芩丹胶囊对血管外膜成纤维细胞TGF-β1/Smad信号转导通路影响的研究
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摘要
研究背景
心血管的重构贯穿于血管内膜损伤后再狭窄、动脉粥样硬化(Atherosclerosis, AS)、高血压等多种心血管疾病的发生、发展过程中,是各种病变维持、恶化的病理结构基础。近年来,血管外膜在损伤后血管重构过程中所起的作用得到了空前的关注。研究证明,外膜对血压升高的反应是最敏感的,并且早于血管中膜和内膜,而且外膜的这种变化是高血压血管结构改变的最早征象之一。血管外膜成纤维细胞(Adventitial fibroblasts, AF)在血管损伤后发生增殖、迁移、分泌细胞外基质,是参与血管损伤后新内膜形成和血管重塑的重要内容。
转化生长因子β1 (Transforming growth factorβ1, TGF-β1)是迄今为止所发现的功能最为复杂的生长因子之一,被公认为是影响AF生物功能最直接,也是最重要的细胞因子。Smad信号通路是TGF-β1的经典下游信号通路,与众多组织如心肌、肝脏、肺及肾脏等的纤维化密切相关。根据Smad在信号转导中的作用可将其分为三类：(1)受体调节型Smad(Receptor activated Smad, R-Smad),包括Smad 1、2、3、5、8,为Ⅰ型受体激酶的底物,在特定的TGF-β超家族成员的信号转导通路中发挥作用；(2)共同介质型Smad(Common Smad, Co-Samd),主要是Smad4。Smad4被磷酸化后可以与R-Smad形成杂聚体,然后进入细胞核内对靶基因的转录进行调节；(3)抑制型Smad(Inhibitory Smad, I-Smad),包括Smad6、7,可以阻断R-Smad与受体或与Co-Samd的结合,从而对Smad信号通路发挥负调控作用。
RNA干扰(RNA interference, RNAi)是新兴的生物技术,被Science评为2002年十大科学成就之首。RNA干扰是一种由目的基因所对应的小分子双链RNA (Double-stranded, dsRNA)启动的序列特异的基因沉默过程,从而导致相应的mRNA被降解,进而阻断基因的表达。人工合成的siRNA可特异性地抑制哺乳类细胞中内源性或外源性基因的表达,使相应基因保持沉默或休眠状态。SiRNA所诱发的基因抑制具有高效性和高度的序列特异性。
本实验通过观察TGF-β1诱导AF生物活性的改变,并用RNA干扰的方法,研究在外膜成纤维细胞增殖、迁移、表型转化和细胞外基质合成中TGF-β1/Smad信号通路的作用机制,且进一步探讨Smad2和Smad3在上述过程中的不同作用。
目的
1.观察TGF-β1对外膜成纤维细胞增殖、迁移、表型转化和细胞外基质合成的影响。
2.探讨TGF-β1/Smad信号通路在外膜成纤维细胞增殖、迁移、表型转化和细胞外基质合成中的作用机制,并研究Smad2和Smad3在这些过程中的不同作用。
方法
1.体外原代成纤维细胞的培养与鉴定：采用组织贴块法体外原代培养大鼠胸主动脉成纤维细胞。大概3-7天可见细胞从组织块周围爬出；SABC法行平滑肌肌动蛋白-α(alpha smooth muscle actin,α-SMA)和波形蛋白(Vimentin)免疫细胞化学染色鉴定。取第3-8代细胞用于实验。
2.小干扰RNA链的合成、有效浓度的筛选：设计并分别合成3条Smad2和Smad3特异性siRNA,同时合成荧光标记的阴性对照siRNA(FAM-siRNA)1条和Smad2/3非特异性阴性对照siRNA 1条。细胞密度达50%-70%时转染FAM-siRNA。转染后荧光显微镜观察荧光阳性细胞,计算转染效率,根据转染效率选择siRNA和Lipofectamine 2000最佳转染浓度。
3.细胞转染和小干扰有效链的筛选：根据Lipofectamin 2000的转染步骤,用western-blot检测各组siRNA的干扰效率,进行有效链的筛选。
4.MTT法检测细胞的增殖能力：接种细胞,按照说明进行细胞转染,24h后,加入含20 ng/ml TGF-β1的正常培养液,继续刺激24h。于刺激结束前4h每孔加入MTT,然后吸弃上清,加入DMSO,比色。
5. Transwell检测细胞迁移能力：应用6孔板transwell小室行体外迁移实验。细胞分组和处理同前,处理过的细胞消化后用DMEM制成细胞悬液,取0.6 ml悬液以5×105/ml的密度接种于上室,下室中加入1.5 ml含10%FCS的正常培养基。37℃培养6h,将上室内侧附着的细胞用湿棉签擦去,固定,伊红和苏木素染色,倒置显微镜下随意取5个视野进行细胞计数(×100)。
6.实时定量PCR检测相关因子mRNA的表达：细胞处理同前,采用Trizol法提取总RNA,并在PCR热循环仪上将RNA逆转录为cDNA。观察基因沉默和TGF-β1处理后,Smad2、Smad3、Smad7、α-SMA、Ⅰ型及Ⅲ型前胶原肽mRNA的表达。采用2-ΔΔCt方法对mRNA表达进行相对定量分析。
7. Western-blot检测相关因子蛋白的表达：取对数生长期细胞消化计数,转染24h后,加入TGF-β1孵育24h,提取蛋白并定量,western-blot检测p-Smad2、p-Smad3、Smad2、Smad3、Smad7、α-SMA、Ⅰ型前胶原肽蛋白表达。
8.统计学处理所有数据资料均采用SPSS 16.0进行统计处理。实验数据用x±s表示,组间差异采用one-way ANOVA分析和t检验,P<0.05为差异有统计学意义。
结果
1.原代细胞的生长形态及鉴定
贴壁3-7d后可见细胞从不同的组织块边缘爬出,取第3代细胞,用α-SMA和Vimentin行免疫细胞化学染色,阳性率达100%。
2.针对Smad2或Smad3的siRNA片段均可以有效下调Smad2或Smad3的表达
SiRNA转染并经TGF-β1刺激后,干扰组Smad2或Smad3蛋白的表达较单纯刺激组下降80%以上。且小干扰具有高度特异性和选择性,即siRNA-Smad3只降低Smad3蛋白的表达,而对Smad2蛋白的表达没有影响,同样siRNA-Smad2只作用于Smad2,而对Smad3蛋白表达没有作用。
3.基因沉默Smad2或Smad3对外膜成纤维细胞增殖影响
(1)与正常对照组相比,TGF-β1刺激组增殖显著增强(P<0.01); Smad2基因被干扰之后,细胞增殖较单纯TGF-β1刺激组减弱(P<0.01)。基因沉默Smad2能显著抑制成纤维细胞的增殖能力。
(2) Smad3基因被沉默后,细胞增殖能力较TGF-β1刺激组减弱(P<0.01)。SiRNA介导的Smad3表达下调可以显著抑制成纤维细胞的增殖。
(3) SiRNA-Smad2和siRNA-Smad3均能显著抑制成纤维细胞的增殖,但两者差异没有统计学意义(P>0.05)。
4.基因沉默Smad2或Smad3对外膜成纤维细胞迁移的影响
(1)与正常对照组相比,TGF-β1刺激后细胞迁移能力显著增强(P<0.01)；下调Smad2基因表达之后,细胞迁移较TGF-β1刺激组减弱(P<0.01)。基因沉默Smad2能显著抑制成纤维细胞的迁移能力。
(2) Smad3基因被沉默后,细胞迁移能力较TGF-β1刺激组减弱(P<0.01)。SiRNA介导的Smad3表达下调亦可以显著抑制成纤维细胞的迁移能力。
(3) SiRNA-Smad2和siRNA-Smad3均能显著抑制成纤维细胞迁移能力,然而两者之间差别无统计学意义(P>0.05)。
5.基因沉默Smad2或Smad3对TGF-β1信号转导通路相关因子mRNA表达的影响
(1)经TGF-β1刺激后,Smad2、Smad3、α-SMA、Ⅰ及Ⅲ型前胶原mRNA表达均较正常组显著增强(P<0.05或P<0.01),而Smad7 mRNA表达则无显著差异(P>0.05).SiRNA介导Smad2表达下调后,与TGF-β1刺激组相比,α-SMA、Ⅰ及Ⅲ型前胶原mRNA表达显著降低,差异具有统计学意义(P<0.05或P<0.01), Smad7 mRNA表达则无显著差异(P>0.05)。
(2) Smad3基因被沉默后,α-SMA, I及Ⅲ型前胶原mRNA较TGF-β1刺激组减弱,其差异具有统计学意义(P<0.05或P<0.01)。Smad7 mRNA表达无显著差异(P>0.05)。
(3)SiRNA-Smad2和siRNA-Smad3均能显著α-SMA、Ⅰ及Ⅲ型前胶原mRNA表达,但两者之间差别无统计学意义(P均>0.05)。
6.基因沉默Smad2或Smad3对TGF-β1信号转导通路相关因子蛋白表达的影响
(1)经TGF-β1刺激后,Smad2、Smad3、p-Smad2、p-Smad3、α-SMA、Ⅰ型前胶原蛋白表达均较正常组显著增强(P<0.01),而Smad7蛋白表达则无显著差异(P>0.05)。SiRNA介导Smad2表达下调后,与TGF-β1刺激组相比,p-Smad2、α-SMA、I型前胶原蛋白表达显著降低,差异具有统计学意义(P<0.05或P< 0.01),Smad7蛋白表达则无显著差异(P>0.05)。
(2) Smad3基因被沉默后,p-Smad3、α-SMA、Ⅰ型前胶原蛋白较TGF-β1刺激组减弱,其差异具有统计学意义(P<0.01)。Smad7蛋白表达无显著差异(P>0.05)。
(3)SiRNA-Smad2和siRNA-Smad3均能显著α-SMA,Ⅰ型前胶原蛋白表达,但两者之间差别无统计学意义(P>0.05)。
结论
1.实验中所选用siRNA可以高效并特异地阻断Smad2或Smad3的表达。
2. TGF-β1对外膜成纤维细胞的增殖有促进作用,Smad2和Smad3信号通路共同参与其中。
3. TGF-β1促进外膜成纤维细胞的迁移,这一过程需要Smad2和Smad3的共同参与。
4. TGF-β1诱导成纤维细胞转分化和胶原沉积,是依赖Smad2和Smad3信号转导通路的共同作用。
研究背景
高血压病是严重危害人类健康的常见病和多发病,其发病率逐年升高,目前我国成人发病率高达18.8%。随着对高血压病发病机理的研究和降压药的发展,患者血压水平多能得到有效控制,但高血压所伴发的靶器官损害的发病率、致残率和致死率仍然高居不下。血管重构(Vascular remodeling, VR)既是高血压状态下机体对各种改变的适应性行为,又是高血压病持续发展恶化的重要病理学基础。研究血管重构的发生和逆转机制,对有效控制血压、预防和减轻各种并发症具有非常重要的意义。
血管外膜在高血压血管重构中的作用越来越受到重视,血压升高是促使外膜成纤维细胞(Adventitial fibroblast, AF)变为肌成纤维细胞(Myofibroblast, MF)的重要因素,后者具有增殖及迁移活性,可由外膜向中膜和内膜下间隙迁移,同时能够促进细胞外基质(Extracellular matrix, ECM)合成,导致晚期纤维化,管腔缩窄,参与了血管重构。
转化生长因子β1(Transforming growth factorβ1, TGF-β1)是重要的促纤维化细胞因子之一,可以通过激活一系列的细胞内信号传导通路,引起靶基因的转录。Smad是其主要的细胞内信号转导途径,与众多组织如心肌、肺及肾脏等的纤维化密切相关。
中药血清药理学方法是指给动物灌服中药一定时间后,用其血清进行实验研究的药理学研究方法。这种实验方法不仅体现了药物在生物体内的生物转化,同时克服了中药复方研究中其他因素的干扰。
芩丹胶囊(Qindan capsule, QC)是导师临床治疗高血压病的经验方,有清热平肝、化瘀和络的功效。以前的研究表明该药能够改善SHR大鼠主动脉壁形态学指标,降低主动脉中膜胶原含量,改善SHR大鼠主动脉中膜血管平滑肌细胞(vascular smooth muscle cell, VSMC)表型改变。但是其对血管外膜及TGF-β1/Smad信号系统的影响尚不明了。本研究探讨了芩丹胶囊对TGF-β1诱导的AF增殖、迁移、表型转化以及细胞外基质合成的影响,并探讨在此过程中TGF-β1/Smad信号转导通路的作用机制,以期为临床防治高血压病血管损害提供更多科学依据。
目的
1.观察芩丹胶囊含药血清对TGF-β1诱导的AF增殖、迁移、平滑肌肌动蛋白-α(alpha smooth muscle actin,α-SM actin)、Ⅰ和Ⅲ型前胶原mRNA和蛋白表达的改变,探讨芩丹胶囊对TGF-β1诱导的AF的生物活性的影响。
2.观察芩丹胶囊含药血清对TGF-β1诱导的AF Smad2、Smad3、Smad7、磷酸化Smad2 (phosphorylation-Smad2, p-Smad2)和磷酸化Smad3 (phosphorylation-Smad3, p-Smad3) mRNA和蛋白表达的影响,探讨芩丹胶囊对外膜成纤维细胞TGF-β1/Smad信号转导通路的作用。
方法
1.芩丹胶囊的制备及质量标准的研究：采用高效液相色谱法(High performance liquid chromatography, HPLC)和薄层层析法(Thin-layer chromatography, TLC),明确芩丹胶囊的有效成分。
2.成纤维细胞的复苏与传代：取出冻存管,快速解冻,用正常培养基悬浮细胞,离心后转移至培养瓶。待细胞长至90%融合时进行传代。
3.芩丹胶囊含药血清的制备：60只健康雄性Wistar大鼠随机分为3组：芩丹胶囊大剂量组(QC大剂量组)给予QC 750mg/(kg·d);芩丹胶囊小剂量组(QC小剂量组)给予QC 150mg/(kg·d);氯沙坦组给予氯沙坦30 mg/(kg-d)。各组均大鼠灌胃给药,最后一日禁食24h,末次给药后1h(氯沙坦组)和2h(芩丹胶囊组)后乙醚麻醉,腹主动脉取血,37℃水浴1 h,3000 rpm离心15min,取上清,过滤除菌,然后在-57℃条件下真空干燥,-20℃保存备用。
4.MTT比色法检测细胞增殖能力：制备细胞悬液,接种于96孔板,各组加入相应血清继续孵育48h,换用含20 ng/ml TGF-β1的培养基诱导24h,在刺激结束前4h加入MTT,弃上清,加入DMSO,振荡,比色。
5. Transwell法检测细胞迁移能力：细胞分组和处理同前,经过处理的细胞被消化,然后用DMEM制成细胞悬液,取0.6ml细胞悬液接种于上室,1.5ml含10%FCS的正常培养基加入下室。置入孵箱内培养6h,取出小室用湿棉签擦内侧的细胞,然后固定染色,计数。
6.实时定量PCR技术检测相关因子mRNA的表达：细胞处理同前,提取总RNA,将RNA逆转录为cDNA。观察芩丹胶囊和氯沙坦含药血清对TGF-β1诱导的Smad2、Smad3、Smad7、α-SMA、Ⅰ型及Ⅲ型前胶原肽mRNA的表达。用2-△△Ct的方法对mRNA的表达进行相对定量分析。
7. Western-blot技术检测相关因子蛋白的表达：细胞分组和处理方法同前,提取蛋白并定量,western-blot检测p-Smad2、p-Smad3、Smad2、Smad3、Smad7、α-SMA、Ⅰ型前胶原肽蛋白表达。
8.统计学处理所有实验数据用x±s表示,数据资料均用SPSS 16.0进行统计处理。组间差异采用one-way ANOVA分析和t检验,设P<0.05为有统计学差异。
结果
1.各实验组细胞增殖情况的比较
与正常对照组相比,TGF-β1刺激组的AF增殖活性明显加强(P<0.01)；各药物处理组增殖与TGF-β1刺激组相比,有非常显著性差异(P<0.05或P<0.01),其中以氯沙坦组减弱最为显著(P<0.01),其次为QC大剂量组(P<0.01),QC大剂量组和氯沙坦组较QC小剂量组有显著差异(P<0.05)。
2.各实验组细胞迁移数目的比较
TGF-β1刺激组的AF的迁移数目明显多于正常对照组,且差异具有统计学意义(P<0.01)；经药物处理以后,与TGF-β1刺激组相比,药物组细胞迁移数目均明显下降(P<0.05或P<0.01)。
3.各实验组细胞Smad2、Smad3、Smad7、α-SMA、Ⅰ及Ⅲ型前胶原mRNA表达的比较
(1) Smad2 mRNA表达比较：与正常对照组相比,TGF-β1刺激组细胞的Smad2 mRNA表达显著升高(P<0.01)；各药物处理组Smad2 mRNA表达较TGF-β1刺激组均有不同程度下降,其中氯沙坦组下降最为显著(P<0.01),其次为QC大剂量组(P<0.01)；QC大剂量组、氯沙坦组与QC小剂量组比较有显著差异(P<0.05)。
(2) Smad3 mRNA表达比较：与正常对照组相比,TGF-β1刺激组细胞Smad3 mRNA表达显著升高(P<0.01)；与TGF-β1刺激组相比,QC大剂量组和氯沙坦组Smad3 mRNA表达均显著下降(P均<0.05),QC小剂量组差异没有显著性(P>0.05)；与氯沙坦组相比,QC小剂量组Smad3 mRNA表达差异有显著性(P<0.05)。
(3) Smad7 mRNA表达比较：与正常对照组相比,TGF-β1刺激组细胞Smad7 mRNA表达没有明显变化(P>0.05)；与TGF-β1刺激组相比,各药物处理组Smad7 mRNA表达均显著提高(P<0.01)；QC大剂量组、氯沙坦组与QC小剂量组比较,差异具有统计学意义(P<0.05)。
(4)α-SMA mRNA表达比较：与正常对照组相比,TGF-β1刺激组α-SMA mRNA表达显著升高(P<0.01)；各药物处理组α-SMA mRNA表达较TGF-β1刺激组均有不同程度下降,其中氯沙坦组下降最为显著(P<0.01),其次为QC大剂量组(P<0.01),小剂量组与氯沙坦组比较差异具有统计学意义(P<0.05)。
(5)Ⅰ型前胶原mRNA表达比较：与正常对照组相比,TGF-β1刺激组细胞Ⅰ型前胶原mRNA表达显著升高(P<0.01)；与TGF-β1刺激组相比,QC大剂量组和氯沙坦组Ⅰ型前胶原mRNA表达均显著下降(P<0.05)；QC小剂量组与TGF-β1刺激组相比差异没有统计学意义(P>0.05)。
(6)Ⅲ型前胶原mRNA表达比较：与正常对照组相比,TGF-β1刺激组细胞Ⅲ型前胶原mRNA表达表达显著升高(P<0.01)；与TGF-β1刺激组相比,各药物处理组Ⅲ型前胶原mRNA表达均显著降低(P<0.05或P<0.01)；其中氯沙坦组下降最为显著(P<0.01),其次为QC大剂量组(P<0.01)；氯沙坦组与QC小剂量组比较有显著差异(P<0.05)。
4.各实验组细胞Smad2、Smad3、p-Smad2、p-Smad3、α-SMA、Ⅰ型前胶原、Smad7蛋白表达的比较
(1) Smad2蛋白表达比较：与正常对照组相比,TGF-β1刺激组细胞Smad2蛋白表达明显升高(P<0.01)。与TGF-β1刺激组相比,各药物组Smad2蛋白表达有所下降(P均<0.01)；氯沙坦组显著下降(P<0.01),且下降幅度大于QC大剂量组与QC小剂量组(P<0.05或P<0.01)。
(2) p-Smad2蛋白表达比较：与正常对照组相比,TGF-β1刺激组细胞p-Smad2蛋白表达显著升高(P<0.01)；与TGF-β1刺激组相比,各药物处理组p-Smad2蛋白表达均显著下降(P<0.01)；QC大剂量组和氯沙坦组表达低于QC小剂量组(P<0.05或P<0.01)。
(3) Smad3蛋白表达比较：与正常对照组相比,TGF-β1刺激组细胞Smad3蛋白表达明显升高(P<0.01)。与TGF-β1刺激组相比,氯沙坦组表达水平下降最明显(P<0.05),其次为QC大剂量组(P<0.05)。且氯沙坦组和QC小剂量组相比有显著性差异(P<0.05)。
(4) p-Smad3蛋白表达比较：与正常对照组相比,TGF-β1刺激组细胞p-Smad3蛋白表达表达显著升高(P<0.01)；与TGF-β1刺激组相比,各药物处理组p-Smad3蛋白表达均显著降低(P<0.01)；氯沙坦组与QC小剂量组比较差异具有统计学意义(P<0.05)。
(5)α-SMA蛋白表达比较：与正常对照组相比,TGF-β1刺激组α-SMA蛋白表达显著升高(P<0.01)；各药物处理组α-SMA蛋白表达较TGF-β1刺激组均有不同程度下降,其中氯沙坦组下降最为显著(P<0.01),其次为QC大剂量组(P<0.01)；QC小剂量组和氯沙坦组比较有显著差异(P<0.01)。
(6)Ⅰ型前胶原蛋白表达比较：与正常对照组相比,TGF-β1刘激组细胞Ⅰ型前胶原蛋白表达显著升高(P<0.01)；与TGF-β1刺激组相比,各药物处理组Ⅰ型前胶原蛋白表达均显著下降(P<0.01)；氯沙坦组Ⅰ型前胶原白表达低于QC小剂量组(P<0.05)。
(7) Smad7蛋白表达比较：与正常对照组相比,TGF-β1刺激组细胞Smad7蛋白表达没有明显变化(P>0.05);与TGF-β1刺激组相比,各药物处理组Smad7蛋白表达均显著提高(P<0.01)；其中氯沙坦组升高最为显著(P<0.01),其次为QC大剂量组(P<0.01)。
结论
1.芩丹胶囊能够通过TGF-β1/Smad信号转导通路抑制TGF-β1诱导的AF增殖和迁移。
2.芩丹胶囊能够抑制TGF-β1诱导的AF表型转化,其机制与降低TGF-β1信号因子Smad2、Smad3、p-Smad2、p-Smad3 mRNA和蛋白表达,并升高Smad7 mRNA和蛋白表达上调有关。
3.芩丹胶囊能够抑制TGF-β1诱导的细胞外基质的合成,其机制与降低TGF-β1信号因子Smad2、Smad3、p-Smad2、p-Smad3 mRNA和蛋白表达,并升高Smad7 mRNA和蛋白表达有关。
研究背景
高血压性血管重构是心血管事件的重要危险因素,转化生长因子β1(Transforming growth factorβ1, TGF-β1),能够激活外膜成纤维细胞(Adventitial fibroblasts, AF),使之迁移、增殖、表型转变及合成细胞外基质的能力增强,参与高血压性血管重构。
槲皮素(3,3',4',5,7-pentahydroxyflavone, Que),是自然界中分布最广的生物黄酮类化合物。大量研究已证实槲皮素是一种有效的降压药,同时还具有抗氧化、抗血栓、抗炎、减轻心肌肥厚等多种心血管保护作用。槲皮素是芩丹胶囊中桑寄生的有效成分,我们通过检测槲皮素对TGF-β1诱导的AFs增殖的影响,同时应用荧光实时定量RT-PCR检测Smad2、α-SMA、Ⅰ/Ⅲ型前胶原mRNA表达情况以及western blot检测Smad2和p-Smad2蛋白表达的改变,探讨了槲皮素逆转TGF-β1诱导的AFs生物活性的机制,为其临床防治高血压性血管重构提供科学依据。
目的
观察槲皮素对TGF-β1诱导的AFs增殖的影响,以及对Smad2、α-SMA、Ⅰ/Ⅲ型前胶原、p-Smad2 mRNA和蛋白的影响,探讨槲皮素逆转TGF-β1诱导的AFs生物活性的机制。
方法
1.AFs的复苏与传代：液氮罐中取出冻存管,快速解冻,悬浮细胞,离心,转移至培养瓶。待细胞长至90%融合时进行传代。
2.MTT法检测细胞增殖情况：以细胞密度为2×104接种于96孔板,培养24h,换无血清培养基培养4h,使细胞同步生长,吸弃上清,各组加入(6.25μmol/L、12.5μmol/L和25μmol/L)槲皮素预处理细胞24h,换用含20ng/ml TGF-β1的培养基刺激24h,在刺激结束前4h加入MTT,弃上清,加入DMSO,振荡,比色。
3.实时定量RT-PCR技术检测相关因子mRNA的表达：细胞饥饿4h后换用正常培养基,用各浓度的槲皮素预处理细胞24 h后,再给予TGF-β1刺激24h,提取总RNA,将RNA逆转录为cDNA。实时定量RT-PCR检测Smad2、α-SMA、Ⅰ型及Ⅲ型前胶原肽mRNA的表达。
4. Western-blot技术检测相关因子蛋白的表达：细胞分组和处理方法同前,提取蛋白并定量,western-blot检测p-Smad2和Smad2蛋白表达情况。
5.统计学处理：所有实验数据用x±s表示,数据资料均用SPSS 16.0进行统计处理。组间差异采用one-way ANOVA分析和t检验,设P<0.05为有统计学意义。
结果
1.各实验组细胞增殖情况的比较
与正常对照组相比,TGF-β1刺激组的AF的增殖活性明显加强(P<0.01)；大、中剂量组增殖与TGF-β1刺激组相比,有非常显著性差异(P<0.01),其中以大剂量组减弱最为显著(P<0.01),其次为中剂量组(P<0.01),小剂量组和刺激组相比没有显著差异(P>0.05)。
2.各实验组细胞Smad2、α-SMA、Ⅰ及Ⅲ型前胶原mRNA表达的比较
(1) Smad2 mRNA表达比较：与正常对照组相比,TGF-β1刺激组细胞的Smad2 mRNA表达显著升高(P<0.01)；大、中剂量组Smad2 mRNA表达较TGF-β1刺激组均有不同程度下降,其中大剂量组下降最为显著(P<0.01),其次为中剂量组(P<0.01),小剂量组没有显著差异(P>0.05)。
(2)α-SMA mRNA表达比较：与正常对照组相比,TGF-β1刺激组α-SMA mRNA表达显著升高(P<0.01)；大、中剂量组α-SMA mRNA表达较TGF-β1刺激组均有不同程度下降(P<0.05或P<0.01),小剂量组与刺激组比较差异没有统计学意义(P>0.05)。
(3)Ⅰ型前胶原mRNA表达比较：与正常对照组相比,TGF-β1刺激组细胞Ⅰ型前胶原mRNA表达显著升高(P<0.01)；与TGF-β1刺激组相比,大剂量组和中剂量组Ⅰ型前胶原mRNA表达均显著下降(P<0.05)；小剂量组与TGF-β1刺激组相比差异没有统计学意义(P>0.05)。
(4)Ⅲ型前胶原mRNA表达比较：与正常对照组相比,TGF-β1刺激组细胞Ⅲ型前胶原mRNA表达表达显著升高(P<0.01)；与TGF-β1刺激组相比,大、中剂量组Ⅲ型前胶原mRNA表达均显著降低(P<0.05)；其中大剂量组下降最为显著(P<0.05),其次为中剂量组(P<0.05),小剂量组没有显著差异(P>0.05)。
3.各实验组细胞Smad2和(?)-Smad2蛋白表达的比较
(1) Smad2蛋白表达比较：与正常对照组相比,TGF-β1刺激组细胞Smad2蛋白表达明显升高(P<0.01)。与TGF-β1刺激组相比,大、中剂量组显著下降(P均<0.01),小剂量组差异没有显著性(P>0.05)。
(2) p-Smad2蛋白表达比较：与正常对照组相比,TGF-β1刺激组细胞p-Smad2蛋白表达显著升高(P<0.01)；与TGF-β1刺激组相比,大、中剂量组p-Smad2蛋白表达均显著下降(P<0.05或P<0.01)；小剂量组蛋白表达下降没有统计学意义(P>0.05)。
结论
1.槲皮素能够显著抑制TGF-β1诱导的成纤维细胞的增殖。
2.槲皮素抑制了TGF-β1诱导的成纤维细胞Smad2、p-Smad、α-SMA、Ⅰ/Ⅲ型前胶原mRNA和蛋白的高表达。
3.槲皮素能够通过TGF-β1/Smad2信号通路改善TGF-β1诱导的成纤维细胞的生物活性的改变。
Background
Vascular remodeling runs through the process of various cardiovascular diseases, such as the restenosis after percutaneous transluminal coronary angioplasty (PTCA), atherosclerosis(AS) and hypertension. And vascular remodeling is considered the major pathological base for maintaining and worsening of hypertension. Recently the adventitia has attracted more and more attention for the importance during vascular remodeling after vascular injury. The adventitia is the most sensitive layer responding to elevated blood pressure compared with intimal and medial. Adventitial fibroblasts (AFs), the main adventitial cell type, drive remodeling and may initiate other changes subsequently, such as alterations in arrangements of neointimal proliferation, smooth muscle cells and extracellular matrix (ECM), which is expected to lead to vascular remodeling.
Transforming growth factorβ1 (TGF-β1), a pleiotropic and multifunctional cytokine, is known as the most important and direct one to regulate AFs bioactivity. The classical signal transduction pathway of the TGF-βfamily is through Smad, which is related to the fibrosis of many parts, such as heart, liver, lung and kidney. Smads are divided into three subfamilies:(1) receptor-activated Smads (R-Smads: Smad1, Smad2, Smad3, Smad5, Smad8), which become phosphorylated receptor I and play an important role in transforming growth factor-β(TGF-β) family; (2)common mediator Smads (Co-Smads:Smad4), which become phosphorylated and oligomerise with activated R-Smads to practice their intracellular effects; (3) inhibitory Smads (I-Smads:Smad6 and Smad7), which exert a negative feedback effect by competing with R-Smads.
RNA interference (RNAi) is a new biotechnology, which was considered the top of the ten scientific achievements in 2002 by Science. RNAi is a sequence-specific gene silencing procedure started by double-stranded (dsRNA) RNA corresponding to target gene. Synthetical siRNA could inhibit the expression of gene, endogenic and ectogenic specifically in mammalian cells. And it could keep the target genes in scilence or resting state. The inhibition of genes started by siRNA is of high efficacy and specificity.
In the present study, we observed the biological activity of AFs induced by TGF-β1 and tried to investigate the mechanism of TGF-β1/Smad signaling pathway on the migration, proliferation, transdifferentiation and ECM deposition of AFs. And moreover, the different roles of Smad2 and Smad3 were studied during the process.
Aims
1. To investigate the effect of TGF-β1 on the migration, proliferation, transdifferentiation and ECM deposition of AFs.
2. To investigate the possible mechanism of TGF-β1/Smad signaling pathway during the activity of AFs induced by TGF-β1. And to study the different roles of Smad2 and Smad3 during the procedure.
Method
1. Primary cell culture and identification in vitro
AFs were cultured by tissue explant in vitro. AFs were observed to grow out from the tissues 3-7 days later. The AF and its purity were identified by immunocytochemistry of Vimentin and a-smooth muscle actin (SMA). Cells of the 3-8 passages were used in assays.
2. Synthesis of siRNA and selection of the effective concentration
Three specific Smad2-siRNAs and Smad3-siRNAs, one FAM-siRNA and one Smad2/3-negative siRNA were designed and chemically synthesized, so were Smad3 siRNAs. Transfection of FAM-siRNA was performed when the AFs were at 50%-70% density. Under light microscopy after transfection, the positive cells were observed. The best concentration of siRNA and Lipofectamine 2000 was chosen according to the transfection efficiency.
3. Transfection and selection of the effective siRNAs
The transfection was performed according to the manufacturer's instructions. Western-blot was used to detect the efficiency of gene silence and the effective siRNAs
4. MTT assay for proliferation
Twenty-four h after transfection, AFs in the experiment of TGF-β1 were treated with TGF-β1 for 24 h.20μl MTT (5 mg/ml) was added, followed by incubation for 4 h at 37℃. Finally, medium was removed and cells were lysed with DMSO. Absorbance of the samples was recorded.
5. Transwell assay for migration
Migration of AFs was measured using transwell chamber apparatus in 6-well plates. AFs were grouped and treated as described above. Briefly, cells were trypsinized and counted,600μl cell suspension at 5×105/ml in DMEM was added to the upper compartment of the chamber, and 1.5 ml DMEM containing 10% FCS was added to the lower compartment. After incubation for 6 h, cells on the upper face of the membrane were removed with a cotton-tipped applicator carefully. Then the membranes were fixed in methanol and stained with hematoxylin and eosin (HE). The number of migrated cells was counted in 5 random fields in each membrane.
6. Real-time RT-PCR
After treatment, total RNA was isolated from AFs with use of Trizol Reagent according to the manufacturer's instructions. RNA was reversely transcribed by a standard protocol. The expression of Smad2, Smad3, Smad7,α-SMA, procollagenⅠandⅢmRNA was studied after treatment of siRNA and TGF-β1. The relative mRNA expression of the genes was determined by the 2-△△Ct method.
7. Western blot analysis
Cells were harvested after transfection for 24 h and stimulation by TGF-β1 for another 24 h. Protein was extracted and detected. Western-blot was used to detect the protein expression of p-Smad2, p-Smad3, Smad2, Smad3, Smad7,α-SMA, procollagenⅠ.
8. Statistical analysis
The data are presented as mean±SD and analyzed with SPSS 16.0. Comparisons among groups were analyzed by ANOVA. An unpaired Student's t-test was applied, when only two groups were compared. P< 0.05 was considered to be statistically significant.
Results
1. Appearance and identification of AFs
AFs were observed to grow out from different tissues 3-7 days later. Immunocytochemistry analysis demonstrated that the multiclonal antibody staining for SMA was negative and the monoclonal antibody staining for vimentin was positive, suggesting 100% purity of AFs.
2. siRNAs targeting Smad2 and Smad3 downregulation the expression of Smad2 and Smad3 efficiently
After transfection and stimulation by TGF-β1, the protein expression of Smad2 or Smad3 in siRNA-Smad2 or siRNA-Smad3 group was decreased more than 80% compared with TGF-β1 group. Moreover, the knockdown was specific and selective, as Smad3 protein levels were decreased by Smad3 siRNA only, so were Smad2 protein levels.
3. Proliferation of AFs induced by TGF-β1 was inhibited by siRNA-Smad2 and-Smad3
(1) Cell proliferation in TGF-β1 group was significantly increased compared with the control group (P<0.01). However, the proliferation decreased when Smad2 expression was knocked down by siRNA (P<0.01). Knockdown of Smad2 could inhibit the proliferation of AFs significantly.
(2) The proliferation decreased when Smad3 expression was knocked down by siRNA (P<0.01). Knockdown of Smad3 by siRNA could inhibit the proliferation of AFs significantly.
(3) SiRNA-Smad2 and siRNA-Smad3 could both inhibit the proliferation of AFs significantly, with no significant difference between Smad2 and Smad3 knockdown (P>0.05).
4. Migration of AFs induced by TGF-β1 was inhibited by siRNA-Smad2 and-Smad3
(1) After stimulation by TGF-β1 for 24 h, the migration of AFs was significantly increased compared with the control group(P<0.01). However, the blockade of Smad2 expression by siRNA could reduce the migration of AFs induced by TGF-β1 (P<0.01). Knockdown of Smad2 by siRNA could inhibit the migration of AFs significantly.
(2) The migration decreased when Smad3 expression was knocked down by siRNA (P<0.01). Knockdown of Smad3 by siRNA could inhibit the migration of AFs significantly.
(3) SiRNA-Smad2 and siRNA-Smad3 could both inhibit the migration of AFs significantly, with no significant difference between Smad2 and Smad3 knockdown (P>0.05).
5. mRNA expression of the related gene induced by TGF-β1 was inhibited by siRNA-Smad2 and -Smad3
(1) After stimulation by TGF-β1, the mRNA expression of Smad2, Smad3,α-SMA, procollagenⅠandⅢall increased compared with the control group (P<0.05 or P<0.01), but not Smad7 (P>0.05). Knockdown of Smad2 by siRNA, the mRNA expression ofα-SMA, procollagenⅠandⅢwas decreased significantly compared with TGF-β1 group (P< 0.05 or P<0.01). However, Smad7 mRNA expression did not differ from that of the control (P> 0.05).
(2) The mRNA expression ofα-SMA, procollagenⅠandⅢdecreased when Smad3 expression was knocked down by siRNA compared with TGF-β1 group (P< 0.05 or P< 0.01), but not Smad7 (P> 0.05).
(3) SiRNA-Smad2 and siRNA-Smad3 could both reduce the mRNA expression ofα-SMA, procollagenⅠandⅢof AFs significantly, with no significant difference between Smad2 and Smad3 knockdown(P>0.05).
6. Protein expression of the related gene induced by TGF-β1 was inhibited by siRNA-Smad2 and -Smad3
(1) After stimulation by TGF-β1, the protein expression of Smad2, Smad3,α-SMA, procollagenⅠwas all increased compared with the control group (P<0.01), but not Smad7 (P>0.05). Knockdown of Smad2 by siRNA, the protein expression ofα-SMA, procollagenⅠwas decreased significantly compared with TGF-β1 group (P< 0.05 or P<0.01). However, Smad7 protein expression did not differ from that of the control (P>0.05).
(2) The protein expression ofα-SMA, procollagenⅠdecreased when Smad3 expression was knocked down by siRNA compared with TGF-β1 group (P<0.01), but not Smad7 (P>0.05).
(3) SiRNA-Smad2 and siRNA-Smad3 could both reduce the protein expression ofα-SMA, procollagenⅠof AFs significantly, with no significant difference between Smad2 and Smad3 knockdown (P>0.05).
Conclusion
(1) SiRNA in the experiment could knockdown the expression of Smad2 and Smad3 effectively and specifically.
(2) TGF-β1 could promote the proliferation of AFs both Smad2 and Smad3-dependently.
(3) TGF-β1 could promote the migration of AFs both Smad2 and Smad3-dependently.
(4) Both Smad2 and Smad3 work as mediators of phenotypic transition and collagen synthesis of AFs induced by TGF-β1
Background
Hypertension is the familiar and risk factor for human health. The attack rate is growing year by year and now the rate is up to 18.8%. Blood pressure could be controlled effectively thanks to the intensive study of mechanism of hypertension and development of hypotensor. But the rate of morbidity, fatality and deformity is still very high. Vascular remodeling is the the adaptive reaction to the disorders in hypertension. Moreover, it is the major pathological base of deterioration in the development of hypertension. Therefore, it is very important to investigate the mechanism of vascular remodeling for blood pressure control, prevention and treatment of hypertension and the complications.
Growing experimental evidence shows that the adventitia plays an critical role in vascular remodeling. High blood pressure is an important factor for adventitial fibroblasts (AFs) transdifferentiation to Myofibroblasts (MF). The migratory and proliferative responses of myofibroblasts, in addition to synthesis of ECM, lead to fibrosis and lumen stenosis, which play important roles during vascular remodeling.
Transforming growth factorβ1 (TGF-β1) is an important multifunctional cytokine that regulates fibrosis by activating a series intracellular signaling pathways, which might lead to the transcription of target genes. Smad is the classical one among the pathways activated by TGF-(31. And Smad signaling pathway is closely related to the fibrosis of cardiac, lung, renal and so on.
Serum pharmacological method is a useful method for the research of traditional Chinese medicine. After fed with traditional Chinese medicine for a long time, the serum was get from the animals and was used in the experiment instead of drugs. This method presents the biotransformation in vivo and also overcomes confusion of other substances.
Qindan capsule (QC), a traditional Chinese medicine presciptions, has been used as an anti-hypertensive drug in clinic by the tutor. It could calm the liver and dominate heat and remove blood stasis. Previous studies have shown that QC could improve the morphological index of the artery, downregulate volume fraction of collagen (VFC) in the media and inhibit the transformation of Smooth muscle cells (VSMCs). But its effect on TGF-β1 signaling pathway in AFs remains unclear. In the present study, we studied the effect of QC on the proliferation, migration, transdifferentiation and collagen synthesis induced by TGF-β1, and investigated the mechanism of QC on TGF-β1/Smad pathway in the process, which might provide more scientific evidence for the treatment of hypertensive vascular remodeling clinically.
Aims
1. To investigate the the effect of QC on the proliferation, migration, and the variation of alpha smooth muscle actin (α-SM actin), procollagenⅠandⅢexpression, and to study the effect of QC on the biological activity of AFs induced by TGF-β1.
2. To investigate the the effect of QC on the variation of Smad2, Smad3, Smad7, phosphorylation-Smad2 (p-Smad2) and phosphorylation-Smad3 (p-Smad3) expression, and to study the possible mechanism of QC on TGF-β1/Smad signaling pathway in AFs.
Methods
1. Preparation of QC and research on QC quality standard
High performance liquid chromatography (HPLC) and Thin-layer chromatography (TLC) methods were used for the research on quality standard, in order to ascertain the active principle in QC.
2. Cell thawing and subculture
Freezing tubes were get out from the nitrogen canister and a thaw was taken as soon as possible. After suspension in culture medium and centrifugation, cells were placed on culture flasks and cultured. When it was 90% in density, cells were subcultured.
3. Preparation of QC-containing serum
Sixty male WKY rats were divided into three groups randomly:the high dosage QC group (QCHD,750 mg/kg), the low dosage QC group (QCLD,150 mg/kg) and Losartan group (30 mg/kg). Rats were starved for 24 h and anaesthetised 1 h (Losartan group) and 2 h (QC groups) after administration of whole-day drugs on the last day. Blood was drawn from the abdominal aorta, separated on a centrifugation (3000 rpm for 15 min). And then mixed as in the same group. After being dried at -57℃for 48 h, the serum was stored at-20℃.
4. MTT colorimetry assay for cell proliferation
AFs were seeded in 96-well plates. AFs in the experiment were treated with QC containing serum of different doses for 48 h prior to stimulation by TGF-β1 for an additional 24 h.20μl MTT was added, followed by incubation for 4 h at 37℃. Finally, cells were lysed with DMSO. Absorbance of the samples was recorded.
5. Transwell assay for migration
After treatment, cells were trypsinized and 600μl cell suspension was added to the upper compartment of the chamber, while 1.5 ml culture medium containing 10% FCS was added to the lower. After 6 h incubation, the chambers were taken out. Cells on the upper face of the membrane were removed by a cotton-tipped applicator carefully. Membranes were then fixed and stained. Cells migrated were counted in each membrane.
6. Real-time RT-PCR for mRNA expression
AFs were grouped and treated as described above. Total RNA was isolated from AFs with use of Trizol Reagent. The expression of Smad2, Smad3, Smad7,α-SMA, procollagenⅠandⅢmRNA was studied after treatment of drug-containing serum and TGF-β1. The relative mRNA expression of the genes was determined by the 2-△△Ct method.
7. Western blot analysis for protein expression
AFs were grouped and treated as described above. Cells were harvested. Protein was extracted and detected. Western-blot was used to detect the protein expression of p-Smad2, p-Smad3, Smad2, Smad3, Smad7,α-SMA, procollagenⅠ.
8. Statistical analysis
The data are presented as mean±SD and analyzed with SPSS 16.0. An unpaired Student's t-test was applied, when only two groups were compared. Comparisons among groups were analyzed by ANOVA. P<0.05 was considered to be statistically significant.
Results
1. Comparison of proliferation of AFs induced by TGF-β1
The proliferation in the TGF-β1 group was higher than that in the contrl group (P<0.01). The proliferation in the treatment groups were decreased compared with that in TGF-β1 group (P<0.05 or P<0.01). And among the treatment groups, the Losartan group had the lowest proliferation (P<0.01). Then it was the QCHD group (P<0.01). The proliferation in the QCLD group was higher than that in the QCHD and Losartan groups (P<0.05).
2. Comparison of migration of AFs induced by TGF-β1
The mumber of cell migration in the TGF-β1 group was higher than that in the contrl group (P<0.01). The migration in the treatment groups were decreased compared with that in TGF-β1 group (P<0.05 or P<0.01).
3. Comparison of Smad2, Smad3, Smad7,α-SMA, procollagenⅠandⅢmRNA expression induced by TGF-β1
(1) Comparison of Smad2 mRNA expression:The expression of Smad2 mRNA was higher in TGF-β1 group than that in the control group (P<0.01). And the mRNA expression in the treatment groups were decreased compared with that in TGF-β1 group (P<0.01). And among the treatment groups, the Losartan group had the lowest expression (P<0.01). Then it was the QCHD group (P<0.01). The Smad2 mRNA expression in the QCLD group was higher than that in the QCHD and Losartan groups (P<0.05).
(2) Comparison of Smad3 mRNA expression:The expression of Smad3 mRNA was higher in TGF-β1 group than that in the control group (P<0.01). The expression of Smad3 mRNA was decreased significantly in the QCHD group and Losartan group compared with that in the TGF-β1 group (all P<0.05). There was no significant difference in comparison between the QCLD group and the TGF-β1 group (P>0.05). There was a significant difference between the QCLD group and the Losartan group (P<0.05).
(3) Comparison of Smad7 mRNA expression:There was no significant difference in comparison between the TGF-β1 group and the control group (P>0.05). And the Smad7 mRNA expression in the treatment groups was increased compared with that in TGF-β1 group (P<0.01). The expression of Smd7 mRNA was inreased significantly in the QCHD group and the Losartan group compared with that in the QCLD group (P<0.05).
(4) Comparison ofα-SMA mRNA expression:The expression ofα-SMA mRNA was higher in TGF-β1 group than that in the control group (P<0.01). And the mRNA expression in the treatment groups was decreased compared with that in TGF-β1 group (P<0.01). Among the treatment groups, the Losartan group had the lowest expression (P<0.01). Then it was the QCHD group (P<0.01). Theα-SMA mRNA expression in the QCLD group was higher than that in the Losartan groups (P<0.05).
(5) Comparison of procollagenⅠmRNA expression:The expression of procollagenⅠmRNA was higher in TGF-β1 group than that in the control group (P<0.01). The expression of procollagenⅠmRNA was decreased significantly in the QCHD group and Losartan group compared with that in the TGF-β1 group (P<0.05). There was no significant difference in comparison between the QCLD group and the TGF-β1 group (P>0.05).
(6) Comparison of procollagenⅢmRNA expression:The expression of procollagenⅢmRNA was higher in TGF-β1 group than that in the control group (P<0.01). And the mRNA expression in the treatment groups was decreased compared with that in TGF-β1 group (P<0.05 or P<0.01). And among the treatment groups, the Losartan group had the lowest proliferation (P<0.01). Then it was the QCHD group (P<0.01). The procollagenⅢmRNA expression in the QCLD group was higher than that in the Losartan group (P<0.05).
3. Comparison of Smad2, Smad3, p-Smad2, p-Smad3,α-SMA, procollagenⅠ, Smad7 protein expression induced by TGF-β1
(1) Comparison of expression of Smad2 protein:The expression of Smad2 protein was higher in TGF-β1 group than that in the control group (P<0.01). And the protein expression in the treatment groups was decreased compared with that in TGF-β1 group (P<0.01). And among the treatment groups, the Losartan group had the lowest expression (P<0.01). And it was lower in the Losartan group than that in the QCHD and QCLD groups (P<0.05 or P<0.01).
(2) Comparison of p-Smad2 protein expression:The expression of p-Smad2 protein was higher in TGF-β1 group than that in the control group (P<0.01). And the protein expression in the treatment groups was decreased compared with that in TGF-β1 group (P<0.01). And it was lower in the Losartan group and QCHD group than that in the QCLD group (P<0.05).
(3) Comparison of Smad3 protein expression:The expression of Smad3 protein was higher in TGF-β1 group than that in the control group (P<0.01). The expression of Smad3 protein was decreased significantly in the QCHD group and Losartan group compared with that in the TGF-β1 group (all P<0.05). There was a significant difference between the QCLD group and the Losartan group (P<0.05).
(4) Comparison of p-Smad3 protein expression:The expression of p-Smad3 protein was higher in TGF-β1 group than that in the control group (P<0.01). And the protein expression in the treatment groups was decreased compared with that in TGF-β1 group (P<0.01). And it was lower in the Losartan group than that in the QCLD group (P<0.05).
(5) Comparison of expression ofα-SMA protein:The expression of a-SMA protein was higher in TGF-β1 group than that in the control group (P<0.01). And the protein expression in the treatment groups was decreased compared with that in TGF-β1 group. Among the treatment groups, the Losartan group had the lowest expression (P<0.01). And then it was QCHD group (P<0.01). It was lower in the Losartan group than that in the QCLD group (P<0.01).
(6) Comparison of expression of procollagenⅠprotein:The expression of procollagenⅠprotein was higher in TGF-β1 group than that in the control group (P<0.01). And the protein expression in the treatment groups was decreased compared with that in TGF-β1 group (P<0.01). It was lower in the Losartan group than that in the QCLD group (P<0.05).
(7) Comparison of Smad7 protein expression:There was no significant difference in comparison between the TGF-β1 group and the control group (P>0.05). And the Smad7 protein expression in the treatment groups was increased compared with that in TGF-β1 group (P<0.01). And among the treatment groups, the Losartan group had the highest expression (P<0.01). And then it was QCHD group(P<0.01).
Conclusion
(1) QC could inhibit the proliferation and migration induced by TGF-β1 in AFs through TGF-β1/Smad signaling pathway.
(2) QC could inhibit the transdifferentiation induced by TGF-β1 in AFs, and the mechanisms might be related to the down-regulation of the mRNA and protein expression of Smad2, Smad3, p-Smad2, p-Smad3 and up-regulation of the mRNA and protein expression of Smad7.
(3) QC could inhibit the extracellular matrix synthesis induced by TGF-β1 in AFs, and the mechanisms might be related to the down-regulation of the mRNA and protein expression of Smad2, Smad3, p-Smad2, p-Smad3 and up-regulation of the mRNA and protein expression of Smad7.
Background
Hypertensive vascular remodeling is the major adverse cardiac event. Transforming growth factorβ1 (TGF-β1) can stimulate the proliferation, migration, extracellular matrix production, and induce the transdifferentiation of AF into myofibroblasts during hypertensive vascular remodeling.
Quercetin (3,3',4',5,7-pentahydroxyflavone, Que), one of the most widely distributed bioflavonoids in the nature, has been reported to possess multiple properties in cardiovascular diseases, such as anti-hypertensive, anti-oxidative, anti-inflammatory, anti-coagulative and attenuating cardiac hypertrophy. Moreover, Que is one of the active principles in QC. In the present study, the effect of Que on the proliferation of AFs stimulated by TGF-β1 was observed. And the variation of Smad2,α-SMA and precollagenⅠandⅢmRNA expression was detected by real-time RT-PCR, while the protein expression of Smad2 and p-Smad2 was detected by western blot. The possible mechanism of Que in reversing biological function of AFs induced by TGF-β1 was investigated, which might provide scientific evidence for the treatment of vascular remodeling in clinic.
Aims
To investigate the effects of Que on the proliferation and the variation of Smad2,α-SMA, precollagenⅠandⅢ, p-Smad2 mRNA and protein expression. And to investigate the possible mechanism of Que in reversing the biological function of AFs induced by TGF-β1.
Methods
1. Cell thawing and subculture
Freezing tubes were taken out from the nitrogen canister. After thaw, suspension and centrifugation, cells were placed on culture flasks. When the density reached 90%, cells were subcultured.
2. MTT assay was used to detect the proliferation
AFs (2×104) were seeded in 96-well plate and incubated overnight. Then cells were serum-starved and synchronized for 4 h. AFs in the experiment were treated with (6.25μmol/L,12.5μmol/L and 25μmol/L) Que for 24 h, then stimulated by TGF-β1 for an additional 24 h.20μl MTT was added and incubated for 4 h prior to harvest. Supernatant was removed and the DMSO was added. Absorbance of the samples was recorded by a microplate reader.
3. Real-time RT-PCR analysis
After starvation and synchronization for 4 h, cells were treated with Que of different doses for 24 h prior to induction by TGF-β1 for another 24 h. Total RNA was isolated using Trizol Reagent and reverse transcribed. The mRNA expression of Smad2,α-SMA, procollagenⅠandⅢwas detected.
4. Western blot analysis
AFs were grouped and treated as described above. Protein was extracted and detected. Western-blot was used to detect the protein expression of p-Smad2 and Smad2.
5. Statistical analysis
Data are expressed as mean±SD. Independent-samples t-test and ANOVA was used with SPSS 6.0. A P-value of less than 0.05 was considered statistical significantly.
Results
1. Comparison of proliferation of AFs induced by TGF-β1
The proliferation in the TGF-β1 group was higher than that in the contrl group (P<0.01). The proliferation in the high and medium dose groups was decreased compared with that in TGF-β1 group (P<0.01). And the high dose group had the lowest proliferation (P<0.01). Then it was the medium dose group (P<0.01). There was no significant difference in comparison between the low dose group and the TGF-β1 group (P>0.05).
2. Comparison of Smad2,α-SMA, procollagenⅠandⅢmRNA expression induced by TGF-β1
(1) Comparison of Smad2 mRNA expression:The expression of Smad2 mRNA was higher in TGF-β1 group than that in the control group (P<0.01). And the mRNA expression in the high dose and medium dose groups was decreased compared with that in TGF-β1 group (P<0.01). The high dose group had the lowest expression (P<0.01). Then it was the medium dose group (P<0.01). There was no significant difference in comparison between the low dose group and the TGF-β1 group (P>0.05).
(2) Comparison of a-SMA mRNA expression:The expression of a-SMA mRNA was higher in TGF-β1 group than that in the control group (P<0.01). And the mRNA expression in the high dose and medium dose groups was decreased compared with that in TGF-β1 group (P<0.05 or P<0.01). No significant difference was shown in comparison between the low dose group and the TGF-β1 group (P>0.05).
(3) Comparison of procollagenⅠmRNA expression:The expression of procollagenⅠmRNA was higher in TGF-β1 group than that in the control group (P<0.01). And the mRNA expression in the high dose and medium dose groups was decreased compared with that in TGF-β1 group (P<0.05). There was no significant difference in comparison between the low dose group and the TGF-β1 group (P>0.05).
(4) Comparison of procollagenⅢmRNA expression:The expression of procollagenⅢmRNA was higher in TGF-β1 group than that in the control group (P<0.01). And the mRNA expression in the high dose and medium dose groups was decreased compared with that in TGF-β1 group (P<0.05). The high dose group had the lowest expression (P<0.05). Then it was the medium dose group (P<0.05). There was no significant difference in comparison between the low dose group and the TGF-β1 group (P>0.05).
3. Comparison of Smad2 and p-Smad2 protein expression induced by TGF-β1
(1) Comparison of expression of Smad2 protein:The expression of Smad2 protein was higher in TGF-β1 group than that in the control group (P<0.01). And the protein expression in high dose and medium dose groups was decreased compared with that in TGF-β1 group (all P<0.01). There was no significant difference in comparison between the low dose group and the TGF-β1 group (P>0.05).
(2) Comparison of p-Smad2 protein expression:The expression of p-Smad2 protein was higher in TGF-β1 group than that in the control group (P<0.01). And the protein expression in the high dose and medium dose groups was decreased compared with that in TGF-β1 group (P<0.01 or P<0.05). No significant difference was shown in comparison between the lowdose group and the TGF-β1 group (P>0.05).
Conclusion
(1) Que could inhibit the proliferation stimulated by TGF-β1 in AFs.
(2) Que could down-regulate the mRNA expression of Smad2,α-SMA and procollagenⅠandⅢ, as well as the protein expression of Smad2 and p-Smad2.
(3) Que could improve the harmful biological activity of AFs induced by TGF-β1 through TGF-β1/Smad2 signaling pathway.
心血管的重构贯穿于血管内膜损伤后再狭窄、动脉粥样硬化(Atherosclerosis, AS)、高血压等多种心血管疾病的发生、发展过程中,是各种病变维持、恶化的病理结构基础。近年来,血管外膜在损伤后血管重构过程中所起的作用得到了空前的关注。研究证明,外膜对血压升高的反应是最敏感的,并且早于血管中膜和内膜,而且外膜的这种变化是高血压血管结构改变的最早征象之一。血管外膜成纤维细胞(Adventitial fibroblasts, AF)在血管损伤后发生增殖、迁移、分泌细胞外基质,是参与血管损伤后新内膜形成和血管重塑的重要内容。
转化生长因子β1 (Transforming growth factorβ1, TGF-β1)是迄今为止所发现的功能最为复杂的生长因子之一,被公认为是影响AF生物功能最直接,也是最重要的细胞因子。Smad信号通路是TGF-β1的经典下游信号通路,与众多组织如心肌、肝脏、肺及肾脏等的纤维化密切相关。根据Smad在信号转导中的作用可将其分为三类：(1)受体调节型Smad(Receptor activated Smad, R-Smad),包括Smad 1、2、3、5、8,为Ⅰ型受体激酶的底物,在特定的TGF-β超家族成员的信号转导通路中发挥作用；(2)共同介质型Smad(Common Smad, Co-Samd),主要是Smad4。Smad4被磷酸化后可以与R-Smad形成杂聚体,然后进入细胞核内对靶基因的转录进行调节；(3)抑制型Smad(Inhibitory Smad, I-Smad),包括Smad6、7,可以阻断R-Smad与受体或与Co-Samd的结合,从而对Smad信号通路发挥负调控作用。
RNA干扰(RNA interference, RNAi)是新兴的生物技术,被Science评为2002年十大科学成就之首。RNA干扰是一种由目的基因所对应的小分子双链RNA (Double-stranded, dsRNA)启动的序列特异的基因沉默过程,从而导致相应的mRNA被降解,进而阻断基因的表达。人工合成的siRNA可特异性地抑制哺乳类细胞中内源性或外源性基因的表达,使相应基因保持沉默或休眠状态。SiRNA所诱发的基因抑制具有高效性和高度的序列特异性。
本实验通过观察TGF-β1诱导AF生物活性的改变,并用RNA干扰的方法,研究在外膜成纤维细胞增殖、迁移、表型转化和细胞外基质合成中TGF-β1/Smad信号通路的作用机制,且进一步探讨Smad2和Smad3在上述过程中的不同作用。
目的
1.观察TGF-β1对外膜成纤维细胞增殖、迁移、表型转化和细胞外基质合成的影响。
2.探讨TGF-β1/Smad信号通路在外膜成纤维细胞增殖、迁移、表型转化和细胞外基质合成中的作用机制,并研究Smad2和Smad3在这些过程中的不同作用。
方法
1.体外原代成纤维细胞的培养与鉴定：采用组织贴块法体外原代培养大鼠胸主动脉成纤维细胞。大概3-7天可见细胞从组织块周围爬出；SABC法行平滑肌肌动蛋白-α(alpha smooth muscle actin,α-SMA)和波形蛋白(Vimentin)免疫细胞化学染色鉴定。取第3-8代细胞用于实验。
2.小干扰RNA链的合成、有效浓度的筛选：设计并分别合成3条Smad2和Smad3特异性siRNA,同时合成荧光标记的阴性对照siRNA(FAM-siRNA)1条和Smad2/3非特异性阴性对照siRNA 1条。细胞密度达50%-70%时转染FAM-siRNA。转染后荧光显微镜观察荧光阳性细胞,计算转染效率,根据转染效率选择siRNA和Lipofectamine 2000最佳转染浓度。
3.细胞转染和小干扰有效链的筛选：根据Lipofectamin 2000的转染步骤,用western-blot检测各组siRNA的干扰效率,进行有效链的筛选。
4.MTT法检测细胞的增殖能力：接种细胞,按照说明进行细胞转染,24h后,加入含20 ng/ml TGF-β1的正常培养液,继续刺激24h。于刺激结束前4h每孔加入MTT,然后吸弃上清,加入DMSO,比色。
5. Transwell检测细胞迁移能力：应用6孔板transwell小室行体外迁移实验。细胞分组和处理同前,处理过的细胞消化后用DMEM制成细胞悬液,取0.6 ml悬液以5×105/ml的密度接种于上室,下室中加入1.5 ml含10%FCS的正常培养基。37℃培养6h,将上室内侧附着的细胞用湿棉签擦去,固定,伊红和苏木素染色,倒置显微镜下随意取5个视野进行细胞计数(×100)。
6.实时定量PCR检测相关因子mRNA的表达：细胞处理同前,采用Trizol法提取总RNA,并在PCR热循环仪上将RNA逆转录为cDNA。观察基因沉默和TGF-β1处理后,Smad2、Smad3、Smad7、α-SMA、Ⅰ型及Ⅲ型前胶原肽mRNA的表达。采用2-ΔΔCt方法对mRNA表达进行相对定量分析。
7. Western-blot检测相关因子蛋白的表达：取对数生长期细胞消化计数,转染24h后,加入TGF-β1孵育24h,提取蛋白并定量,western-blot检测p-Smad2、p-Smad3、Smad2、Smad3、Smad7、α-SMA、Ⅰ型前胶原肽蛋白表达。
8.统计学处理所有数据资料均采用SPSS 16.0进行统计处理。实验数据用x±s表示,组间差异采用one-way ANOVA分析和t检验,P<0.05为差异有统计学意义。
结果
1.原代细胞的生长形态及鉴定
贴壁3-7d后可见细胞从不同的组织块边缘爬出,取第3代细胞,用α-SMA和Vimentin行免疫细胞化学染色,阳性率达100%。
2.针对Smad2或Smad3的siRNA片段均可以有效下调Smad2或Smad3的表达
SiRNA转染并经TGF-β1刺激后,干扰组Smad2或Smad3蛋白的表达较单纯刺激组下降80%以上。且小干扰具有高度特异性和选择性,即siRNA-Smad3只降低Smad3蛋白的表达,而对Smad2蛋白的表达没有影响,同样siRNA-Smad2只作用于Smad2,而对Smad3蛋白表达没有作用。
3.基因沉默Smad2或Smad3对外膜成纤维细胞增殖影响
(1)与正常对照组相比,TGF-β1刺激组增殖显著增强(P<0.01); Smad2基因被干扰之后,细胞增殖较单纯TGF-β1刺激组减弱(P<0.01)。基因沉默Smad2能显著抑制成纤维细胞的增殖能力。
(2) Smad3基因被沉默后,细胞增殖能力较TGF-β1刺激组减弱(P<0.01)。SiRNA介导的Smad3表达下调可以显著抑制成纤维细胞的增殖。
(3) SiRNA-Smad2和siRNA-Smad3均能显著抑制成纤维细胞的增殖,但两者差异没有统计学意义(P>0.05)。
4.基因沉默Smad2或Smad3对外膜成纤维细胞迁移的影响
(1)与正常对照组相比,TGF-β1刺激后细胞迁移能力显著增强(P<0.01)；下调Smad2基因表达之后,细胞迁移较TGF-β1刺激组减弱(P<0.01)。基因沉默Smad2能显著抑制成纤维细胞的迁移能力。
(2) Smad3基因被沉默后,细胞迁移能力较TGF-β1刺激组减弱(P<0.01)。SiRNA介导的Smad3表达下调亦可以显著抑制成纤维细胞的迁移能力。
(3) SiRNA-Smad2和siRNA-Smad3均能显著抑制成纤维细胞迁移能力,然而两者之间差别无统计学意义(P>0.05)。
5.基因沉默Smad2或Smad3对TGF-β1信号转导通路相关因子mRNA表达的影响
(1)经TGF-β1刺激后,Smad2、Smad3、α-SMA、Ⅰ及Ⅲ型前胶原mRNA表达均较正常组显著增强(P<0.05或P<0.01),而Smad7 mRNA表达则无显著差异(P>0.05).SiRNA介导Smad2表达下调后,与TGF-β1刺激组相比,α-SMA、Ⅰ及Ⅲ型前胶原mRNA表达显著降低,差异具有统计学意义(P<0.05或P<0.01), Smad7 mRNA表达则无显著差异(P>0.05)。
(2) Smad3基因被沉默后,α-SMA, I及Ⅲ型前胶原mRNA较TGF-β1刺激组减弱,其差异具有统计学意义(P<0.05或P<0.01)。Smad7 mRNA表达无显著差异(P>0.05)。
(3)SiRNA-Smad2和siRNA-Smad3均能显著α-SMA、Ⅰ及Ⅲ型前胶原mRNA表达,但两者之间差别无统计学意义(P均>0.05)。
6.基因沉默Smad2或Smad3对TGF-β1信号转导通路相关因子蛋白表达的影响
(1)经TGF-β1刺激后,Smad2、Smad3、p-Smad2、p-Smad3、α-SMA、Ⅰ型前胶原蛋白表达均较正常组显著增强(P<0.01),而Smad7蛋白表达则无显著差异(P>0.05)。SiRNA介导Smad2表达下调后,与TGF-β1刺激组相比,p-Smad2、α-SMA、I型前胶原蛋白表达显著降低,差异具有统计学意义(P<0.05或P< 0.01),Smad7蛋白表达则无显著差异(P>0.05)。
(2) Smad3基因被沉默后,p-Smad3、α-SMA、Ⅰ型前胶原蛋白较TGF-β1刺激组减弱,其差异具有统计学意义(P<0.01)。Smad7蛋白表达无显著差异(P>0.05)。
(3)SiRNA-Smad2和siRNA-Smad3均能显著α-SMA,Ⅰ型前胶原蛋白表达,但两者之间差别无统计学意义(P>0.05)。
结论
1.实验中所选用siRNA可以高效并特异地阻断Smad2或Smad3的表达。
2. TGF-β1对外膜成纤维细胞的增殖有促进作用,Smad2和Smad3信号通路共同参与其中。
3. TGF-β1促进外膜成纤维细胞的迁移,这一过程需要Smad2和Smad3的共同参与。
4. TGF-β1诱导成纤维细胞转分化和胶原沉积,是依赖Smad2和Smad3信号转导通路的共同作用。
研究背景
高血压病是严重危害人类健康的常见病和多发病,其发病率逐年升高,目前我国成人发病率高达18.8%。随着对高血压病发病机理的研究和降压药的发展,患者血压水平多能得到有效控制,但高血压所伴发的靶器官损害的发病率、致残率和致死率仍然高居不下。血管重构(Vascular remodeling, VR)既是高血压状态下机体对各种改变的适应性行为,又是高血压病持续发展恶化的重要病理学基础。研究血管重构的发生和逆转机制,对有效控制血压、预防和减轻各种并发症具有非常重要的意义。
血管外膜在高血压血管重构中的作用越来越受到重视,血压升高是促使外膜成纤维细胞(Adventitial fibroblast, AF)变为肌成纤维细胞(Myofibroblast, MF)的重要因素,后者具有增殖及迁移活性,可由外膜向中膜和内膜下间隙迁移,同时能够促进细胞外基质(Extracellular matrix, ECM)合成,导致晚期纤维化,管腔缩窄,参与了血管重构。
转化生长因子β1(Transforming growth factorβ1, TGF-β1)是重要的促纤维化细胞因子之一,可以通过激活一系列的细胞内信号传导通路,引起靶基因的转录。Smad是其主要的细胞内信号转导途径,与众多组织如心肌、肺及肾脏等的纤维化密切相关。
中药血清药理学方法是指给动物灌服中药一定时间后,用其血清进行实验研究的药理学研究方法。这种实验方法不仅体现了药物在生物体内的生物转化,同时克服了中药复方研究中其他因素的干扰。
芩丹胶囊(Qindan capsule, QC)是导师临床治疗高血压病的经验方,有清热平肝、化瘀和络的功效。以前的研究表明该药能够改善SHR大鼠主动脉壁形态学指标,降低主动脉中膜胶原含量,改善SHR大鼠主动脉中膜血管平滑肌细胞(vascular smooth muscle cell, VSMC)表型改变。但是其对血管外膜及TGF-β1/Smad信号系统的影响尚不明了。本研究探讨了芩丹胶囊对TGF-β1诱导的AF增殖、迁移、表型转化以及细胞外基质合成的影响,并探讨在此过程中TGF-β1/Smad信号转导通路的作用机制,以期为临床防治高血压病血管损害提供更多科学依据。
目的
1.观察芩丹胶囊含药血清对TGF-β1诱导的AF增殖、迁移、平滑肌肌动蛋白-α(alpha smooth muscle actin,α-SM actin)、Ⅰ和Ⅲ型前胶原mRNA和蛋白表达的改变,探讨芩丹胶囊对TGF-β1诱导的AF的生物活性的影响。
2.观察芩丹胶囊含药血清对TGF-β1诱导的AF Smad2、Smad3、Smad7、磷酸化Smad2 (phosphorylation-Smad2, p-Smad2)和磷酸化Smad3 (phosphorylation-Smad3, p-Smad3) mRNA和蛋白表达的影响,探讨芩丹胶囊对外膜成纤维细胞TGF-β1/Smad信号转导通路的作用。
方法
1.芩丹胶囊的制备及质量标准的研究：采用高效液相色谱法(High performance liquid chromatography, HPLC)和薄层层析法(Thin-layer chromatography, TLC),明确芩丹胶囊的有效成分。
2.成纤维细胞的复苏与传代：取出冻存管,快速解冻,用正常培养基悬浮细胞,离心后转移至培养瓶。待细胞长至90%融合时进行传代。
3.芩丹胶囊含药血清的制备：60只健康雄性Wistar大鼠随机分为3组：芩丹胶囊大剂量组(QC大剂量组)给予QC 750mg/(kg·d);芩丹胶囊小剂量组(QC小剂量组)给予QC 150mg/(kg·d);氯沙坦组给予氯沙坦30 mg/(kg-d)。各组均大鼠灌胃给药,最后一日禁食24h,末次给药后1h(氯沙坦组)和2h(芩丹胶囊组)后乙醚麻醉,腹主动脉取血,37℃水浴1 h,3000 rpm离心15min,取上清,过滤除菌,然后在-57℃条件下真空干燥,-20℃保存备用。
4.MTT比色法检测细胞增殖能力：制备细胞悬液,接种于96孔板,各组加入相应血清继续孵育48h,换用含20 ng/ml TGF-β1的培养基诱导24h,在刺激结束前4h加入MTT,弃上清,加入DMSO,振荡,比色。
5. Transwell法检测细胞迁移能力：细胞分组和处理同前,经过处理的细胞被消化,然后用DMEM制成细胞悬液,取0.6ml细胞悬液接种于上室,1.5ml含10%FCS的正常培养基加入下室。置入孵箱内培养6h,取出小室用湿棉签擦内侧的细胞,然后固定染色,计数。
6.实时定量PCR技术检测相关因子mRNA的表达：细胞处理同前,提取总RNA,将RNA逆转录为cDNA。观察芩丹胶囊和氯沙坦含药血清对TGF-β1诱导的Smad2、Smad3、Smad7、α-SMA、Ⅰ型及Ⅲ型前胶原肽mRNA的表达。用2-△△Ct的方法对mRNA的表达进行相对定量分析。
7. Western-blot技术检测相关因子蛋白的表达：细胞分组和处理方法同前,提取蛋白并定量,western-blot检测p-Smad2、p-Smad3、Smad2、Smad3、Smad7、α-SMA、Ⅰ型前胶原肽蛋白表达。
8.统计学处理所有实验数据用x±s表示,数据资料均用SPSS 16.0进行统计处理。组间差异采用one-way ANOVA分析和t检验,设P<0.05为有统计学差异。
结果
1.各实验组细胞增殖情况的比较
与正常对照组相比,TGF-β1刺激组的AF增殖活性明显加强(P<0.01)；各药物处理组增殖与TGF-β1刺激组相比,有非常显著性差异(P<0.05或P<0.01),其中以氯沙坦组减弱最为显著(P<0.01),其次为QC大剂量组(P<0.01),QC大剂量组和氯沙坦组较QC小剂量组有显著差异(P<0.05)。
2.各实验组细胞迁移数目的比较
TGF-β1刺激组的AF的迁移数目明显多于正常对照组,且差异具有统计学意义(P<0.01)；经药物处理以后,与TGF-β1刺激组相比,药物组细胞迁移数目均明显下降(P<0.05或P<0.01)。
3.各实验组细胞Smad2、Smad3、Smad7、α-SMA、Ⅰ及Ⅲ型前胶原mRNA表达的比较
(1) Smad2 mRNA表达比较：与正常对照组相比,TGF-β1刺激组细胞的Smad2 mRNA表达显著升高(P<0.01)；各药物处理组Smad2 mRNA表达较TGF-β1刺激组均有不同程度下降,其中氯沙坦组下降最为显著(P<0.01),其次为QC大剂量组(P<0.01)；QC大剂量组、氯沙坦组与QC小剂量组比较有显著差异(P<0.05)。
(2) Smad3 mRNA表达比较：与正常对照组相比,TGF-β1刺激组细胞Smad3 mRNA表达显著升高(P<0.01)；与TGF-β1刺激组相比,QC大剂量组和氯沙坦组Smad3 mRNA表达均显著下降(P均<0.05),QC小剂量组差异没有显著性(P>0.05)；与氯沙坦组相比,QC小剂量组Smad3 mRNA表达差异有显著性(P<0.05)。
(3) Smad7 mRNA表达比较：与正常对照组相比,TGF-β1刺激组细胞Smad7 mRNA表达没有明显变化(P>0.05)；与TGF-β1刺激组相比,各药物处理组Smad7 mRNA表达均显著提高(P<0.01)；QC大剂量组、氯沙坦组与QC小剂量组比较,差异具有统计学意义(P<0.05)。
(4)α-SMA mRNA表达比较：与正常对照组相比,TGF-β1刺激组α-SMA mRNA表达显著升高(P<0.01)；各药物处理组α-SMA mRNA表达较TGF-β1刺激组均有不同程度下降,其中氯沙坦组下降最为显著(P<0.01),其次为QC大剂量组(P<0.01),小剂量组与氯沙坦组比较差异具有统计学意义(P<0.05)。
(5)Ⅰ型前胶原mRNA表达比较：与正常对照组相比,TGF-β1刺激组细胞Ⅰ型前胶原mRNA表达显著升高(P<0.01)；与TGF-β1刺激组相比,QC大剂量组和氯沙坦组Ⅰ型前胶原mRNA表达均显著下降(P<0.05)；QC小剂量组与TGF-β1刺激组相比差异没有统计学意义(P>0.05)。
(6)Ⅲ型前胶原mRNA表达比较：与正常对照组相比,TGF-β1刺激组细胞Ⅲ型前胶原mRNA表达表达显著升高(P<0.01)；与TGF-β1刺激组相比,各药物处理组Ⅲ型前胶原mRNA表达均显著降低(P<0.05或P<0.01)；其中氯沙坦组下降最为显著(P<0.01),其次为QC大剂量组(P<0.01)；氯沙坦组与QC小剂量组比较有显著差异(P<0.05)。
4.各实验组细胞Smad2、Smad3、p-Smad2、p-Smad3、α-SMA、Ⅰ型前胶原、Smad7蛋白表达的比较
(1) Smad2蛋白表达比较：与正常对照组相比,TGF-β1刺激组细胞Smad2蛋白表达明显升高(P<0.01)。与TGF-β1刺激组相比,各药物组Smad2蛋白表达有所下降(P均<0.01)；氯沙坦组显著下降(P<0.01),且下降幅度大于QC大剂量组与QC小剂量组(P<0.05或P<0.01)。
(2) p-Smad2蛋白表达比较：与正常对照组相比,TGF-β1刺激组细胞p-Smad2蛋白表达显著升高(P<0.01)；与TGF-β1刺激组相比,各药物处理组p-Smad2蛋白表达均显著下降(P<0.01)；QC大剂量组和氯沙坦组表达低于QC小剂量组(P<0.05或P<0.01)。
(3) Smad3蛋白表达比较：与正常对照组相比,TGF-β1刺激组细胞Smad3蛋白表达明显升高(P<0.01)。与TGF-β1刺激组相比,氯沙坦组表达水平下降最明显(P<0.05),其次为QC大剂量组(P<0.05)。且氯沙坦组和QC小剂量组相比有显著性差异(P<0.05)。
(4) p-Smad3蛋白表达比较：与正常对照组相比,TGF-β1刺激组细胞p-Smad3蛋白表达表达显著升高(P<0.01)；与TGF-β1刺激组相比,各药物处理组p-Smad3蛋白表达均显著降低(P<0.01)；氯沙坦组与QC小剂量组比较差异具有统计学意义(P<0.05)。
(5)α-SMA蛋白表达比较：与正常对照组相比,TGF-β1刺激组α-SMA蛋白表达显著升高(P<0.01)；各药物处理组α-SMA蛋白表达较TGF-β1刺激组均有不同程度下降,其中氯沙坦组下降最为显著(P<0.01),其次为QC大剂量组(P<0.01)；QC小剂量组和氯沙坦组比较有显著差异(P<0.01)。
(6)Ⅰ型前胶原蛋白表达比较：与正常对照组相比,TGF-β1刘激组细胞Ⅰ型前胶原蛋白表达显著升高(P<0.01)；与TGF-β1刺激组相比,各药物处理组Ⅰ型前胶原蛋白表达均显著下降(P<0.01)；氯沙坦组Ⅰ型前胶原白表达低于QC小剂量组(P<0.05)。
(7) Smad7蛋白表达比较：与正常对照组相比,TGF-β1刺激组细胞Smad7蛋白表达没有明显变化(P>0.05);与TGF-β1刺激组相比,各药物处理组Smad7蛋白表达均显著提高(P<0.01)；其中氯沙坦组升高最为显著(P<0.01),其次为QC大剂量组(P<0.01)。
结论
1.芩丹胶囊能够通过TGF-β1/Smad信号转导通路抑制TGF-β1诱导的AF增殖和迁移。
2.芩丹胶囊能够抑制TGF-β1诱导的AF表型转化,其机制与降低TGF-β1信号因子Smad2、Smad3、p-Smad2、p-Smad3 mRNA和蛋白表达,并升高Smad7 mRNA和蛋白表达上调有关。
3.芩丹胶囊能够抑制TGF-β1诱导的细胞外基质的合成,其机制与降低TGF-β1信号因子Smad2、Smad3、p-Smad2、p-Smad3 mRNA和蛋白表达,并升高Smad7 mRNA和蛋白表达有关。
研究背景
高血压性血管重构是心血管事件的重要危险因素,转化生长因子β1(Transforming growth factorβ1, TGF-β1),能够激活外膜成纤维细胞(Adventitial fibroblasts, AF),使之迁移、增殖、表型转变及合成细胞外基质的能力增强,参与高血压性血管重构。
槲皮素(3,3',4',5,7-pentahydroxyflavone, Que),是自然界中分布最广的生物黄酮类化合物。大量研究已证实槲皮素是一种有效的降压药,同时还具有抗氧化、抗血栓、抗炎、减轻心肌肥厚等多种心血管保护作用。槲皮素是芩丹胶囊中桑寄生的有效成分,我们通过检测槲皮素对TGF-β1诱导的AFs增殖的影响,同时应用荧光实时定量RT-PCR检测Smad2、α-SMA、Ⅰ/Ⅲ型前胶原mRNA表达情况以及western blot检测Smad2和p-Smad2蛋白表达的改变,探讨了槲皮素逆转TGF-β1诱导的AFs生物活性的机制,为其临床防治高血压性血管重构提供科学依据。
目的
观察槲皮素对TGF-β1诱导的AFs增殖的影响,以及对Smad2、α-SMA、Ⅰ/Ⅲ型前胶原、p-Smad2 mRNA和蛋白的影响,探讨槲皮素逆转TGF-β1诱导的AFs生物活性的机制。
方法
1.AFs的复苏与传代：液氮罐中取出冻存管,快速解冻,悬浮细胞,离心,转移至培养瓶。待细胞长至90%融合时进行传代。
2.MTT法检测细胞增殖情况：以细胞密度为2×104接种于96孔板,培养24h,换无血清培养基培养4h,使细胞同步生长,吸弃上清,各组加入(6.25μmol/L、12.5μmol/L和25μmol/L)槲皮素预处理细胞24h,换用含20ng/ml TGF-β1的培养基刺激24h,在刺激结束前4h加入MTT,弃上清,加入DMSO,振荡,比色。
3.实时定量RT-PCR技术检测相关因子mRNA的表达：细胞饥饿4h后换用正常培养基,用各浓度的槲皮素预处理细胞24 h后,再给予TGF-β1刺激24h,提取总RNA,将RNA逆转录为cDNA。实时定量RT-PCR检测Smad2、α-SMA、Ⅰ型及Ⅲ型前胶原肽mRNA的表达。
4. Western-blot技术检测相关因子蛋白的表达：细胞分组和处理方法同前,提取蛋白并定量,western-blot检测p-Smad2和Smad2蛋白表达情况。
5.统计学处理：所有实验数据用x±s表示,数据资料均用SPSS 16.0进行统计处理。组间差异采用one-way ANOVA分析和t检验,设P<0.05为有统计学意义。
结果
1.各实验组细胞增殖情况的比较
与正常对照组相比,TGF-β1刺激组的AF的增殖活性明显加强(P<0.01)；大、中剂量组增殖与TGF-β1刺激组相比,有非常显著性差异(P<0.01),其中以大剂量组减弱最为显著(P<0.01),其次为中剂量组(P<0.01),小剂量组和刺激组相比没有显著差异(P>0.05)。
2.各实验组细胞Smad2、α-SMA、Ⅰ及Ⅲ型前胶原mRNA表达的比较
(1) Smad2 mRNA表达比较：与正常对照组相比,TGF-β1刺激组细胞的Smad2 mRNA表达显著升高(P<0.01)；大、中剂量组Smad2 mRNA表达较TGF-β1刺激组均有不同程度下降,其中大剂量组下降最为显著(P<0.01),其次为中剂量组(P<0.01),小剂量组没有显著差异(P>0.05)。
(2)α-SMA mRNA表达比较：与正常对照组相比,TGF-β1刺激组α-SMA mRNA表达显著升高(P<0.01)；大、中剂量组α-SMA mRNA表达较TGF-β1刺激组均有不同程度下降(P<0.05或P<0.01),小剂量组与刺激组比较差异没有统计学意义(P>0.05)。
(3)Ⅰ型前胶原mRNA表达比较：与正常对照组相比,TGF-β1刺激组细胞Ⅰ型前胶原mRNA表达显著升高(P<0.01)；与TGF-β1刺激组相比,大剂量组和中剂量组Ⅰ型前胶原mRNA表达均显著下降(P<0.05)；小剂量组与TGF-β1刺激组相比差异没有统计学意义(P>0.05)。
(4)Ⅲ型前胶原mRNA表达比较：与正常对照组相比,TGF-β1刺激组细胞Ⅲ型前胶原mRNA表达表达显著升高(P<0.01)；与TGF-β1刺激组相比,大、中剂量组Ⅲ型前胶原mRNA表达均显著降低(P<0.05)；其中大剂量组下降最为显著(P<0.05),其次为中剂量组(P<0.05),小剂量组没有显著差异(P>0.05)。
3.各实验组细胞Smad2和(?)-Smad2蛋白表达的比较
(1) Smad2蛋白表达比较：与正常对照组相比,TGF-β1刺激组细胞Smad2蛋白表达明显升高(P<0.01)。与TGF-β1刺激组相比,大、中剂量组显著下降(P均<0.01),小剂量组差异没有显著性(P>0.05)。
(2) p-Smad2蛋白表达比较：与正常对照组相比,TGF-β1刺激组细胞p-Smad2蛋白表达显著升高(P<0.01)；与TGF-β1刺激组相比,大、中剂量组p-Smad2蛋白表达均显著下降(P<0.05或P<0.01)；小剂量组蛋白表达下降没有统计学意义(P>0.05)。
结论
1.槲皮素能够显著抑制TGF-β1诱导的成纤维细胞的增殖。
2.槲皮素抑制了TGF-β1诱导的成纤维细胞Smad2、p-Smad、α-SMA、Ⅰ/Ⅲ型前胶原mRNA和蛋白的高表达。
3.槲皮素能够通过TGF-β1/Smad2信号通路改善TGF-β1诱导的成纤维细胞的生物活性的改变。
Background
Vascular remodeling runs through the process of various cardiovascular diseases, such as the restenosis after percutaneous transluminal coronary angioplasty (PTCA), atherosclerosis(AS) and hypertension. And vascular remodeling is considered the major pathological base for maintaining and worsening of hypertension. Recently the adventitia has attracted more and more attention for the importance during vascular remodeling after vascular injury. The adventitia is the most sensitive layer responding to elevated blood pressure compared with intimal and medial. Adventitial fibroblasts (AFs), the main adventitial cell type, drive remodeling and may initiate other changes subsequently, such as alterations in arrangements of neointimal proliferation, smooth muscle cells and extracellular matrix (ECM), which is expected to lead to vascular remodeling.
Transforming growth factorβ1 (TGF-β1), a pleiotropic and multifunctional cytokine, is known as the most important and direct one to regulate AFs bioactivity. The classical signal transduction pathway of the TGF-βfamily is through Smad, which is related to the fibrosis of many parts, such as heart, liver, lung and kidney. Smads are divided into three subfamilies:(1) receptor-activated Smads (R-Smads: Smad1, Smad2, Smad3, Smad5, Smad8), which become phosphorylated receptor I and play an important role in transforming growth factor-β(TGF-β) family; (2)common mediator Smads (Co-Smads:Smad4), which become phosphorylated and oligomerise with activated R-Smads to practice their intracellular effects; (3) inhibitory Smads (I-Smads:Smad6 and Smad7), which exert a negative feedback effect by competing with R-Smads.
RNA interference (RNAi) is a new biotechnology, which was considered the top of the ten scientific achievements in 2002 by Science. RNAi is a sequence-specific gene silencing procedure started by double-stranded (dsRNA) RNA corresponding to target gene. Synthetical siRNA could inhibit the expression of gene, endogenic and ectogenic specifically in mammalian cells. And it could keep the target genes in scilence or resting state. The inhibition of genes started by siRNA is of high efficacy and specificity.
In the present study, we observed the biological activity of AFs induced by TGF-β1 and tried to investigate the mechanism of TGF-β1/Smad signaling pathway on the migration, proliferation, transdifferentiation and ECM deposition of AFs. And moreover, the different roles of Smad2 and Smad3 were studied during the process.
Aims
1. To investigate the effect of TGF-β1 on the migration, proliferation, transdifferentiation and ECM deposition of AFs.
2. To investigate the possible mechanism of TGF-β1/Smad signaling pathway during the activity of AFs induced by TGF-β1. And to study the different roles of Smad2 and Smad3 during the procedure.
Method
1. Primary cell culture and identification in vitro
AFs were cultured by tissue explant in vitro. AFs were observed to grow out from the tissues 3-7 days later. The AF and its purity were identified by immunocytochemistry of Vimentin and a-smooth muscle actin (SMA). Cells of the 3-8 passages were used in assays.
2. Synthesis of siRNA and selection of the effective concentration
Three specific Smad2-siRNAs and Smad3-siRNAs, one FAM-siRNA and one Smad2/3-negative siRNA were designed and chemically synthesized, so were Smad3 siRNAs. Transfection of FAM-siRNA was performed when the AFs were at 50%-70% density. Under light microscopy after transfection, the positive cells were observed. The best concentration of siRNA and Lipofectamine 2000 was chosen according to the transfection efficiency.
3. Transfection and selection of the effective siRNAs
The transfection was performed according to the manufacturer's instructions. Western-blot was used to detect the efficiency of gene silence and the effective siRNAs
4. MTT assay for proliferation
Twenty-four h after transfection, AFs in the experiment of TGF-β1 were treated with TGF-β1 for 24 h.20μl MTT (5 mg/ml) was added, followed by incubation for 4 h at 37℃. Finally, medium was removed and cells were lysed with DMSO. Absorbance of the samples was recorded.
5. Transwell assay for migration
Migration of AFs was measured using transwell chamber apparatus in 6-well plates. AFs were grouped and treated as described above. Briefly, cells were trypsinized and counted,600μl cell suspension at 5×105/ml in DMEM was added to the upper compartment of the chamber, and 1.5 ml DMEM containing 10% FCS was added to the lower compartment. After incubation for 6 h, cells on the upper face of the membrane were removed with a cotton-tipped applicator carefully. Then the membranes were fixed in methanol and stained with hematoxylin and eosin (HE). The number of migrated cells was counted in 5 random fields in each membrane.
6. Real-time RT-PCR
After treatment, total RNA was isolated from AFs with use of Trizol Reagent according to the manufacturer's instructions. RNA was reversely transcribed by a standard protocol. The expression of Smad2, Smad3, Smad7,α-SMA, procollagenⅠandⅢmRNA was studied after treatment of siRNA and TGF-β1. The relative mRNA expression of the genes was determined by the 2-△△Ct method.
7. Western blot analysis
Cells were harvested after transfection for 24 h and stimulation by TGF-β1 for another 24 h. Protein was extracted and detected. Western-blot was used to detect the protein expression of p-Smad2, p-Smad3, Smad2, Smad3, Smad7,α-SMA, procollagenⅠ.
8. Statistical analysis
The data are presented as mean±SD and analyzed with SPSS 16.0. Comparisons among groups were analyzed by ANOVA. An unpaired Student's t-test was applied, when only two groups were compared. P< 0.05 was considered to be statistically significant.
Results
1. Appearance and identification of AFs
AFs were observed to grow out from different tissues 3-7 days later. Immunocytochemistry analysis demonstrated that the multiclonal antibody staining for SMA was negative and the monoclonal antibody staining for vimentin was positive, suggesting 100% purity of AFs.
2. siRNAs targeting Smad2 and Smad3 downregulation the expression of Smad2 and Smad3 efficiently
After transfection and stimulation by TGF-β1, the protein expression of Smad2 or Smad3 in siRNA-Smad2 or siRNA-Smad3 group was decreased more than 80% compared with TGF-β1 group. Moreover, the knockdown was specific and selective, as Smad3 protein levels were decreased by Smad3 siRNA only, so were Smad2 protein levels.
3. Proliferation of AFs induced by TGF-β1 was inhibited by siRNA-Smad2 and-Smad3
(1) Cell proliferation in TGF-β1 group was significantly increased compared with the control group (P<0.01). However, the proliferation decreased when Smad2 expression was knocked down by siRNA (P<0.01). Knockdown of Smad2 could inhibit the proliferation of AFs significantly.
(2) The proliferation decreased when Smad3 expression was knocked down by siRNA (P<0.01). Knockdown of Smad3 by siRNA could inhibit the proliferation of AFs significantly.
(3) SiRNA-Smad2 and siRNA-Smad3 could both inhibit the proliferation of AFs significantly, with no significant difference between Smad2 and Smad3 knockdown (P>0.05).
4. Migration of AFs induced by TGF-β1 was inhibited by siRNA-Smad2 and-Smad3
(1) After stimulation by TGF-β1 for 24 h, the migration of AFs was significantly increased compared with the control group(P<0.01). However, the blockade of Smad2 expression by siRNA could reduce the migration of AFs induced by TGF-β1 (P<0.01). Knockdown of Smad2 by siRNA could inhibit the migration of AFs significantly.
(2) The migration decreased when Smad3 expression was knocked down by siRNA (P<0.01). Knockdown of Smad3 by siRNA could inhibit the migration of AFs significantly.
(3) SiRNA-Smad2 and siRNA-Smad3 could both inhibit the migration of AFs significantly, with no significant difference between Smad2 and Smad3 knockdown (P>0.05).
5. mRNA expression of the related gene induced by TGF-β1 was inhibited by siRNA-Smad2 and -Smad3
(1) After stimulation by TGF-β1, the mRNA expression of Smad2, Smad3,α-SMA, procollagenⅠandⅢall increased compared with the control group (P<0.05 or P<0.01), but not Smad7 (P>0.05). Knockdown of Smad2 by siRNA, the mRNA expression ofα-SMA, procollagenⅠandⅢwas decreased significantly compared with TGF-β1 group (P< 0.05 or P<0.01). However, Smad7 mRNA expression did not differ from that of the control (P> 0.05).
(2) The mRNA expression ofα-SMA, procollagenⅠandⅢdecreased when Smad3 expression was knocked down by siRNA compared with TGF-β1 group (P< 0.05 or P< 0.01), but not Smad7 (P> 0.05).
(3) SiRNA-Smad2 and siRNA-Smad3 could both reduce the mRNA expression ofα-SMA, procollagenⅠandⅢof AFs significantly, with no significant difference between Smad2 and Smad3 knockdown(P>0.05).
6. Protein expression of the related gene induced by TGF-β1 was inhibited by siRNA-Smad2 and -Smad3
(1) After stimulation by TGF-β1, the protein expression of Smad2, Smad3,α-SMA, procollagenⅠwas all increased compared with the control group (P<0.01), but not Smad7 (P>0.05). Knockdown of Smad2 by siRNA, the protein expression ofα-SMA, procollagenⅠwas decreased significantly compared with TGF-β1 group (P< 0.05 or P<0.01). However, Smad7 protein expression did not differ from that of the control (P>0.05).
(2) The protein expression ofα-SMA, procollagenⅠdecreased when Smad3 expression was knocked down by siRNA compared with TGF-β1 group (P<0.01), but not Smad7 (P>0.05).
(3) SiRNA-Smad2 and siRNA-Smad3 could both reduce the protein expression ofα-SMA, procollagenⅠof AFs significantly, with no significant difference between Smad2 and Smad3 knockdown (P>0.05).
Conclusion
(1) SiRNA in the experiment could knockdown the expression of Smad2 and Smad3 effectively and specifically.
(2) TGF-β1 could promote the proliferation of AFs both Smad2 and Smad3-dependently.
(3) TGF-β1 could promote the migration of AFs both Smad2 and Smad3-dependently.
(4) Both Smad2 and Smad3 work as mediators of phenotypic transition and collagen synthesis of AFs induced by TGF-β1
Background
Hypertension is the familiar and risk factor for human health. The attack rate is growing year by year and now the rate is up to 18.8%. Blood pressure could be controlled effectively thanks to the intensive study of mechanism of hypertension and development of hypotensor. But the rate of morbidity, fatality and deformity is still very high. Vascular remodeling is the the adaptive reaction to the disorders in hypertension. Moreover, it is the major pathological base of deterioration in the development of hypertension. Therefore, it is very important to investigate the mechanism of vascular remodeling for blood pressure control, prevention and treatment of hypertension and the complications.
Growing experimental evidence shows that the adventitia plays an critical role in vascular remodeling. High blood pressure is an important factor for adventitial fibroblasts (AFs) transdifferentiation to Myofibroblasts (MF). The migratory and proliferative responses of myofibroblasts, in addition to synthesis of ECM, lead to fibrosis and lumen stenosis, which play important roles during vascular remodeling.
Transforming growth factorβ1 (TGF-β1) is an important multifunctional cytokine that regulates fibrosis by activating a series intracellular signaling pathways, which might lead to the transcription of target genes. Smad is the classical one among the pathways activated by TGF-(31. And Smad signaling pathway is closely related to the fibrosis of cardiac, lung, renal and so on.
Serum pharmacological method is a useful method for the research of traditional Chinese medicine. After fed with traditional Chinese medicine for a long time, the serum was get from the animals and was used in the experiment instead of drugs. This method presents the biotransformation in vivo and also overcomes confusion of other substances.
Qindan capsule (QC), a traditional Chinese medicine presciptions, has been used as an anti-hypertensive drug in clinic by the tutor. It could calm the liver and dominate heat and remove blood stasis. Previous studies have shown that QC could improve the morphological index of the artery, downregulate volume fraction of collagen (VFC) in the media and inhibit the transformation of Smooth muscle cells (VSMCs). But its effect on TGF-β1 signaling pathway in AFs remains unclear. In the present study, we studied the effect of QC on the proliferation, migration, transdifferentiation and collagen synthesis induced by TGF-β1, and investigated the mechanism of QC on TGF-β1/Smad pathway in the process, which might provide more scientific evidence for the treatment of hypertensive vascular remodeling clinically.
Aims
1. To investigate the the effect of QC on the proliferation, migration, and the variation of alpha smooth muscle actin (α-SM actin), procollagenⅠandⅢexpression, and to study the effect of QC on the biological activity of AFs induced by TGF-β1.
2. To investigate the the effect of QC on the variation of Smad2, Smad3, Smad7, phosphorylation-Smad2 (p-Smad2) and phosphorylation-Smad3 (p-Smad3) expression, and to study the possible mechanism of QC on TGF-β1/Smad signaling pathway in AFs.
Methods
1. Preparation of QC and research on QC quality standard
High performance liquid chromatography (HPLC) and Thin-layer chromatography (TLC) methods were used for the research on quality standard, in order to ascertain the active principle in QC.
2. Cell thawing and subculture
Freezing tubes were get out from the nitrogen canister and a thaw was taken as soon as possible. After suspension in culture medium and centrifugation, cells were placed on culture flasks and cultured. When it was 90% in density, cells were subcultured.
3. Preparation of QC-containing serum
Sixty male WKY rats were divided into three groups randomly:the high dosage QC group (QCHD,750 mg/kg), the low dosage QC group (QCLD,150 mg/kg) and Losartan group (30 mg/kg). Rats were starved for 24 h and anaesthetised 1 h (Losartan group) and 2 h (QC groups) after administration of whole-day drugs on the last day. Blood was drawn from the abdominal aorta, separated on a centrifugation (3000 rpm for 15 min). And then mixed as in the same group. After being dried at -57℃for 48 h, the serum was stored at-20℃.
4. MTT colorimetry assay for cell proliferation
AFs were seeded in 96-well plates. AFs in the experiment were treated with QC containing serum of different doses for 48 h prior to stimulation by TGF-β1 for an additional 24 h.20μl MTT was added, followed by incubation for 4 h at 37℃. Finally, cells were lysed with DMSO. Absorbance of the samples was recorded.
5. Transwell assay for migration
After treatment, cells were trypsinized and 600μl cell suspension was added to the upper compartment of the chamber, while 1.5 ml culture medium containing 10% FCS was added to the lower. After 6 h incubation, the chambers were taken out. Cells on the upper face of the membrane were removed by a cotton-tipped applicator carefully. Membranes were then fixed and stained. Cells migrated were counted in each membrane.
6. Real-time RT-PCR for mRNA expression
AFs were grouped and treated as described above. Total RNA was isolated from AFs with use of Trizol Reagent. The expression of Smad2, Smad3, Smad7,α-SMA, procollagenⅠandⅢmRNA was studied after treatment of drug-containing serum and TGF-β1. The relative mRNA expression of the genes was determined by the 2-△△Ct method.
7. Western blot analysis for protein expression
AFs were grouped and treated as described above. Cells were harvested. Protein was extracted and detected. Western-blot was used to detect the protein expression of p-Smad2, p-Smad3, Smad2, Smad3, Smad7,α-SMA, procollagenⅠ.
8. Statistical analysis
The data are presented as mean±SD and analyzed with SPSS 16.0. An unpaired Student's t-test was applied, when only two groups were compared. Comparisons among groups were analyzed by ANOVA. P<0.05 was considered to be statistically significant.
Results
1. Comparison of proliferation of AFs induced by TGF-β1
The proliferation in the TGF-β1 group was higher than that in the contrl group (P<0.01). The proliferation in the treatment groups were decreased compared with that in TGF-β1 group (P<0.05 or P<0.01). And among the treatment groups, the Losartan group had the lowest proliferation (P<0.01). Then it was the QCHD group (P<0.01). The proliferation in the QCLD group was higher than that in the QCHD and Losartan groups (P<0.05).
2. Comparison of migration of AFs induced by TGF-β1
The mumber of cell migration in the TGF-β1 group was higher than that in the contrl group (P<0.01). The migration in the treatment groups were decreased compared with that in TGF-β1 group (P<0.05 or P<0.01).
3. Comparison of Smad2, Smad3, Smad7,α-SMA, procollagenⅠandⅢmRNA expression induced by TGF-β1
(1) Comparison of Smad2 mRNA expression:The expression of Smad2 mRNA was higher in TGF-β1 group than that in the control group (P<0.01). And the mRNA expression in the treatment groups were decreased compared with that in TGF-β1 group (P<0.01). And among the treatment groups, the Losartan group had the lowest expression (P<0.01). Then it was the QCHD group (P<0.01). The Smad2 mRNA expression in the QCLD group was higher than that in the QCHD and Losartan groups (P<0.05).
(2) Comparison of Smad3 mRNA expression:The expression of Smad3 mRNA was higher in TGF-β1 group than that in the control group (P<0.01). The expression of Smad3 mRNA was decreased significantly in the QCHD group and Losartan group compared with that in the TGF-β1 group (all P<0.05). There was no significant difference in comparison between the QCLD group and the TGF-β1 group (P>0.05). There was a significant difference between the QCLD group and the Losartan group (P<0.05).
(3) Comparison of Smad7 mRNA expression:There was no significant difference in comparison between the TGF-β1 group and the control group (P>0.05). And the Smad7 mRNA expression in the treatment groups was increased compared with that in TGF-β1 group (P<0.01). The expression of Smd7 mRNA was inreased significantly in the QCHD group and the Losartan group compared with that in the QCLD group (P<0.05).
(4) Comparison ofα-SMA mRNA expression:The expression ofα-SMA mRNA was higher in TGF-β1 group than that in the control group (P<0.01). And the mRNA expression in the treatment groups was decreased compared with that in TGF-β1 group (P<0.01). Among the treatment groups, the Losartan group had the lowest expression (P<0.01). Then it was the QCHD group (P<0.01). Theα-SMA mRNA expression in the QCLD group was higher than that in the Losartan groups (P<0.05).
(5) Comparison of procollagenⅠmRNA expression:The expression of procollagenⅠmRNA was higher in TGF-β1 group than that in the control group (P<0.01). The expression of procollagenⅠmRNA was decreased significantly in the QCHD group and Losartan group compared with that in the TGF-β1 group (P<0.05). There was no significant difference in comparison between the QCLD group and the TGF-β1 group (P>0.05).
(6) Comparison of procollagenⅢmRNA expression:The expression of procollagenⅢmRNA was higher in TGF-β1 group than that in the control group (P<0.01). And the mRNA expression in the treatment groups was decreased compared with that in TGF-β1 group (P<0.05 or P<0.01). And among the treatment groups, the Losartan group had the lowest proliferation (P<0.01). Then it was the QCHD group (P<0.01). The procollagenⅢmRNA expression in the QCLD group was higher than that in the Losartan group (P<0.05).
3. Comparison of Smad2, Smad3, p-Smad2, p-Smad3,α-SMA, procollagenⅠ, Smad7 protein expression induced by TGF-β1
(1) Comparison of expression of Smad2 protein:The expression of Smad2 protein was higher in TGF-β1 group than that in the control group (P<0.01). And the protein expression in the treatment groups was decreased compared with that in TGF-β1 group (P<0.01). And among the treatment groups, the Losartan group had the lowest expression (P<0.01). And it was lower in the Losartan group than that in the QCHD and QCLD groups (P<0.05 or P<0.01).
(2) Comparison of p-Smad2 protein expression:The expression of p-Smad2 protein was higher in TGF-β1 group than that in the control group (P<0.01). And the protein expression in the treatment groups was decreased compared with that in TGF-β1 group (P<0.01). And it was lower in the Losartan group and QCHD group than that in the QCLD group (P<0.05).
(3) Comparison of Smad3 protein expression:The expression of Smad3 protein was higher in TGF-β1 group than that in the control group (P<0.01). The expression of Smad3 protein was decreased significantly in the QCHD group and Losartan group compared with that in the TGF-β1 group (all P<0.05). There was a significant difference between the QCLD group and the Losartan group (P<0.05).
(4) Comparison of p-Smad3 protein expression:The expression of p-Smad3 protein was higher in TGF-β1 group than that in the control group (P<0.01). And the protein expression in the treatment groups was decreased compared with that in TGF-β1 group (P<0.01). And it was lower in the Losartan group than that in the QCLD group (P<0.05).
(5) Comparison of expression ofα-SMA protein:The expression of a-SMA protein was higher in TGF-β1 group than that in the control group (P<0.01). And the protein expression in the treatment groups was decreased compared with that in TGF-β1 group. Among the treatment groups, the Losartan group had the lowest expression (P<0.01). And then it was QCHD group (P<0.01). It was lower in the Losartan group than that in the QCLD group (P<0.01).
(6) Comparison of expression of procollagenⅠprotein:The expression of procollagenⅠprotein was higher in TGF-β1 group than that in the control group (P<0.01). And the protein expression in the treatment groups was decreased compared with that in TGF-β1 group (P<0.01). It was lower in the Losartan group than that in the QCLD group (P<0.05).
(7) Comparison of Smad7 protein expression:There was no significant difference in comparison between the TGF-β1 group and the control group (P>0.05). And the Smad7 protein expression in the treatment groups was increased compared with that in TGF-β1 group (P<0.01). And among the treatment groups, the Losartan group had the highest expression (P<0.01). And then it was QCHD group(P<0.01).
Conclusion
(1) QC could inhibit the proliferation and migration induced by TGF-β1 in AFs through TGF-β1/Smad signaling pathway.
(2) QC could inhibit the transdifferentiation induced by TGF-β1 in AFs, and the mechanisms might be related to the down-regulation of the mRNA and protein expression of Smad2, Smad3, p-Smad2, p-Smad3 and up-regulation of the mRNA and protein expression of Smad7.
(3) QC could inhibit the extracellular matrix synthesis induced by TGF-β1 in AFs, and the mechanisms might be related to the down-regulation of the mRNA and protein expression of Smad2, Smad3, p-Smad2, p-Smad3 and up-regulation of the mRNA and protein expression of Smad7.
Background
Hypertensive vascular remodeling is the major adverse cardiac event. Transforming growth factorβ1 (TGF-β1) can stimulate the proliferation, migration, extracellular matrix production, and induce the transdifferentiation of AF into myofibroblasts during hypertensive vascular remodeling.
Quercetin (3,3',4',5,7-pentahydroxyflavone, Que), one of the most widely distributed bioflavonoids in the nature, has been reported to possess multiple properties in cardiovascular diseases, such as anti-hypertensive, anti-oxidative, anti-inflammatory, anti-coagulative and attenuating cardiac hypertrophy. Moreover, Que is one of the active principles in QC. In the present study, the effect of Que on the proliferation of AFs stimulated by TGF-β1 was observed. And the variation of Smad2,α-SMA and precollagenⅠandⅢmRNA expression was detected by real-time RT-PCR, while the protein expression of Smad2 and p-Smad2 was detected by western blot. The possible mechanism of Que in reversing biological function of AFs induced by TGF-β1 was investigated, which might provide scientific evidence for the treatment of vascular remodeling in clinic.
Aims
To investigate the effects of Que on the proliferation and the variation of Smad2,α-SMA, precollagenⅠandⅢ, p-Smad2 mRNA and protein expression. And to investigate the possible mechanism of Que in reversing the biological function of AFs induced by TGF-β1.
Methods
1. Cell thawing and subculture
Freezing tubes were taken out from the nitrogen canister. After thaw, suspension and centrifugation, cells were placed on culture flasks. When the density reached 90%, cells were subcultured.
2. MTT assay was used to detect the proliferation
AFs (2×104) were seeded in 96-well plate and incubated overnight. Then cells were serum-starved and synchronized for 4 h. AFs in the experiment were treated with (6.25μmol/L,12.5μmol/L and 25μmol/L) Que for 24 h, then stimulated by TGF-β1 for an additional 24 h.20μl MTT was added and incubated for 4 h prior to harvest. Supernatant was removed and the DMSO was added. Absorbance of the samples was recorded by a microplate reader.
3. Real-time RT-PCR analysis
After starvation and synchronization for 4 h, cells were treated with Que of different doses for 24 h prior to induction by TGF-β1 for another 24 h. Total RNA was isolated using Trizol Reagent and reverse transcribed. The mRNA expression of Smad2,α-SMA, procollagenⅠandⅢwas detected.
4. Western blot analysis
AFs were grouped and treated as described above. Protein was extracted and detected. Western-blot was used to detect the protein expression of p-Smad2 and Smad2.
5. Statistical analysis
Data are expressed as mean±SD. Independent-samples t-test and ANOVA was used with SPSS 6.0. A P-value of less than 0.05 was considered statistical significantly.
Results
1. Comparison of proliferation of AFs induced by TGF-β1
The proliferation in the TGF-β1 group was higher than that in the contrl group (P<0.01). The proliferation in the high and medium dose groups was decreased compared with that in TGF-β1 group (P<0.01). And the high dose group had the lowest proliferation (P<0.01). Then it was the medium dose group (P<0.01). There was no significant difference in comparison between the low dose group and the TGF-β1 group (P>0.05).
2. Comparison of Smad2,α-SMA, procollagenⅠandⅢmRNA expression induced by TGF-β1
(1) Comparison of Smad2 mRNA expression:The expression of Smad2 mRNA was higher in TGF-β1 group than that in the control group (P<0.01). And the mRNA expression in the high dose and medium dose groups was decreased compared with that in TGF-β1 group (P<0.01). The high dose group had the lowest expression (P<0.01). Then it was the medium dose group (P<0.01). There was no significant difference in comparison between the low dose group and the TGF-β1 group (P>0.05).
(2) Comparison of a-SMA mRNA expression:The expression of a-SMA mRNA was higher in TGF-β1 group than that in the control group (P<0.01). And the mRNA expression in the high dose and medium dose groups was decreased compared with that in TGF-β1 group (P<0.05 or P<0.01). No significant difference was shown in comparison between the low dose group and the TGF-β1 group (P>0.05).
(3) Comparison of procollagenⅠmRNA expression:The expression of procollagenⅠmRNA was higher in TGF-β1 group than that in the control group (P<0.01). And the mRNA expression in the high dose and medium dose groups was decreased compared with that in TGF-β1 group (P<0.05). There was no significant difference in comparison between the low dose group and the TGF-β1 group (P>0.05).
(4) Comparison of procollagenⅢmRNA expression:The expression of procollagenⅢmRNA was higher in TGF-β1 group than that in the control group (P<0.01). And the mRNA expression in the high dose and medium dose groups was decreased compared with that in TGF-β1 group (P<0.05). The high dose group had the lowest expression (P<0.05). Then it was the medium dose group (P<0.05). There was no significant difference in comparison between the low dose group and the TGF-β1 group (P>0.05).
3. Comparison of Smad2 and p-Smad2 protein expression induced by TGF-β1
(1) Comparison of expression of Smad2 protein:The expression of Smad2 protein was higher in TGF-β1 group than that in the control group (P<0.01). And the protein expression in high dose and medium dose groups was decreased compared with that in TGF-β1 group (all P<0.01). There was no significant difference in comparison between the low dose group and the TGF-β1 group (P>0.05).
(2) Comparison of p-Smad2 protein expression:The expression of p-Smad2 protein was higher in TGF-β1 group than that in the control group (P<0.01). And the protein expression in the high dose and medium dose groups was decreased compared with that in TGF-β1 group (P<0.01 or P<0.05). No significant difference was shown in comparison between the lowdose group and the TGF-β1 group (P>0.05).
Conclusion
(1) Que could inhibit the proliferation stimulated by TGF-β1 in AFs.
(2) Que could down-regulate the mRNA expression of Smad2,α-SMA and procollagenⅠandⅢ, as well as the protein expression of Smad2 and p-Smad2.
(3) Que could improve the harmful biological activity of AFs induced by TGF-β1 through TGF-β1/Smad2 signaling pathway.
引文
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