【作者】 张冀东；

【导师】 崔炜；

【作者基本信息】 河北医科大学 ， 内科学， 2008， 博士

【摘要】 在临床上心肌重构是指心脏大小、形态、功能的异常改变。心肌重构的组织病理学特点是正常心腔室壁组织的结构重排,包括心肌肥厚、心肌成纤维细胞增殖、细胞外基质沉积和细胞调亡。心肌成纤维细胞过度增殖,胶原合成增加,排列紊乱是心肌纤维化的病理学基础。心肌纤维化参与多种急、慢性心血管疾病的病理过程,可引起心功能不全、心律失常甚至心脏性猝死,因此对心肌纤维化的研究有着重要的理论与现实意义。心脏成纤维细胞占心脏容积的25%和心脏细胞总数的70%,是心肌间质细胞的主要成分。心肌纤维化的主要影响因素有肾素-血管紧张素-醛固酮(aldosterone,Ald)系统、成纤维细胞生长因子(basic fibroblast growth factor,bFGF)、转化生长因子、内皮素、缓激肽、一氧化氮、去甲肾上腺素等。其中醛固酮是目前公认的促心肌纤维化因素,可促进心肌成纤维细胞增殖和胶原合成增加,从而导致心肌纤维化。心脏有独立的醛固酮系统,自身就能合成醛固酮,而且存在大量的、特异性的和高亲和力的盐皮质激素受体(mineralocorticoid receptor,MR)。资料显示慢性心力衰竭病人醛固酮水平升高,并且高水平的醛固酮是其死亡率增加的独立预测因子;MR拮抗剂可减轻高血压大鼠模型和血管紧张素Ⅱ诱导的心脏重塑的进展,表明醛固酮在心力衰竭的损伤中起重要作用。他汀类药物即3-羟基-3-甲基-戊二酸单酰辅酶A还原酶抑制剂(HMG-CoA reductase inhibitors)作为调脂药物的一种,广泛应用于心血管疾病的预防和治疗中。近来关于他汀类药物的非降脂依赖性抗心肌纤维化的研究报道日益增多,其作用涉及心肌纤维化的多个信号转导环节,通过调控细胞外的刺激信号、细胞内的信号转导和细胞核内基因的转录活化等,阻止心肌纤维化的发生、发展,降低心脏疾病的死亡率。他汀类药物抗心肌纤维化的主要机制是抑制胆固醇合成中的甲羟戊酸通路。甲羟戊酸是几种类异戊二烯衍生物的前体,包括焦磷酸法尼酯(farnesyl pyropho- sphate,FPP)和焦磷酸牛儿基牛儿酯(geranylgeranyl pyrophosphate,GGPP)。FPP和GGPP是小GTP蛋白(包括Ras和Rho家族)翻译后异戊二烯化(包括法尼基化作用和牛基化作用)所必需。小GTP蛋白调控许多胞内信号通路,如细胞构架的改建、基因表达、细胞的增殖、渗入、分化和凋亡和细胞周期等重要功能。新合成的小GTP蛋白是与GDP结合的灭活状态,游离于细胞浆中,必须和GTP结合后才有生物活性,而异戊二烯化是它们膜定位和发挥生物学作用所必需。其中FPP是Ras家族蛋白异戊二烯化所必需的,而GGPP是Rho家族蛋白异戊二烯化所必需的。他汀类药物竞争性抑制HMG CoA还原酶的作用,减少细胞内甲羟戊酸合成,使其下游的类异戊二烯化作用产物FPP和GGPP减少,阻碍了小GTP蛋白的异戊二烯化,使与浆膜结合的激活的小GTP蛋白减少,阻止其发挥生物学活性。他汀类的上述作用可被加入甲羟戊酸或FPP、GGPP所逆转。真核生物的心肌细胞内有许多调控细胞生存和凋亡的酶和蛋白质,它们通过各种信号转导通路发挥作用,保持细胞生存和凋亡的动态平衡, PTEN-PI3K/Akt和钙调神经磷酸酶(Calcineurin,CaN)信号通路是其中非常重要的两个途径。PTEN-PI3K/Akt信号通路可调节心肌细胞的增殖、分化、存活等,磷酸酶张力蛋白同源物(phosphatase and tensin homology deleted on chromosome ten,PTEN)是PIP3磷酸酶的一种,可负性调节PI3K/Akt信号通路。有研究表明PTEN与心肌肥厚之间存在密切关系,而且可能参与了他汀类药物抗心肌细胞肥大的作用;另外,RhoA/ ROCK(RhoA的主要靶蛋白)可以调节PTEN的细胞内定位和磷酸化,激活PTEN的磷脂磷酸酶活性,从而逆转PI3K /Akt信号通路。1998年Molkentin等构建过表达CaN和NFAT3的转基因鼠出现了与人类心肌肥大相似的病理变化,提出CaN介导的信号通路在心肌肥大的发生中发挥重要作用,CaN是胞内Ca2+信号诱导核内肥大基因活化的中心环节。有资料显示,心脏特异性过度表达CaN所引起的心肌肥厚模型中,PTEN表达降低;而PTEN基因表达阴性的大鼠心肌细胞可通过PI3Kα增加Akt活性,从而增加细胞内L型钙通道(L-type Ca2+ channels,ICa,L)离子流;另外PTEN过度表达能够明显抑制血管紧张素Ⅱ(angiotoninⅡ,AngⅡ)引起的心肌细胞内Ca2 +浓度、CaN mRNA与蛋白表达以及CaN活性增高,负性调控AngⅡ刺激所致的心肌细胞肥大,提示PTEN可能负性调节Ca2+/CaN;以上这些研究结果提示在心肌肥厚中CaN和PTEN至少在表达水平上存在某种联系。目前有关PTEN和CaN的研究多局限于心肌细胞肥大中,在心肌纤维化上的研究很少;在他汀类药物抗心肌纤维化中PTEN和CaN是否参与未见相关报道。故本研究以体外培养的新生SD大鼠心肌成纤维细胞为研究对象,以醛固酮诱导其心肌成纤维细胞增殖和胶原合成,并加入阿托伐他汀(atorvastatin,Ato)干预,观察在阿托伐他汀逆转心肌成纤维细胞增殖的过程中PTEN mRNA和蛋白、CaN mRNA和蛋白的表达变化,并进一步观察加入FPP、GGPP分别逆转阿托伐他汀对Ras和Rho蛋白的作用后上述指标的表达变化,以探讨PTEN和CaN是否参与了他汀类药物抗心肌纤维化的作用,并明确PTEN和CaN在甲羟戊酸途径中与Rho及Ras蛋白的关系,从而深入了解他汀类药物抗心肌纤维化的信号转导机制。第一部分醛固酮对心肌成纤维细胞钙调神经磷酸酶表达的影响目的:研究醛固酮对心肌成纤维细胞增殖、胶原合成和钙调神经磷酸酶表达的影响。方法:用胰酶消化法分离并培养新生SD大鼠的心肌成纤维细胞,以醛固酮诱导其增殖和胶原合成,并加入螺内酯(spironolactone,Spi)干预,采用MTT检测心肌成纤维细胞的增殖,羟脯氨酸检测其胶原合成,半定量RT-PCR测定心肌成纤维细胞CaN mRNA的表达,Western blot测定其CaN蛋白的表达变化。结果:1醛固酮和螺内酯对心肌成纤维细胞增殖的影响:与对照组比较,10-7mol/L、10-8mol/L、10-9mol/L Ald组的平均吸光度值均显著升高(P<0.05),但三组之间没有差异性;10-7mol/L Ald+10-8 mol/L、10-9 mol/L Spi组的平均吸光度值均明显低于10-7mol/L Ald组(P<0.05),而与对照组比较没有差异性,两组之间比较也没有差异性。2醛固酮和螺内酯对心肌成纤维细胞胶原合成的影响:与对照组比较,10-7mol/L、10-8mol/L、10-9mol/L Ald组的羟脯氨酸含量均显著升高(P<0.05),但三组之间没有差异性;10-7mol/L Ald+10-8mol/L、10-9mol/L Spi组的羟脯氨酸含量均明显低于10-7mol/L Ald组(P<0.05),而与对照组比较没有差异性,两组之间比较也没有差异性。3醛固酮对心肌成纤维细胞CaN mRNA表达的影响:与对照组相比,10-7mol/L、10-8mol/L、10-9mol/L Ald组的CaN mRNA水平明显升高(P<0.05),并呈浓度依赖性,10-7mol/L Ald组的CaN mRNA水平明显高于10-8mol/L和10-9mol/L Ald组(P<0.05)。4螺内酯对心肌成纤维细胞CaN mRNA表达的影响:10-7mol/L Ald+ 10-8mol/L、10-9mol/L Spi组的CaN mRNA水平明显低于10-7mol/L Ald组(P<0.05),而与对照组比较没有差异性,两组之间比较也没有差异性。5醛固酮对心肌成纤维细胞CaN蛋白表达的影响:10-7mol/L、10-8mol /L、10-9mol/L Ald组的CaN蛋白水平明显高于对照组(P<0.05),并呈浓度依赖性,10-7mol/L Ald组的CaN蛋白水平明显高于10-8mol/L和10-9 mol/L Ald组(P<0.05)。6螺内酯对心肌成纤维细胞CaN蛋白表达的影响:10-7mol/L Ald+ 10-8mol/L、10-9mol/L Spi组的CaN蛋白水平明显低于10-7mol/L Ald组(P<0.05),而与对照组比较没有差异性,两组之间比较也没有差异性。结论:外源性醛固酮可诱导心肌成纤维细胞增殖和胶原合成,发挥其致心肌纤维化作用,此作用与其通过盐皮质激素受体(MR)介导的基因组通路升高CaN mRNA、蛋白水平的表达有关。第二部分醛固酮对心肌成纤维细胞PTEN表达的影响目的:研究醛固酮对心肌成纤维细胞PTEN表达的影响。方法:以体外培养的新生SD大鼠心肌成纤维细胞为研究对象,加入醛固酮和螺内酯干预后,采用MTT检测心肌成纤维细胞的增殖,羟脯氨酸检测其胶原合成,半定量RT-PCR测定心肌成纤维细胞PTEN mRNA的表达,Western blot测定其PTEN蛋白的表达。结果:1醛固酮和螺内酯对心肌成纤维细胞增殖的影响:与对照组比较,10-7mol/L、10-8mol/L、10-9mol/L Ald组的平均吸光度值均显著升高(P<0.05),但三组之间没有差异性;10-7mol/L Ald+10-8mol/L、10-9mol/L Spi组的平均吸光度值均明显低于10-7mol/L Ald组(P<0.05),而与对照组比较没有差异性,两组之间比较也没有差异性。2醛固酮和螺内酯对心肌成纤维细胞胶原合成的影响:与对照组比较,10-7mol/L、10-8mol/L、10-9mol/L Ald组的羟脯氨酸含量均显著升高(P<0.05),但三组之间没有差异性;10-7mol/L Ald+10-8mol/L、10-9mol/L Spi组的羟脯氨酸含量均明显低于10-7mol/L Ald组(P<0.05),而与对照组比较没有差异性,两组之间比较也没有差异性。3醛固酮对心肌成纤维细胞PTEN mRNA表达的影响:与对照组相比,10-7mol/L、10-8mol/L、10-9mol/L Ald组的PTEN mRNA水平明显降低(P<0.05),并呈浓度依赖性,10-7mol/L Ald组的PTEN mRNA水平明显低于10-8mol/L和10-9mol/L Ald组(P<0.05)。4螺内酯对心肌成纤维细胞PTEN mRNA表达的影响:10-7mol/L Ald +10-8mol/L、10-9mol/L Spi组的PTEN mRNA水平均明显高于10-7mol/L Ald组(P<0.05),但与对照组比较没有差异性,两组之间比较也没有差异性。5醛固酮对心肌成纤维细胞PTEN蛋白表达的影响:10-7mol/L、10-8mol/L、10-9mol/L Ald组的PTEN蛋白水平明显低于对照组(P<0.05),并呈浓度依赖性,10-7mol/L Ald组的PTEN蛋白水平明显低于10-8mol/L和10-9mol/L Ald组(P<0.05)。6螺内酯对心肌成纤维细胞PTEN蛋白表达的影响: 10-7mol/L Ald+ 10-8mol/L、10-9mol/L Spi组的PTEN蛋白水平均明显高于10-7mol/L Ald组(P<0.05),但与对照组比较没有差异性,两组之间比较也没有差异性。结论:醛固酮可通过MR介导的基因组通路降低PTEN mRNA、蛋白水平的表达发挥其致心肌纤维化作用。第三部分阿托伐他汀对醛固酮诱导的心肌成纤维细胞钙调神经磷酸酶表达的影响目的:研究阿托伐他汀对醛固酮诱导的心肌成纤维细胞增殖、胶原合成和CaN表达的影响。方法:用醛固酮诱导新生SD大鼠的心肌成纤维细胞增殖和胶原合成,并加入阿托伐他汀、FPP和GGPP干预,采用MTT检测心肌成纤维细胞的增殖,羟脯氨酸检测其胶原合成,半定量RT-PCR测定心肌成纤维细胞CaN mRNA的表达,Western blot测定其CaN蛋白的表达变化。结果:1阿托伐他汀对醛固酮诱导的心肌成纤维细胞增殖的影响:10-7mol/L Ald组的平均吸光度值显著高于对照组(P<0.05);10-7mol/LAld+10-7、10-6、10-5mol/L Ato组的平均吸光度值均明显小于10-7mol/L Ald组(P<0.05),但3组之间比较没有差异性。2阿托伐他汀对醛固酮诱导的心肌成纤维细胞胶原合成的影响:与对照组相比,10-7mol/L Ald组可明显增加心肌成纤维细胞的羟脯氨酸含量(P<0.05);与10-7mol/L Ald组相比,10-7mol/L Ald+10-7、10-6、10-5mol/L Ato组则可以明显减少心肌成纤维细胞的羟脯氨酸含量(P<0.05),但3组之间比较没有统计学差异。3阿托伐他汀对醛固酮诱导的心肌成纤维细胞CaN mRNA表达的影响:与对照组相比,10-7mol/L Ald组的CaN mRNA水平明显升高(P<0.05);10-7mol/L Ald+10-7、10-6、10-5mol/L Ato组的CaN mRNA水平则均明显低于10-7mol/L Ald组(P<0.05),并且呈浓度依赖性,10-7mol/L Ald+ 10-5mol/L Ato组的CaN mRNA水平明显低于10-7mol/L Ald+10-7、10-6mol/L Ato组(P<0.05)。4阿托伐他汀对醛固酮诱导的心肌成纤维细胞CaN蛋白表达的影响:与对照组相比,10-7mol/L Ald组的CaN蛋白水平明显升高(P<0.05);与10-7mol/L Ald组相比,10-7mol/L Ald+10-7、10-6、10-5mol/L Ato组的CaN蛋白水平则均明显下降(P<0.05),并呈浓度依赖性,10-7mol/L Ald+10-5 mol/L Ato组的CaN蛋白水平明显低于10-7mol/L Ald+10-7、10-6mol/L Ato组(P<0.05)。5 FPP、GGPP对心肌成纤维细胞CaN mRNA的影响:与10-7mol/L Ald+ 10-5mol/L Ato组相比,10-7mol/L Ald+10-5mol/L Ato+10-5mol/L FPP组的CaN mRNA水平明显升高(P<0.05),而10-7mol/L Ald+10-5 mol/L Ato+10-5mol/L GGPP组的CaN mRNA水平则没有明显改变。6 FPP、GGPP对心肌成纤维细胞CaN蛋白的影响:与10-7mol/L Ald+ 10-5mol/L Ato组相比,10-7mol/L Ald+10-5mol/L Ato+10-5mol/L FPP组的CaN蛋白水平明显升高(P<0.05),而10-7mol/L Ald+10-5mol/L Ato+10-5 mol/L GGPP组的CaN蛋白水平则没有明显改变。结论:阿托伐他汀可明显抑制醛固酮诱导的心肌成纤维细胞增殖和胶原合成,其抗心肌纤维化作用与其通过抑制甲羟戊酸通路中的Ras蛋白活性来降低CaN mRNA、蛋白的表达有关。第四部分阿托伐他汀对醛固酮诱导的心肌成纤维细胞PTEN表达的影响目的:研究阿托伐他汀对醛固酮诱导的心肌成纤维细胞PTEN表达的影响。方法:用醛固酮诱导新生SD大鼠的心肌成纤维细胞增殖,并加入阿托伐他汀、FPP和GGPP干预,采用MTT检测心肌成纤维细胞的增殖,羟脯氨酸检测其胶原合成,半定量RT-PCR测定心肌成纤维细胞PTEN mRNA的表达,Western blot测定其PTEN蛋白的表达变化。结果:1阿托伐他汀对醛固酮诱导的心肌成纤维细胞增殖的影响:10-7mol/L Ald组的平均吸光度值显著高于对照组(P<0.05);10-7mol/L Ald+ 10-7、10-6、10-5mol/L Ato组的平均吸光度值均明显小于10-7mol/L Ald组(P<0.05),但3组之间比较没有差异性。2阿托伐他汀对醛固酮诱导的心肌成纤维细胞胶原合成的影响:与对照组相比,10-7mol/L Ald组可明显增加心肌成纤维细胞的羟脯氨酸含量(P<0.05);与10-7mol/L Ald组相比,10-7mol/L Ald+10-7、10-6、10-5mol/L Ato组则可以明显减少心肌成纤维细胞的羟脯氨酸含量(P<0.05),但3组之间比较没有统计学差异。3阿托伐他汀对醛固酮诱导的心肌成纤维细胞PTEN mRNA表达的影响:与对照组相比,10-7mol/L Ald组的PTEN mRNA水平明显降低(P<0.05)。而10-7mol/L Ald+10-7、10-6、10-5mol/L Ato组的PTEN mRNA水平则均明显高于10-7mol/L Ald组(P<0.05),并且呈浓度依赖性,10-7mol/L Ald+10-5mol/L Ato组的PTEN mRNA水平明高于10-7mol/L Ald+10-7、10-6 mol/L Ato组(P<0.05)。4阿托伐他汀对醛固酮诱导的心肌成纤维细胞PTEN蛋白表达的影响:与对照组相比,10-7mol/L Ald组的PTEN蛋白水平明显降低(P<0.05)。与10-7mol/L Ald组相比,10-7mol/L Ald+10-7、10-6、10-5mol/L Ato组的PTEN蛋白水平则均明显升高(P<0.05),并呈浓度依赖性,10-7mol/L Ald+10-5 mol/L Ato组的PTEN蛋白水平明显高于10-7mol/L Ald+10-7、10-6 mol/L Ato组(P<0.05)。5 FPP、GGPP对心肌成纤维细胞的PTEN mRNA的影响:与10-7mol/LAld+10-5mol/L Ato组相比,10-7mol/L Ald+10-5mol/L Ato+10-5mol/L FPP组和10-7mol/L Ald+10-5mol/L Ato+10-5mol/L GGPP组的PTEN mRNA水平均明显降低(P<0.05)。6 FPP、GGPP对心肌成纤维细胞PTEN蛋白的影响:与10-7mol/L Ald+10-5mol/L Ato组相比,10-7mol/L Ald+10-5mol/L Ato+10-5mol/L FPP组和10-7mol /L Ald+10-5 mol/L Ato+10-5mol/L GGPP组的PTEN蛋白水平均明显降低(P<0.05)。结论:阿托伐他汀在逆转醛固酮致心肌纤维化同时,可抑制甲羟戊酸通路中的Ras和Rho蛋白活性来升高PTEN mRNA、蛋白的表达。第五部分在醛固酮诱导和阿托伐他汀干预的心肌成纤维细胞中CaN和PTEN表达的相关性分析目的:探讨CaN和PTEN在醛固酮诱导和阿托伐他汀干预的心肌成纤维细胞中表达水平上的相关性。方法:用醛固酮诱导新生SD大鼠的心肌成纤维细胞增殖,并加入阿托伐他汀干预,半定量RT-PCR测定心肌成纤维细胞CaN和PTEN mRNA的表达,Western blot测定其CaN和PTEN蛋白的表达,运用Spearman等级相关分析处理CaN和PTEN表达的相关性分析,检验水准取α=0.05。结果:1在醛固酮诱导的心肌成纤维细胞中CaN和PTEN mRNA表达的相关性分析:经Spearman等级相关分析,在醛固酮诱导的心肌成纤维细胞中CaN和PTEN mRNA表达呈显著负相关,其相关系数r=﹣0.860(P<0.01)。2在醛固酮诱导的心肌成纤维细胞中CaN和PTEN蛋白表达的相关性分析:经Spearman等级相关分析,在醛固酮诱导的心肌成纤维细胞中CaN和PTEN蛋白表达呈显著负相关(r=﹣0.868,P<0.01)。3在阿托伐他汀干预的心肌成纤维细胞中CaN和PTEN mRNA表达的相关性分析:经Spearman等级相关分析,阿托伐他汀干预的心肌成纤维细胞中CaN和PTEN mRNA表达呈显著负相关(r=﹣0.808,P<0.01)。4在阿托伐他汀干预的心肌成纤维细胞中CaN和PTEN蛋白表达的相关性分析:经Spearman等级相关分析,在阿托伐他汀干预的心肌成纤维细胞中CaN和PTEN mRNA表达呈显著负相关(r=﹣0.799,P<0.01)。结论:在醛固酮致心肌纤维化和阿托伐他汀逆转过程中,CaN和PTEN mRNA、蛋白表达均呈显著负相关,提示CaN和PTEN在表达水平上存在密切关系。总结1外源性醛固酮可诱导心肌成纤维细胞增殖和胶原合成,发挥其致心肌纤维化作用,与其通过MR介导的基因组通路升高CaN表达有关。2醛固酮可通过MR介导的基因组通路降低PTEN表达发挥其致心肌纤维化作用。3阿托伐他汀可明显抑制醛固酮诱导的心肌成纤维细胞增殖和胶原合成,其抗心肌纤维化作用与其通过抑制甲羟戊酸通路中的Ras蛋白活性来降低CaN的表达有关。4阿托伐他汀在逆转醛固酮致心肌纤维化同时,可抑制甲羟戊酸通路中的Ras和Rho蛋白活性来升高PTEN的表达。5在醛固酮致心肌纤维化和阿托伐他汀逆转过程中,CaN和PTEN mRNA、蛋白表达均呈显著负相关,提示CaN和PTEN在表达水平上存在密切关系。更多还原

【Abstract】 Cardiac remodeling is manifested clinically as changes in the size, shape, and function of the heart. Histopathologically, it is characterized by a structural rearrangement of components of the normal chamber wall that involves cardiomyocyte hypertrophy, cardiac fibroblast proliferation, fibrosis, and cell death. Cardiac fibrosis, which is a disproportionate accumulation of fibrillar collagen, is an integral feature of the remodeling characteristic of the failing heart. Cardiac fibroblasts are crucially involved in the processes of cardiac fibrosis, producing growth factors and cytokines that act as autocrine and paracrine factors, as well as extracellular matrix proteins and proteinases. Humoral factors that affect the phenotype and function of cardiac fibroblasts include renin-angiotensin-aldosterone system, basic fibroblast growth factor (bFGF), transforming growth factor-β(TGF-β), endothelin, bradykinin, nitric oxide, catecholamines, et al. Aldosterone, which is recognized as vital promoter of myocardial fibrosis, can promote the proliferation of cardiac fibroblast and collagen synthesis, resulting in myocardial fibrosis. Normal cardiac tissue, which has independent aldosterone system, contains abundant, specific, high-affinity mineralocorticoid receptor (MR). Several studies showed that there are increased aldosterone levels found in heart failure patients and high levels of aldosterone is the independent predictor of increased mortality; the MR antagonists can alleviate the progression of cardiac remodeling induced by angiotensinⅡ. So aldosterone plays an important role on heart failure.3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins), which are the best way to reduce cholesterol and the risk of heart attack as a lipid-lowering drug, are widely used in the prevention and treatment of cardiovascular disease. In recent years a substantial quantity of data has accumulated showing that statins exert anti-fibrosis effects on myocardium, which are independent of their plasma cholesterol lowering properties. Statins have been shown to inhibit Angiotensin II-mediated cardiac fibrosis as well as to block various intracellular signaling pathways including downregulation of the activity of small GTP-binding proteins. Statins inhibit HMG-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis in the liver, which catalyzes the conversion of HMG-CoA to mevalonic acid. In addition to inhibiting cholesterol synthesis, statins also block the synthesis of isoprenoid intermediates such as farnesyl pyrophosphate (FPP) and geranyl- geranyl pyrophosphate (GGPP). Both FPP and GGPP serve as important lipid attachments for the posttranslational modification of a variety of proteins, including heterotrimeric G proteins and small GTP-binding proteins belonging to the family of Ras, Rho GTPases. Isoprenylation is critical for intracellular trafficking and function of small GTP-binding proteins. In general, modification with FPP is necessary for proper localization of Ras family proteins, whereas GGPP is required for Rho family proteins. By inhibiting mevalonate synthesis, statins inhibit the synthesis of isoprenoid intermediates thereby preventing isoprenylation of small GTPases, leading to the inhibition of these signaling molecules, which contributing to the prevention of cardiac fibrosis and heart failure.There are various intracellular signaling pathways participate in the regulation of cell survival and apoptosis, including PI3K/Akt and calcineurin (CaN) pathways. PTEN( phosphatase and tensin homology deleted on chromosome ten), which act as a negative regulator of PI3K/ Akt pathway, is proved have a close relationship with cardiomyocyte hypertrophy. Calcineurin, which is the key link to activated intranuclear hypertrophic gene induced by intra-cellular Ca2+, play the vital role in the development of cardiomyocyte hypertrophy. Recently, sun et al reported that inactivation of PTEN enhances L-type Ca2+ channel (ICa,L) via PI3K-dependent increase in PKB activation and provided powerful evidence for a critical interface between cardiomyocyte Ca2+ signaling and PI3K-regulated pathway. Some domestic studies also showed that cardiac hypertrophy induced by AngⅡcould be blocked by PTEN overexpression via suppressing Ca2+ /Calcineurin pathway. Those data suggested that there may exist some“crosstalking”between PTEN-PI3K/Akt and Ca2+/ Calcineurin pathways.Now, most studies on the role of calcineurin or PTEN are limited to cardiomyocyte hypertrophy, whether calcineurin or PTEN participate in anti-fibrosis effects of statins is still unknown. So we cultured cardiac fibroblasts of neonatal Sprague-Dawley rats induced by aldosterone, treated them with spironolactone, atorvastatin, FPP, GGPP respectively, tested the expression of calcineurin、PTEN mRNA and protein by RT-PCR and Western Blot, to investigate the role of calcineurin and PTEN on the anti-fibrosis effects of atorvastatin and to explore the signal transduction mechanisms.Part 1 The effects of aldosterone on the expression of calcineurin in cultured neonatal cardiac fibroblastsObjective: To investigate the role of calcineurin (CaN) in aldosterone (Ald)–induced proliferation of cardiac fibroblasts and to explore the effects of spironolactone (Spi) on the expression of CaN.Method: In cultured cardiac fibroblasts of neonatal Sprague-Dawley (S-D) rats, the effects of Ald and Spi on proliferation were measured by MTT colorimetric assay, synthesis of collagen was observed by the hydroxyproline concentration determined, CaN mRNA and protein were tested by RT-PCR and Western Blot, respectively.Results: 1. Effects of Ald and Spi on the proliferation: Compared with the control group, 10-7mol/L、10-8mol/L、10-9mol/L Ald groups significantly increased the proliferation of fibroblasts respectively (P<0.05), but there were no difference among those three groups. Compared with 10-7mol/L Ald group, the proliferation of fibroblasts in 10-7mol/L Ald+10-8mol/L、10-9mol/L Spi groups were significantly decreased respectively (P<0.05) , which were no difference with the control group. 2. Effects of Ald and Spi on the hydroxyproline concentration: Compared with the control group, 10-7mol/L、10-8mol/L、10-9mol/L Ald groups significantly increased the hydroxyproline concentration respectively (P<0.05), but there were no difference among those three groups. Compared with 10-7mol/L Ald group, the hydroxyproline concentration in 10-7mol/L Ald+10-8 mol/L、10-9mol/L Spi groups were significantly decreased respectively (P<0.05) , which were no difference with the control group.3. Effects of Ald on the expression of CaN mRNA: Compared with the control group, the expression of CaN mRNA in 10-7mol/L、10-8mol/L、10-9mol /L Ald groups were significantly increased in a concentration-dependent fashion respectively (P<0.05).4. Effects of Spi on the expression of CaN mRNA: Compared with 10-7mol/L Ald group, the expression of CaN mRNA in 10-7mol/L Ald+10-8 mol/L、10-9mol/L Spi groups were significantly decreased respectively (P<0.05), which were no difference with the control group.5. Effects of Ald on the expression of CaN protein: Compared with the control group, the expression of CaN protein in 10-7mol/L、10-8mol/L、10-9mol /L Ald groups were significantly increased in a concentration-dependent fashion respectively (P<0.05).6. Effects of Spi on the expression of CaN protein: Compared with 10-7mol/L Ald group, the expression of CaN protein in 10-7mol/L Ald+10-8 mol/L、10-9mol/L Spi groups were significantly decreased respectively (P<0.05), which were no difference with the control group.Conclusions: Exogenous aldosterone stimulated cardiac fibroblasts proliferation、collagen synthesis and increased the expression of CaN mRNA、protein. The effects of Ald on calcineurin were realized by genomic way.Part 2 The effects of aldosterone on the expression of PTEN in cultured neonatal cardiac fibroblastsObjective: To investigate the role of PTEN in aldosterone (Ald)–induced proliferation of cardiac fibroblasts and to explore the effects of spironolactone (Spi) on the expression of PTEN.Method: In cultured cardiac fibroblasts of neonatal Sprague-Dawley (S-D) rats, the effects of Ald and Spi on proliferation were measured by MTT colorimetric assay, synthesis of collagen was observed by the hydroxyproline concentration determined, PTEN mRNA and protein were tested by RT-PCR and Western Blot, respectively.Results: 1. Effects of Ald and Spi on the proliferation: Compared with the control group, 10-7mol/L、10-8mol/L、10-9mol/L Ald groups significantly increased the proliferation of fibroblasts respectively (P<0.05), but there were no difference among those three groups. Compared with 10-7mol/L Ald group, the proliferation of fibroblasts in 10-7mol/L Ald+10-8mol/L、10-9mol/L Spi groups were significantly decreased respectively (P<0.05) , which were no difference with the control group.2. Effects of Ald and Spi on the hydroxyproline concentration: Compared with the control group, 10-7mol/L、10-8mol/L、10-9mol/L Ald groups significantly increased the hydroxyproline concentration respectively (P<0.05), but there were no difference among those three groups. Compared with 10-7mol/L Ald group, the hydroxyproline concentration in 10-7mol/L Ald+10-8 mol/L、10-9mol/L Spi groups were significantly decreased respectively (P<0.05) , which were no difference with the control group.3. Effects of Ald on the expression of PTEN mRNA: Compared with the control group, the expression of PTEN mRNA in 10-7mol/L、10-8mol/L、10-9 mol/L Ald groups were significantly decreased in a concentration-dependent fashion respectively (P<0.05).4. Effects of Spi on the expression of PTEN mRNA: Compared with 10-7mol/L Ald group, the expression of PTEN mRNA in 10-7mol/L Ald+10-8 mol/L、10-9mol/L Spi groups were significantly increased respectively (P<0.05), which were no difference with the control group.5. Effects of Ald on the expression of PTEN protein: Compared with the control group, the expression of PTEN protein in 10-7mol/L、10-8mol/L、10-9 mol/L Ald groups were significantly decreased in a concentration-dependent fashion respectively (P<0.05).6. Effects of Spi on the expression of PTEN protein: Compared with 10-7mol/L Ald group, the expression of PTEN protein in 10-7mol/L Ald+10-8 mol/L、10-9mol/L Spi groups were significantly increased respectively (P<0.05), which were no difference with the control group.Conclusions: Exogenous aldosterone stimulated cardiac fibroblasts proliferation、collagen synthesis and decreased the expression of PTEN mRNA、protein. The effects of Ald on PTEN were realized by genomic way.Part 3 The effects of atorvastatin on the expression of calcineurin in aldosterone–induced cardiac fibroblastsObjective: To investigate the effects of atorvastatin(Ato)on calcineurin in aldosterone–induced cardiac fibroblasts.Method: In aldosterone–induced cardiac fibroblasts of neonatal Sprague- Dawley(S-D) rats, the effects of Ato、FPP、GGPP on proliferation were measured by MTT colorimetric assay, synthesis of collagen was observed by the hydroxyproline concentration determined, CaN mRNA and protein were tested by RT-PCR and Western Blot, respectively.Results: 1. Effects of Ato on the proliferation in aldosterone–induced cardiac fibroblasts: Compared with the control group, 10-7mol/L Ald group significantly increased the proliferation of fibroblasts(P<0.05). Compared with 10-7mol/L Ald group, the proliferation of fibroblasts in 10-7mol/L Ald+ 10-7、10-6、10-5mol/L Ato groups were significantly decreased respectively (P<0.05) , which were no difference among those three groups.2. Effects of Ato on the hydroxyproline concentration in aldosterone–induced cardiac fibroblasts: Compared with the control group, 10-7 mol/L Ald group significantly increased the hydroxyproline concentration (P<0.05). Compared with 10-7mol/L Ald group, the hydroxyproline concentration in 10-7mol/L Ald+10-7、10-6、10-5mol/L Ato groups were significantly decreased respectively (P<0.05) , which were no difference among those three groups.3. Effects of Ato on the expression of CaN mRNA in aldosterone–induced cardiac fibroblasts: Compared with the control group, the expression of CaN mRNA in 10-7mol/L Ald group was significantly increased (P<0.05). Compared with 10-7mol/L Ald group, the expression of CaN mRNA in 10-7 mol/L Ald+10-7、10-6、10-5mol/L Ato groups were significantly decreased in a concentration-dependent fashion respectively (P<0.05).4. Effects of Ato on the expression of CaN protein in aldosterone–induced cardiac fibroblasts: Compared with the control group, the expression of CaN protein in 10-7mol/L Ald group was significantly increased (P<0.05). Compared with 10-7mol/L Ald group, the expression of CaN mRNA in 10-7 mol/L Ald+10-7、10-6、10-5mol/L Ato groups were significantly decreased in a concentration-dependent fashion respectively (P<0.05).5. Effects of FPP、GGPP on the expression of CaN mRNA in aldosterone–induced cardiac fibroblasts: The expression of CaN mRNA in 10-7mol/L Ald+10-5mol/L Ato+10-5mol/L FPP group was significantly increased compared to 10-7mol/L Ald+10-5mol/L Ato group (P<0.05), while the expression of CaN mRNA in 10-7mol/L Ald+10-5mol/L Ato+10-5mol/L GGPP group was no difference with 10-7mol/L Ald+ 10-5mol/L Ato group.6. Effects of FPP、GGPP on the expression of CaN protein in aldosterone–induced cardiac fibroblasts: The expression of CaN protein in 10-7mol/L Ald+10-5mol/L Ato+10-5mol/L FPP group was significantly increased compared to 10-7mol/L Ald+10-5mol/L Ato group (P<0.05), while the expression of CaN mRNA in 10-7mol/L Ald+10-5mol/L Ato+10-5mol/L GGPP group was no difference with 10-7mol/L Ald+10-5mol/L Ato group.Conclusions: Atorvastatin could decrease the expression of calcineurin by inhibiting Ras activity, which might result in the reversion of cardiac fibrosis induced by aldosterone.Part 4 The effects of atorvastatin on the expression of PTEN in aldosterone–induced cardiac fibroblasts Objective: To investigate the effects of atorvastatin(Ato)on PTEN in aldosterone–induced cardiac fibroblasts.Method: In aldosterone–induced cardiac fibroblasts of neonatal Sprague- Dawley(S-D) rats, the effects of Ato、FPP、GGPP on proliferation were measured by MTT colorimetric assay, synthesis of collagen was observed by the hydroxyproline concentration determined, PTEN mRNA and protein were tested by RT-PCR and Western Blot, respectively.Results: 1. Effects of Ato on the proliferation in aldosterone–induced cardiac fibroblasts: Compared with the control group, 10-7mol/L Ald group significantly increased the proliferation of fibroblasts(P<0.05). Compared with 10-7mol/L Ald group, the proliferation of fibroblasts in 10-7mol/L Ald+ 10-7、10-6、10-5mol/L Ato groups were significantly decreased respectively (P<0.05) , which were no difference among those three groups.2. Effects of Ato on the hydroxyproline concentration in aldosterone–induced cardiac fibroblasts: Compared with the control group, 10-7 mol/L Ald group significantly increased the hydroxyproline concentration (P<0.05). Compared with 10-7mol/L Ald group, the hydroxyproline concentration in 10-7 mol/L Ald+10-7、10-6、10-5mol/L Ato groups were significantly decreased respectively (P<0.05) , which were no difference among those three groups.3. Effects of Ato on the expression of PTEN mRNA in aldosterone–induced cardiac fibroblasts: Compared with the control group, the expression of PTEN mRNA in 10-7mol/L Ald group was significantly decreased (P<0.05). Compared with 10-7mol/L Ald group, the expression of PTEN mRNA in 10-7mol/L Ald+10-7、10-6、10-5mol/L Ato groups were significantly increased in a concentration-dependent fashion respectively (P<0.05).4. Effects of Ato on the expression of PTEN protein in aldosterone– induced cardiac fibroblasts: Compared with the control group, the expression of PTEN protein in 10-7mol/L Ald group was significantly decreased (P<0.05). Compared with 10-7mol/L Ald group, the expression of PTEN mRNA in 10-7mol/L Ald+10-7、10-6、10-5mol/L Ato groups were significantly increased in a concentration-dependent fashion respectively (P<0.05).5. Effects of FPP、GGPP on the expression of PTEN mRNA in aldosterone–induced cardiac fibroblasts: Compared with 10-7mol/L Ald+10-5 mol/L Ato group, the expression of PTEN mRNA in 10-7mol/L Ald+10-5mol/L Ato+10-5mol/L FPP group and 10-7mol/L Ald+10-5mol/L Ato+10-5mol/L GGPP group were significantly decreased respectively (P<0.05).6. Effects of FPP、GGPP on the expression of PTEN protein in aldosterone–induced cardiac fibroblasts: Compared with 10-7mol/L Ald+10-5 mol/L Ato group, the expression of PTEN protein in 10-7mol/L Ald+10-5mol/L Ato+10-5mol/L FPP group and 10-7mol/L Ald+10-5mol/L Ato+10-5mol/L GGPP group were significantly decreased respectively (P<0.05).Conclusions: Atorvastatin could increase the expression of PTEN by inhibiting Ras and Rho activity, which might result in the reversion of cardiac fibrosis induced by aldosterone.Part 5 The Correlation between the expression of calcineurin and PTEN in aldosterone–induced and atorvastatin-interfered cardiac fibroblastsObjective: To investigate the correlation between the expression of calcineurin and PTEN in aldosterone–induced and atorvastatin-interfered cardiac fibroblasts.Method: In aldosterone–induced and atorvastatin-interfered cardiac fibroblasts of neonatal Sprague-Dawley rats, the effects of Ald、Ato on the expression of calcineurin and PTEN were tested by RT-PCR and Western Blot, respectively, the correlation between the expression of CaN and PTEN were analyzed by Spearman rank correlation test.Results: 1. The correlation between the expression of CaN and PTEN mRNA in aldosterone–induced cardiac fibroblasts: The expression of CaN mRNA were negatively correlated with the expression of PTEN mRNA (r=﹣0.860, P<0.01).2. The correlation between the expression of CaN and PTEN protein in aldosterone–induced cardiac fibroblasts: The expression of CaN protein were negatively correlated with the expression of PTEN protein (r=﹣0.868, P<0.01).3. The correlation between the expression of CaN and PTEN mRNA in atorvastatin-interfered cardiac fibroblasts: The expression of CaN mRNA were negatively correlated with the expression of PTEN mRNA (r=﹣0.808, P<0.01).4. The correlation between the expression of CaN and PTEN protein in atorvastatin-interfered cardiac fibroblasts: The expression of CaN protein were negatively correlated with the expression of PTEN protein (r=﹣0.799, P<0.01).Conclusions: In aldosterone–induced and atorvastatin-interfered cardiac fibroblasts, the expression of CaN mRNA、protein were negatively correlated with the expression of PTEN mRNA、protein respectively, suggested there were a close relation between the expression of CaN and PTEN.Conclusions1. Exogenous aldosterone stimulated cardiac fibroblasts proliferation、collagen synthesis and increased the expression of CaN mRNA、protein. The effects of Ald on calcineurin were realized by genomic way.2. Exogenous aldosterone stimulated cardiac fibroblasts proliferation、collagen synthesis and decreased the expression of PTEN mRNA、protein. The effects of Ald on PTEN were realized by genomic way.3. Atorvastatin could decrease the expression of calcineurin by inhibiting Ras activity, which might result in the reversion of cardiac fibrosis induced by aldosterone.4. Atorvastatin could increase the expression of PTEN by inhibiting Ras and Rho activity, which might result in the reversion of cardiac fibrosis induced by aldosterone.5. In aldosterone–induced and atorvastatin-interfered cardiac fibroblasts, the expression of CaN mRNA、protein were negatively correlated with the expression of PTEN mRNA、protein respectively, suggested there were a close relation between the expression of CaN and PTEN.更多还原