce increasing AngⅡstandard of blood plasma and left ventricular on SD rats. Garlicn could decrease circulating and regional abnormal AngⅡ. SOD activity of models decreased and MDA content of models increased in comparison with sham group. Pressure overload resulted in peroxidative damage in myocardial fibrosis. Garlicn could inhibit MDA and enhance SOD.
Conclusion: Garlicn could decrease circulating and regional abnormal AngⅡ. Pressure overload resulted in peroxidative damage in myocardial fibrosis. Garlicn could inhibit MDA and enhance SOD.
PartⅣEffects of Garlicin on Fibroblasts Proliferation and Collagen Excrete
Objectiv: Investigating the effects of Garlicin on fibroblasts proliferation and collagen excretion to explore its anti-fibrosis mechanisms. the effects of garlicin on fibroblasts proliferation and collagen synthesis, to explore its anti-fibrosis mechanism.
Methods: The rate of proliferation is determined by the incorporation of H3-thymidine into NIH3T3 cellular nucleic acids. We observed apoptosis on NIH3T3 cells through DNA ladder experiment. TypeⅠcollagen expression was detected by immunofluorescent microscopy. Analysising of hydroxyproline in cell culture medium based alkaline hydrolysis.
Results: Garlicin could inhibit DNA synthesis in NIH3T3 fibroblasts and TypeⅠcollagen expression from 0.2 ug/ml to 5ug/ml. Garlicin also reduced hydroxyproline in cell culture medium. But we could not obserse apoptosis through DNA ladder experiment.
Conclusion: Garlicin could depress fibroblasts expressing typeⅠcollagen and proliferating.
PartⅤ: Influence of Garlicin on TGF-β1 Signal Transmit Passway Involvement in Myocardial Fibrosis
Objectiv: Investigating the influence of garlicin on TGF-β1 signal transmit passway involvement in myocardial fibrosis.
Methods: detecting TGF-β1 of serum by ELISA and TGF-β1 protein expression on rats’heart by immunohistochemistry. Testing Smad2 and Smad7 mRNA expression in heart by Real-time PCR. At the same time, garlicin effected on TGF-β1 signal transmit passway was evaluated by a luciferase assay using Mv1Lu-(CAGA)12-Luc cell line responsing to TGF-β.
Results: TGF-β1 of serum and TGF-β1 protein expression on rats’heart all increased in model group than sham group. Garlicin could inhibited the abnormal enhancement. Real-time RT- PCR result manifested Smad2 mRNA up-regulated and Smad7 mRNA down-regulated. Garlicin treatment could inhibit Smad2 and promoted Smad7 and lead to TGF-β1 inclining to normal standard. In vitro experiment, the luciferase activity responding to TGF-βwas inbibited obviously in 1ug/mL garlicin group under the stimulation of 2ng/mL TGF-β1 and Its inhibition ratio was about 20%. the larger dose (5ug/mL) weakened this effect.
Conclusion: Pressure overload activated TGF-β1 signal transmit passway of rats. Garlicin could relieve this passway in certain degree.
心肌发生纤维化时最主要的病理改变为细胞外基质(ECM)的大量积聚。在心脏中,ECM含量最多的是胶原蛋白,其不成比例的堆积或纤维化对组织硬度具有决定作用。心脏成纤维细胞在局部神经体液因子的作用下过度激活,是胶原分泌增多的主要原因,但其发生机制尚未完全阐明。
本研究致力于探讨左室重构过程中心肌纤维化的发生机制并寻求有效的药物干预手段。全文共分为以下五部分:
第一部分大蒜素对压力负荷大鼠左室重构的影响
目的:观察压力负荷大鼠左室重构模型情况及大蒜素对其的影响。
方法:将90只SD雄性大鼠随机分为6组:假手术组(Sham)、模型组(Control)、大蒜素注射液组( Garlicin 1 )、大蒜素口服组( Garlicin 2 )、川芎嗪组(Tetramethylpyrazine,TMP)、氯沙坦组( Losartan ),每组15只。以腹主动脉缩窄方法制备压力负荷性左室重构模型。于术后3天开始给药,给药30天。给药结束后行心脏超声检查;其后采血样用于后续的生化检测,取心脏作组织形态学检查。
结果:腹主动脉缩窄30天造成的压力超负荷大鼠左室重构模型经超声心动检测证实腹主动脉缩窄引起的心肌肥厚属于向心性肥厚,室壁增厚而室腔容积减少,心室的收缩功能尚处于代偿阶段。各给药组同模型组比较室壁有不同程度的减轻(室壁厚度:Control组>Garlicin 2组>Garlicin 1组/TMP组>Losartan组>sham组)但是除losartan组收缩期左室后壁厚度外(P<0.05),均未出现明显统计学差异,增大样本量有可能出现。模型组心脏体重指数高于假手术组(P<0.05),氯沙坦组明显低于模型组(P<0.05),两个大蒜素组和川芎嗪组较模型组有降低趋势,但没有出现统计学差异。从病理形态来看,光镜下假手术组心肌纤维排列整齐,心肌细胞大小正常,模型组心肌纤维排列紊乱,细胞横径稍大,各药物治疗组有不同程度的改善。电镜下见假手术组细胞为正常超微结构,模型组的细胞超微结构的改变主要为:心肌细胞变大、线粒体变性、肌原纤维变性、细胞核不规则等,各治疗组也有所改善。
结论:大蒜素注射液与川芎嗪注射液有相似的抑制左室重构作用,但其效果不如氯沙坦。大蒜素口服组的疗效不如大蒜素注射液。
第二部分大蒜素对压力负荷大鼠心肌纤维化的影响
目的:观察压力负荷大鼠左室重构过程中的心肌纤维化及大蒜素对其的影响。
方法:检测左室心肌羟脯氨酸(HOP)、血清胶原代谢物Ⅰ型前胶原羧基端肽(PⅠCP)及Ⅲ型前胶原氨基端肽(PⅢNP)、血清透明质酸(HA)及层粘蛋白(LN)。对心肌组织进行苦味酸天狼猩红染色,计算左室心肌胶原容积分数(CVF,%)和血管周围胶原面积(PVCA,%);制备电镜标本观察心肌胶原纤维和超微结构。
结果:模型组心肌羟脯氨酸明显增多,说明胶原的合成增加;同时,通过天狼猩红染色观察到心肌纤维被胶原纤维栅栏状包绕,CVF和PVCA较假手术组明显增高,呈间质纤维化和血管周围纤维化现象,属反应性纤维化阶段。血清中PⅠCP、PⅢNP、LN、HA较假手术组均明显增高,,也说明腹主动脉缩窄造成的压力负荷大鼠存在心肌纤维化现象。阳性药物氯沙坦与模型组相比,可抑制心肌羟脯氨酸,减少心肌胶原(P<0.05),并降低血清PⅠCP、PⅢNP、LN(P<0.001或P<0.05),降低CVF和PVCA(P<0.05),减少心肌胶原的排列紊乱。大蒜素注射液和川芎嗪注射液均可降低血清PⅠCP、PⅢNP和HA的增多(P<0.05),降低PVCA(P<0.05),可改善血管周围胶原的增生和排列紊乱。大蒜素口服仅能改善PⅠCP、PⅢNP(同模型组相比P<0.05)。
结论:大蒜素注射液对腹主动脉缩窄导致的心肌反应性纤维化有一定的抑制作用。大蒜素口服后对心肌纤维化也有一定的作用。
第三部分大蒜素对压力负荷大鼠AngⅡ和SOD、MDA的影响
目的:观察大蒜素对压力负荷大鼠AngⅡ和SOD、MDA的影响。
方法:通过制备腹主动脉缩窄造成的压力负荷大鼠动物模型,检测大蒜素对血浆和心肌中AngⅡ,血清中SOD活性和MDA含量。
结果:腹主动脉缩窄后,模型组比假手术组的血浆和心肌AngⅡ含量升高(P<0.05),其均值可达假手术组的2倍以上。氯沙坦治疗后血浆中AngⅡ均值水平较模型组升高;心肌组织中的AngⅡ却出现降低趋势(P>0.05)。川芎嗪组、Garlicin 1组的血浆中AngⅡ水平较模型组明显降低(P<0.05),余未出现统计学差异。SOD活力在模型组中有降低趋势(Control组:Sham组,P=0.06);各药物治疗后均有不同程度的提高,其中,Losartan组和TMP组较模型组的总SOD活力明显提高(P<0.05),Garlicin 1组也有较为明显的提高,但是未出现统计学差异(P=0.07)。MDA含量的变化在模型组较假手术组增高(P<0.05),四个治疗组与模型组相比出现明显的降低(P<0.05)。
结论:压力负荷使大鼠AngⅡ增高,导致RAAS的激活。大蒜素可降低压力负荷导致的循环和心肌局部的AngⅡ异常增高,而口服大蒜素使AngⅡ降低的程度不大。大蒜素能够通过抑制血清MDA、提高SOD活力,减轻左室重构过程中的过氧化损伤。
第四部分大蒜素对成纤维细胞增殖和胶原分泌的影响
目的:观察大蒜素对成纤维细胞增殖和胶原分泌的影响,探讨抗纤维化作用机理。
方法:以NIH3T3细胞为模型,通过H3-thymidine DNA掺入量测定大蒜素对NIH3T3细胞DNA合成的影响;DNA ladder检测细胞凋亡情况;碱消化法检测细胞培养液中羟脯氨酸含量;荧光免疫染色方法检测NIH3T3细胞Ⅰ型胶原蛋白的表达。
结果:大蒜素作用于NIH3T3细胞24h,在0.2-5ug/ml范围内剂量依赖的抑制NIH3T3细胞的DNA合成;但没有观察到DNA ladder。大蒜素可使细胞培养液中的羟脯氨酸含量减少,Ⅰ型胶原蛋白的表达减少。
结论:大蒜素对NIH3T3细胞生长增殖有抑制作用,减少细胞胶原的分泌,减少Ⅰ型胶原的表达,可能是大蒜素抗纤维化的作用机理之一。
第五部分大蒜素对TGF-β1及其信号传导系统的影响
目的:探讨大蒜素对心肌纤维化过程中TGF-β1及其信号传导的影响。
方法:采用ELISA方法检测压力负荷大鼠模型血清TGF-β1含量,通过免疫组化方法检测心肌TGF-β1表达,并通过图像分析计算阳性表达的面积(Area)、平均光密度(Density)和IOD值。采用Real-time PCR方法检测TGF-β1下游信号心肌Smad2、Smad7 mRNA表达的变化。同时通过体外实验Mv1Lu-(CAGA)12-Luc(稳定转染TGF-β1信号响应基因片段的细胞系)荧光酶活性的变化进一步验证大蒜素对TGF-β1信号传导系统的影响。
结果:模型组同假手术组比较血清TGF-β1水平明显升高,氯沙坦组同模型组相比TGF-β1水平明显下降, Garlicin 1、Garlicin 2和TMP组的血清同模型组相比TGF-β1水平明显下降,同假手术组比较仍存在明显的升高。免疫组化观察TGF-β1蛋白阳性表达为棕黄色沉积,主要在大鼠心肌间质中表达。经图像分析模型组同假手术组比较存在统计学意义上的升高。氯沙坦组、大蒜素注射液组、川芎嗪组同模型组相比Area和IOD两个方面都出现了明显的降低,而大蒜素口服组仅Area值表现出统计学意义上的降低。
Real-time PCR结果显示模型组的Smad2表达量比假手术组上调了1.18倍,而Smad7下调了0.55倍。氯沙坦组与模型组相比Smad2下调至0.63倍,而Smad7仍下调至0.88倍;川芎嗪组与模型组相比Smad2下调至0.49倍, Smad7上调至1.40倍;大蒜素注射液组与模型组相比Smad2下调至0.75倍,Smad7上调至1.27倍;大蒜素口服组与模型组相比Smad2下调至0.71倍,而Smad7却下调至0.72倍。体外实验Mv1Lu-(CAGA)12-Luc细胞荧光酶活性在2ng/mL TGF-β刺激的情况下,大蒜素的中剂量(1ug/mL)组表现出对荧光素酶活性的明显抑制,其抑制率达20%左右,增大剂量至5ug/mL时此作用减弱。大蒜素药物血清在大中小三个剂量组与药物直接作用表现出相同的趋势,大剂量组甚至有增强荧光素酶活性的趋势。
结论:压力负荷可使大鼠TGF-β1信号传导通路活化,心肌TGF-β1蛋白、Smad2 mRNA的表达上调,而Smad7 mRNA表达下调;大蒜素可在一定程度上缓解该信号传导通路的异常。大蒜素可调节细胞TGF-β1报告系统的荧光素酶活性,提示大蒜素可调节TGF-β1信号传导通路,在一定剂量时可抑制TGF-β1信号传导通路的活化。
Hypertension is one kind of common, frequently-occurring disease, which seriously impairs people's healthy in mind and body. The heart is one of impaired target organ in hypertension-related complications. Lasting pressure overload leads to adaptive change of heart structure and function, that is heart remodeling which mainly character is left ventricular hypertrophy (LVH). The adaptational process at initial stages of LVH is arm to balance increased stress on cardiac muscle, but extended overload stress leads to maladjust.
Scholars and researchers consider LVH not only changes on cardiac myocytes, but also on cardiac interstitium and blood vessels. Inproportional proliferation of Cardiac interstitium constituent relative to substance constructs myocardium structural hetertogneity, that is myocardial fibrosis. Myocardial fibrosis is fibrous tissue accumulation or collagen contents alteration in heart tissue, and is the characteristic pathological change in LVH and hypertension heart disease(HHD). Excess cardiac fibrosis is a major determinant that contributes to the progression of arrhythmia, heart failure and cardiac sudden death in HHD. However, mechanisms of which are not well addressed.
The present study was carried out to investigate the mechanisms responsible for myocardial fibrosis in left ventricular remodeling (LVR) and seek for effective pharmaceutical intervention. The whole study included five parts as follows:
PartⅠ: Effects of Garlicin on Cardiac Hypertrophy of Pressure Overload Rat
Objective: To establish animal models of cardiac hypertrophy of pressure overload rats and observe effects of garlicin treated.
Methods: Pressure overloaded myocardial hypertrophy was produced by banding of aorta abdominalis in male SD rats.The 90 rats randomized to 6 groups,they were groups of Sham, Control, Garlicin 1, Garlicin 2, Tetramethylpyrazine(TMP), Losartan, respectively. The rats were administered with drugs 3d after operation, They were examined by echocardiography on after 30 days treatment, then were sacrificed. Portions of heart were processed for histological and morphological examination,blood samples were obtained to conduct subsequent biochemical analysis.
Results:LVH and dysfunction were verified by echocardiography. systolic function of left ventricular is on compensation phase, myocardial hypertrophy of this model belongs to concentric hypertrophy, the thickness of ventricular wall increased and volume decreased. Garlicin treatment could lead to certain lessening. Heart weight/body weight lowered in comparison with controls, LVH was seen in hearts of rats on 30 days after operation, Garlicin treated could ameliorate this pathological change.
Conclusion:It showed that Garlicin can prevent cardiac remodeling of hypertrophy cardium induced by pressure over-load including myocardial hypertrophy.。
PartⅡ:Effects of Garlicin on Myocardial Fibrosis of Pressure Overload Rats
Objective: To observ effects of garlicin on myocardial fibrosis of pressure overload rats.
Methods: We detected hydroxyproline(HOP) of left ventricular and carboxy terminal propeptide of procollagen type I (PⅠCP), amino terminal propeptide of procollagen type III(PⅢNP), hyaluronic acid (HA), laminin(LN) of serum. hearts were processed for histological and morphological examination.
Results: Histological and serological findings showed that was reparative fibrosis, promotion of synthesis and lightened degradation of cardiac collagens were presented in heart tissue of model rats. Garlicin treatment could reduce HOP of left ventricular, PⅠCP, PⅢNP and HA of serum in comparison with controls. Garlicin could also ameliorate collagen surrounding blood vessel in heart histologically.
Conclusion: It showed that Garlicin can prevent myocardial fibrosis of hypertrophy cardium induced by pressure over-load.
PartⅢ: Decreasing AngⅡand Ameliorating Peroxidative Stress of Pressure Overload Rats by Garlicin treatment
Objective: To observ effects of Garlicin on AngⅡand SOD, MDA of pressure overload rats.
Methods:we detected AngⅡo f blood plasma and left ventricular and superoxide anion (SOD) and malondialdehyde (MDA) of serum.
Results: Pressure overload could induce increasing AngⅡstandard of blood plasma and left ventricular on SD rats. Garlicn could decrease circulating and regional abnormal AngⅡ. SOD activity of models decreased and MDA content of models increased in comparison with sham group. Pressure overload resulted in peroxidative damage in myocardial fibrosis. Garlicn could inhibit MDA and enhance SOD.
Conclusion: Garlicn could decrease circulating and regional abnormal AngⅡ. Pressure overload resulted in peroxidative damage in myocardial fibrosis. Garlicn could inhibit MDA and enhance SOD.
PartⅣEffects of Garlicin on Fibroblasts Proliferation and Collagen Excrete
Objectiv: Investigating the effects of Garlicin on fibroblasts proliferation and collagen excretion to explore its anti-fibrosis mechanisms. the effects of garlicin on fibroblasts proliferation and collagen synthesis, to explore its anti-fibrosis mechanism.
Methods: The rate of proliferation is determined by the incorporation of H3-thymidine into NIH3T3 cellular nucleic acids. We observed apoptosis on NIH3T3 cells through DNA ladder experiment. TypeⅠcollagen expression was detected by immunofluorescent microscopy. Analysising of hydroxyproline in cell culture medium based alkaline hydrolysis.
Results: Garlicin could inhibit DNA synthesis in NIH3T3 fibroblasts and TypeⅠcollagen expression from 0.2 ug/ml to 5ug/ml. Garlicin also reduced hydroxyproline in cell culture medium. But we could not obserse apoptosis through DNA ladder experiment.
Conclusion: Garlicin could depress fibroblasts expressing typeⅠcollagen and proliferating.
PartⅤ: Influence of Garlicin on TGF-β1 Signal Transmit Passway Involvement in Myocardial Fibrosis
Objectiv: Investigating the influence of garlicin on TGF-β1 signal transmit passway involvement in myocardial fibrosis.
Methods: detecting TGF-β1 of serum by ELISA and TGF-β1 protein expression on rats’heart by immunohistochemistry. Testing Smad2 and Smad7 mRNA expression in heart by Real-time PCR. At the same time, garlicin effected on TGF-β1 signal transmit passway was evaluated by a luciferase assay using Mv1Lu-(CAGA)12-Luc cell line responsing to TGF-β.
Results: TGF-β1 of serum and TGF-β1 protein expression on rats’heart all increased in model group than sham group. Garlicin could inhibited the abnormal enhancement. Real-time RT- PCR result manifested Smad2 mRNA up-regulated and Smad7 mRNA down-regulated. Garlicin treatment could inhibit Smad2 and promoted Smad7 and lead to TGF-β1 inclining to normal standard. In vitro experiment, the luciferase activity responding to TGF-βwas inbibited obviously in 1ug/mL garlicin group under the stimulation of 2ng/mL TGF-β1 and Its inhibition ratio was about 20%. the larger dose (5ug/mL) weakened this effect.
Conclusion: Pressure overload activated TGF-β1 signal transmit passway of rats. Garlicin could relieve this passway in certain degree.
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