【作者】 鲁静朝；

【导师】 崔炜；

【作者基本信息】 河北医科大学 ， 内科学， 2009， 博士

【摘要】 左室肥厚是高血压患者未来心血管病死亡和致残的独立危险因素。左室肥厚最初是对压力负荷过重的代偿性应答,但逐渐演变为失代偿性心肌重构,诱发心脏舒张及收缩功能下降、冠脉储量减少、心律失常及心脏自主神经活性失调及心律失常,最终导致充血性心力衰竭和猝死。逆转左室肥厚能够明显改善高血压患者的预后。近年来,探讨预防和逆转左室肥厚的优化治疗是高血压治疗的一处活跃领域。左室肥厚的病理特征主要包括心肌细胞肥大、心脏成纤维细胞增殖及细胞外基质沉积、心肌壁内小血管重构。引发左室肥厚的致病因素主要包括压力负荷过重,肾素-血管紧张素-醛固酮系统(Renin-angiotensin-aldosterone system, RAAS)激活,氧化应激,炎症因子上调(如TNF-α),转化生长因子(TGF-β1)、内皮素(ET-1)、心钠素(ANP)等细胞因子和血管活性物质表达增多等。此外,新近研究发现,作为肿瘤坏死因子超家族成员,RANKL/RANK/OPG系统不仅参与骨的形成,还与胚胎心脏发育、心肌梗死后心室重构、免疫-炎症性心肌病等有密切关系,是一组多功能的细胞因子系统。临床资料显示RANKL/RANK/OPG系统可能参与了高血压左室肥厚的进程。研究显示,尽管降压治疗是逆转左室肥厚的重要因素,但并不是唯一因素。实验和临床证据均显示,抗氧化及抗炎治疗可以延缓心室肥厚及心力衰竭的进程。在诸多降压药物中,氨氯地平,一种长效的二氢吡啶类钙离子拮抗剂,已被证实在降压作用之外,还可能通过抗氧化及抗炎作用逆转心室肥厚。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),除强效降脂外,还具有不依赖于降脂的多效性作用,如直接抗氧化、抗炎及保护血管内皮等,在临床上广泛应用于预防和治疗心血管疾病。近来研究报道他汀可能通过多效性作用预防心肌肥厚和心力衰竭的发生。越来越多的证据显示氨氯地平和阿托伐他汀联合应用可能通过协同抑制氧化应激和前炎症因子表达而改善动脉硬化,增强一氧化氮(NO)生物利用度,保护血管内皮。在ASCOT-LLA研究中,无明显血脂增高的高血压患者,在服用氨氯地平的基础上加用小剂量阿托伐他汀能够有效减少卒中和未来心血管病事件的发生率。这些结果显示联合应用氨氯地平及阿托伐他汀对于心血管疾病具有协同或叠加效应。虽然联合应用氨氯地平及阿托伐他汀对于血管的协同保护效应已逐步被认可,但在氨氯地平治疗的基础上,加用阿托伐他汀能否进一步改善心室肥厚及可能的药理机制目前并不清楚。随着年龄的增长,自发性高血压大鼠(Spontaneously hypertensive rats, SHR)由代偿性心室肥厚发展为心力衰竭,在心脏血流动力学及神经激素激活方面和人类高血压性左室重构的发生、发展极为相似。于出生后6~8周SHR大鼠血压开始升高,于16周~48周存在代偿性心室肥厚,约于36周时开始出现心脏舒张功能失调,于70周左右时出现失代偿性心室肥厚,心室腔扩张明显,心脏收缩功能和舒张功能明显下降。一般认为,SHR大鼠是探讨高血压性心脏病的理想动物模型。本研究以36周龄SHR大鼠为研究对象,通过观察氨氯地平和阿托伐他汀单药和联合用药对SHR大鼠心肌肥厚、心脏舒张功能失调、心肌组织氧化应激状态、炎症因子调控、MMPS/TIMPS系统平衡、OPG/RANKL/RANK系统表达等的干预效应,了解在氨氯地平治疗的基础上,加用阿托伐他汀能否进一步改善心室肥厚及心脏功能,并探讨这一联合效应的潜在药理机制,为二者联合用药治疗高血压左室肥厚提供新的思路和实验依据。第一部分联合应用氨氯地平及阿托伐他汀对高血压大鼠心室肥厚的干预效应目的:了解氨氯地平、阿托伐他汀单药及联合用药对高血压大鼠左室肥厚及心脏功能的干预效应,对血浆及心肌AngⅡ水平的影响,探讨联合用药对高血压心室肥厚及心脏功能的改善是否具有协同效应。方法:雄性Wistar-Kyoto大鼠(WKY大鼠)作为正常血压WKY对照组,雄性SHR大鼠随机分为4组,即SHR对照组、氨氯地平组(10 mg/kg/d)、阿托伐他汀组(10 mg/kg/d)、联合用药组(氨氯地平10 mg/kg/d及阿托伐他汀10 mg/kg/d)。灌胃给药12 wk后,应用形态测量学测定左室质量指数(LVMI),经胸超声心动图测定心室壁厚度、左室重量(LVW)及心脏功能,血流动力学测定左室压力及心功能,ELASA法测定血浆BNP含量。用HE染色及Masson三色染色观察心肌细胞肥大程度及心肌间质胶原沉积情况,生化法检测心肌羟脯氨酸含量。用RT-PCR法检测心肌组织ANP、β-MHC、Procollagen I、TGF-βmRNA表达,放免法测定大鼠血浆及心肌AngⅡ含量。结果:1各组大鼠尾动脉血压、体重、心率及血脂的变化给药后氨氯地平组及联合用药组的SBP均明显低于SHR对照组,有统计学差异(P均< 0.05),但两组间相比基本一致,无统计学差异(P > 0.05)。阿托伐他汀组和SHR对照组相比,SBP略有下降,但无统计学差异(P > 0.05)。氨氯地平或(和)阿托伐他汀治疗后并不影响大鼠的体重、心率及血脂含量(均P > 0.05)。2各组大鼠心室肥厚指标的变化SHR对照组大鼠的LVMI、血浆BNP含量、心肌细胞横截面面积、心肌间质纤维化及羟脯氨酸含量均明显高于WKY大鼠(P均< 0.05),经氨氯地平或(和)阿托伐他汀治疗后,SHR大鼠的上述指标明显下降,以联合用药组最为明显(P < 0.05)。3各组大鼠心肌胚胎基因及促纤维化基因的变化和同周龄的WKY大鼠相比,48周龄的SHR大鼠心肌ANP、β-MHC、procollagen I和TGF-β1的mRNA表达明显增强(P均< 0.05)。氨氯地平或(和)阿托伐他汀显著削弱了左室ANP、β-MHC、procollagen I和TGF-β1 mRNA的表达(P均< 0.05),且以联合用药最为明显(P均< 0.05)。4各组大鼠超声心动图的指标变化48周龄时,SHR对照组大鼠的LVW明显高于WKY大鼠(P < 0.05);氨氯地平组和阿托伐他汀组的LVW较SHR大鼠对照组的LVW明显下降(P均< 0.05),并以联合用药组的LVW下降最为明显(P < 0.05)。各组大鼠的LVEF及LVFS基本一致,无明显统计学差异(P均> 0.05),提示心脏整体收缩功能正常。和WKY大鼠相比,48周龄时SHR大鼠对照组的IVRT显著延长(P < 0.05),提示左室舒张功能受损。经氨氯地平或阿托伐他汀治疗后,IVRT有所缩短,但无明显统计学差异(P均> 0.05),仅联合用药时IVRT明显缩短(P < 0.05)。5各组大鼠血流动力学指标的比较SHR大鼠的LVEDP增高、τ延长及dp/dtmin/LVSP降低(P均< 0.05),但dp/dtmax和WKY大鼠基本一致(P > 0.05),提示48周龄时SHR大鼠主要表现为左室舒张功能受损,而心脏整体收缩功能正常。氨氯地平或(和)阿托伐他汀治疗后,SHR大鼠的LVEDP和τ明显降低,dp/dtmin/LVSP明显升高(P均<0.05),改善效应以联合用药最为显著(P均< 0.05)。6各组大鼠外周血及心肌AngⅡ含量的比较48周龄时,SHR对照组血浆AngⅡ浓度与WKY对照组相比无明显差别(P > 0.05),而心肌AngⅡ浓度明显高于WKY对照组(P < 0.05)。用药12周后,氨氯地平组血浆AngⅡ浓度明显高于SHR对照组(P < 0.05),而阿托伐他汀组、联合用药组的血浆AngⅡ浓度和SHR对照组无显著差别(P均> 0.05),提示阿托伐他汀抑制了氨氯地平对SHR大鼠循环RAS的激活。氨氯地平组的心肌AngⅡ浓度和SHR对照组基本一致,无显著差别(P > 0.05),而阿托伐他汀组和联合用药组的心肌AngⅡ浓度却较SHR对照组明显降低(P均< 0.05)。结论:氨氯地平和阿托伐他汀联合用药对高血压左室肥厚及舒张功能的改善具有协同效应,其机制可能与联合用药降低循环及心肌AngII含量有关。第二部分联合应用氨氯地平及阿托伐他汀对高血压大鼠心肌氧化应激的干预作用目的:观察氨氯地平和阿托伐他汀单药及联合用药对SHR大鼠心肌组织氧化应激的影响,探讨联合用药改善高血压左室肥厚及舒张功能障碍的可能机制。方法:采用生化法测定血清TG、TC、HDL、LDL含量,双抗体夹心ELISA法测定血清氧化低密度脂蛋白(oxidized LDL,oxLDL)含量。采用超氧化物阴离子荧光探针染色法测定心肌组织原位的超氧阴离子水平,通过测定心肌组织丙二醛(Maleic Dialdehyde, MDA)水平来间接评估心肌组织的氧化应激状态。采用光泽精化学增强发光法测定左室心肌组织的NADPH氧化酶活动度。采用黄嘌呤氧化酶法测定心肌组织铜/锌超氧化物歧化酶(Cu/Zn SOD)活性。Western blot技术分析心肌组织NADPH氧化酶亚基p22phox、p40phox、p47phox、Rac-1及Cu/ZnSOD的蛋白表达。结果:1血脂及血清oxLDL含量测定WKY大鼠和各组SHR大鼠的血清TG、TC、HDL、LDL含量基本一致,未见明显差别(P > 0.05)。和WKY大鼠相比,SHR大鼠的血清oxLDL含量明显升高,氨氯地平及阿托伐他汀治疗组血清oxLDL含量较同期SHR大鼠均显著下降(P均< 0.05),且双药组较单药组降低更明显(P < 0.05)。2各组大鼠心肌原位超氧化物水平及MDA水平和同龄WKY大鼠相比,48周龄SHR大鼠DHE染色强度和MDA水平明显增加(P均< 0.05)。氨氯地平和阿托伐他汀显著削弱了左室DHE染色强度和MDA水平(P均< 0.05)。联合用药则进一步降低了心脏的DHE染色强度和MDA水平(P均< 0.05)。3各组大鼠心肌组织NADPH氧化酶和Cu/Zn SOD活动度的影响SHR大鼠的左室NADPH氧化酶活动度显著强于WKY大鼠( P < 0.05)。氨氯地平和阿托伐他汀单药显著降低了SHR大鼠左室NADPH氧化酶活动度(P均< 0.05)。联合用药则进一步降低了左室NADPH氧化酶活动度(P < 0.05)。相反,SHR大鼠的Cu/Zn SOD活动度明显降低(P < 0.05),氨氯地平和阿托伐他汀及联合用药轻微增强了Cu/Zn SOD的活动度,但与SHR相比,并无明显统计学差异(P均> 0.05)。4各组大鼠心脏NADPH氧化酶亚基及Cu/Zn SOD的蛋白表达和同龄WKY大鼠相比,SHR大鼠心肌组织膜蛋白提取物的p22phox、p40phox、p47phox和Rac-1蛋白表达显著增强(P均< 0.05)。氨氯地平或阿托伐他汀显著降低了心肌组织p22phox、p40phox、p47phox、及Rac-1的蛋白表达(P均< 0.05)。和氨氯地平单药相比,联合用药进一步降低了心肌组织p22phox、p47phox及Rac-1的蛋白表达(P均< 0.05),但对p40phox表达的抑制效应未见明显差异(P > 0.05)。和同龄WKY大鼠相比,SHR大鼠Cu/Zn SOD的蛋白表达有所降低(P < 0.05),但氨氯地平、阿托伐他汀及联合用药未能改善降低的Cu/Zn SOD水平(P均> 0.05)。结论:SHR大鼠血清oxLDL含量、心肌组织DHE染色强度及MDA水平明显高于WKY大鼠,表明氧化应激参与了SHR大鼠心肌肥厚及心脏舒张功能失调的发生与发展。氨氯地平和阿托伐他汀可能通过抑制NADPH氧化酶亚基转位和活性,降低氧化应激,改善SHR大鼠心肌肥厚及心脏功能。联合应用氨氯地平和阿托伐他汀可能通过对氧化应激的协同干预效应,改善心肌肥厚和心脏功能。第三部分联合应用氨氯地平及阿托伐他汀对高血压大鼠心肌促炎症因子的干预作用目的:观察氨氯地平和阿托伐他汀单药及联合用药对SHR大鼠循环hs-CRP、TNF-α、IL-1β水平的影响,及对心肌组织TNF-α、IL-1β、NF-κBp65、IκB-α蛋白表达的影响,探讨联合用药改善高血压左室肥厚及舒张功能障碍的潜在药理机制。方法:用ELISA法测定大鼠血清hs-CRP含量,放免法测定血清TNF-α、IL-1含量。HE染色观察炎症细胞浸润情况,用Western blot技术检测大鼠心肌TNF-α、IL-1β、NF-κB、IκB-α的蛋白表达,免疫组化观察NF-κB p65的核移位情况。结果:1氨氯地平、阿托伐他汀及联合用药对SHR大鼠心肌组织炎症细胞浸润的影响:和WKY大鼠相比,SHR大鼠心肌有较多炎症细胞浸润。氨氯地平或(和)阿托伐他汀治疗后,可见少量炎症细胞浸润。联合用药效果最为明显。2氨氯地平、阿托伐他汀及联合用药对SHR大鼠血清hs-CRP、TNF-α、IL-1β含量的影响:SHR对照组大鼠血清hs-CRP、TNF-α、IL-1β含量明显高于WKY大鼠(P均< 0.05)。氨氯地平、阿托伐他汀及联合用药治疗后,SHR大鼠血浆hs-CRP、TNF-α含量显著下降(P均< 0.05);联合用药组下降尤为显著(P < 0.05)。氨氯地平、阿托伐他汀轻度降低SHR大鼠血浆IL-1β含量,但和SHR对照组大鼠相比,差异无明显统计学意义(P均> 0.05),仅联合用药显著降低SHR大鼠血浆IL-1β含量(P < 0.05)。3氨氯地平、阿托伐他汀及联合用药对SHR大鼠心肌组织TNF-α、IL-1β蛋白表达的影响:Western Blot结果显示:SHR大鼠心肌组织TNF-α、IL-1β的蛋白表达与同龄WKY大鼠比较明显升高(P均< 0.05);氨氯地平、阿托伐他汀干预后心肌组织TNF-α、IL-1β的蛋白表达明显下降(P均< 0.05)。联合用药干预后SHR大鼠心肌组织TNF-α、IL-1β的蛋白表达较单药干预进一步下降(P均< 0.05)。4氨氯地平、阿托伐他汀及联合用药对SHR大鼠心肌组织NF-κB p65蛋白表达的影响:Western Blot结果显示:SHR大鼠心肌组织NF-κB p65蛋白表达与同龄WKY大鼠比较明显升高(P < 0.05);氨氯地平、阿托伐他汀干预后心肌组织NF-κB p65的蛋白表达明显下降(P均< 0.05)。联合用药干预后SHR大鼠心肌组织NF-κB p65的蛋白表达较单药组降低更加明显(P均< 0.05)。免疫组化结果显示:WKY大鼠NF-κB p65蛋白主要在心肌细胞及心脏成纤维细胞胞浆中表达,细胞核中弱表达;SHR大鼠心肌组织NF-κB p65蛋白表达增加,且在心肌细胞核及心脏成纤维细胞核中表达增加,核染色阳性细胞数目增多。氨氯地平、阿托伐他汀干预后心肌组织NF-κB p65表达水平明显减低,核移位减少。联合用药组较单药组降低更加明显。5氨氯地平、阿托伐他汀及联合用药对SHR大鼠心肌组织IκB-α蛋白表达的影响::Western Blot结果显示:和同龄WKY大鼠相比,SHR大鼠心肌组织IκB-α蛋白表达明显减少(P < 0.05);氨氯地平、阿托伐他汀干预后心肌组织IκB-α的蛋白表达增加,但和SHR大鼠相比无统计学差异(P均> 0.05)。联合用药干预后SHR大鼠心肌组织IκB-α的蛋白表达明显增加(P < 0.05)。免疫组化结果显示:和Western Blot结果一致。结论:氨氯地平或(和)阿托伐他汀可能通过下调心肌组织TNF-α、IL-1β、NF-κB p65的蛋白表达,上调IκB-α的蛋白表达,改善心肌肥厚和心脏舒张功能失调,联合用药对心肌组织炎症应答的抑制效果最佳。第四部分联合应用氨氯地平和阿托伐他汀对高血压大鼠心肌MMPs/TIMPs的干预作用目的:了解MMP-2、MMP-9、TIMP-1及TIMP-2在自发性高血压大鼠心肌组织中的表达情况,并探讨氨氯地平和阿托伐他汀单药和联合用药对MMP-2、MMP-9、TIMP-1及TIMP-2的干预作用。方法:用明胶酶图法测定明胶酶MMP-2及MMP-9的活性,用Western blot技术检测大鼠心肌MMP-2、MMP-9、TIMP-1及TIMP-2的蛋白表达,用RT-PCR检测MMP-2、MMP-9、TIMP-1及TIMP-2的mRNA水平。结果:1氨氯地平、阿托伐他汀及联合用药对SHR大鼠心肌组织MMP-2、MMP-9活性的影响:明胶酶谱结果显示:SHR大鼠心肌MMP-2、MMP-9明胶酶活性与同龄WKY大鼠比较明显升高(P均< 0.05)。氨氯地平或阿托伐他汀干预后MMP-2、MMP-9的明胶酶活性明显下降(P均< 0.05),且双药组较单药组降低更加明显(P均< 0.05)。2氨氯地平、阿托伐他汀及联合用药对SHR大鼠心肌组织MMP-2、MMP-9蛋白及mRNA表达的影响:Western Blot及RT-PCR结果显示:SHR大鼠心肌MMP-2、MMP-9的蛋白及mRNA表达水平与同龄WKY大鼠比较明显升高(P均< 0.05)。氨氯地平或阿托伐他汀干预后MMP-2、MMP-9蛋白及mRNA表达水平明显下降(P均< 0.05),且双药组较单药组降低更加明显(P均< 0.05)。3氨氯地平、阿托伐他汀及联合用药对SHR大鼠心肌组织TIMP-1蛋白及mRNA表达的影响:Western Blot及RT-PCR结果显示:SHR大鼠心肌TIMP-1蛋白及mRNA表达水平与同龄WKY大鼠比较有所升高(P均< 0.05)。氨氯地平或阿托伐他汀及联合用药干预后SHR大鼠心肌TIMP-1的蛋白及mRNA表达略有减弱,但与SHR对照组之间无明显统计差异,且三个药物干预组之间亦无明显统计学差异(P均> 0.05)。4氨氯地平、阿托伐他汀及联合用药对SHR大鼠心肌组织TIMP-2蛋白及mRNA表达的影响:Western Blot及RT-PCR结果显示:SHR大鼠心肌组织TIMP-2蛋白及mRNA表达水平与同龄WKY大鼠基本一致,无明显统计学差异(P均> 0.05)。氨氯地平、阿托伐他汀及联合用药对SHR大鼠心肌TIMP-2的蛋白及mRNA表达无明显的增强和抑制效应(P均> 0.05)。结论:氨氯地平或(和)阿托伐他汀降低了SHR大鼠心肌组织MMP-2、MMP-9的活性、蛋白及mRNA表达,以联合用药效果最佳。氨氯地平或(和)阿托伐他汀可能通过干预MMP-2/TIMP-2和MMP-9/TIMP-1系统失衡,改善高血压大鼠的心肌间质纤维化。第五部分RANKL/RANK/OPG系统在高血压大鼠心肌肥厚中的作用及氨氯地平、阿托伐他汀的干预效应目的:了解SHR大鼠心肌肥厚过程中RANKL/RANK/OPG系统的变化及氨氯地平和阿托伐他汀对它的干预作用。方法:用免疫组化法检测RANKL、RANK、OPG的蛋白表达及组织定位,用Western blot技术检测大鼠心肌RANKL、RANK、OPG的蛋白表达,用RT-PCR检测RANKL、RANK、OPG的mRNA表达,结果:1氨氯地平、阿托伐他汀及联合用药对SHR大鼠心肌组织RANKL蛋白及mRNA表达的影响:免疫组化显示:WKY大鼠的心肌细胞、心脏成纤维细胞、血管内皮细胞及血管平滑肌细胞均可见RANKL少量表达。SHR大鼠心肌细胞、心脏成纤维细胞、血管内皮细胞及血管平滑肌细胞表达增强,阳性染色细胞数明显增多,染色加深。此外,SHR大鼠小动脉内附壁的单核细胞及心肌间质内炎症细胞可见RANKL表达。氨氯地平或阿托伐他汀干预后SHR大鼠心肌RANKL表达减弱,阳性染色细胞数减少,染色变浅(P均< 0.05)。联合用药组较单药组对RANKL表达的降低效应更加明显(P均< 0.05)。Western Blot及RT-PCR结果显示:SHR大鼠心肌RANKL蛋白及mRNA表达水平与同龄WKY大鼠比较明显升高(P均< 0.05)。氨氯地平或(和)阿托伐他汀干预后RANKL蛋白及mRNA表达水平明显下降(P均< 0.05),且联合用药组较单药组降低更加明显(P均< 0.05)。2氨氯地平、阿托伐他汀及联合用药对SHR大鼠心肌组织RANK蛋白及mRNA表达的影响:免疫组化显示: WKY大鼠的心肌细胞、心脏成纤维细胞、血管内皮细胞及血管平滑肌细胞均可见RANK少量表达。SHR大鼠心肌细胞、心脏成纤维细胞、血管内皮细胞及血管平滑肌细胞表达增强,阳性染色细胞数明显增多,染色加深。氨氯地平或阿托伐他汀干预后SHR大鼠心肌RANK表达减弱,阳性染色细胞数减少,染色变浅(P均< 0.05)。联合用药较阿托伐他汀降低RANK表达的效应更加明显(P均< 0.05)。Western Blot及RT-PCR结果显示:SHR大鼠心肌RANK蛋白及mRNA表达水平与同龄WKY大鼠比较明显升高(P均< 0.05)。氨氯地平或阿托伐他汀干预后RANK的蛋白及mRNA表达水平明显下降(P均< 0.05),且双药组较阿托伐他汀组降低更加明显(P均< 0.05)。3氨氯地平、阿托伐他汀及联合用药对SHR大鼠心肌组织OPG蛋白及mRNA表达的影响:免疫组化显示: WKY大鼠的心肌细胞、心脏成纤维细胞均可见少量OPG表达,血管内皮细胞及血管平滑肌细胞未见表达。SHR大鼠心肌细胞、心脏成纤维细胞OPG表达增强,阳性染色细胞数明显增多,染色加深;血管内皮细胞及血管平滑肌细胞OPG可见少量表达。氨氯地平、阿托伐他汀及联合用药组干预后SHR大鼠心肌OPG表达减弱(P均< 0.05),但三个药物干预组之间无明显统计学差异(P > 0.05)。Western Blot及RT-PCR结果显示:SHR大鼠心肌OPG蛋白及mRNA表达水平与同龄WKY大鼠比较明显升高(P均< 0.05)。氨氯地平、阿托伐他汀及联合用药干预后SHR大鼠心肌OPG的蛋白及mRNA表达水平降低(P均< 0.05),但三个药物干预组之间无明显统计学差异(P均> 0.05)。结论: SHR大鼠心肌组织RANKL、RANK、OPG蛋白及mRNA表达增高,RANKL/RANK/OPG系统活化可能是SHR大鼠心室重构的重要原因。氨氯地平或(和)阿托伐他汀降低了RANKL、RANK、OPG蛋白及mRNA表达,以联合用药效果最佳。更多还原

【Abstract】 Cardiac hypertrophy is an independent risk factor for cardiovascular morbidity and mortality in patients with hypertension. Cardiac hypertrophy initially occurs as a compensatory response to pressure overload, but gradually leads to inadequate remodeling, cardiac diastolic and systolic dysfunction, impaired coronary preservation, cardiac arrhythmia, dysregulated cardiac sympathetic nervous activity, and finally induces congestive heart failure and sudden death. Therefore, investigation of novel treatments to prevent and reverse cardiac hypertrophy is an area of intense activity in treatment of hypertension.The main pathologic characteristics of left ventricular hypertrophy (LVH) include cardiomyocyte hypertrophy, cardiacfibroblast proliferation, interstitial fibrosis and intramyocardial small coronary artery remodeling. Several pathogenic factors was proved to be related with LVH such as pressure overload, activated Renin-angiotensin -aldesterone system (RAS), oxygen stress, inflammatory cytokine upregulation, transforming growth factors (TGF-β1), endothelin (ET-1), atrial natriuretic peptide (ANP) and so on. In addition, as members of tumor necrosis factor super family, RANKL/RANK/OPG system was reported to be a multi-functional cell cytokine system, not only to be involved in the formation of bone but also closely related with the development of embryonic heart, post-infarcted ventricular remodeling and immune-inflammatory cardiomyopathy.The regression of left ventricular hypertrophy (LVH) by antihypertensive treatment is associated with improvement in the prognosis of patients with hypertension. However, previous studies have shown that lowering of blood pressure is an important, but not the sole factor for the reversal of cardiac hypertrophy. Recently, accumulating experimental and clinical evidence has indicated that inhibition of oxidative stress (excessive production of ROS) and inflammatory response may delay the progression of cardiac hypertrophy and heart failure. In addition to its antihypertensive activity, amlodipine, a long-acting dihydropyridine calcium channel blocker (CCB), has been shown to exert a favorable effect on regression of cardiac hypertrophy, via inhibition of oxidative stress. Statins, HMG-COA reductase inhibitors, are potent inhibitors of cholesterol biosynthesis, and exert direct antioxidative and anti-inflammatory effects and improve vascular endothelial function, which may result in significant prevention and treatment of cardiovascular disease (CVD), independently of their lipid-lowering effect. A growing body of evidence supports the notion that statins might prevent cardiac hypertrophy and the development of heart failure, through pleiotropic effects.Accumulating evidence has shown that co-administration of amlodipine and statins has additive beneficial effects on inhibition of atherosclerosis and preservation of NO bioavailability, through inhibition of oxidative stress and proinflammatory cytokine expression. Moreover, in the ASCOT-LLA study, low-dose atorvastatin (10 mg/day) was shown to reduce significantly stroke and some cardiac end points when added to amlodipine in patients with hypertension and average cholesterol levels. These results suggest a possible synergetic or additive beneficial effect of combined amlodipine and atorvastatin on cardio vascular disease (CVD). The beneficial effects on vascular protection have been investigated extensively. However, to the best of our knowledge, whether co-administration of amlodipine and atorvastatin has an additive beneficial effect on advanced cardiac hypertrophy, and the underlying pharmacological mechanisms, remain poorly understood.Spontaneously hypertensive rats (SHR) have been used frequently as an ideal model of genetic hypertension and hypertensive heart disease, which develops heart failure with aging, similar to that in humans. SHR with heart failure mimic hypertension-induced cardiac remodeling and heart failure in humans, caused by cardiac and hemodynamic as well as neurohormonal abnormalities during the transition from compensated cardiac hypertrophy to heart failure. In general, SHR usually has elevated blood pressure at the age of 6~8 week, presented with compensatory cardiac hypertrophy at 16~48 week old and cardiac diastolic dysfunction at 36 week old roughly, and finally accompanied with decompensated cardiac hypertrophy, obvious ventricular dilation, diminished cardiac systolic and diastolic function at the age of 70 week.In the present study, we used 36-week-old SHR as a model of hypertensive cardiac hypertrophy complicated with early-stage diastolic dysfunction, to examine the effects of amlodipine and atorvastatin alone and combination of both drugs on advanced cardiac hypertrophy, cardiac diastolic dysfunction, myocardial oxidative stress, regulation of inflammatory cytokine, balance of MMPs/TIMPs system, and expression of RANKL/RANK/OPG system. Thereby, the purpose of the presented study was to investigate the following: whether addition of atorvastatin exerts further beneficial effects on advanced cardiac hypertrophy and diastolic dysfunction compared with amlodipine monotherapy; the possible underlying pharmacologic mechanism of the additive beneficial effect of combination therapy; and proposing new idea and experimental evidence for combination therapy on hypertensive cardiac hypertrophy.Section 1. The effects of combined amlodipine and atorvastatin on cardiac hypertrophy in SHRObjective: To examine the effects of amlodipine, atorvastatin, and their combination on cardiac hypertrophy, cardiac function, circulatory and myocardial ANG II level in SHR, and explore whether combination therapy has synergestic effects on hypertensive cardiac hypertrophy and improvement of cardiac function.Methods: male Wistar-Kyoto (WKY) rats were chosen as WKY control group with normal pressure, male SHR rats were divided into four groups including vehicle-treated SHR, amlodipine-treated SHR (10 mg/kg/d), atorvastatin-treated SHR (10 mg/kg/d), combination of amlodipine and atorvastatin-treated SHR (both 10 mg/kg/d). Drugs were administered by oral gavage over 12 weeks. Left ventricular mass index (LVMI) was assessed by morphology measurement. The thicknesses of left ventricle walls, left ventricle weight (LVW) and cardiac function were measured by transthoracic echocardiography. Left ventricular pressure and function were assessed by hemodynamic examination. Plasma brain natriuretic peptide (BNP) was measured by ELISA assay. Cardiomyocyte hypertrophy and collagen accumulation in cardiac tissue were measured by HE and Masson staining respectively. The hydroxyproline content of cardiac tissue was examined by biochemistry technique. The mRNA expression of ANP,β-MHC, Procollagen I and TGF-βwere measured with RT-PCR. Plasma and myocardial angiotensin II level were analyzed by radio-immune assay.Results:1 Effects on blood pressure, body weight, heart rate and serum lipid level:Amlodipine alone or combined with atorvastatin decreased SBP significantly to a similar degree (P < 0.05), whereas the same dose of atorvastatin alone had little effect on SBP during 12 weeks treatment in SHR (P > 0.05). Twelve weeks treatment with amlodipine, atorvastatin or their combination did not significantly affect body weight, heart rate or serum lipid level of SHR (P > 0.05).2 Effects on cardiac hypertrophy:In contrast to WKY, SHR were presented with increased LVMI, increased plasma BNP level, as well as cardiomyocyte cross-sectional area and interstitial fibrosis in SHR (each P < 0.05). Treatment with amlodipine or atorvastatin alone significantly decreased LVMI, plasma BNP level, as well as cardiomyocyte cross-sectional area and interstitial fibrosis in SHR (each P < 0.05). Moreover, combined amlodipine and atorvastatin treatment induced significant reversal of LVH and attenuation of plasma BNP level, as well as decreased cardiomyocyte cross-sectional area and interstitial fibrosis in SHR to a greater extent than each agent alone (each P < 0.05).3 Effects on expression of cardiac hypertrophic and profibrotic gene markers:Increased expressions of the LV ANP,β-MHC, collagen I and TGF-β1 mRNA were observed in 48 weeks SHR compared with WKY with same age (each P < 0.05). Amlodipine or atorvastatin alone significantly attenuated the increase of LV ANP,β-MHC, pro-collagen I and TGF-β1 mRNA expression in SHR (each P < 0.05), and combination therapy suppressed the expression even further (each P < 0.05).4 Echocardiography:Echocardiographic examination showed that LVW were higher in 48-week-old SHR than that in WKY with same age (P < 0.05). There was diastolic dysfunction in SHR as compared with WKY as illustrated by prolonged IVRT (P < 0.05), but LV end-diastolic dimension as well as LVEF and LVSF were not different from WKY (P > 0.05), Which indicated systolic function remained unaltered in 48-week-old SHR. Treatment with amlodipine or atorvastatin alone tended to decrease IVRT in SHR, but only combination therapy significantly attenuated IVRT (P < 0.05).5 Hemodynamic examinations:There was diastolic dysfunction in SHR as compared with WKY as illustrated by increased LVEDP, prolongedτ, and decreased dP/dtmin/LVSP (each P < 0.05), but dP/dtmax were not different from WKY, Which indicated systolic function remained unaltered in 48-week-old SHR. Both amlodipine and atorvastatin reduced LVEDP andτ, increased dP/dtmin/LVSP (each P < 0.05). Furthermore, in contrast to amlodipine monotherapy, the combination therapy further reduced LVEDP,τand increased dP/dtmin/LVSP in SHR (each P < 0.05).6 Effects on plasma and myocardial Ang II level:There was no difference in plasma Ang II level between SHR and WKY (P > 0.05), however, myocardial Ang II level was obviously higher in SHR than that in WKY (P < 0.05). After 12-week drug administration, amlodipine increased plasma Ang II level (P < 0.05) but atorvastatin and combination therapy did not alter plasma Ang II level in SHR (P > 0.05). This outcome indicated that atorvastatin inhibited the elevation of plasma angiotensin II level induced by amlodipine. Myocardial AngII concentration of amlodipine group is similar to SHR control group, however, Myocardial AngII concentration of atorvastatin and combination therapy group were lower than that of SHR control group (both P < 0.05).Conclusions: Combined amlodipine and atorvastatin treatment had synergistic effects on improvement of hypertensive cardiac hypertrophy and diastolic dysfunction, which was related with decreased myocardial angiotensin II level.Section 2. The effects of combined amlodipine and atorvastatin on myocardial oxidative stress in SHRObjective: To examine the effects of amlodipine, atorvastatin and their combination on myocardial oxidative stress, and explore the probable mechanism of the additive beneficial effects on hypertensive cardiac hypertrophy and diastolic dysfunction.Methods: The serum lipid level was measured by biochemical assay. The concentration of serum oxLDL was detected with ELISA methold. Cardiac oxidative stresses were detected using two approaches. The oxidative fluorescence dye dihydroethidium (DHE) was used to evaluate in situ ROS generation in the LV; the levels of malondialdehyde (MDA) were measured as indirect oxidative marker. NADPH oxidase activity of LV tissue homogenate was measured by lucigenin chemiluminescence. Cu/Zn SOD activity was evaluated by xanthine oxidase methold. Western blot was used to evaluate the protein expression of both NADPH oxidase subunits including p22phox, p40phox, p47phox, Rac-1 and Cu/Zn SOD.Results:1 The levels of serum lipid and oxLDL: There are no differences of lipid levels between WKY and SHR (P > 0.05). Serum oxLDL concentration of SHR control group was significantly higher than that of the control group (P < 0.05). Serum oxLDL concentration both in amlodipine and atorvastatin administration group were significantly dropped compared with that of same aged WKY rats ( each P < 0.05), and those in the combined drug group were even dropped down further than that of the single drug groups( each P < 0.05).2 Effects on cardiac superoxide and MDA level: the intensity of DHE staining and cardiac MDA level of 48-week-old SHR was significantly enhanced compared with WKY (each P < 0.05). Both amlodipine and atorvastatin reduced intensity of DHE staining and LV MDA level in SHR to a similar degree (each P < 0.05). Furthermore, combination treatment decreased cardiac intensity of DHE staining and MDA level of SHR to a greater extent compared with amlodipine alone (each P < 0.05).3 Effects on cardiac NADPH oxidase and Cu/Zn SOD activity: LV NADPH oxidase activity was significantly higher in SHR control group compared with that in WKY group (P < 0.05). Amlodipine or atorvastatin monotherapy significantly reduced LV NADPH oxidase activity in SHR (each P < 0.05), and activity was further decreased by combination therapy (each P < 0.05). Conversely, Cu/Zn SOD activity was significantly lower in the vehicle SHR group than that in the WKY group (P < 0.05). Amlodipine and atorvastatin, alone and in combination, only slightly but not significantly enhanced Cu/Zn SOD activity (P > 0.05).4 Effects on cardiac NADPH oxidase subunits and Cu/Zn SOD expression: p22phox, p40phox, p47phox and Rac-1 levels in membrane fraction of cardiac tissues in SHR were significantly higher than those in WKY (each P < 0.05). p22phox, p40phox, p47phox and Rac-1 levels in membrane fraction from cardiac tissues were significantly reduced by amlodipine or atorvastatin (each P < 0.05). Combination therapy reduced p22phox, p47phox, and Rac-1 levels rather than p40phox further compared with amlodipine monotherapy (each P < 0.05). Cu/Zn SOD levels in SHR were significantly higher than that in WKY (P < 0.05). Cu/Zn SOD levels in SHR were not altered by amlodipine, atorvastatin, or their combination (each P > 0.05).Conclusion: Serum oxLDL concentration, myocardial DHE intensity and MDA level were significantly higher in SHR than those in WKY. These findings suggested that oxidative stress may take part in genesis and development of hypertensive cardiac hypertrophy and cardiac diastolic dysfunction. Inhibition of cardiac hypertrophy and preservation of cardiac function by amlodipine and atorvastatin might be attributed to their inhibition of NADPH oxidase subunits expression and enzyme activity. The addition of atorvastatin to amlodipine achieved more benefits as compared with each monotherapy on advanced cardiac hypertrophy and early diastolic dysfunction. These additive benefits were likely to be related with prominent attenuation on NADPH oxidase-mediated ROS generation in left ventricle from SHR.Section 3. The effects of combined amlodipine and atorvastatin on myocardial proinflammatory cytokine in SHRObjective: To explore whether activated proinflammatory cytokine network may play some role in the additive beneficial effects of amlodipine, atorvastatin, and their combination on cardiac hypertrophy and cardiac diastolic dysfunction, through examining the effects of each drug and co-administration therapy on circulatory levels of hs-CRP, TNF-α, IL-1β, and the protein expressions of cardiac TNF-α, IL-1β, NF-κBp65, IκBαin SHR.Methods: The levels of serum hs-CRP were detected with ELISA. The serums TNF-α, IL-1βwere detected by radioimmunity assay (RIA). Cardiac inflammatory cell infiltration was observed by HE staining. The protein expressions of TNF-α, IL-1β, subunit P65 of NF-κB and IκBαwere detected with Western blot technique. The translocation of NF-κB p65 from cytoplasm to nucleus was observed using immunnohistochemistry.Results:1 The effects on cardiac inflammatory cell infiltration by amlodipine, atorvastatin and combination therapy: cardiac inflammatory cell infiltration in SHR was higher than that in WKY. Both amlodipine and atorvastatin inhibit local inflammatory cell infiltration in cardiac tissue, and combination therapy inhibits further compared with each monotherapy.2 The effects on serum concentration of hs-CRP, TNF-α, IL-1βby amlidipine, atorvastatin, and their combination: the serum levels of hs-CRP, TNF-α, IL-1βwere significantly higher in SHR control group than that of WKY control group (each P < 0.05). The serum levels of hs-CRP and TNF-αin the amlodipine or atorvastatin administration group were significantly dropped compared with SHR control group (each P< 0.05), and those in the combined drug group were even dropped down further than that of the single drug groups (P < 0.05). Both amlodipine and atorvastatin slightly but not significantly decreased serum IL-1βconcentration (each P > 0.05), and combination therapy significantly decreased serum IL-1βconcentration compared with SHR control group (P < 0.05). 3 The effects on protein expression of TNF-α, IL-1βby amlodipine, atorvastatin, and their combination: Compared with that of same aged WKY, the protein expressions of TNF-α, IL-1βwere proved to be enhanced in SHR control group by western blot (P < 0.05). Furthermore, all the proteins were reduced markedly after amlodipine or atorvastatin intervention (each P < 0.05), and that of the combined drug group was even lower than that of the single drug groups (each P < 0.05).4 The effects on protein expression of NF-κB p65 by amlodipine, atorvastatin, and their combination:Western Blot: Compared with that of same aged WKY, the protein expressions of NF-κB p65 were significantly enhanced in SHR control group (each P < 0.05). Furthermore, the protein expressions of NF-κB p65 were reduced markedly after amlodipine or atorvastatin intervention (each P < 0.05), and that of the combined drug group was even lower than that of the single drug groups (P < 0.05).Immunohistochemistry: The expression of NF-κB p65 was weaker within the nucleus of cardiocmyocyte and cardiac fibroblast cells in WKY group, whereas was significantly enhanced in SHR control group, that of the amlodipine and atorvastatin were obviously reduced compared with that of SHR control group, and that in the combined drug group was even lower than that of the single drug groups.5 The effects on protein expression of IκB-αby amlodipine, atorvastatin, and their combination: Western Blot: The protein expression of IκB-αin SHR group was obviously reduced (P < 0.05), whereas that in amlodipine and atorvastatin groups were only slightly enhanced compared with that in SHR group (each P > 0.05), and that in the combined drug group was significantly higher than that in the SHR group (P < 0.05).Immunohistochemistry: just as that of Western Blot.Conclusions: By way of down regulation for myocardial TNF-α, IL-1β, NF-κBp65 as well as up regulation for IκB-α, amlodipine and atorvastatin may obviously reverse cardiac hypertrophy and improve cardiac diastolic dysfunction. Amlodipine combined with atorvastatin may have additive effect on inhibiting of inflammatory response.Section 4. The effects of combined amlodipine and atorvastatin on the balance of myocardial MMPs/TIMPs system in SHRObjective: To explore the effect of amlodipine, atorvastatin, and their combination on the balance of myocardial MMPs/TIMPs system, by investigating the changes of MMP-2, MMP-9 as well as TIMP-1, TIMP-2 expression in SHR.Methods: Geltin zemography was used to evaluate the activity of MMP-2 and MMP-9. The protein expressions of MMP-2, MMP-9 as well as TIMP-1, TIMP-2 were detected with Western blot. RT-PCR was used to observe the gene expression of MMP-2, MMP-9 as well as TIMP-1, TIMP-2.Results:1 The effects on activity of MMP-2, MMP-9 by amlodipine, atorvastatin and their combination:Gelatin zemography: The activity of MMP-2, MMP-9 was higher in SHR than that in WKY (each P < 0.05). Either amlodipine or atorvastatin lowered the activity of MMP-2 and MMP-9 of SHR (each P < 0.05); furthermore, combination therapy had the best lowering effect (P < 0.05).2 The effects on protein and mRNA expression of MMP-2, MMP-9 by amlodipine, atorvastatin and their combination:Western Blot and RT-PCR: The protein and mRNA expression levels of MMP-2 and MMP-9 in SHR were all obviously increased in contrast to that in WKY (each P < 0.05). The protein and mRNA expression levels of MMP-2 and MMP-9 in the amlodipine or atorvastatin administration groups were obviously reduced compared with that in SHR group (each P < 0.05). The protein and mRNA expression levels of MMP-2 and MMP-9 in the combined amlodipine and atorvastatin group were even lower than that in the single amlodipine or atorvastatin groups (each P < 0.05).3 The effects on protein and mRNA expression of TIMP-1 by amlodipine, atorvastatin and their combination:Western Blot and RT-PCR: The protein and mRNA expression levels of TIMP-1 in SHR were obviously increased in contrast to that of WKY (each P < 0.05). Nevertheless, either amlodipine or atorvastatin as well as combination therapy did not alter myocardial TIMP-1 levels in SHR (each P > 0.05).4 The effects on protein and mRNA expressio of TIMP-2 by amlodipine, atorvastatin and their combination:Western Blot and RT-PCR: The protein and mRNA expression levels of TIMP-2 in SHR were not different from that in WKY (each P > 0.05). Either amlodipine or atorvastatin as well as combination therapy did not alter myocardial TIMP-2 levels in SHR (each P > 0.05).Conclusion: Both amlodipine and atorvastatin decreased the activity, protein and mRNA expression levels of MMP-2, MMP-9 in SHR, and combination therapy had the best lowering effects. Thereby, by way of maintaining the balance of MMP-2/TIMP-2 and MMP-9/TIMP-1, amlodipine and atorvastatin may obviously relieve interstitial fibrosis.Section 5. The role of RANKL/RANK/OPG system in cardiac hypertrophy and the effects of combined amlodipine and atorvastatinObjective: To explore the role of RANKL/RANK/OPG system in cardiac hypertrophy, by investigating the changes of RANKL, RANK, and OPG expression in SHR and WKY. Furthermore, to elucidate the effects of amlodipine, atorvastatin, and their combination on the balance of myocardial RANKL/RANK/OPG system.Methods: The immunohistochemistry was used to evaluate the protein expression and tissue localization of RANKL, RANK, and OPG. The protein expressions of RANKL, RANK, and OPG were detected with Western blot. RT-PCR was used to observe the mRNA expression of RANKL, RANK, and OPG.Results:1 The effects on protein and mRNA expression of RANKL by amlodipine, atorvastatin and their combination:Immunohistochemistry: The small amounts of positive expressions of RANKL were present in cardiomyocytes, cardiac fibroblasts, and vascular endothelial as well as smooth mucle cells in WKY. The positive expressions of RANKL in SHR were even darker and larger in size compared with that in WKY in view of their optic density (P < 0.05). In addition, monocytes adhered to coronary artery wall and some inflammatory cells infiltrated in the interstitial area were also stained with RANKL expression. Both amlodipine and atorvastatin decreased the average integrated optic density obviously compared with that of SHR control group (each P < 0.05), and combination therapy reduced it further (each P < 0.05).Western Blot and RT-PCR: The protein and mRNA expression levels of RANKL in SHR control group were all obviously increased in contrast to that in WKY (each P < 0.05). The protein and mRNA expression levels of RANKL in either amlodipine or atorvastatin alone groups were obviously reduced compared with that in SHR control group (each P < 0.05), and that of the combined amlodipine and atorvastatin group was even lower than that of the amlodipine or atorvastatin alone groups (each P < 0.05).2 The effects on protein and mRNA expression of RANK by amlodipine, atorvastatin and their combination:Immunohistochemistry: The small amounts of positive expressions of RANKL were present in cardiomyocytes, cardiac fibroblasts, and vascular endothelial as well as smooth mucle cells in WKY. The positive expressions of RANKL in SHR were even darker and larger in size compared with that in WKY in view of their optic density (P < 0.05). Both amlodipine and atorvastatin decreased the average integrated optic density obviously compared with that of SHR control group (each P < 0.05), and combination therapy reduced it further (each P < 0.05).Western Blot and RT-PCR: The protein and mRNA expression levels of RANK in SHR control group were all obviously increased in contrast to that of WKY (each P < 0.05). The protein and mRNA expression levels of RANK in either amlodipine or atorvastatin alone groups were obviously reduced compared with that in SHR control group (each P < 0.05), and that in the combined amlodipine and atorvastatin group was even lower than that of atorvastatin alone group (each P < 0.05).3 The effects on protein and mRNA expression of OPG by amlodipine, atorvastatin and their combination:Immunohistochemistry: The positive expressions of OPG were present in cardiomyocytes, cardiac fibroblasts but not in vascular endothelial and smooth mucle cells in WKY. The positive expressions of OPG in SHR were even darker and larger in size compared with that in WKY in view of their optic density (P < 0.05). Amlodipine and atorvastatin as well as combination therapy decreased the average integrated optic density obviously compared with that of SHR control group (each P < 0.05), but there was no difference among three different treatment (P > 0.05).Western Blot and RT-PCR: The protein and mRNA expression levels of OPG in SHR control group were all obviously increased in contrast to that of WKY (each P < 0.05). The protein and mRNA expression levels of OPG in either amlodipine or atorvastatin alone groups were obviously reduced compared with that in SHR control group (each P < 0.05), but there was no difference among three different treatment (P > 0.05).Conclusion: Both mRNA and protein expression RANKL, RANK, and OPG in SHR were significantly enhanced compared with WKY, suggesting that the pathologic changes of ventricular remodeling may be associated with the activiation of RANKL/RANK/OPG system. Both amlodipine and atorvastatin inhibit the activation of RANKL/ RANK/OPG system in myocardium and combination therapy inhibits it to a further degree.更多还原