【作者】 关胜江；

【导师】 楚立；

【作者基本信息】 河北医科大学 ， 药理学， 2009， 博士

【摘要】 心肌肥厚是多种心血管病的共同病理结局,其主要特征是细胞体积增大、蛋白合成增加、胚胎型基因表达上调。早期的心肌肥厚可能是心肌细胞对内外界刺激的一种适应性改变,有一定的代偿意义,有利于维持心输出量,但持续的心肌肥厚最终会不可避免地导致心力衰竭,甚至猝死。因此,探明心肌肥厚信号转导通路,从而有效的预防和逆转心肌肥厚具有重要的意义。心肌肥厚是肥大刺激诱导核内基因异常表达的结果,细胞内信号转导通路是肥大刺激与核内基因转录活化的偶联环节。然而,不同刺激诱导的心肌肥大可能具有不同的“分子表型”,这主要取决于它们启动的信号转导通路。对心肌肥大信号转导通路的深入认识,不仅有助于阐明心肌肥厚的细胞分子机制,而且可为药物干预防治心肌肥厚提供新思路。RhoA/Rho激酶信号通路介导了多种细胞功能,包括细胞的收缩、细胞的粘附与迁移、细胞的增殖与凋亡等。而这些效应与临床上多种心血管疾病如高血压、再狭窄、动脉粥样硬化、肺动脉高压、脑血管痉挛、血管瘤、缺血再灌注损伤的发生有关。因此RhoA/Rho激酶信号通路在心血管疾病的发病中起着重要作用。近年来有关RhoA/Rho激酶信号通路与自发性高血压、压力超负荷、血管紧张素II（angiotensin II,Ang II）所导致的心肌肥大的关系虽然已有一些报道,但是,到目前为止,还没有研究能够阐明抑制Rho激酶对异丙肾上腺素（isoproterenol,Iso）诱发的心肌肥大以及糖尿病所诱发的心肌病的影响。本实验以Rho激酶抑制药法舒地尔（fasudil,Fas）为工具药,研究了:（1）RhoA/Rho激酶信号通路在Iso诱导的大鼠心肌肥厚中的作用;（2）RhoA/Rho激酶信号通路对苯肾上腺素（phenylephrine,PE）、Iso和Ang II诱导的心肌细胞肥大的影响;（3）RhoA/Rho激酶信号通路对链脲佐菌素（streptozotocin,STZ）糖尿病心肌病的影响。第一部分RhoA/Rho激酶信号通路在Iso诱导的大鼠心肌肥厚中的作用目的:研究RhoA/Rho激酶信号通路在Iso诱导的大鼠心肌肥厚中的作用。方法:清洁级雄性Wistar大鼠40只,体重200～220 g,随机分成5组:空白对照组、Iso模型组、Fas低剂量组、Fas高剂量组及卡托普利（captopril,Cap）组,每组8只。除空白对照组外,其余各组大鼠背部皮下注射Iso 5 mg·kg^-1·d^-1,连续7 d。给Iso同时,Fas低、高剂量组分别给予Fas 2、10 mg·kg^-1·d^-1,分两次腹腔注射给药;Cap组给予Cap 30 mg·kg^-1·d^-1,灌胃;Iso组、空白对照组给予等容积生理盐水。停药24 h后,测定下列指标:（1）血流动力学:包括心率（HR）、左室收缩压（LVSP）、左室舒张末压（LVEDP）、左室内压最大上升速率（+dp/dtmax）、左室内压最大下降速率（-dp/dtmax）等;（2）血液生化:经颈总动脉取血,测定Ang II、丙二醛（MDA）含量及超氧化物歧化酶（SOD）、乳酸脱氢酶（LDH）和肌酸激酶（CK）的活性;（3）心脏重量指数（HWI）;（4）病理形态学:观察心肌的病理学改变并测量心肌细胞直径（CMD）和心肌胶原容积分数（CVF）;（5）RT-PCR测定RhoA、Rho激酶mRNA的表达。结果:1血流动力学:与空白对照组比较,Iso模型组大鼠LVEDP升高34.3%（P<0.01）,而LVSP、+dp/dtmax和-dp/dtmax分别下降16.8%（P<0.01）、16.2%（P<0.01）和14.4%（P<0.01）。与Iso模型组相比,Fas高剂量组和Cap组大鼠LVEDP分别下降了16.1%（P<0.05）和28.0%（P<0.01）;Fas高剂量组大鼠LVSP、+dp/dtmax和-dp/dtmax分别升高了14.7%（P<0.05）、10.2%（P<0.05）和9.9%（P<0.05）;Cap组大鼠LVSP、+dp/dtmax和-dp/dtmax分别升高了15.8%（P<0.05）、14.4%（P<0.01）和9.9%（P<0.05）。各组大鼠心率无显著性差异（P>0.05）。2血液生化:与空白对照组比较,Iso模型组大鼠血浆Ang II含量升高了34.2%（P<0.01）。与Iso模型组比较,Fas低、高剂量组大鼠血浆Ang II含量无明显变化（P>0.05）,而Cap组大鼠血浆Ang II下降了34.8%（P<0.01）。与空白对照组比较,Iso模型组大鼠血清SOD降低了60.4%（P<0.01）,而MDA升高了117.0%（P<0.01）。与Iso模型组比较,Fas低、高剂量组及Cap组大鼠血清SOD分别升高了30.7%（P<0.05）、64.8%（P<0.01）、85.7%（P<0.01）;而MDA分别下降了9.8%（P<0.05）、25.1%（P<0.01）和28.0%（P<0.01）。与空白对照组比较,Iso模型组血清LDH和CK分别升高了20.7%（P<0.01）和46.4%（P<0.01）。经Fas低、高剂量及Cap治疗后,LDH分别下降了14.6%（P<0.05）、36.1%（P<0.01）和42.8%（P<0.01）;CK分别下降了18.3%（P<0.05）、32.0%（P<0.01）和41.4%（P<0.01）。3 HWI:各组动物体重无明显差异（P>0.05）。与空白对照组比较,Iso模型组HWI升高了19.2%（P<0.01）。经Fas低、高剂量及Cap治疗后,HWI分别下降了6.9%（P<0.05）,7.7%（P<0.01）和9.0%（P<0.01）。4病理形态学:空白对照组大鼠心肌纤维排列整齐,横纹清晰,胞核明显,无细胞肿胀;Iso模型组大鼠心肌纤维排列紊乱、肿胀、断裂、横纹消失,甚至溶解,经Fas及Cap治疗后,心肌的病理学改变均有不同程度的改善。与空白对照组比较,Iso模型组大鼠心肌CMD和CVF分别升高了16.9%（P<0.01）和32.9%（P<0.05）。与Iso模型组比较,Fas低、高剂量组和Cap组大鼠心肌CMD分别下降了8.1%（P<0.05）、12.3%（P<0.01）和13.5%（P<0.01）;Fas高剂量组和Cap组大鼠心肌CVF分别下降了21.6%（P<0.05）和26.2%（P<0.05）。5 RhoA、Rho激酶mRNA表达:与空白对照组比较,Iso模型组RhoA、Rho激酶mRNA表达上调（P<0.01和P<0.05）。而经Fas干预后,RhoA、Rho激酶mRNA表达显著下降。小结:Iso诱导的心肌肥厚的发病过程中,伴有RhoA/Rho激酶信号通路的激活;Fas可抑制该通路的激活,改善Iso诱导的心肌肥厚的心肌病理变化及心脏功能。Fas可以抑制脂质过氧化反应,提高机体抗氧化能力,发挥对心肌的保护作用。第二部分RhoA/Rho激酶信号通路对PE、Iso和Ang II诱导的心肌细胞肥大的影响目的:研究RhoA/Rho激酶信号通路对PE、Iso和Ang II所致大鼠心肌肥大的影响。方法:用消化法分离并培养新生大鼠的心肌细胞,随机分为8组:空白对照组,Fas对照组(Fas 10-5 mol·L^-1),Iso组(Iso 10-5 mol·L^-1)Iso+Fas组(Iso 10-5 mol·L^-1+Fas 10-5 mol·L^-1),Ang II组(Ang II 10-7 mol·L^-1),AngII+Fas组(Ang II10-7 mol·L^-1+Fas 10-5 mol·L^-1),PE组(PE 10-5 mol·L^-1)PE+Fas组(PE 10-5 mol·L^-1+Fas 10-5 mol·L^-1)。Simpson法测各组心肌细胞大小,Lowrys法测心肌细胞蛋白质含量,RT-PCR测定RhoA/Rho激酶mRNA表达,Western blot测定c-fos蛋白的表达。采用钙调神经磷酸酶（CaN）试剂盒检测CaN的活性,李田昌等的方法检测丝裂素活化蛋白激酶（MAPK）的活性。结果:1心肌细胞大小:PE组、Iso组和Ang II组心肌细胞大小明显高于对照组（P<0.01）,而Fas组与对照组相比无明显差异（P>0.05）。而给予Fas干预后,PE+Fas组心肌细胞大小明显低于PE组（P<0.01）;Ang II+Fas组心肌细胞大小明显低于Ang II组（P<0.01）;Iso+Fas组与Iso组相比无明显差异（P>0.05）。2心肌细胞蛋白质合成:PE组、Iso组和Ang II组心肌细胞蛋白质合成明显高于对照组（P<0.01）,而Fas组与对照组相比无明显差异（P>0.05）。而给予Fas干预后,PE+Fas组心肌细胞蛋白质合成明显低于PE组（P<0.01）;Ang II+Fas组心肌细胞蛋白质合成明显低于Ang II组（P<0.01）;而Iso+Fas组与Iso组相比无明显差异（P>0.05）。3 RhoA、Rho激酶mRNA的表达:PE组和Ang II组心肌细胞RhoA/Rho激酶mRNA的表达明显高于对照组（P<0.01）,而Iso组和Fas组与对照组相比无明显差异（P>0.05）。而给予Fas干预后,PE+Fas组心肌细胞RhoA/Rho激酶mRNA的表达明显低于PE组（P<0.01）;Ang II+Fas组心肌细胞RhoA/Rho激酶mRNA的表达明显低于Ang II组（P<0.01）;而Iso+Fas组与Iso组相比无明显差异（P>0.05）。4 MAPK活性:PE组、Iso组和Ang II组心肌细胞MAPK活性明显高于对照组（P<0.01）,而Fas组与对照组相比无明显差异（P>0.05）。而给予Fas干预后,PE+Fas组与PE相比,Iso+Fas组与Iso组相比,Ang II+Fas组与Ang II组相比均未见明显差异（P>0.05）。5 CaN活性:PE组、Iso组和Ang II组心肌细胞CaN活性均明显高于对照组（P<0.01）,而Fas组与对照组相比无明显差异（P>0.05）。而给予Fas干预后,PE+Fas组与PE相比,Iso+Fas组与Iso组相比,Ang II+Fas组与Ang II组相比CaN蛋白活性均未见明显差异（P>0.05）。6 c-fos蛋白表达:PE组、Iso组和Ang II组心肌细胞c-fos蛋白表达均明显高于对照组（P<0.01）,而Fas组与对照组相比无明显差异（P>0.05）。而给予Fas干预后,PE+Fas组心肌细胞c-fos蛋白表达明显低于PE组（P<0.01）;Ang II+Fas组心肌细胞c-fos蛋白表达明显低于Ang II组（P<0.01）;而Iso+Fas组与Iso组相比无明显差异（P>0.05）。小结:RhoA/Rho激酶通路参与了PE、Ang II所致大鼠心肌细胞肥大,Rho激酶抑制剂Fas通过抑制该通路的激活,抑制c-fos蛋白的表达,改善PE、Ang II所致大鼠心肌细胞肥大,而与CaN通路、MAPK通路无关。Iso所致大鼠心肌细胞肥大不伴有RhoA/Rho激酶通路的激活。第三部分RhoA/Rho激酶信号通路对大鼠STZ糖尿病心肌病的影响目的:研究RhoA/Rho激酶信号通路对大鼠STZ糖尿病心肌病的影响。方法:清洁级雄性Wistar大鼠50只,体重200～250 g,禁食不禁水12 h后,一次性尾静脉注射0.3%的STZ溶液60 mg·kg-1;筛选血糖≥16.7 mmol·L^-1为糖尿病大鼠,随机分成3组:STZ模型组（n=11）、Fas组（n=11）及Cap组（n=10）。另外10只给予等容积缓冲液（0.1 mol/L柠檬酸钠-柠檬酸缓冲液,pH 4.5）设为空白对照组。饲养至给药8 w后,各组分别给予干预药物:Fas组给予Fas 10 mg·kg^-1·d^-1分两次腹腔注射;Cap组给予Cap 30 mg·kg^-1·d^-1分两次灌胃给药;空白对照组和STZ模型组腹腔注射等容积的生理盐水;给药持续4 w。给药期间密切观察各组大鼠活动、进食、饮水、大小便等一般情况,在给药前及给药后每2 w测体重及血糖一次。停药24 h后,测定下列指标:（1）血流动力学:包括HR、LVSP、LVEDP和±dp/dtmax;（2）血液生化:测定Ang II、MDA含量及SOD、LDH、CK活性;（3）HWI;（4）病理形态学:观察心肌的病理学改变并测量CMD和CVF;（5）RT-PCR测定RhoA,Rho激酶mRNA的表达;（6）Western blot测定心肌组织Bax、Bcl-2蛋白表达。结果:1血流动力学:与空白对照组比较,STZ模型组大鼠LVEDP升高75.9%（P<0.01）,而LVSP、+dp/dtmax和-dp/dtmax分别下降23.9%（P<0.01）、25.9%（P<0.01）和19.7%（P<0.01）。与STZ模型组相比,Fas组和Cap组大鼠LVEDP分别下降了20.9%（P<0.05）和27.1%（P<0.01）;Fas组大鼠LVSP、+dp/dtmax和-dp/dtmax分别升高了17.9%（P<0.05）、13.6%（P<0.01）和13.9%（P<0.05）;Cap组大鼠LVSP、+dp/dtmax和-dp/dtmax分别升高了24.4%（P<0.01）、19.6%（P<0.01）和16.6%（P<0.05）。各组大鼠心率无显著性差异（P>0.05）。2血液生化:与空白对照组比较,STZ模型组大鼠血浆Ang II含量升高了127.3%（P<0.01）。与STZ模型组比较,Fas组大鼠血浆Ang II含量无明显变化（P>0.05）,而Cap组大鼠血浆Ang II下降了38.5%（P<0.01）。与空白对照组比较,STZ模型组大鼠血清SOD降低了51.5%（P<0.01）,而MDA升高了95.8%（P<0.01）。与STZ模型组比较,Fas组及Cap组大鼠血清SOD分别升高了40.5%（P<0.05）和62.6%（P<0.01）;而MDA分别下降了14.8%（P<0.01）和24.3%（P<0.01）。与空白对照组比较,STZ模型组血清LDH和CK分别升高了149.4%（P<0.01）和149.5%（P<0.01）。与STZ模型组比较,Fas组及Cap组大鼠血清LDH分别下降了45.1%（P<0.01）和42.8%（P<0.01）;CK分别下降了30.9%（P<0.01）和43.6%（P<0.01）。3 HWI:与空白对照组比较,STZ模型组、Fas组及Cap组大鼠体重均显著下降（P<0.01）。而STZ模型组大鼠HWI与对照组比较升高了13.1%（P<0.01）,经Fas及Cap治疗后,HWI分别下降了6.3%（P<0.05）和8.7%（P<0.01）。4病理形态学:电镜下观察可见:空白对照组大鼠心肌肌原纤维结构清晰,排列整齐,胶原纤维较少,线粒体排列整齐,无肿胀变性;STZ模型组大鼠心肌细胞明显肥大,肌纤维增粗,明显增多,结构紊乱,典型肌小节结构破坏,可见断裂现象,线粒体变性肿胀,嵴较稀疏。Fas组和Cap组与模型组相比心肌细胞明显减小,线粒体结构基本正常,胶原含量减少,排列较整齐。与空白对照组比较,STZ模型组大鼠心肌CMD和CVF分别升高了18.3%（P<0.01）和50.9%（P<0.01）。与STZ模型组比较,Fas组和Cap组大鼠CMD分别下降了8.7%（P<0.05）和12.0%（P<0.01）;心肌CVF分别下降了19.8%（P<0.05）和26.9%（P<0.01）。5 RhoA,Rho激酶mRNA的表达:与空白对照组比较,STZ模型组大鼠心肌组织RhoA、Rho激酶mRNA表达上调（P<0.01）。与STZ模型组比较Fas组及Cap组大鼠心肌组织RhoA、Rho激酶mRNA表达下调（P<0.01）。结果提示,STZ诱导后其RhoA、Rho激酶mRNA表达显著上调,而经Fas干预后,RhoA、Rho激酶mRNA表达显著下降。6 Bax、Bcl-2蛋白表达:与空白对照组比较,STZ模型组大鼠心肌组织Bax蛋白表达升高（P<0.01）,而Bcl-2蛋白表达下降（P<0.01）,与STZ模型组比较,Fas组及Cap组大鼠心肌组织Bax蛋白表达下降（P<0.01）,而Bcl-2蛋白表达升高（P<0.01）。结果提示,STZ诱导的糖尿病大鼠心肌组织Bax蛋白表达升高而Bcl-2蛋白表达下降,经Fas和Cap的干预,可逆转这种病理变化。小结:在糖尿病心肌病的发病过程中,伴有RhoA/Rho激酶信号通路的激活;Fas可抑制该通路的激活,改善了糖尿病心肌病的心肌病理变化及心脏功能,延缓了向心力衰竭的发展;其机制可能与抑制Ang II/Gq/RhoA/Rho激酶信号途径,抑制脂质过氧化反应,提高机体抗氧化能力,调节凋亡基因的表达有关。结论1 RhoA/Rho激酶信号通路参与了Iso诱导的心肌肥厚的发病过程;Rho激酶抑制剂Fas可抑制该通路的激活,改善Iso诱导的心肌肥厚的心肌病理变化及心脏功能。Fas还可以抑制脂质过氧化反应,提高机体抗氧化能力,发挥对心肌的保护作用。2 RhoA/Rho激酶通路参与了PE、Ang II所致大鼠心肌细胞肥大;Rho激酶抑制剂Fas通过抑制该通路的激活,抑制c-fos蛋白的表达,改善PE、Ang II所致大鼠心肌细胞肥大,而与CaN通路、MAPK通路无关。Iso所致大鼠心肌细胞肥大不伴有RhoA/Rho激酶通路的激活。3 RhoA/Rho激酶信号通路参与了糖尿病心肌病的发病过程;Rho激酶抑制剂Fas可抑制该通路的激活,改善了糖尿病心肌病的心肌病理变化及心脏功能,延缓了向心力衰竭的发展;其机制可能与抑制Ang II/Gq/RhoA/Rho激酶信号途径,抑制脂质过氧化反应,提高机体抗氧化能力,调节凋亡基因的表达有关。更多还原

【Abstract】 Cardiac hypertrophy is the common pathology of a variety of cardiovascular diseases, which is characterized by the increase in cell volume and protein synthesis and fetal gene expression up-regulation. Although hypertrophy is initially a compensatory mechanism that helps sustain cardiac output, prolonged hypertrophy will inevitably give rise to heart failure and subsequent cardiac death. Therefore, it is very important to prevent or reverse cardiac hypertrophy.Cardiac hypertrophy is the consequence of hypertrophic stimulus-induced changes in gene expression, which is linked by intracellular signal transduction. It is likely, however, that there are“molecular phenotype”differences underlying cardiac hypertrophy triggered by different stimuli, which is caused by the different signal pathways that they initiated. Studying on the signaling pathways in cardiomyocyte hypertrophy contributed to elucidate the cellular and molecular mechanisms of cardiac hypertrophy and might find some new strategies for the prevention of cardiac hypertrophy.RhoA/Rho kinase signaling pathway plays an important role in mediating various cellular functions such as contraction, adhesion,motility, proliferation and apoptosis, and all of which are involved in the pathogenesis of cardiovascular diseases such as hypertension, restenosis, atherosclerosis, pulmonary hypertension, cerebral vasospasm, vascular aneurysms, myocardial ischemia reperfusion injury and cardiac hypertrophy. RhoA/Rho kinase signaling pathway plays an important role in cardiovascular diseases. However, it has not been evaluated the roles of RhoA/Rho kinase signaling pathway in cardiac hypertrophy induced by isoproterenol （Iso） and diabetic cardiomyopathy induced by streptozotocin （STZ）.In this study, we used Rho kinase inhibitor fasudil （Fas） to investigate: （1） Roles of RhoA/Rho kinase signaling pathway in cardiac hypertrophy induced by Iso in rats; （2） Roles of RhoA/Rho kinase signaling pathway in cardiomyocyte hypertrophy induced by phenylephrine （PE）, Iso and angiotensin II （Ang II）; （3） Roles of RhoA/Rho kinase signaling pathway in diabetic cardiomyopathy induced by STZ in rats.Part 1 Roles of RhoA/Rho kinase signaling pathway in cardiac hypertrophy induced by Iso in ratsObjective: To investigate the roles of RhoA/Rho kinase signaling pathway in cardiac hypertrophy induced by Iso in rats.Methods: Forty male Wistar rats （200-220 g） were supplied by the Experimental Animal Center of Hebei Medical University. Rats were randomly and equally divided into 5 groups （n=8）: control group, Iso model group, low-dose Fas group, high-dose Fas group and captopril （Cap） group. Except for control group, cardiac hypertrophy models rats were induced by injection of Iso (5 mg·kg^-1·d^-1 ih.) for 7 d. At the same time, Fas group and Cap group were treated with Fas (2, 10 mg·kg^-1·d^-1 ip.) and Cap (30 mg·kg^-1·d^-1 ig.) respectively; control group and Iso model group were given an equal volume of onormal saline. Twenty-four hours after the treatment, the following indices were determined: （1） Haemodynamic parameters, including heart rate （HR）, maximum ascending and descending rate of left ventricular pressure （±dp/dtmax）, left-ventricular systolic pressure （LVSP） and left-ventricular end diastolic pressure （LVEDP）, were recorded by using MS4000U-1C biological signal quantitative recording and analyzing system; （2） Biochemical indices, including the contents of Ang II and malondialdehyde （MDA）, the activities of superoxide dismutase （SOD）, creatine kinase （CK） and lactate dehydrogenase （LDH）; （3） Heart weight index （HWI）; （4） Histopathological examinations: cardiomyocyte diameter （CMD） and collagen volume fraction （CVF） were analyzed; （5） Expressions of RhoA and Rho kinase mRNA were tested by reverse transcription polymerase chain reaction （RT-PCR）.Results: 1 Detection of haemodynamic changes: In this experiment, there was a significant increase in LVEDP and significant decrease in LVSP, +dp/dtmax and -dp/dtmax in Iso model group compared with control group. The values of LVEDP increased by 34.3% （P<0.01）, LVSP, +dp/dtmax and -dp/dtmax decreased by 16.8% （P<0.01）, 16.2% （P<0.01） and 14.4% （P<0.01） respectively in Iso model group compared with control group. Fas (10 mg·kg^-1·d^-1) and Cap treatment reduced the values of LVEDP by 16.1% （P<0.05） and 28% （P<0.01） respectively from Iso model levels. Fas (10 mg·kg^-1·d^-1) treatment increased the values of LVSP, +dp/dtmax and -dp/dtmax by 14.7% （P<0.05）, 10.2% （P<0.05） and 9.9% （P<0.05） respectively from Iso model levels. Cap treatment increased the values of LVSP, +dp/dtmax and -dp/dtmax by 15.8% （P<0.05）, 14.4% （P<0.01） and 9.9% （P<0.05） respectively from Iso model levels. Heart rate was not significantly different in all groups （P>0.05）.2 Analysis of biochemical indices in blood: The content of Ang II was significantly increased by 34.2% in Iso model group compared with control group （P<0.01）. Cap treatment reduced the content of Ang II markedly by 34.8% （P<0.01） from Iso model levels. The content of MDA was significantly increased by 117.0% in Iso model group compared with control group （P<0.01）. Fas (2, 10 mg·kg^-1·d^-1) and Cap treatment reduced the content of MDA by 9.8% （P<0.05）, 25.1% （P<0.01） and 28.0% （P<0.01） respectively. The activity of SOD was greatly decreased by 60.4% （P<0.01） in Iso model group compared with control group. Fas (2, 10 mg·kg^-1·d^-1) and Cap treatment elevated the activity of SOD by 30.7% （P<0.05）, 64.8% （P<0.01） and 85.7% （P<0.01） respectively. The activities of LDH and CK were greatly increased by 20.7% （P<0.01）, 46.4% （P<0.01） respectively in Iso model group compared with control group. Fas (2, 10 mg·kg^-1·d^-1) and Cap treatment reduced the activity of LDH by 14.6% （P<0.05）, 36.1% （P<0.01） and 42.8% （P<0.01） respectively, and reduced the activity of CK by by 18.3% （P<0.05）, 32.2% （P<0.01） and 41.4% （P<0.01） respectively.3 Assessment of HWI:Body weight was not significantly different in all groups at the end of the study （P>0.05）. However HWI was significantly increased by 19.2% in Iso model group compared with control group （P<0.01）. Fas (2, 10 mg·kg^-1·d^-1) and Cap treatment reduced HWI by 6.9% （P<0.05）, 7.7% （P<0.01） and 9.0% （P<0.01） respectively from Iso model levels.4 Histopathological examinations: The myocardial structure of rats in control group was clearly evident. Myofibrils are the main components of cytoplasm, and they appeared orderly, with bright and dark areas clearly evident. The myocardial structure in Iso model group was characterized by obvious reductions in myofibril content, breaking, and disordered myofilaments. The pathological changes of rats in Fas and Cap groups were slighter than that of Iso model group. CMD and CVF were significantly increased by 16.9% （P<0.01） and 32.9% （P<0.05） in Iso model group compared with control group （P<0.01）. Fas (2, 10 mg·kg^-1·d^-1) and Cap treatment reduced CMD by 8.1% （P<0.05）, 12.3% （P<0.01） and 13.5% （P<0.01） respectively from Iso model levels, and reduced CVF by 21.6% （P<0.05） and 26.2% （P<0.05） respectively from Iso model levels.5 Expressions of RhoA and ROCK mRNA:Expression levels of RhoA mRNA and Rho kinase mRNA were both significantly higher in Iso group than that in control group （all P<0.01）. Fas (10 mg·kg^-1·d^-1) and Cap treatment significantly down-regulated expression levels of RhoA mRNA and Rho kinase mRNA （P<0.01 and P<0.05 respectively）.Conclusions: RhoA/Rho kinase signaling pathway may play an important role in cardiomyocyte hypertrophy induced byβ-adrenergic receptor stimulation. Rho kinase inhibitor Fas could improve the cardiac dysfunction and the pathological changes in Iso induced cardiac hypertrophy, which is associated with the inhibition of Ang II/Gq/RhoA/Rho kinase signaling pathway and enhancement of antioxidative ability.Part 2 Roles of RhoA/Rho kinase signaling pathway in cardiomyocyte hypertrophy induced by PE, Iso and Ang IIObjective: To investigate the roles of RhoA/Rho kinase signaling pathway in cardiomyocyte hypertrophy induced by PE, Iso and Ang II Methods: Cardiomyocytes were obtained from ventricles of neonatal rats. Bromodeoxyuridine (0.1 mol·L^-1) was present to prevent cardiac fibroblast proliferation. Cultured cardiomyocytes were divided into 8 groups: control group, Fas group (Fas 10-5 mol·L^-1), Iso group (Iso 10-5 mol·L^-1), Iso + Fas group (Iso 10-5 mol·L^-1 + Fas 10-5 mol·L^-1), Ang II group (Ang II 10-7 mol·L^-1), Ang II +Fas group (Ang II 10-7 mol·L^-1+Fas 10-5 mol·L^-1), PE group(PE 10-5 mol·L^-1), PE+Fas group (PE 10 -5mol·L^-1+Fas 10-5 mol·L^-1).Cell size was obtained by measuring the diameter of trypsinized cell. The protein content, which was measured by the method of Lowry et al, was divided by total cell number to give cell protein concentration （pg/cell）. Expressions of RhoA and Rho kinase mRNA were tested by RT-PCR. Expression of c-fos protein was determined with Western blot. Calcineurin （CaN） assessment kit was employed to detect the acitivity of CaN. The acitivity of mitogen-activated protein kinases （MAPK） was measured by method of Tianchang Li.Results:1 Cell size: The diameter of cardiomyocyte was significantly increased in PE, Iso and Ang II groups compared with control group （all P<0.01）. Compared with PE group, the diameter of cardiomyocyte was significantly reduced in PE+Fas group （P<0.01）. Compared with Ang II group, the diameter of cardiomyocyte was significantly reduced in Ang II+Fas group （P<0.01）. There were no significant differences of diameters of cardiomyocyte between Iso and Iso+Fas groups （P>0.05）. The single use of Fas had no effect on diameter of cardiomyocyte （P>0.05）.2 Cell protein concentration: The cell protein concentration was significantly increased in PE, Iso and Ang II groups compared with control group （all P<0.01）. Compared with PE group, the cell protein concentration was significantly reduced in PE+Fas group （P<0.01）. Compared with Ang II group, the cell protein concentration was significantly reduced in Ang II+Fas group （P<0.01）. There were no significant differences of the cell protein concentration between Iso and Iso+Fas group （P>0.05）. The single use of Fas had no effect on the cell protein concentration （P>0.05）.3 Expressions of RhoA and Rho kinase mRNA: Expressions of RhoA and Rho kinase mRNA were significantly increased in PE and Ang II groups compared with control group （all P<0.01）. Compared with PE group, expressions of RhoA and Rho kinase mRNA were significantly reduced in PE+Fas group （all P<0.01）. Compared with Ang II group, expressions of RhoA and Rho kinase mRNA were significantly reduced in Ang II+Fas group （all P<0.01）. There were no significant differences among them in control, Iso and Iso+Fas groups （all P>0.05）.4 The activity of MAPK: The activity of MAPK was significantly increased in PE, Iso and Ang II groups compared with control group （all P<0.01）. There were no significant differences of the activities of MAPK between PE and PE+Fas groups, Iso and Iso+Fas groups, Ang II and Ang II+Fas groups （all P>0.05）.5 The activity of CaN: The activity of CaN was significantly increased in PE, Iso and Ang II groups compared with control group （all P<0.01）. There were no significant differences of the activities of CaN between PE and PE+Fas groups, Iso and Iso+Fas group, Ang II and Ang II+Fas group （all P>0.05）.6 Expression of c-fos protein: Expression of c-fos protein was significantly increased in PE, Iso and Ang II groups compared with control group （all P<0.01）. Compared with PE group, expression of c-fos protein was significantly reduced in PE+Fas group （P<0.01）. Compared with Ang II group, expression of c-fos protein was significantly reduced in Ang II+Fas group （all P<0.01）. There were no significant differences of c-fos protein expression between Iso and Iso+Fas groups （P>0.05）.Conclusions: RhoA/Rho kinase signaling pathway may play an important role in the cardiomyocyte hypertrophy induced by PE and Ang II. Fas could improve the cardiomyocyte hypertrophy induced by PE and Ang II by inhibiting expression of c-fos protein. Part 3 Roles of RhoA/Rho kinase signaling pathway in STZ induced diabetic cardiomyopathyObjective: To investigate the roles of RhoA/Rho kinase signaling pathway in STZ induced diabetic cardiomyopathy and whether administration of Fas, a relatively specific Rho kinase inhibitor, would prevent the diabetic cardiomyopathy as well as the mechanism involved.Methods: Fifty male Wistar rats （200-250 g） were supplied by the Experimental Animal Center of Hebei Medical University. Diabetes was induced in rats fasted for 12 h by a single injection of STZ (60 mg·kg-1, dissolved in fresh 0.1 mol·L^-1 citrate buffer, pH 4.5) via the caudal vein of animals. Age-matched rats （n=10） were used as controls and were administered with an equivalent volume of citrate buffer alone via the same route. Induction of diabetes was confirmed by measuring blood glucose levels. Blood sample was taken from the caudal vein 1 week after STZ injection and the blood glucose level exceeded 16.7 mmol·L^-1 was considered diabetic. After 8 weeks, diabetic rats in good conditions were randomized into 3 groups, diabetic （n=11）, Fas （n=11） and Cap （n=10） groups. Rats of Fas and Cap groups were administered with Fas （5 mg·kg-1 bid, ip.） or Cap （15 mg·kg-1 bid, ig.） respectively, while control and diabetic rats were treated with vehicle (saline, 1 ml·kg^-1 bid, ip.) for 4 weeks. Blood glucose levels and body weights were then measured biweekly in both the control and diabetic rats and the general characteristics of all rats were observed throughout the treatment. Twenty-four hours after the treatment, the following indices were determined. （1） Haemodynamic parameters, including heart rate, LVSP, LVEDP and±dp/dtmax, were recorded by using MS4000U-1C biological signal quantitative recording and analyzing system; （2） Biochemical indices, including the contents of Ang II and MDA, the activities of SOD, CK and LDH; （3） Heart weight index （HWI）; （4） Histopathological examinations: CMD and CVF were analyzed; （5） Expressions of RhoA and Rho kinase mRNA were tested by RT-PCR; （6） Expressions of Bax and Bcl-2 proteins were quantified by Western blot. Results:1 Detection of haemodynamic changes: In this experiment, there was a significant increase in LVEDP and significant decrease in LVSP, +dp/dtmax and -dp/dtmax in STZ model group compared with control group. The values of LVEDP was increased by 75.9% （P<0.01）, LVSP, +dp/dtmax and -dp/dtmax were decreased by 23.9% （P<0.01）, 25.9% （P<0.01） and 19.7% （P<0.01） respectively in Iso model group compared with control group. Fas and Cap treatment reduced the values of LVEDP by 20.9% （P<0.05）, 27.1% （P<0.01） respectively from STZ model levels. Fas treatment increased the values of LVSP, +dp/dtmax and -dp/dtmax by 17.9% （P<0.05）, 13.6% （P<0.05） and 13.9% （P<0.05） respectively from STZ model levels. Cap treatment increased the values of LVSP, +dp/dtmax and -dp/dtmax by 24.4% （P<0.01）, 19.6% （P<0.01） and 16.6% （P<0.05） respectively from STZ model levels. Heart rate was not significantly different in all groups （P>0.05）.2 Analysis of biochemical indices in blood: The content of Ang II was significantly increased by 127.3% in STZ model group compared with control group （P<0.01）. Cap treatment reduced the content of Ang II by 38.5% （P<0.01） from STZ model levels. The content of MDA was significantly increased by 95.8% in STZ model group compared with control group （P<0.01）. Fas and Cap treatment reduced the content of MDA by 14.8% （P<0.01） and 24.3% （P<0.01） respectively. The activity of SOD was greatly decreased by 51.5% （P<0.01） in STZ model group compared with control group. Fas and Cap treatment increased the activity of SOD by 40.5% （P<0.01） and 62.6% （P<0.01） respectively. The activities of LDH and CK were greatly increased by 149.4% （P<0.01） and 149.5% （P<0.01）, respectively in STZ model group compared with control group. Fas and Cap treatment reduced the activity of LDH by 45.1% （P<0.01） and 47.1% （P<0.01） respectively; and reduced the activity of CK by 30.9% （P<0.01） and 43.6% （P<0.01） respectively.3 Assessment of HWI: Compared with control group, body weight was significantly decreased in STZ model, Fas and Cap groups at the end of the study （all P<0.01）. However HWI was significantly increased by 13.1% in STZ model group compared with control group （P<0.01）. Fas and Cap treatment reduced HWI by 6.3% （P<0.05） and 8.7% （P<0.01） respectively from STZ model levels.4 Histopathological examinations: The myocardial structure of rats in control group was clearly evident. Myofibrils are the main components of cytoplasm, and they appeared orderly, with bright and dark areas clearly evident. The myocardial structure in STZ model group was characterized by obvious reductions in myofibril content, breaking and disordered myofilaments. The pathological changes of rats in Fas and Cap groups were slighter than that of STZ model group. CMD and CVF were significantly increased by 18.3% （P<0.01） and 50.9% （P<0.01） in Iso model group compared with control group. Fas and Cap treatment reduced CMD by 8.7% （P<0.05） and 12.0% （P<0.01）, and reduced CVF by 19.8% （P<0.05） and 26.9% （P<0.01） respectively from Iso model levels.5 Determination of RhoA and ROCK mRNA expressions:Expressions of RhoA and Rho kinase mRNA were both significantly higher in STZ group than that in control group （all P<0.01）. Fas and Cap treatment down-regulated the expression levels of RhoA mRNA （P<0.01 and P<0.05 respectively） and Rho kinase mRNA （P<0.01 and P<0.05 respectively）.Conclusions: RhoA/Rho kinase signaling pathway may play a critical role in the development of diabetic cardiomyopathy. Fas could improve the cardiac dysfunction and the pathological changes in heart of the diabetic animals. This may partly attribute to the suppression of caridac hypertrophy, myocardial fibrosis, and subsequent cardiac remodeling, which is associated with the inhibition of RhoA/Rho kinase signaling pathway, enhancement of antioxidative ability and regulation of expressions of apoptosis-related genes.Conclusions1 RhoA/Rho kinase signaling pathway may play an important role in cardiomyocyte hypertrophy induced byβ-adrenergic receptor stimulation. Rho kinase inhibitor Fas could improve the cardiac dysfunction and the pathological changes in Iso-induced cardiac hypertrophy, which is associated with the inhibition of Ang II/Gq/RhoA/Rho kinase signaling pathway and enhancement of antioxidative ability.2 RhoA/Rho kinase signaling pathway may play an important role in the cardiomyocyte hypertrophy induced by PE and Ang II. Fas could improve the cardiomyocyte hypertrophy induced by PE and Ang II by inhibiting the expression of c-fos protein.3 RhoA/Rho kinase signaling pathway may play a critical role in the development of diabetic cardiomyopathy. Fas could improve the cardiac dysfunction and the pathological changes in heart of the diabetic animals. This may partly attribute to the suppression of caridac hypertrophy, myocardial fibrosis, and subsequent cardiac remodeling, which is associated with the inhibition of Ang II/Gq/RhoA/Rho kinase signaling pathway, enhancement of antioxidative ability, and regulation of expressions of apoptosis-related genes.更多还原