牛磺酸抑制心肌成纤维细胞的增殖作用及机制研究
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摘要
心肌纤维化(MF)发生于多种心血管疾病,是许多心血管疾病中一个重要的病理改变。由于长期心肌供血不足，引起心肌组织发生营养障碍和萎缩，或大面积心肌梗死后，以至正常心肌组织结构中胶原纤维过量积聚、胶原浓度显著升高或胶原成分发生改变。现代细胞分子医学研究证实：心肌纤维化病变是众多心脏病患者共同面临的最大威胁，包括风湿性心脏病、冠状动脉硬化性心脏病和心肌炎等重大心脏疾病，都是以心肌纤维化病变为基础的，心肌纤维化不断发展会引起一系列临床症状，最终以心功能完全衰竭致患者死亡。此病理过程是心功能由代偿期向失代偿期转变的关键，其消退可恢复心肌组织和间质成分之间的平衡，使僵硬程度逐渐软化，有利于心功能的改善。故如何防治心肌纤维化是当前世界医学研究的重点和热点。
牛磺酸是正常存在于体内的含硫氨基酸，于1827年由Tiedemann等从牛胆汁中分离提取。人体内含量丰富，主要存在于心肌，具有广泛的生物学效应。研究表明，牛磺酸在维持细胞内外渗透压平衡、调节细胞钙稳态、清除氧自由基，减轻过氧化损伤和直接膜稳定等方面发挥作用。近年来，本实验室对牛磺酸调节心血管疾病的药理作用及机制进行了初步研究，证实牛磺酸具有抑制心肌成纤维细胞的增殖及逆转心肌重塑的作用，但具体的作用机制至今尚未阐明。
本研究分为以下几部分：首先研究牛磺酸抑制在体异丙肾上腺素(Isoprenaline，Iso)诱导MF作用的实验研究；其次研究牛磺酸抑制心肌成纤维细胞的增殖作用与PKCα、iNOS、ERK1/2和ROS等有关，再进一步研究牛磺酸逆转心肌重塑的细胞内信号转导机制。
1、牛磺酸抑制Iso诱导MF作用的实验研究
Wistar大鼠，体重200~250g，雌雄各半，随机分成5组：对照组、Iso模型组和Tau (60、120和240) mg·kg~(-1)·d~(-1)各剂量组。Iso组于大鼠背部的皮下注射Iso(5mg·kg~(-1)·d~(-1))，连续7d；对照组注射相同体积的生理盐水；Tau各剂量组于造模的第2d开始腹腔注射给药，每天一次，连续14d。通过检测HMI、LVMI、心肌组织Hyp的含量、MDA含量和SOD活性及免疫组织化学染色检测心肌组织中TGF-β1的表达。
结果可见：Tau在60~240mg·kg~(-1)·d~(-1)可明显降低HMI、LVMI和心肌组织中Hyp含量；Tau60~240mg·kg~(-1)·d~(-1)组都出现MDA含量减少，SOD活性提高；不同剂量的Tau不同程度地抑制了TGF-β1表达，说明Tau能够抑制TGF-β1蛋白表达，提示Iso通过提高TGF-β1蛋白表达，进而引起心肌组织的纤维化。Tau可通过提高SOD活性，降低TGF-β1蛋白的表达，进而减轻心肌组织的纤维化。
2、牛磺酸对myoFbs增殖的抑制作用
观察牛磺酸(Tau)对血管紧张素II (Angiotensin II, AngII)诱导大鼠乳鼠myoFbs增殖的抑制作用，确定Tau抑制myoFbs增殖作用的有效剂量。分离出生1~3d大乳鼠myoFbs，培养72h后传代培养2-3代后随机分为：正常对照组、AngII (终浓度为10-7mol·L~(-1))模型组和Tau (120、60、30) mmol·L~(-1)组，继续培养24/48h后，观察细胞形态学的变化，应用MTT比色法检测细胞存活率、试剂盒测定细胞培养基中羟脯氨酸(I和III)含量、NO含量及iNOS的活性、ELISA检测细胞上清液中TGF-β1和CTGF的含量，荧光共聚焦检测ERK1/2核转位、PKCα膜转位和蛋白表达，Western blot检测p-ERK1/2、p-PKCα和iNOS蛋白表达。
结果表明，与对照组比较，AngII组myoFbs存活数量明显增多，myoFbs培养液中羟脯氨酸(I和III)含量、NO含量及iNOS的活性、TGF-β1和CTGF的含量、ERK1/2核转位、PKCα膜转位和蛋白表达均增加(P<0.01)；与模型组比较，Tau (120、60和30)mmol·L~(-1)组myoFbs存活数量减少(P<0.05，P<0.01)，myoFbs培养液中羟脯氨酸(I和III)含量、NO含量及iNOS的活性、TGF-β1和CTGF的含量明显降低(P<0.05，P<0.01)。荧光共聚焦检测ERK1/2核转位、PKCα膜转位和蛋白表达，Western blot检测p-ERK1/2、p-PKCα和iNOS蛋白表达明显减少。流式细胞仪检测，与模型组比较，Tau (120、60、30) mmol·L~(-1)各组myoFbs的周期G0/G1的百分比明显增加，S的百分比降低。
由以上可知，Tau能抑制AngII引起的myoFbs的增殖、减少羟脯氨酸(I和III)的生成、降低TGF-β1和CTGF的含量，此作用可能与NO、iNOS、ERK1/2、PKCα等因素有关，其中Tau在剂量为60mmol·L~(-1)时作用较为显著。
3、Tau抑制myoFbs增殖机制的研究
(1) Tau通过抑制p-PKCα的膜转位和表达调控myoFbs的增殖
分离出生1~3d大乳鼠的myoFbs，培养72h后传代培养2-3代随机分为正常对照组、AngII (10-7mol·L~(-1))模型组、Tau (60mmol·L~(-1))组、D4681(0.625μg·L~(-1))组、AngII+D4681组和AngII+D4681+Tau组，继续培养24/48h后，应用MTT比色法检测细胞存活率、流式细胞仪检测细胞周期的分布、免疫细胞化学法、荧光共聚焦法和Western blot检测p-PKCα的膜转位和蛋白的表达。
结果显示：Tau可抑制myoFbs的增殖，与D4681合用后抑制作用更加明显，并降低S期和升高G0/G1期的比例；同时Tau可抑制p-PKCα的膜转位和蛋白的表达，D4681与Tau共同使用后抑制p-PKCα膜转位和蛋白表达作用更加明显。由此可见Tau抑制myoFbs的增殖是通过抑制p-PKCα的膜转位和表达作用实现的。应用特异性PKCα抑制剂D4681可加强Tau抑制myoFbs的增殖作用。
(2)Tau通过提高iNOS活性和NO含量抑制myoFbs的增殖
分离出生1~3d大鼠乳鼠的myoFbs，培养72h后传代培养2-3代随机分为正常对照组、AngII (10-7mol·L~(-1))模型组、Tau (60mmol·L~(-1))组、D4681(0.625μg·L~(-1))组、D4681+AngII组、D4681+Tau+AngII、AG (10μg·L~(-1))组、AngII+AG组和AngII+AG+Tau组，继续培养24/48h后，应用MTT比色法检测细胞存活率、测定细胞培养基中iNOS活性和NO含量、并采用流式细胞仪检测细胞周期分布、应用免疫细胞化学法、荧光共聚焦和Western blot检测iNOS在胞浆的表达。
结果显示：与模型组比较，D4681+Tau+AngII和AngII+AG+Tau组myoFbs的增殖明显减少，细胞培养液iNOS活性下降、NO含量降低(P<0.01，P<0.001)，S期的百分比下降、G0/G1期的比例明显升高(P<0.05，P<0.01)，iNOS在胞浆的表达明显升高。D4681+Tau+AngII组iNOS活性与Tau组比较有所升高，提示Tau可通过抑制PKCα的膜转位及表达进而提高iNOS活性，抑制myoFbs的增殖。
(3) Tau通过抑制p-ERK1/2的核转位和表达抑制myoFbs的增殖
分离出生1~3d大乳鼠的myoFbs，培养72h后传代培养2-3代随机分为正常对照组、AngII (10-7mol·L~(-1))模型组、Tau (60mmol·L~(-1))组、D4681(0.625μg·L~(-1))组、AngII+D4681组、AngII+D4681+Tau组、PD98059(10μmol·L~(-1))+AngII组和PD98059+Tau+Ang II组,继续培养24/48h后，应用免疫细胞化学法、荧光共聚焦和Western blot检测p-ERK1/2的核转位和表达。
结果显示：Tau可抑制p-ERK1/2的核转位和表达，D4681与Tau共同使用后抑制p-ERK1/2的核转位和表达作用更加明显，而PD98059对p-PKCα的膜转位及表达没有影响。由此可见Tau抑制myoFbs的增殖是通过抑制p-ERK1/2的核转位和表达作用实现的。应用特异性PKCα的抑制剂D4681可加强Tau抑制p-ERK1/2的核转位和表达作用。
(4) Tau通过ROS调控myoFbs的增殖
研究中发现在AngII诱导myoFbs增殖中有ROS量的变化，为进一步检测Tau是否通过减少ROS的生成进而抑制myoFbs的增殖。采用体外培养myoFbs，并应用特异性抗氧化剂N-乙酰半胱氨酸(N-acetyl cysteine，NAC)，检测Tau对ROS及p-PKCα的作用，并探讨其作用机制。
分离出生1~3d大乳鼠的myoFbs，培养72h后传代培养2-3代随机分为正常对照组、AngII(10-7mol·L~(-1))模型组、Tau (30、60、120)mmol·L~(-1)组和NAC (10-4mol·L~(-1))组，继续培养24/48h后，应用MTT比色法检测细胞存活率，并采用ELISA试剂盒检测细胞培养液中·OH含量，荧光共聚焦检测ROS的表达，同时采用Western blot检测myoFbs中p-PKCα蛋白的表达。
结果与对照组比较，AngII组myoFbs的增殖率、细胞培养液中·OH含量、myoFbs中ROS的含量和p-PKCα的表达都明显增加；应用抗氧化剂N-乙酰半胱氨酸(NAC)后，与模型组相比，Tau组和NAC组myoFbs的增殖率、细胞培养液中·OH含量、ROS的含量及p-PKCα的表达均明显降低。由此可知，Tau抑制myoFbs的增殖作用与其减少ROS的生成进而使p-PKCα的表达下降，从而启动抗心肌纤维化的作用有关。
综上所述，Tau能抑制myoFbs的增殖，逆转心肌重塑，从而达到对心肌细胞的保护作用，其细胞内信号转导机制可能是通过激活ROS-PKCα-ERK1/2和iNOS通路，从而改变TGF-β1和CTGF的生成量而实现的。
Myocardial fibrosis (MF) occurs in a variety of cardiovascular disease, for manycardiovascular disease in an important pathological changes. As a result of long-termmyocardial blood supply, myocardial histogenesis dystrophy and atrophy, or large area aftermyocardial infarction, so that normal myocardial tissue structure of collagen fiber excessaccumulation, collagen concentration significantly increased or collagen compositionchange. Modern cell and molecular medicine research confirmed: myocardial fibrosislesions is many heart patients common biggest threats, including rheumatic heart disease,coronary heart disease and myocarditis and other major heart disease, are the basis ofmyocardial fibrosis disease, myocardial fibrosis development would cause a series ofsymptoms, and ultimately to cardiac function complete failure cause patients died. Thepathological process is cardiac function by compensatory period to decompensated phaseshift key, its extinction can restore the myocardial tissue and stroma between balance,ankylose degree gradually softening, which is beneficial to the improvement of heartfunction. So how to the prevention of myocardial fibrosis is the hot spot and focus ofmedical research.
Taurine (Tau) is normal exist in the body of the sulfur amino acid, in1827by theTiedemann ox bile from the extraction separation. The human body rich content, mainlyexists in myocardial tissue, has extensive biological effect. Research shows that taurine canmaintain cell osmotic balance, regulating cell calcium steady state, clear oxygen freeradicals, reducing the peroxidation damage and direct membrane stability and function. Inrecent years, the laboratory of taurine regulation of cardiovascular disease pharmacologicalaction and mechanism was studied, confirmed that taurine can inhibit myocardial fibroblastproliferation and reversal myocardial remodeling effect, but the specific mechanism ofaction has not bright.
This research is divided into three parts, first research taurine inhibiting effects ofmyocardial fibroblasts proliferation, PKC alpha, iNOS and ERK1/2, and then further studyTau reversal myocardial remodeling of intracellular signal transduction mechanism.
1. Tau effects on inhibition proliferation of myoFbs
Aim to observe that Tau effects on inhibition proliferation of myoFbs induced byAngiotensinII (AngII) in neonatal rats, determine the Tau inhibition myoFbs proliferationeffect of effective dose. Separation was born1~3d big lactation rats myoFbs, culture72hafter subculture2-3generations randomly divided into normal control group, AngII(10-7mol·L~(-1)) model group and Tau (120,60and30)mmol·L~(-1)group, continue to cultivate24/48h,observe the change of cell morphology, the application of MTT colorimetric method todetect cell survival, kit test cell medium hydroxyproline (I and III) content, NO content andiNOS activity, ELISA detection cell supernatant fluid of TGF-β1and CTGF content,fluorescent confocal detection ERK1/2nuclear transfer, PKCα membrane inversion andprotein expression, Western blot test p-ERK1/2, p-PKCa and iNOS protein expression.
The results show that, compared with controls, myoFbs proliferation live quantityobviously increased in AngII group, myoFbs medium, hydroxyproline (I and III) content,NO content and iNOS activity, TGF-β1and CTGF content, ERK1/2nuclear transfer, PKCαmembrane inversion and protein expression increased (P<0.01); Compared with modelgroup, Tau (120,60and30)mmol·L~(-1)group, proliferation of myoFbs quantity reduce(P<0.05, P<0.01), myoFbs medium, hydroxyproline (I and III) content, NO content andiNOS activity, TGF-β1and CTGF was obviously lower,(P<0.05, P<0.01). Fluorescentconfocal detection ERK1/2nuclear transfer, PKCα membrane inversion and proteinexpression, Western blot testing p-ERK1/2, p-PKCα and iNOS protein expressionsignificantly reduced. Flow cytometry instrument detection Tau(120,60and30)mmol L~(-1)group myoFbs apoptosis number no significant reduce.
Tau inhibits AngII cause myoFbs proliferation, reduce hydroxyproline (I and III)generation, reduce the TGF-β1and CTGF content, this effect may and NO, iNOS, ERK1/2,PKCα wait for an element to concern, including Tau in dose for60mmol·L~(-1)effect issignificant.
2. Study on mechanism of Tau inhibits proliferation of myoFbs
(1) Tau by inhibiting p-PKCα membrane inversion and expression regulation myoFbsproliferation Separation was born1~3d rat myoFbs, culture72h after subculture2-3generation randomly divided into normal control group, AngII (final concentration for10-7mol·L~(-1)) model group, Tau (120,60and30) mmol·L~(-1)group, D4681(0.625ug·L~(-1)) group,AngII+D4681group and AngII+D4681+Tau group, continue to cultivate24/48h, applicationMTT colorimetric method to detect cell survival rate, flow cytometry instrument detectioncell cycle distribution, immunocytochemistry method, fluorescent confocal method and Western blot detection p-PKCα membrane inversion and expression. The results show that itcan inhibit the proliferation myoFbs, Tau and D4681after share inhibition effect moreapparent, and reduce the proportion of S phase, rise G0/G1phase rate. At the same time Tauinhibits p-PKCα membrane inversion and expression, D4681and Tau common after useinhibition p-PKCα membrane inversion and the expression effect more apparent, thus it canbe seen that Tau can inhibit proliferation of myoFbs is through the inhibition of p-PKCαmembrane inversion and express the realization of function. Application specific PKCαinhibitor D4681can strengthen Tau effect son inhibition proliferation of myoFbs.
(2) Tau can improve activity of iNOS and content of NO
Separation myoFbs were born1~3d rat, myoFbs were cultured72h after subculture,2-3generation randomly divided into normal control group, AngII (10-7mol·L~(-1)) modelgroup, Tau (60mmol·L~(-1)) group, AG(10μg·L~(-1)) group, AngII+AG group and AngII+AG+Taugroup, continue to cultivate24/48h, application MTT colorimetric method to detect cellsurvival, measuring cell medium iNOS activity and NO content by flow cytometry, and thedetection instrument cell cycle distribution, application immunocytochemistry method,fluorescent confocal and Western blotting detection in the expression of iNOS cytoplasm.
Results show that AngII+AG+Tau group myoFbs proliferation significantly reduced,cell culture fluid iNOS activity decline, NO content lower (P<0.01, P<0.001), S periodpercentage decline, G0/G1phase proportion increased significantly(P<0.05, P<0.01), and theexpression of iNOS in cytoplasm increased significantly comparison with the model group,Tau can improve iNOS activity, inhibition proliferation of myoFbs.
(3) Tau by inhibiting p-ERK1/2nuclear transfer and expression to inhibit proliferationof myoFbs. Separation myoFbs was born1~3d rat, which were cultured72h after subculture2-3generation,then randomly divided into normal control group, AngII (10-7mol·L~(-1)) modelgroup, Tau(60mmol·L~(-1))group, D4681(0.625ug·L~(-1))group, AngII+D4681group andAngII+D4681+Tau group, continue to cultivate24/48h, application immunocytochemistrymethod, fluorescent confocal and Western blotting testing p-ERK1/2nuclear transfer andexpression. The results show that Tau inhibits p-ERK1/2nuclear transfer and expression,D4681and Tau common after use inhibition p-ERK1/2nuclear transfer and the expressioneffect more apparent, thus it can be seen myoFbs Tau inhibition of proliferation is throughthe inhibition of p-ERK1/2nuclear transfer and express the realization of function.Application specific PKCα inhibitor D4681can strengthen Tau inhibition p-ERK1/2nucleartransfer and the expression effect.
(4) Tau regulate proliferation of myoFbs through ROS
Research now myoFbs found AngII induced proliferation of ROS quantity changes, inorder to further testing whether Tau by reducing the generation of ROS and myoFbsinhibition of proliferation. The myoFbs were cultured in vitro, and application specificantioxidant to detect the content of ROS and the expression of p-PKCα, discuss themechanism of action. Separation myoFbs were born1~3d rat, culture72h after subculture2-3generation randomly divided into normal control group, AngII (10-7mol·L~(-1)) modelgroup, Tau (120,60and30)mmol·L~(-1)group, NAC (10-4mol·L~(-1)) and Tau+NAC group,continue to cultivate24/48h, application MTT colorimetric method to detect cell survivalrate, and use the ELISA kit to detecti content of·OH in cell medium, laser confocaldetection the expression of ROS, Western blot detection of p-PKCα expression of myoFbs.
Results comparing with control group, AngII group myoFbs proliferation, contentof·OH in cell medium, ROS and p-PKCα expression are increased significantly;Application of antioxidant n-acetyl cysteine (NAC), compared with the model group, Taugroup and NAC group myoFbs proliferation, content of·OH in cell medium, ROS andp-PKCα expression are all decreased. Therefore, effect of Tau on inhibition of proliferationmyoFbs and reduce the generation of ROS and p-PKCα expression, which can start againstmyocardial fibrosis effect about.
To sum up, Tau can inhibit the proliferation of myoFbs and reversal myocardialremodeling, thus to attain the myocardial cell of protection, the intracellular signaltransduction mechanism may be through the activation of PKCα-ROS, ERK1/2and iNOSpath, so as to change the TGF-β1and CTGF of generation and the realization.
牛磺酸是正常存在于体内的含硫氨基酸，于1827年由Tiedemann等从牛胆汁中分离提取。人体内含量丰富，主要存在于心肌，具有广泛的生物学效应。研究表明，牛磺酸在维持细胞内外渗透压平衡、调节细胞钙稳态、清除氧自由基，减轻过氧化损伤和直接膜稳定等方面发挥作用。近年来，本实验室对牛磺酸调节心血管疾病的药理作用及机制进行了初步研究，证实牛磺酸具有抑制心肌成纤维细胞的增殖及逆转心肌重塑的作用，但具体的作用机制至今尚未阐明。
本研究分为以下几部分：首先研究牛磺酸抑制在体异丙肾上腺素(Isoprenaline，Iso)诱导MF作用的实验研究；其次研究牛磺酸抑制心肌成纤维细胞的增殖作用与PKCα、iNOS、ERK1/2和ROS等有关，再进一步研究牛磺酸逆转心肌重塑的细胞内信号转导机制。
1、牛磺酸抑制Iso诱导MF作用的实验研究
Wistar大鼠，体重200~250g，雌雄各半，随机分成5组：对照组、Iso模型组和Tau (60、120和240) mg·kg~(-1)·d~(-1)各剂量组。Iso组于大鼠背部的皮下注射Iso(5mg·kg~(-1)·d~(-1))，连续7d；对照组注射相同体积的生理盐水；Tau各剂量组于造模的第2d开始腹腔注射给药，每天一次，连续14d。通过检测HMI、LVMI、心肌组织Hyp的含量、MDA含量和SOD活性及免疫组织化学染色检测心肌组织中TGF-β1的表达。
结果可见：Tau在60~240mg·kg~(-1)·d~(-1)可明显降低HMI、LVMI和心肌组织中Hyp含量；Tau60~240mg·kg~(-1)·d~(-1)组都出现MDA含量减少，SOD活性提高；不同剂量的Tau不同程度地抑制了TGF-β1表达，说明Tau能够抑制TGF-β1蛋白表达，提示Iso通过提高TGF-β1蛋白表达，进而引起心肌组织的纤维化。Tau可通过提高SOD活性，降低TGF-β1蛋白的表达，进而减轻心肌组织的纤维化。
2、牛磺酸对myoFbs增殖的抑制作用
观察牛磺酸(Tau)对血管紧张素II (Angiotensin II, AngII)诱导大鼠乳鼠myoFbs增殖的抑制作用，确定Tau抑制myoFbs增殖作用的有效剂量。分离出生1~3d大乳鼠myoFbs，培养72h后传代培养2-3代后随机分为：正常对照组、AngII (终浓度为10-7mol·L~(-1))模型组和Tau (120、60、30) mmol·L~(-1)组，继续培养24/48h后，观察细胞形态学的变化，应用MTT比色法检测细胞存活率、试剂盒测定细胞培养基中羟脯氨酸(I和III)含量、NO含量及iNOS的活性、ELISA检测细胞上清液中TGF-β1和CTGF的含量，荧光共聚焦检测ERK1/2核转位、PKCα膜转位和蛋白表达，Western blot检测p-ERK1/2、p-PKCα和iNOS蛋白表达。
结果表明，与对照组比较，AngII组myoFbs存活数量明显增多，myoFbs培养液中羟脯氨酸(I和III)含量、NO含量及iNOS的活性、TGF-β1和CTGF的含量、ERK1/2核转位、PKCα膜转位和蛋白表达均增加(P<0.01)；与模型组比较，Tau (120、60和30)mmol·L~(-1)组myoFbs存活数量减少(P<0.05，P<0.01)，myoFbs培养液中羟脯氨酸(I和III)含量、NO含量及iNOS的活性、TGF-β1和CTGF的含量明显降低(P<0.05，P<0.01)。荧光共聚焦检测ERK1/2核转位、PKCα膜转位和蛋白表达，Western blot检测p-ERK1/2、p-PKCα和iNOS蛋白表达明显减少。流式细胞仪检测，与模型组比较，Tau (120、60、30) mmol·L~(-1)各组myoFbs的周期G0/G1的百分比明显增加，S的百分比降低。
由以上可知，Tau能抑制AngII引起的myoFbs的增殖、减少羟脯氨酸(I和III)的生成、降低TGF-β1和CTGF的含量，此作用可能与NO、iNOS、ERK1/2、PKCα等因素有关，其中Tau在剂量为60mmol·L~(-1)时作用较为显著。
3、Tau抑制myoFbs增殖机制的研究
(1) Tau通过抑制p-PKCα的膜转位和表达调控myoFbs的增殖
分离出生1~3d大乳鼠的myoFbs，培养72h后传代培养2-3代随机分为正常对照组、AngII (10-7mol·L~(-1))模型组、Tau (60mmol·L~(-1))组、D4681(0.625μg·L~(-1))组、AngII+D4681组和AngII+D4681+Tau组，继续培养24/48h后，应用MTT比色法检测细胞存活率、流式细胞仪检测细胞周期的分布、免疫细胞化学法、荧光共聚焦法和Western blot检测p-PKCα的膜转位和蛋白的表达。
结果显示：Tau可抑制myoFbs的增殖，与D4681合用后抑制作用更加明显，并降低S期和升高G0/G1期的比例；同时Tau可抑制p-PKCα的膜转位和蛋白的表达，D4681与Tau共同使用后抑制p-PKCα膜转位和蛋白表达作用更加明显。由此可见Tau抑制myoFbs的增殖是通过抑制p-PKCα的膜转位和表达作用实现的。应用特异性PKCα抑制剂D4681可加强Tau抑制myoFbs的增殖作用。
(2)Tau通过提高iNOS活性和NO含量抑制myoFbs的增殖
分离出生1~3d大鼠乳鼠的myoFbs，培养72h后传代培养2-3代随机分为正常对照组、AngII (10-7mol·L~(-1))模型组、Tau (60mmol·L~(-1))组、D4681(0.625μg·L~(-1))组、D4681+AngII组、D4681+Tau+AngII、AG (10μg·L~(-1))组、AngII+AG组和AngII+AG+Tau组，继续培养24/48h后，应用MTT比色法检测细胞存活率、测定细胞培养基中iNOS活性和NO含量、并采用流式细胞仪检测细胞周期分布、应用免疫细胞化学法、荧光共聚焦和Western blot检测iNOS在胞浆的表达。
结果显示：与模型组比较，D4681+Tau+AngII和AngII+AG+Tau组myoFbs的增殖明显减少，细胞培养液iNOS活性下降、NO含量降低(P<0.01，P<0.001)，S期的百分比下降、G0/G1期的比例明显升高(P<0.05，P<0.01)，iNOS在胞浆的表达明显升高。D4681+Tau+AngII组iNOS活性与Tau组比较有所升高，提示Tau可通过抑制PKCα的膜转位及表达进而提高iNOS活性，抑制myoFbs的增殖。
(3) Tau通过抑制p-ERK1/2的核转位和表达抑制myoFbs的增殖
分离出生1~3d大乳鼠的myoFbs，培养72h后传代培养2-3代随机分为正常对照组、AngII (10-7mol·L~(-1))模型组、Tau (60mmol·L~(-1))组、D4681(0.625μg·L~(-1))组、AngII+D4681组、AngII+D4681+Tau组、PD98059(10μmol·L~(-1))+AngII组和PD98059+Tau+Ang II组,继续培养24/48h后，应用免疫细胞化学法、荧光共聚焦和Western blot检测p-ERK1/2的核转位和表达。
结果显示：Tau可抑制p-ERK1/2的核转位和表达，D4681与Tau共同使用后抑制p-ERK1/2的核转位和表达作用更加明显，而PD98059对p-PKCα的膜转位及表达没有影响。由此可见Tau抑制myoFbs的增殖是通过抑制p-ERK1/2的核转位和表达作用实现的。应用特异性PKCα的抑制剂D4681可加强Tau抑制p-ERK1/2的核转位和表达作用。
(4) Tau通过ROS调控myoFbs的增殖
研究中发现在AngII诱导myoFbs增殖中有ROS量的变化，为进一步检测Tau是否通过减少ROS的生成进而抑制myoFbs的增殖。采用体外培养myoFbs，并应用特异性抗氧化剂N-乙酰半胱氨酸(N-acetyl cysteine，NAC)，检测Tau对ROS及p-PKCα的作用，并探讨其作用机制。
分离出生1~3d大乳鼠的myoFbs，培养72h后传代培养2-3代随机分为正常对照组、AngII(10-7mol·L~(-1))模型组、Tau (30、60、120)mmol·L~(-1)组和NAC (10-4mol·L~(-1))组，继续培养24/48h后，应用MTT比色法检测细胞存活率，并采用ELISA试剂盒检测细胞培养液中·OH含量，荧光共聚焦检测ROS的表达，同时采用Western blot检测myoFbs中p-PKCα蛋白的表达。
结果与对照组比较，AngII组myoFbs的增殖率、细胞培养液中·OH含量、myoFbs中ROS的含量和p-PKCα的表达都明显增加；应用抗氧化剂N-乙酰半胱氨酸(NAC)后，与模型组相比，Tau组和NAC组myoFbs的增殖率、细胞培养液中·OH含量、ROS的含量及p-PKCα的表达均明显降低。由此可知，Tau抑制myoFbs的增殖作用与其减少ROS的生成进而使p-PKCα的表达下降，从而启动抗心肌纤维化的作用有关。
综上所述，Tau能抑制myoFbs的增殖，逆转心肌重塑，从而达到对心肌细胞的保护作用，其细胞内信号转导机制可能是通过激活ROS-PKCα-ERK1/2和iNOS通路，从而改变TGF-β1和CTGF的生成量而实现的。
Myocardial fibrosis (MF) occurs in a variety of cardiovascular disease, for manycardiovascular disease in an important pathological changes. As a result of long-termmyocardial blood supply, myocardial histogenesis dystrophy and atrophy, or large area aftermyocardial infarction, so that normal myocardial tissue structure of collagen fiber excessaccumulation, collagen concentration significantly increased or collagen compositionchange. Modern cell and molecular medicine research confirmed: myocardial fibrosislesions is many heart patients common biggest threats, including rheumatic heart disease,coronary heart disease and myocarditis and other major heart disease, are the basis ofmyocardial fibrosis disease, myocardial fibrosis development would cause a series ofsymptoms, and ultimately to cardiac function complete failure cause patients died. Thepathological process is cardiac function by compensatory period to decompensated phaseshift key, its extinction can restore the myocardial tissue and stroma between balance,ankylose degree gradually softening, which is beneficial to the improvement of heartfunction. So how to the prevention of myocardial fibrosis is the hot spot and focus ofmedical research.
Taurine (Tau) is normal exist in the body of the sulfur amino acid, in1827by theTiedemann ox bile from the extraction separation. The human body rich content, mainlyexists in myocardial tissue, has extensive biological effect. Research shows that taurine canmaintain cell osmotic balance, regulating cell calcium steady state, clear oxygen freeradicals, reducing the peroxidation damage and direct membrane stability and function. Inrecent years, the laboratory of taurine regulation of cardiovascular disease pharmacologicalaction and mechanism was studied, confirmed that taurine can inhibit myocardial fibroblastproliferation and reversal myocardial remodeling effect, but the specific mechanism ofaction has not bright.
This research is divided into three parts, first research taurine inhibiting effects ofmyocardial fibroblasts proliferation, PKC alpha, iNOS and ERK1/2, and then further studyTau reversal myocardial remodeling of intracellular signal transduction mechanism.
1. Tau effects on inhibition proliferation of myoFbs
Aim to observe that Tau effects on inhibition proliferation of myoFbs induced byAngiotensinII (AngII) in neonatal rats, determine the Tau inhibition myoFbs proliferationeffect of effective dose. Separation was born1~3d big lactation rats myoFbs, culture72hafter subculture2-3generations randomly divided into normal control group, AngII(10-7mol·L~(-1)) model group and Tau (120,60and30)mmol·L~(-1)group, continue to cultivate24/48h,observe the change of cell morphology, the application of MTT colorimetric method todetect cell survival, kit test cell medium hydroxyproline (I and III) content, NO content andiNOS activity, ELISA detection cell supernatant fluid of TGF-β1and CTGF content,fluorescent confocal detection ERK1/2nuclear transfer, PKCα membrane inversion andprotein expression, Western blot test p-ERK1/2, p-PKCa and iNOS protein expression.
The results show that, compared with controls, myoFbs proliferation live quantityobviously increased in AngII group, myoFbs medium, hydroxyproline (I and III) content,NO content and iNOS activity, TGF-β1and CTGF content, ERK1/2nuclear transfer, PKCαmembrane inversion and protein expression increased (P<0.01); Compared with modelgroup, Tau (120,60and30)mmol·L~(-1)group, proliferation of myoFbs quantity reduce(P<0.05, P<0.01), myoFbs medium, hydroxyproline (I and III) content, NO content andiNOS activity, TGF-β1and CTGF was obviously lower,(P<0.05, P<0.01). Fluorescentconfocal detection ERK1/2nuclear transfer, PKCα membrane inversion and proteinexpression, Western blot testing p-ERK1/2, p-PKCα and iNOS protein expressionsignificantly reduced. Flow cytometry instrument detection Tau(120,60and30)mmol L~(-1)group myoFbs apoptosis number no significant reduce.
Tau inhibits AngII cause myoFbs proliferation, reduce hydroxyproline (I and III)generation, reduce the TGF-β1and CTGF content, this effect may and NO, iNOS, ERK1/2,PKCα wait for an element to concern, including Tau in dose for60mmol·L~(-1)effect issignificant.
2. Study on mechanism of Tau inhibits proliferation of myoFbs
(1) Tau by inhibiting p-PKCα membrane inversion and expression regulation myoFbsproliferation Separation was born1~3d rat myoFbs, culture72h after subculture2-3generation randomly divided into normal control group, AngII (final concentration for10-7mol·L~(-1)) model group, Tau (120,60and30) mmol·L~(-1)group, D4681(0.625ug·L~(-1)) group,AngII+D4681group and AngII+D4681+Tau group, continue to cultivate24/48h, applicationMTT colorimetric method to detect cell survival rate, flow cytometry instrument detectioncell cycle distribution, immunocytochemistry method, fluorescent confocal method and Western blot detection p-PKCα membrane inversion and expression. The results show that itcan inhibit the proliferation myoFbs, Tau and D4681after share inhibition effect moreapparent, and reduce the proportion of S phase, rise G0/G1phase rate. At the same time Tauinhibits p-PKCα membrane inversion and expression, D4681and Tau common after useinhibition p-PKCα membrane inversion and the expression effect more apparent, thus it canbe seen that Tau can inhibit proliferation of myoFbs is through the inhibition of p-PKCαmembrane inversion and express the realization of function. Application specific PKCαinhibitor D4681can strengthen Tau effect son inhibition proliferation of myoFbs.
(2) Tau can improve activity of iNOS and content of NO
Separation myoFbs were born1~3d rat, myoFbs were cultured72h after subculture,2-3generation randomly divided into normal control group, AngII (10-7mol·L~(-1)) modelgroup, Tau (60mmol·L~(-1)) group, AG(10μg·L~(-1)) group, AngII+AG group and AngII+AG+Taugroup, continue to cultivate24/48h, application MTT colorimetric method to detect cellsurvival, measuring cell medium iNOS activity and NO content by flow cytometry, and thedetection instrument cell cycle distribution, application immunocytochemistry method,fluorescent confocal and Western blotting detection in the expression of iNOS cytoplasm.
Results show that AngII+AG+Tau group myoFbs proliferation significantly reduced,cell culture fluid iNOS activity decline, NO content lower (P<0.01, P<0.001), S periodpercentage decline, G0/G1phase proportion increased significantly(P<0.05, P<0.01), and theexpression of iNOS in cytoplasm increased significantly comparison with the model group,Tau can improve iNOS activity, inhibition proliferation of myoFbs.
(3) Tau by inhibiting p-ERK1/2nuclear transfer and expression to inhibit proliferationof myoFbs. Separation myoFbs was born1~3d rat, which were cultured72h after subculture2-3generation,then randomly divided into normal control group, AngII (10-7mol·L~(-1)) modelgroup, Tau(60mmol·L~(-1))group, D4681(0.625ug·L~(-1))group, AngII+D4681group andAngII+D4681+Tau group, continue to cultivate24/48h, application immunocytochemistrymethod, fluorescent confocal and Western blotting testing p-ERK1/2nuclear transfer andexpression. The results show that Tau inhibits p-ERK1/2nuclear transfer and expression,D4681and Tau common after use inhibition p-ERK1/2nuclear transfer and the expressioneffect more apparent, thus it can be seen myoFbs Tau inhibition of proliferation is throughthe inhibition of p-ERK1/2nuclear transfer and express the realization of function.Application specific PKCα inhibitor D4681can strengthen Tau inhibition p-ERK1/2nucleartransfer and the expression effect.
(4) Tau regulate proliferation of myoFbs through ROS
Research now myoFbs found AngII induced proliferation of ROS quantity changes, inorder to further testing whether Tau by reducing the generation of ROS and myoFbsinhibition of proliferation. The myoFbs were cultured in vitro, and application specificantioxidant to detect the content of ROS and the expression of p-PKCα, discuss themechanism of action. Separation myoFbs were born1~3d rat, culture72h after subculture2-3generation randomly divided into normal control group, AngII (10-7mol·L~(-1)) modelgroup, Tau (120,60and30)mmol·L~(-1)group, NAC (10-4mol·L~(-1)) and Tau+NAC group,continue to cultivate24/48h, application MTT colorimetric method to detect cell survivalrate, and use the ELISA kit to detecti content of·OH in cell medium, laser confocaldetection the expression of ROS, Western blot detection of p-PKCα expression of myoFbs.
Results comparing with control group, AngII group myoFbs proliferation, contentof·OH in cell medium, ROS and p-PKCα expression are increased significantly;Application of antioxidant n-acetyl cysteine (NAC), compared with the model group, Taugroup and NAC group myoFbs proliferation, content of·OH in cell medium, ROS andp-PKCα expression are all decreased. Therefore, effect of Tau on inhibition of proliferationmyoFbs and reduce the generation of ROS and p-PKCα expression, which can start againstmyocardial fibrosis effect about.
To sum up, Tau can inhibit the proliferation of myoFbs and reversal myocardialremodeling, thus to attain the myocardial cell of protection, the intracellular signaltransduction mechanism may be through the activation of PKCα-ROS, ERK1/2and iNOSpath, so as to change the TGF-β1and CTGF of generation and the realization.
引文
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