低剂量螺内酯与厄贝沙坦联合应用对大鼠心肌肥厚性重构的干预研究
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摘要
心肌肥厚是心脏对急、慢性血流动力学超负荷和非血流动力学因素的一种基本的适应性反应,是高血压、瓣膜病、急性心肌梗死及先天性心脏病等心血管疾病的一种常见的并发症。心肌肥厚使心力衰竭和心脏性猝死的风险显著增加。心肌肥厚的发生过程中肾素-血管紧张素-醛固酮系统被激活。应用血管紧张素转化酶抑制剂（ACEI）或血管紧张素受体阻断剂（ARB）可降低心衰病人的死亡率和发病率。然而仅仅抑制血管紧张素Ⅱ并不能完全阻断醛固酮的生成，大约有40%的心衰病人在应用血管紧张素转化酶抑制剂治疗时仍伴有醛固酮增高。越来越多的研究表明，醛固酮参与了心肌肥厚、心衰过程。随机临床实验表明，醛固酮受体拮抗剂螺内酯与血管紧张素转换酶抑制剂合用降低了心肌梗死后充血性心力衰竭患者的死亡率。因此，心力衰竭治疗的临床实践指南推荐醛固酮受体拮抗剂作为心衰患者的一线治疗药物。然而，应用醛固酮受体拮抗剂进行治疗的同时增加了并发症风险，特别是高钾血症，可引起严重的心律失常事件。因此，在临床上联合使用醛固酮受体拮抗剂引发高血钾的担忧减少了醛固酮受体拮抗剂在心衰治疗中的应用，特别是高血压伴早期心肌肥厚性重构的治疗是否应用醛固酮受体阻断剂尚无定论。
近几年，越来越多的实验研究中采用“低剂量”醛固酮受体拮抗剂，尽管不同的实验研究采用螺内酯（spironolactone，Sp）的剂量各异，一般接受的概念是低剂量指不影响血压的剂量。实验发现，在过表达肾素-血管紧张素-醛固酮系统的转基因大鼠，低剂量Sp（1mg/kg）与传统使用的剂量(30mg/kg)具有相当的心脏保护作用。临床实验发现，低于常规用量下的螺内酯与其他一线降压药合用可以发挥显著肾脏保护作用。上述研究结果提示，低于传统排钠利尿用量的“低剂量”Sp具有脏器保护作用，此作用不依赖于其血流动力学的改变。而临床研究发现低剂量Sp引发高血钾的风险显著减低。由此我们假定，在不影响肾脏排钠、储钾的低剂量下，醛固酮受体拮抗剂与ACEI或ARB抗心衰药物联合应用可以对抗心脏肥厚性重构，同时又可避免高血钾的风险。为验证上述假设，本研究首先利用压力超负荷致心肌肥厚大鼠模型，评价“低剂量” Sp（不影响血钾）与ARB类药物厄贝沙坦（irbesartan，Ir）联用对心肌肥厚性重构的有效性，并与常规剂量下Sp的作用进行比较。同时测定血清钾浓度，评估其诱发高血钾的风险；并观察低剂量Sp与Ir联合应用对心肌氧化应激状态的影响，以探讨两药联合效应的潜在药理机制。此外，本研究利用核因子NF-E2相关因子(nuclear factor erythroid2-related factor2，Nrf2)基因敲除小鼠，观察Nrf2在外源性醛固酮诱导心肌肥厚中的调控作用。研究预期为临床醛固酮受体拮抗剂应用于心肌肥厚、心衰的治疗提供实验依据。
第一部分低剂量螺内酯与厄贝沙坦联合应用对大鼠心肌肥厚性重构的影响
目的：本研究利用压力超负荷致心肌肥厚大鼠模型，评价“低剂量”醛固酮受体拮抗剂螺内酯与血管紧张素受体阻断剂厄贝沙坦联用对心肌肥厚性重构的有效性，并与常规剂量下螺内酯的作用进行比较。同时测定血清K浓度，评估其诱发高血钾的风险。
方法：选用雄性SD大鼠，腹主动脉部分缩窄（PAAC）法制备心肌肥厚模型。造模4周后用超声诊断仪测定心室壁厚度。随后进行随机分组：假手术组，模型组，厄贝沙坦（15mg/kg/d）组，低剂量螺内酯（1mg/kg/d）+厄贝沙坦（15mg/kg/d）组，常规剂量螺内酯（20mg/kg/d）+厄贝沙坦（15mg/kg/d）组。灌胃给药4周后，超声心动图测定心室壁厚度及心脏收缩功能参数；右侧颈动脉插管至心室腔，记录麻醉动物的血压及左室压力变化等血流动力学参数；计算左室质量指数（左心室重/体重，LVW/BW）；HE染色及Masson染色观察心肌细胞肥大程度及心肌间质胶原沉积情况； RT-PCR法检测心肌组织ANP、β-MHC、Procollagen I、TGF-β等心肌肥厚、纤维化相关生物标志物的mRNA表达。
结果：
1低剂量或常规剂量Sp与Ir联合应用对大鼠LVW/BW的影响：药物治疗结束，各组大鼠体重无明显差别。左心室质量指数（LVW/BW）可用来反映由于压力超负荷所致心肌肥厚。和假手术组相比，模型组左心室质量指数明显增大（p <0.01）。与模型组相比，单用Ir使左心室质量指数有所下降，但无明显差异；低剂量或常规剂量Sp与Ir联合应用均可降低左心室质量指数（p <0.01），但低剂量与高剂量之间无明显差别。提示低剂量Sp与Ir联合应用可降低压力超负荷所致左心室质量指数的增加，且其作用与常规剂量Sp相当。
2低剂量或常规剂量Sp与Ir联合应用对大鼠超声心动图参数的影响：腹主动脉狭窄术后4周动物的室间隔厚度（IVs）、左室后壁厚度（LVPW）均明显增大，而左室内径（LVd）明显降低（p <0.05或p <0.01），射血分数（EF）、短轴缩短率（FS）无明显改变。说明PAAC引起心肌肥厚，收缩功能尚正常，处于代偿阶段，模型成功。药物治疗结束后，各组大鼠的EF及FS基本一致(p均>0.05)，提示心脏整体收缩功能尚正常。单用Ir对IVs、LVPW、LVd无明显影响；低剂量或常规剂量Sp与Ir联合应用均可降低IVs、LVPW，而增大LVd，差异有统计学意义（p <0.05），提示低剂量或常规剂量Sp与Ir联合应用均可降低PAAC所致心肌肥厚，二者作用无明显差别，低剂量Sp作用与常规剂量Sp作用相当。
3低剂量或常规剂量Sp与Ir联合应用对大鼠血流动力学参数的影响：模型组收缩压（SBP）、舒张压（DBP）均明显升高，左室舒张末压（LVEDP）增高及dp/dtmin/LVSP降低(p均<0.05)，但dp/dtmax和假手术组大鼠基本一致，说明腹主动脉狭窄术后8周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。此结果与超声心动图所得结果一致。单用厄贝沙坦治疗对SBP、DBP、LVEDP及dp/dtmin/LVSP无明显影响，但低剂量或常规剂量Sp与Ir联合应用均可降低SBP、DBP，且可拮抗PAAC所致LVEDP的增高及dp/dtmin/LVSP的降低。提示低剂量或常规剂量Sp与Ir联合应用不仅逆转了心肌肥厚，而且改善了左室舒张功能，且低剂量Sp可达到与常规剂量相似的作用。
4低剂量或常规剂量Sp与Ir联合应用对各组大鼠心肌组织的病理改变的影响：HE染色结果显示：和假手术组相比，模型组大鼠心肌细胞横截面面积明显增加(p <0.01)。单用Ir对心肌细胞横截面面积无明显影响，但Ir联合应用低剂量或常规剂量Sp均明显降低了心肌细胞横截面面积(p均<0.05)，提示Ir联合应用低剂量或常规剂量Sp治疗改善了心肌细胞的肥厚程度，且低剂量Sp可达到与常规剂量相似的作用。Masson染色结果显示：和假手术组相比，模型组大鼠心肌间质胶原纤维明显增多，排列紊乱。Ir干预组心肌胶原积聚明显减轻，排列也趋于规则。Ir联合应用低剂量或常规剂量Sp较单独Ir对心肌间质纤维化改善程度更加明显(p均<0.05)。提示低剂量Sp与Ir联合应用可降低压力超负荷所致左心室心肌纤维化，且其作用与常规剂量Sp相当。
5低剂量或常规剂量Sp与Ir联合应用对心肌肥大及纤维化相关生物标志物的影响：心肌肥厚不仅表现为心肌细胞肥大及间质纤维化程度增加，还表现为肥大基因及促纤维化基因的上调。因此，我们观察了各组大鼠心肌肥大基因ANP及β-MHC，促纤维化基因procollagen I和TGF-β1的表达情况。结果表明，腹主动脉狭窄术后8周大鼠心肌ANP、β-MHC、procollagen I和TGF-β1的表达明显增强(p均<0.01)。单独应用Ir削弱了左室ANP、β-MHC、procollagen I和TGF-β1mRNA的表达，但统计无显著差异，Ir联合应用低剂量或常规剂量Sp (p均<0.05)可明显降低左室ANP、β-MHC、procollagen I和TGF-β1mRNA的表达。提示低剂量Sp与Ir联合应用可抑制压力超负荷所致左心室心肌肥大基因及促纤维化基因上调，且其作用与常规剂量Sp相当。
6低剂量或常规剂量Sp与Ir联合应用对血钾、钠的影响：应用醛固酮拮抗剂最重要和常见的不良反应就是高血钾症，为了比较低剂量或常规剂量Sp与Ir联合应用的作用，我们进一步观察了各组对血钾、钠的影响。和对照组相比，常规剂量Sp与Ir联合应用明显使血钾浓度增加，但低剂量Sp与Ir联合应用对血钾无明显影响。各组血钠均无明显改变。提示低剂量Sp与Ir联合应用不会引起血钾增高，此点优于常规剂量Sp。
结论：低剂量螺内酯与厄贝沙坦联合应用可改善腹主动脉缩窄所致心肌肥厚、心肌纤维化及心脏舒张功能。此作用与常规剂量螺内酯与厄贝沙坦联合应用相似。常规剂量螺内酯与Ir联合应用明显升高血钾，而低剂量对血钾无影响。上述实验结果表明，低剂量醛固酮受体拮抗剂与ARB合用具有显著的抗心肌肥厚作用，同时可以降低高血钾风险。
第二部分低剂量螺内酯与厄贝沙坦联合应用对大鼠腹主动脉部分缩窄所致氧化应激的影响
目的：观察低剂量螺内酯（Sp）和厄贝沙坦（Ir）联合用药对腹主动脉部分缩窄（PAAC）大鼠心肌组织氧化应激状态的影响，探讨联合用药改善压力超负荷所致左室肥厚及舒张功能障碍的潜在药理机制。
方法：选用雄性SD大鼠，腹主动脉部分缩窄制备心肌肥厚模型。造模4周后进行随机分组：假手术组，模型组，厄贝沙坦（15mg/kg/d）组，低剂量螺内酯（1mg/kg/d）组，低剂量螺内酯（1mg/kg/d）+厄贝沙坦（15mg/kg/d）组。灌胃给药4周后，生化法测定心肌组织丙二醛（MaleicDialdehyde, MDA）水平。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。采用氮蓝四唑显色法法测定心肌组超氧化物歧化酶（SOD）活性。Western blot技术分析心肌组织NADPH氧化酶（NOX）亚基NOX2、NOX4及SOD亚基SOD-1、SOD-2蛋白表达。
结果：
1Ir、低剂量Sp和低剂量Sp与Ir联合应用对心肌MDA含量的影响: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。和假手术组相比，模型组心肌MDA含量明显增多（p<0.01），表明PAAC引起心肌氧化应激损伤。应用Ir单药或低剂量Sp与Ir联合应用均可降低PAAC所致心肌MDA水平的增加（p<0.05或p<0.01）。与单药相比，联合用药进一步降低了心肌的MDA水平（p<0.05）。上述结果提示Ir可显著改善心肌的氧化应激状态，但Ir与低剂量Sp联合应用效果更佳。
2Ir、低剂量Sp和低剂量Sp与Ir联合应用对心肌NADPH氧化酶活性的影响: NADPH氧化酶为心肌组织中活性氧簇生成的主要酶之一，所以我们观察了NADPH氧化酶在心肌中的改变。和假手术组相比，模型组心肌NADPH氧化酶活性明显增强（p<0.01），应用Ir、低剂量Sp单药或低剂量Sp与Ir联合应用均有降低NADPH氧化酶活性的趋势，但只有低剂量Sp与Ir联合应用有统计学意义（p<0.05）。与单独应用低剂量Sp相比，联合用药进一步降低了心肌的NADPH氧化酶活性（p<0.05）。
3Ir、低剂量Sp和低剂量Sp与Ir联合应用对心肌NADPH氧化酶亚型蛋白表达的影响:为了进一步说明NADPH氧化酶活性改变的机制，我们应用免疫印迹的方法观察心肌NADPH氧化酶亚型NOX2、NOX4的改变。和假手术组相比，模型组心肌NADPH氧化酶NOX4亚型蛋白表达明显增多（p<0.01），但NOX2亚型无明显改变。说明PAAC所致心肌氧化应激损伤可能与NADPH氧化酶NOX4亚型表达增加有关。和模型组相比，单用Ir或低剂量Sp均对NOX4亚型无明显影响；但低剂量Sp与Ir联合应用显著降低了NOX4亚型蛋白表达（p<0.05）。上述结果说明Ir与低剂量Sp联合应用对NOX4亚型蛋白表达的抑制效果更佳，提示低剂量Sp与Ir联合应用降低PAAC所致心肌氧化应激损伤的机制可能与其对NOX4的影响有关。
4Ir、低剂量Sp和低剂量Sp与Ir联合应用对心肌SOD活性的影响: SOD为一线的活性氧簇清除酶，推测PAAC所致氧化应激与SOD功能失调有关。所以我们观察了SOD在心肌中的改变。和假手术组相比，模型组心肌SOD活性明显降低（p<0.05）。和模型组相比，单用Ir或低剂量Sp均对SOD活性有增强趋势，但无统计学差异；而低剂量Sp与Ir联合应用显著增加SOD活性（p<0.05）。
5Ir、低剂量Sp和低剂量Sp与Ir联合应用对心肌SOD亚型蛋白表达的影响:为了进一步说明SOD改变的机制，我们应用免疫印迹的方法观察心肌SOD亚型SOD-1、SOD-2的改变。PAAC使心肌SOD-2蛋白表达明显减少（p<0.01），但SOD-1无明显改变。单用Ir或低剂量Sp均对SOD-2表达有增强趋势，但无统计学差异；而低剂量Sp与Ir联合应用则显著增加SOD-2表达（p<0.05）。上述结果说明Ir与低剂量Sp联合应用对SOD-2蛋白表达的增强效果更佳，提示低剂量Sp与Ir联合应用降低PAAC所致心肌氧化应激损伤的机制可能与其对SOD-2的影响有关。
结论：
1腹主动脉缩窄引起大鼠心肌组织MDA含量增高，NADPH氧化酶活性及NOX4亚基表达增强，SOD活性降低及SOD-2亚基表达降低，提示NADPH氧化酶和SOD介导的氧化应激参与了PAAC大鼠心肌肥厚和心脏舒张功能失调的进程。
2低剂量螺内酯与厄贝沙坦联合应用可降低PAAC所致心肌氧化应激损伤，其可能是通过抑制NADPH氧化酶和增强SOD活性而发挥作用。
第三部分Nrf2转录因子在醛固酮所致小鼠心肌肥厚中的调控作用
目的：为进一步深入探讨醛固酮致心肌肥厚的机制，本研究拟采用Nrf2基因敲除小鼠，观察核因子NF-E2相关因子(nuclear factor erythroid2-related factor2，Nrf2)在外源性醛固酮诱导心肌肥厚中的调控作用。
方法：实验中Nrf2+/+、Nrf2-/-ICR小鼠有河北医科大学第二附属医院神经内科李春岩院士惠赠。对所用动物的基因型进行从DNA水平、mRNA水平、蛋白水平进行鉴定。选用健康雄性Nrf2+/+、Nrf2-/-小鼠，按体重随机分为四组，分别为：Nrf2+/+对照组、Nrf2+/+醛固酮组、Nrf2-/-对照组、Nrf2-/-醛固酮组。醛固酮组小鼠采用皮下植泵方式给药，醛固酮用量为1mg/kg/day，连续给药28天。对照组进行假手术。应用小动物超声影像系统（707B，30Hz）对心脏进行超声测定心室壁厚度及心脏收缩功能。碘[125I]醛固酮放射免疫分析药盒放射免疫γ计数器测定各组小鼠血中醛固酮的浓度。计算心脏质量指数（心脏重/体重，HW/BW），急性胰酶法消化心肌细胞，负载DCFH-DA染料观察心肌氧化应激水平。Western blot技术分析心肌组织Nrf2蛋白表达。RT-PCR法检测心肌组织Nrf2及其下游抗氧化酶血红素加氧酶（heme oxygenase， HO-1）,依赖还原型辅酶Ⅰ/Ⅱ:醌氧化还原酶（NAD(P)H:quinone oxidoreductase1，NQO1）的mRNA表达。
结果：
1Nrf2基因敲除小鼠鉴定结果：分别从DNA、mRNA、蛋白水平鉴定小鼠基因型。通过鼠尾提取DNA并进行PCR扩增，Nrf2-/-小鼠扩增产物条带位于400bp左右位置，而野生型（Nrf2+/+）小鼠的PCR扩增产物位于700bp；提取心肌mRNA进行反转录、扩增，野生型小鼠表达Nrf2mRNA， Nrf2-/-小鼠无Nrf2mRNA表达；提取心肌蛋白进行免疫印迹，野生型小鼠表达Nrf2蛋白，Nrf2-/-小鼠无Nrf2蛋白表达。
2小鼠体内醛固酮血药浓度的测定：Nrf2+/+对照组、Nrf2+/+醛固酮组血药浓度为0.32±0.07ng/mL，1.79±0.52ng/mL，Nrf2-/-对照组、Nrf2-/-醛固酮组血药浓度为0.23±0.05ng/mL，1.48±0.44ng/mL，各醛固酮组血药浓度明显高于相应对照组（p<0.05），表明渗透压泵可以将醛固酮释放到小鼠体内，使醛固酮在体内的含量明显增加。
3小鼠心脏超声心动图改变：各组小鼠的射血分数（EF）、短轴缩短率（FS）基本一致(p均>0.05)，提示心脏整体收缩功能正常。Nrf2+/+对照组、Nrf2-/-对照组的左室舒张末期前壁厚度(LVID)、左室舒张末期后壁厚度(LVPW)、EF、FS等指标相差不大，无明显统计学差异（p均>0.05)。提示基因敲除Nrf2对心脏基本形态和功能无明显影响。和相应的对照组相比，醛固酮组LVID、LVPW均明显增大。与Nrf2+/+醛固酮组相比，Nrf2-/-醛固酮组LVID、LVPW明显增大（p<0.05）。结果说明醛固酮可引起左室心肌肥厚，敲除Nrf2可加重醛固酮所致心肌肥厚。
4小鼠心脏质量指数的变化：Nrf2+/+对照组、Nrf2+/+醛固酮组心脏质量指数为3.92±0.08，4.34±0.31，Nrf2-/-对照组、Nrf2-/-醛固酮组心脏质量指数为3.91±0.31，4.99±0.17。显然，与野生型相比，Nrf2基因敲除并不影响心脏质量指数，给予醛固酮后心脏质量指数明显高于相应对照组（p<0.05），表明醛固酮增加心脏重量。与Nrf2+/+醛固酮组相比，Nrf2-/-醛固酮组心脏质量指数明显增大（p<0.05）。提示Nrf2基因敲除可使醛固酮对心脏质量指数的作用增强。
5小鼠心肌肥厚生物标志物ANP、β-MHC mRNA表达变化：与相应的对照组相比，醛固酮可使心肌肥大基因ANP、β-MHC mRNA表达量均明显增加（p<0.01）。与Nrf2+/+醛固酮组相比，Nrf2-/-醛固酮组ANP、β-MHC mRNA表达量增加更为明显（p<0.01）。提示Nrf2基因敲除增强了醛固酮对心肌肥厚生物标志物影响的作用。
6醛固酮对小鼠心肌ROS水平的影响：以上结果说明醛固酮可引起心肌肥厚，已知氧化应激在心肌肥厚发展中有重要作用，所以我们利用荧光探针DCFH-DA进行活性氧检测进一步观察了醛固酮对心肌的氧化应激的影响。与相应的对照组相比，Nrf2+/+醛固酮组、Nrf2-/-醛固酮组心肌细胞ROS水平均明显增加，分别增加约1.7、2.8倍（p<0.01）。与Nrf2+/+醛固酮组相比，Nrf2-/-醛固酮组ROS水平均明显增大（p<0.01）。提示醛固酮可引起左室心肌氧化应激损伤，敲除Nrf2可加重醛固酮所致心肌氧化应激损伤。
7醛固酮对野生型小鼠心脏Nrf2的影响：以上结果说明醛固酮可引起小鼠心肌肥厚、氧化应激增强，且敲除Nrf2可加重醛固酮致心肌肥厚、氧化应激的作用。为了进一步说明Nrf2在醛固酮致心肌肥厚中的作用，我们通过荧光定量PCR、免疫印迹技术观察醛固酮对小鼠心脏Nrf2表达的影响。醛固酮使Nrf2mRNA及蛋白水平均明显增加。提示醛固酮可上调Nrf2转录因子的表达，可能醛固酮激活了Nrf2转录因子及其下游抗氧化酶通路。
8醛固酮对小鼠心脏抗氧化酶HO-1、NQO-1mRNA表达的影响：既然醛固酮可上调Nrf2转录因子的表达，那么醛固酮对Nrf2的下游抗氧化基因有无影响？所以我们进一步观察醛固酮对Nrf2的下游抗氧化酶HO-1、NQO-1mRNA表达的影响。结果表明，醛固酮使心肌HO-1、NQO-1mRNA表达明显增加,但敲除Nrf2-/-后，醛固酮不会引起使HO-1、NQO-1mRNA表达增加，而使NQO-1mRNA表达明显降低。提示醛固酮可通过Nrf2上调抗氧化酶HO-1、NQO-1的表达，从而抑制醛固酮所致心肌氧化应激损伤及心肌肥厚。
结论：敲除Nrf2基因可加重醛固酮所致小鼠心肌肥厚，提示Nrf2参与了醛固酮所致心肌肥厚的调控作用。醛固酮上调心肌Nrf2转录因子，改变其下游抗氧化酶表达，提示醛固酮在致心肌肥厚的同时亦启动Nrf2-抗氧化酶通路，该通路对心肌肥厚发挥抑制作用。
There is emerging evidence that aldosterone is involved in thepathogenesis of cardiac hypertrophy and heart failure. The use of aldosteronereceptor antagonists for the treatment of myocardial failure and selected casesof hypertension, in combination with the current therapy, has been tested inrecent clinical trials. The Randomized Aldactone Evaluation Study (RALES)study has demonstrated that spironolactone, an aldosterone antagonist, addedto an angiotensin-converting enzyme (ACE) inhibitor in patients with severecongestive heart failure, reduces overall mortality. The EPHESUS study hasshown that eplerenone, added to ACEI or angiotensin AT1receptor blocker(ARB) decreases cardiovascular events and death in patients with leftventricular dysfunction following myocardial infarction. Therefore, clinicalpractice guidelines for heart failure management have designated aldosteronereceptor antagonists as first-line therapies for patients with heart failure andreduced left ventricular ejection fraction (LVEF). However, aldosteronereceptor antagonists carry an increased risk of complications, especiallyhyperkalemia may cause serious arrhythmic events. Accordingly, in clinicalpractice, the benefits of aldosterone receptor antagonists may be reduced andthey are used significantly less frequently compared with the otherguideline-recommended medications.
In recent years, low dose of aldosterone receptor antagonists has beenused in increasing number of experimental studies. It is generally acceptedthat “low-dose” refers to a non-pressure-lowering dose, although the doses ofaldosterone receptor antagonists vary with different reports. It was found thatthe low-dose spironolactone (1mg/kg) had no effect on systolic blood pressurebut improved diastolic dysfunction comparable to a conventional dose (30mg/kg) in a transgenic rat model of RAAS over-expression. A subpressordose of eplerenone markedly ameliorated vascular endothelial dysfunction,cardiac inflammation and fibrosis in salt-sensitive hypertensive rats to asimilar degree as an antihypertensive dose of amlodipine. These resultssuggest that the low-dose of aldosterone receptor antagonists exert organprotective effect independent of hemodynamic changes. The clinical study hasshown that low-dose of spironolactone significantly reduce the risk ofhyperkalemia. Thus, we hypothesize that low doses of aldosterone antagonists,added to ACEI or ARB therapy potentiates the cardioprotective effects, andwith little potential of hyperkalemia.
To verify our hypothesis, this study was designed to evaluate theeffectiveness of "low-dose" and conventional-dose aldosterone antagonistspironolactone in combination with ARB irbesartan on cardiac hypertrophicremodeling induced by abdominal aorta coarctation (PartialAbdominalAortic Constriction, PAAC) in rats. Plasma K level was measured to assesshyperkalemia risk. The effects of low-dose spironolactone in combinationwith irbesartan on myocardial oxidative stress were investigated in order tounderstand the pharmacological mechanisms underlying the potentiationeffects of the two-drug combination. To further explore the mechanism ofaldosterone induced myocardial hypertrophy, the possibility thatNF-E2-related factor (nuclear factor erythroid2-related factor2, Nrf2) wasinvolved in the regulation of cardiac hypertrophy induced by exogenousaldosterone was tested by using Nrf2knockout mice. The results will providesome new insights into the clinical application of aldosterone antagonists totreat cardiac hypertrophy and heart failure.
Section1The effects of low-dose spironolactone added to irbesartan onmyocardial remodelling induced by pressure overload inrats
Objective: To evaluate the effectiveness of "low-dose" andconventional-dose aldosterone antagonist spironolactone in combination withARB irbesartan on cardiac hypertrophic remodeling induced by PAAC in rats. Plasma K level was measured to assess hyperkalemia risk.
Methods: Male SD rats were chosen, the rats were prepared the model ofmyocardial hypertrophy by PAAC. At4weeks after surgery, cardiachypertrophy was confirmed by echocardiographic measurements. Then, PAACrats were divided into4groups: model, irbesartan (Ir,15mg/kg daily),conventional dose of spironolactone (20mg/kg daily) added toirbesartan(CSp+Ir) and low dose spironolactone (1mg/kg daily) added toirbesartan(LSp+Ir). At the end of8weeks, Left ventricular mass index(LVW/BW) was assessed by morphology measurement. The thicknesses ofleft ventricle walls and cardiac function were measured by transthoracicechocardiography. Left ventricular pressure and function were assessed byhemodynamic examination. Cardiomyocyte hypertrophy and collagenaccumulation in cardiac tissue were measured by HE and Masson stainingrespectively. The mRNA expression of ANP, β-MHC, Procollagen I andTGF-β were measured with RT-PCR.
Results:
1Effects on cardiac hypertrophy:1) Echocardiography showed that allrats subjected to PAAC developed cardiac hypertrophy at4weeks aftersurgery. Left ventricular posterior wall thickness (LVPW), interventricularseptum thickness (IVs) were increased, while left ventricular end diastolicdimension (LVd) was decreased in PAAC compared with age-matched shamrats. Which indicated that PAAC in this experimental condition result in hearthypertrophy and the model is successful. There was no significant differenceamong the experimental groups before therapy.2) At the end of drugtreatment, body weight was similar among the experimental groups. The ratioof LV weight to body weight (LVW/BW), an index of LV hypertrophy, was34%greater in model rats than in sham rats, and the overload-induced increasein this parameter was not significantly reduced by monotherapy with Ir, but itwas comparably attenuated by low-dose and conventional Sp added to Ir.3)Echocardiography revealed that LVPW, IVs and LVd were greater in modelrats than in sham rats at8weeks. Monotherapy with Ir didn’t affect these parameters. Combination therapy led to significant decrease in theseparameters compared with monotherapy with Ir, whereas there was nodifference in treatment with low-dose and conventional Sp added to Ir.4)Histological analysis revealed that hemodynamic overload had increased thecross-sectional area of cardiac myocytes in model rats by45%compared withthat apparent in sham rats. The extent of load induced cardiomyocytehypertrophy was reduced by treatment with low-dose and conventional Spadded to Ir (p<0.01). Whereas there was no difference in treatment withlow-dose and conventional Sp added to Ir.5) Hemodynamic overload resultedin upregulation of the expression of ANP and β-MHC, molecular markers ofhypertrophy, in the heart of model rats, and this effect was inhibited bylow-dose and conventional Sp added to Ir. Whereas there was no difference intreatment with low-dose and conventional Sp added to Ir. The results indicatedthat low dose Sp added to Ir can reduce PAAC induced cardiac hypertrophy,furthermore, the effect of low dose Sp is comparable with conventional Sp.
2Effects on cardiac fibrosis: Marked interstitial fibrosis was also detectedin the left ventricle of model rats as compared with matched Sham; thisincrease in cardiac fibrosis was also significantly reduced by low-dose andconventional Sp added to Ir. There was no difference in treatment withlow-dose and conventional Sp added to Ir. The rise in collagen type I andTGF-β1gene expression in the myocardium from model rats was attenuatedby low-dose and conventional Sp added to Ir. Whereas there was no differencein treatment with low-dose and conventional Sp added to Ir. The resultsindicated that low dose Sp added to Ir can reduce PAAC induced cardiacfibrosis, and the effect of low dose Sp is comparable with conventional Sp.
3Effects on cardiac Function: Echocardiography revealed left ventricularejection fraction(LVEF), left ventricular fractional shortening (LVSF) werenot different among the experimental groups (p>0.05), which indicatedsystolic function remained unaltered. Haemodynamic values obtained at theend of the treatment period. PAAC increase systolic blood pressure(SBP) anddiastolic blood pressure(DBP). Ir alone had no significant effect on SBP and DBP (p>0.05), but SBP and DBP was significantly reduced by low-dose andconventional Sp added to Ir. There was diastolic dysfunction in model rats ascompared with Sham as illustrated by increased LVEDP and decreasedDP/dtmin/LVSP (each p <0.05), but dP/dtmaxwere not different from Sham,which indicated systolic function remained unaltered in CMC-PAACrats,which is consistent with the result of echocardiography. Both low-doseand conventional Sp added to Ir reduced LVEDP and increased DP/dt min/LVSP(each p <0.05). The results indicated that low dose Sp added to Ir can reducePAAC induced diastolic dysfunction, and the effect of low dose Sp iscomparable with conventional Sp.
4Effects on the concentration of plasma K+and Na+: There was nodifference in the plasma concentration of Na+among all groups of rats.However, conventional dose of Sp added to Ir significantly elevated potassiumlevel in plasma, whereas, low-dose Sp did not affect the plasma K+concentration. The results indicated that low dose Sp added to Ir do not affectthe plasma K+concentration, which is was superior to conventional Sp.
Conclusion: Low doses of aldosterone antagonists spironolactone,added to ARB therapy potentiates the cardioprotective effects, and with littlepotential of hyperkalemia.
Section2The effects of combined low dose of spironolactone andirbesartan on myocardial oxidative stress induced bypressure overload in rats
Objective: To examine the effects of low dose of spironolactone,irbesartan and their combination on myocardial oxidative stress, and explorethe probable mechanism of the additive beneficial effects on cardiachypertrophy and diastolic dysfunction.
Methods: The levels of malondialdehyde (MDA) were measured asindirect oxidative marker. NADPH oxidase activity of left ventricular tissuehomogenate was measured by lucigenin chemiluminescence. SOD activitywas evaluated by nitro-blue tetrazolium methold. Western blot was used toevaluate the protein expression of both NADPH oxidase subunits including NOX2，NOX4and SOD subunits including SOD-1and SOD-2.
Results:
1Effects on MDA level: We evaluate the level of oxidative stress bydeterminating malondialdehyde (Maleic Dialdehyde, MDA) content.Compared with the sham operation group, model group of myocardial MDAcontent increased significantly (p<0.01), which demonstrate thatPAAC induced myocardial oxidative stress injury. Ir monotherapy, but notlow dose Sp, significantly reduced myocardial MDA level (p<0.05). Thecombination of Ir and low dose Sp further reduce the myocardialMDA level (p<0.05). These results suggest that Ir can significantly improvethe oxidative stress in myocardium, but Ir and low dose spironolactonecombined with better results.
2Effects on cardiac NADPH oxidase activity: NADPH oxidase is oneof the main enzyme to generating active oxygen in myocardialtissue, we observed the changes of NADPH oxidase activity in myocardium.Compared with the sham operation group, model group of myocardialNADPH oxidase activity was significantly enhanced (p<0.01).LSp group, Ir+LSp group, Ir decreased the activity of NADPH oxidase, butthere is significant difference only in Ir+LSp group(p<0.05). Compared withLSp group, combined treatment further reduced the activity of NADPHoxidase (p<0.05) in myocardium.
3Effects on cardiac NADPH oxidase subunits expression: In order tofurther illustrate the change mechanism of NADPH oxidase activity, weused immunoblotting to examine myocardial NADPH oxidasesubunit NOX2, NOX4protein expression. PAAC significantly increased theexpression of myocardial NADPH oxidase isoforms of NOX4protein(p<0.01), but no significant changes in NOX2isoforms. Monotherapy with Iror low dose Sp tended to reduce the expression of NOX4protein, but thedifference did not reach statistical significance. However, the combination ofIr and low dose Sp significantly attenuated the increases in the expression ofNOX4protein (p<0.05). These results suggest a better inhibitory effect of Ir combined with low dose of Sp on the expression of NOX4protein.
4Effects on cardiac SOD activity：Compared with the sham operationgroup, model group of myocardial SOD activity decreased significantly(p<0.05)，and it tended to be reduced by monotherapy with Ir or low dose Sp,but the difference did not reach statistical significance. However, thecombination of Ir and low dose Sp significantly attenuated the decreases inSOD activity (p<0.05).
5Effects on cardiac SOD subunits expression: In order to explainthe mechanism of SOD change, we used immunoblotting to examinemyocardial SOD subtype: SOD-1, SOD-2. Compared with the shamoperation group, model group of myocardial SOD-2proteinexpression increased significantly (p<0.01), but SOD-1had noobvious change. Monotherapy with Ir or low dose Sp tended to increase theexpression of SOD-2protein, but the difference did not reach statisticalsignificance. However, the combination of Ir and low dose Sp significantlyattenuated the decreases in the expression of SOD-2.
Conclusions:
1Abdominal aortic constriction caused the increase of MDA content inrat myocardial tissue, enhanced NADPH oxidase activity and the expressionof subunit NOX4, and decreased the activity of SOD and the expression ofSOD-2subunit, suggesting that oxidative stress induced by NADPH oxidaseand SOD is involved in the process of myocardial hypertrophy and diastolicdysfunction in PAAC rats.
2Low dose Sp combined with irbesartan can reduce PAAC inducedmyocardial oxidative stress injury, which may play a role by inhibiting theNADPH oxidase activity and enhancement of SOD activity.
Section3Regulation effect of Nrf2transcription factor on aldosteroneinduced cardiac hypertrophy in mice
Objective: To explore the role of Nrf2in regulation of myocardialhypertrophy induced by external aldosterone, further explorethe mechanism of aldosterone induced myocardial hypertrophy.
Methods: The Nrf2-null mice backcrossed into a ICR background weregifted by academician Li Chunyan of Department of Internal Medicine,Second Affiliated Hospital of Hebei Medical University. The genotypesof the animal were identified from the DNA， mRNA and proteinlevel. Aldosterone-treated wild type(WT) and Nrf2-null mice were implantedwith osmotic mini-pumps for subcutaneous infusion for4wk.(1mg/kg/·day)for4wk. Heart mass index (HW/BW) was assessed by morphologymeasurement. The thicknesses of left ventricle walls and cardiac function weremeasured by transthoracic echocardiography. Blood aldosterone concentrationwere measured using [125I] aldosterone radioimmunoassay. Myocardialcells were digested by trypsin method, and loaded DCFH-DA dye to observethe level of ROS. Western blot was used to evaluate the protein expression ofNrf2. The mRNA expression of Nrf2, HO-1, NQO-1were measured withRT-PCR.
Results:
1Nrf2knockout mice identification: PCR and agarose electrophoresis oftail DNA show that amplified bands of Nrf2-null mice is in400bp position, and the WT mice is in700bp. we confirmed that the Nrf2ispresent in the WT mouse hearts The protein (Western blot) and RNAexpression (RT-PCR) analysis confirmed disruption of Nrf2in the KO mouseheart.
2Effects on plasma aldosterone level: Plasma aldosterone concentrationwere significantly higher than corresponding control group (p<0.05),suggesting that osmotic pump with constant speed can release of aldosteroneinto mice, which significantly increased aldosterone level in the body.
3Echocardiography: Echocardiography revealed left ventricular ejectionfraction(LVEF), left ventricular fractional shortening (LVSF) were notdifferent among the experimental groups (p>0.05), which indicated systolicfunction remained unaltered. The Nrf2-null in baseline have a norma lleftVentricular anterior wall thickness(LVID), left ventricular posterior wallthickness(LVPW), LVEF, LVFS. These results indicate that there is no evidence for cardiac dysfunction due to disruption of Nrf2. Aldosteroneinfusion led to an increase in LVID and LVPW, however, the aldosterone–induced increase in LVID and LVPW were exaggerated in Nrf2-null mice.These results suggest that Nrf2deficiency exaggerates aldosterone-induced thechange of echocardiography parameters.
4Effects on HW/BW: Aldosterone infusion led to an increase in heartweight to body weight ratio (HW/BW), however, the aldosterone–inducedincrease in HW/BW was exaggerated in Nrf2-null mice. These resultssuggest that Nrf2deficiency exaggerates aldosterone-induced the change ofHW/BW.
5Effects on expression of cardiac hypertrophy gene ANP, β-MHC:Aldosterone infusion led to an upregulation of cardiac hypertrophy geneANP, β–MHC. However, the aldosterone-induced molecular markers ofhypertrophy changes were exaggerated in Nrf2-null mice.
6Effects on ROS level: In view of the anti-oxidative defence role of Nrf2in the heart, we investigated the role of Nrf2in regulating aldosterone-inducedmyocardial oxidative stress. Aldosterone infusion increased myocardial levelsof H2-DCFDA, a marker of ROS, indicating elevated levels of oxidative stressin the heart. The aldosterone-induced oxidative stress levels in the heart weremarkedly enhanced as a result of Nrf2deficiency.
7Effects on expression of Nrf2and antioxidative enzymes, HO-1、NQO-1: The chronic aldosterone infusion-enhanced the mRNA and proteinexpression Nrf2in WT myocardium. In response to the sustained aldosteronestimulation, several Nrf2downstream genes, including HO-1, NQO-1, in WTbut not in Nrf2-null hearts were up-regulated at mRNA level.
Conclusion: Our present study demonstrates that Nrf2is a criticalregulator for maintaining structural and functional integrity of the heart in thesetting of sustained aldosterone stimulation. Mechanistically, itis likely thatNrf2protects against maladaptive cardiac remodelling and dysfunction byrepressing ROS production and up-regulating antioxidative enzymes.
近几年，越来越多的实验研究中采用“低剂量”醛固酮受体拮抗剂，尽管不同的实验研究采用螺内酯（spironolactone，Sp）的剂量各异，一般接受的概念是低剂量指不影响血压的剂量。实验发现，在过表达肾素-血管紧张素-醛固酮系统的转基因大鼠，低剂量Sp（1mg/kg）与传统使用的剂量(30mg/kg)具有相当的心脏保护作用。临床实验发现，低于常规用量下的螺内酯与其他一线降压药合用可以发挥显著肾脏保护作用。上述研究结果提示，低于传统排钠利尿用量的“低剂量”Sp具有脏器保护作用，此作用不依赖于其血流动力学的改变。而临床研究发现低剂量Sp引发高血钾的风险显著减低。由此我们假定，在不影响肾脏排钠、储钾的低剂量下，醛固酮受体拮抗剂与ACEI或ARB抗心衰药物联合应用可以对抗心脏肥厚性重构，同时又可避免高血钾的风险。为验证上述假设，本研究首先利用压力超负荷致心肌肥厚大鼠模型，评价“低剂量” Sp（不影响血钾）与ARB类药物厄贝沙坦（irbesartan，Ir）联用对心肌肥厚性重构的有效性，并与常规剂量下Sp的作用进行比较。同时测定血清钾浓度，评估其诱发高血钾的风险；并观察低剂量Sp与Ir联合应用对心肌氧化应激状态的影响，以探讨两药联合效应的潜在药理机制。此外，本研究利用核因子NF-E2相关因子(nuclear factor erythroid2-related factor2，Nrf2)基因敲除小鼠，观察Nrf2在外源性醛固酮诱导心肌肥厚中的调控作用。研究预期为临床醛固酮受体拮抗剂应用于心肌肥厚、心衰的治疗提供实验依据。
第一部分低剂量螺内酯与厄贝沙坦联合应用对大鼠心肌肥厚性重构的影响
目的：本研究利用压力超负荷致心肌肥厚大鼠模型，评价“低剂量”醛固酮受体拮抗剂螺内酯与血管紧张素受体阻断剂厄贝沙坦联用对心肌肥厚性重构的有效性，并与常规剂量下螺内酯的作用进行比较。同时测定血清K浓度，评估其诱发高血钾的风险。
方法：选用雄性SD大鼠，腹主动脉部分缩窄（PAAC）法制备心肌肥厚模型。造模4周后用超声诊断仪测定心室壁厚度。随后进行随机分组：假手术组，模型组，厄贝沙坦（15mg/kg/d）组，低剂量螺内酯（1mg/kg/d）+厄贝沙坦（15mg/kg/d）组，常规剂量螺内酯（20mg/kg/d）+厄贝沙坦（15mg/kg/d）组。灌胃给药4周后，超声心动图测定心室壁厚度及心脏收缩功能参数；右侧颈动脉插管至心室腔，记录麻醉动物的血压及左室压力变化等血流动力学参数；计算左室质量指数（左心室重/体重，LVW/BW）；HE染色及Masson染色观察心肌细胞肥大程度及心肌间质胶原沉积情况； RT-PCR法检测心肌组织ANP、β-MHC、Procollagen I、TGF-β等心肌肥厚、纤维化相关生物标志物的mRNA表达。
结果：
1低剂量或常规剂量Sp与Ir联合应用对大鼠LVW/BW的影响：药物治疗结束，各组大鼠体重无明显差别。左心室质量指数（LVW/BW）可用来反映由于压力超负荷所致心肌肥厚。和假手术组相比，模型组左心室质量指数明显增大（p <0.01）。与模型组相比，单用Ir使左心室质量指数有所下降，但无明显差异；低剂量或常规剂量Sp与Ir联合应用均可降低左心室质量指数（p <0.01），但低剂量与高剂量之间无明显差别。提示低剂量Sp与Ir联合应用可降低压力超负荷所致左心室质量指数的增加，且其作用与常规剂量Sp相当。
2低剂量或常规剂量Sp与Ir联合应用对大鼠超声心动图参数的影响：腹主动脉狭窄术后4周动物的室间隔厚度（IVs）、左室后壁厚度（LVPW）均明显增大，而左室内径（LVd）明显降低（p <0.05或p <0.01），射血分数（EF）、短轴缩短率（FS）无明显改变。说明PAAC引起心肌肥厚，收缩功能尚正常，处于代偿阶段，模型成功。药物治疗结束后，各组大鼠的EF及FS基本一致(p均>0.05)，提示心脏整体收缩功能尚正常。单用Ir对IVs、LVPW、LVd无明显影响；低剂量或常规剂量Sp与Ir联合应用均可降低IVs、LVPW，而增大LVd，差异有统计学意义（p <0.05），提示低剂量或常规剂量Sp与Ir联合应用均可降低PAAC所致心肌肥厚，二者作用无明显差别，低剂量Sp作用与常规剂量Sp作用相当。
3低剂量或常规剂量Sp与Ir联合应用对大鼠血流动力学参数的影响：模型组收缩压（SBP）、舒张压（DBP）均明显升高，左室舒张末压（LVEDP）增高及dp/dtmin/LVSP降低(p均<0.05)，但dp/dtmax和假手术组大鼠基本一致，说明腹主动脉狭窄术后8周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。此结果与超声心动图所得结果一致。单用厄贝沙坦治疗对SBP、DBP、LVEDP及dp/dtmin/LVSP无明显影响，但低剂量或常规剂量Sp与Ir联合应用均可降低SBP、DBP，且可拮抗PAAC所致LVEDP的增高及dp/dtmin/LVSP的降低。提示低剂量或常规剂量Sp与Ir联合应用不仅逆转了心肌肥厚，而且改善了左室舒张功能，且低剂量Sp可达到与常规剂量相似的作用。
4低剂量或常规剂量Sp与Ir联合应用对各组大鼠心肌组织的病理改变的影响：HE染色结果显示：和假手术组相比，模型组大鼠心肌细胞横截面面积明显增加(p <0.01)。单用Ir对心肌细胞横截面面积无明显影响，但Ir联合应用低剂量或常规剂量Sp均明显降低了心肌细胞横截面面积(p均<0.05)，提示Ir联合应用低剂量或常规剂量Sp治疗改善了心肌细胞的肥厚程度，且低剂量Sp可达到与常规剂量相似的作用。Masson染色结果显示：和假手术组相比，模型组大鼠心肌间质胶原纤维明显增多，排列紊乱。Ir干预组心肌胶原积聚明显减轻，排列也趋于规则。Ir联合应用低剂量或常规剂量Sp较单独Ir对心肌间质纤维化改善程度更加明显(p均<0.05)。提示低剂量Sp与Ir联合应用可降低压力超负荷所致左心室心肌纤维化，且其作用与常规剂量Sp相当。
5低剂量或常规剂量Sp与Ir联合应用对心肌肥大及纤维化相关生物标志物的影响：心肌肥厚不仅表现为心肌细胞肥大及间质纤维化程度增加，还表现为肥大基因及促纤维化基因的上调。因此，我们观察了各组大鼠心肌肥大基因ANP及β-MHC，促纤维化基因procollagen I和TGF-β1的表达情况。结果表明，腹主动脉狭窄术后8周大鼠心肌ANP、β-MHC、procollagen I和TGF-β1的表达明显增强(p均<0.01)。单独应用Ir削弱了左室ANP、β-MHC、procollagen I和TGF-β1mRNA的表达，但统计无显著差异，Ir联合应用低剂量或常规剂量Sp (p均<0.05)可明显降低左室ANP、β-MHC、procollagen I和TGF-β1mRNA的表达。提示低剂量Sp与Ir联合应用可抑制压力超负荷所致左心室心肌肥大基因及促纤维化基因上调，且其作用与常规剂量Sp相当。
6低剂量或常规剂量Sp与Ir联合应用对血钾、钠的影响：应用醛固酮拮抗剂最重要和常见的不良反应就是高血钾症，为了比较低剂量或常规剂量Sp与Ir联合应用的作用，我们进一步观察了各组对血钾、钠的影响。和对照组相比，常规剂量Sp与Ir联合应用明显使血钾浓度增加，但低剂量Sp与Ir联合应用对血钾无明显影响。各组血钠均无明显改变。提示低剂量Sp与Ir联合应用不会引起血钾增高，此点优于常规剂量Sp。
结论：低剂量螺内酯与厄贝沙坦联合应用可改善腹主动脉缩窄所致心肌肥厚、心肌纤维化及心脏舒张功能。此作用与常规剂量螺内酯与厄贝沙坦联合应用相似。常规剂量螺内酯与Ir联合应用明显升高血钾，而低剂量对血钾无影响。上述实验结果表明，低剂量醛固酮受体拮抗剂与ARB合用具有显著的抗心肌肥厚作用，同时可以降低高血钾风险。
第二部分低剂量螺内酯与厄贝沙坦联合应用对大鼠腹主动脉部分缩窄所致氧化应激的影响
目的：观察低剂量螺内酯（Sp）和厄贝沙坦（Ir）联合用药对腹主动脉部分缩窄（PAAC）大鼠心肌组织氧化应激状态的影响，探讨联合用药改善压力超负荷所致左室肥厚及舒张功能障碍的潜在药理机制。
方法：选用雄性SD大鼠，腹主动脉部分缩窄制备心肌肥厚模型。造模4周后进行随机分组：假手术组，模型组，厄贝沙坦（15mg/kg/d）组，低剂量螺内酯（1mg/kg/d）组，低剂量螺内酯（1mg/kg/d）+厄贝沙坦（15mg/kg/d）组。灌胃给药4周后，生化法测定心肌组织丙二醛（MaleicDialdehyde, MDA）水平。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。采用氮蓝四唑显色法法测定心肌组超氧化物歧化酶（SOD）活性。Western blot技术分析心肌组织NADPH氧化酶（NOX）亚基NOX2、NOX4及SOD亚基SOD-1、SOD-2蛋白表达。
结果：
1Ir、低剂量Sp和低剂量Sp与Ir联合应用对心肌MDA含量的影响: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。和假手术组相比，模型组心肌MDA含量明显增多（p<0.01），表明PAAC引起心肌氧化应激损伤。应用Ir单药或低剂量Sp与Ir联合应用均可降低PAAC所致心肌MDA水平的增加（p<0.05或p<0.01）。与单药相比，联合用药进一步降低了心肌的MDA水平（p<0.05）。上述结果提示Ir可显著改善心肌的氧化应激状态，但Ir与低剂量Sp联合应用效果更佳。
2Ir、低剂量Sp和低剂量Sp与Ir联合应用对心肌NADPH氧化酶活性的影响: NADPH氧化酶为心肌组织中活性氧簇生成的主要酶之一，所以我们观察了NADPH氧化酶在心肌中的改变。和假手术组相比，模型组心肌NADPH氧化酶活性明显增强（p<0.01），应用Ir、低剂量Sp单药或低剂量Sp与Ir联合应用均有降低NADPH氧化酶活性的趋势，但只有低剂量Sp与Ir联合应用有统计学意义（p<0.05）。与单独应用低剂量Sp相比，联合用药进一步降低了心肌的NADPH氧化酶活性（p<0.05）。
3Ir、低剂量Sp和低剂量Sp与Ir联合应用对心肌NADPH氧化酶亚型蛋白表达的影响:为了进一步说明NADPH氧化酶活性改变的机制，我们应用免疫印迹的方法观察心肌NADPH氧化酶亚型NOX2、NOX4的改变。和假手术组相比，模型组心肌NADPH氧化酶NOX4亚型蛋白表达明显增多（p<0.01），但NOX2亚型无明显改变。说明PAAC所致心肌氧化应激损伤可能与NADPH氧化酶NOX4亚型表达增加有关。和模型组相比，单用Ir或低剂量Sp均对NOX4亚型无明显影响；但低剂量Sp与Ir联合应用显著降低了NOX4亚型蛋白表达（p<0.05）。上述结果说明Ir与低剂量Sp联合应用对NOX4亚型蛋白表达的抑制效果更佳，提示低剂量Sp与Ir联合应用降低PAAC所致心肌氧化应激损伤的机制可能与其对NOX4的影响有关。
4Ir、低剂量Sp和低剂量Sp与Ir联合应用对心肌SOD活性的影响: SOD为一线的活性氧簇清除酶，推测PAAC所致氧化应激与SOD功能失调有关。所以我们观察了SOD在心肌中的改变。和假手术组相比，模型组心肌SOD活性明显降低（p<0.05）。和模型组相比，单用Ir或低剂量Sp均对SOD活性有增强趋势，但无统计学差异；而低剂量Sp与Ir联合应用显著增加SOD活性（p<0.05）。
5Ir、低剂量Sp和低剂量Sp与Ir联合应用对心肌SOD亚型蛋白表达的影响:为了进一步说明SOD改变的机制，我们应用免疫印迹的方法观察心肌SOD亚型SOD-1、SOD-2的改变。PAAC使心肌SOD-2蛋白表达明显减少（p<0.01），但SOD-1无明显改变。单用Ir或低剂量Sp均对SOD-2表达有增强趋势，但无统计学差异；而低剂量Sp与Ir联合应用则显著增加SOD-2表达（p<0.05）。上述结果说明Ir与低剂量Sp联合应用对SOD-2蛋白表达的增强效果更佳，提示低剂量Sp与Ir联合应用降低PAAC所致心肌氧化应激损伤的机制可能与其对SOD-2的影响有关。
结论：
1腹主动脉缩窄引起大鼠心肌组织MDA含量增高，NADPH氧化酶活性及NOX4亚基表达增强，SOD活性降低及SOD-2亚基表达降低，提示NADPH氧化酶和SOD介导的氧化应激参与了PAAC大鼠心肌肥厚和心脏舒张功能失调的进程。
2低剂量螺内酯与厄贝沙坦联合应用可降低PAAC所致心肌氧化应激损伤，其可能是通过抑制NADPH氧化酶和增强SOD活性而发挥作用。
第三部分Nrf2转录因子在醛固酮所致小鼠心肌肥厚中的调控作用
目的：为进一步深入探讨醛固酮致心肌肥厚的机制，本研究拟采用Nrf2基因敲除小鼠，观察核因子NF-E2相关因子(nuclear factor erythroid2-related factor2，Nrf2)在外源性醛固酮诱导心肌肥厚中的调控作用。
方法：实验中Nrf2+/+、Nrf2-/-ICR小鼠有河北医科大学第二附属医院神经内科李春岩院士惠赠。对所用动物的基因型进行从DNA水平、mRNA水平、蛋白水平进行鉴定。选用健康雄性Nrf2+/+、Nrf2-/-小鼠，按体重随机分为四组，分别为：Nrf2+/+对照组、Nrf2+/+醛固酮组、Nrf2-/-对照组、Nrf2-/-醛固酮组。醛固酮组小鼠采用皮下植泵方式给药，醛固酮用量为1mg/kg/day，连续给药28天。对照组进行假手术。应用小动物超声影像系统（707B，30Hz）对心脏进行超声测定心室壁厚度及心脏收缩功能。碘[125I]醛固酮放射免疫分析药盒放射免疫γ计数器测定各组小鼠血中醛固酮的浓度。计算心脏质量指数（心脏重/体重，HW/BW），急性胰酶法消化心肌细胞，负载DCFH-DA染料观察心肌氧化应激水平。Western blot技术分析心肌组织Nrf2蛋白表达。RT-PCR法检测心肌组织Nrf2及其下游抗氧化酶血红素加氧酶（heme oxygenase， HO-1）,依赖还原型辅酶Ⅰ/Ⅱ:醌氧化还原酶（NAD(P)H:quinone oxidoreductase1，NQO1）的mRNA表达。
结果：
1Nrf2基因敲除小鼠鉴定结果：分别从DNA、mRNA、蛋白水平鉴定小鼠基因型。通过鼠尾提取DNA并进行PCR扩增，Nrf2-/-小鼠扩增产物条带位于400bp左右位置，而野生型（Nrf2+/+）小鼠的PCR扩增产物位于700bp；提取心肌mRNA进行反转录、扩增，野生型小鼠表达Nrf2mRNA， Nrf2-/-小鼠无Nrf2mRNA表达；提取心肌蛋白进行免疫印迹，野生型小鼠表达Nrf2蛋白，Nrf2-/-小鼠无Nrf2蛋白表达。
2小鼠体内醛固酮血药浓度的测定：Nrf2+/+对照组、Nrf2+/+醛固酮组血药浓度为0.32±0.07ng/mL，1.79±0.52ng/mL，Nrf2-/-对照组、Nrf2-/-醛固酮组血药浓度为0.23±0.05ng/mL，1.48±0.44ng/mL，各醛固酮组血药浓度明显高于相应对照组（p<0.05），表明渗透压泵可以将醛固酮释放到小鼠体内，使醛固酮在体内的含量明显增加。
3小鼠心脏超声心动图改变：各组小鼠的射血分数（EF）、短轴缩短率（FS）基本一致(p均>0.05)，提示心脏整体收缩功能正常。Nrf2+/+对照组、Nrf2-/-对照组的左室舒张末期前壁厚度(LVID)、左室舒张末期后壁厚度(LVPW)、EF、FS等指标相差不大，无明显统计学差异（p均>0.05)。提示基因敲除Nrf2对心脏基本形态和功能无明显影响。和相应的对照组相比，醛固酮组LVID、LVPW均明显增大。与Nrf2+/+醛固酮组相比，Nrf2-/-醛固酮组LVID、LVPW明显增大（p<0.05）。结果说明醛固酮可引起左室心肌肥厚，敲除Nrf2可加重醛固酮所致心肌肥厚。
4小鼠心脏质量指数的变化：Nrf2+/+对照组、Nrf2+/+醛固酮组心脏质量指数为3.92±0.08，4.34±0.31，Nrf2-/-对照组、Nrf2-/-醛固酮组心脏质量指数为3.91±0.31，4.99±0.17。显然，与野生型相比，Nrf2基因敲除并不影响心脏质量指数，给予醛固酮后心脏质量指数明显高于相应对照组（p<0.05），表明醛固酮增加心脏重量。与Nrf2+/+醛固酮组相比，Nrf2-/-醛固酮组心脏质量指数明显增大（p<0.05）。提示Nrf2基因敲除可使醛固酮对心脏质量指数的作用增强。
5小鼠心肌肥厚生物标志物ANP、β-MHC mRNA表达变化：与相应的对照组相比，醛固酮可使心肌肥大基因ANP、β-MHC mRNA表达量均明显增加（p<0.01）。与Nrf2+/+醛固酮组相比，Nrf2-/-醛固酮组ANP、β-MHC mRNA表达量增加更为明显（p<0.01）。提示Nrf2基因敲除增强了醛固酮对心肌肥厚生物标志物影响的作用。
6醛固酮对小鼠心肌ROS水平的影响：以上结果说明醛固酮可引起心肌肥厚，已知氧化应激在心肌肥厚发展中有重要作用，所以我们利用荧光探针DCFH-DA进行活性氧检测进一步观察了醛固酮对心肌的氧化应激的影响。与相应的对照组相比，Nrf2+/+醛固酮组、Nrf2-/-醛固酮组心肌细胞ROS水平均明显增加，分别增加约1.7、2.8倍（p<0.01）。与Nrf2+/+醛固酮组相比，Nrf2-/-醛固酮组ROS水平均明显增大（p<0.01）。提示醛固酮可引起左室心肌氧化应激损伤，敲除Nrf2可加重醛固酮所致心肌氧化应激损伤。
7醛固酮对野生型小鼠心脏Nrf2的影响：以上结果说明醛固酮可引起小鼠心肌肥厚、氧化应激增强，且敲除Nrf2可加重醛固酮致心肌肥厚、氧化应激的作用。为了进一步说明Nrf2在醛固酮致心肌肥厚中的作用，我们通过荧光定量PCR、免疫印迹技术观察醛固酮对小鼠心脏Nrf2表达的影响。醛固酮使Nrf2mRNA及蛋白水平均明显增加。提示醛固酮可上调Nrf2转录因子的表达，可能醛固酮激活了Nrf2转录因子及其下游抗氧化酶通路。
8醛固酮对小鼠心脏抗氧化酶HO-1、NQO-1mRNA表达的影响：既然醛固酮可上调Nrf2转录因子的表达，那么醛固酮对Nrf2的下游抗氧化基因有无影响？所以我们进一步观察醛固酮对Nrf2的下游抗氧化酶HO-1、NQO-1mRNA表达的影响。结果表明，醛固酮使心肌HO-1、NQO-1mRNA表达明显增加,但敲除Nrf2-/-后，醛固酮不会引起使HO-1、NQO-1mRNA表达增加，而使NQO-1mRNA表达明显降低。提示醛固酮可通过Nrf2上调抗氧化酶HO-1、NQO-1的表达，从而抑制醛固酮所致心肌氧化应激损伤及心肌肥厚。
结论：敲除Nrf2基因可加重醛固酮所致小鼠心肌肥厚，提示Nrf2参与了醛固酮所致心肌肥厚的调控作用。醛固酮上调心肌Nrf2转录因子，改变其下游抗氧化酶表达，提示醛固酮在致心肌肥厚的同时亦启动Nrf2-抗氧化酶通路，该通路对心肌肥厚发挥抑制作用。
There is emerging evidence that aldosterone is involved in thepathogenesis of cardiac hypertrophy and heart failure. The use of aldosteronereceptor antagonists for the treatment of myocardial failure and selected casesof hypertension, in combination with the current therapy, has been tested inrecent clinical trials. The Randomized Aldactone Evaluation Study (RALES)study has demonstrated that spironolactone, an aldosterone antagonist, addedto an angiotensin-converting enzyme (ACE) inhibitor in patients with severecongestive heart failure, reduces overall mortality. The EPHESUS study hasshown that eplerenone, added to ACEI or angiotensin AT1receptor blocker(ARB) decreases cardiovascular events and death in patients with leftventricular dysfunction following myocardial infarction. Therefore, clinicalpractice guidelines for heart failure management have designated aldosteronereceptor antagonists as first-line therapies for patients with heart failure andreduced left ventricular ejection fraction (LVEF). However, aldosteronereceptor antagonists carry an increased risk of complications, especiallyhyperkalemia may cause serious arrhythmic events. Accordingly, in clinicalpractice, the benefits of aldosterone receptor antagonists may be reduced andthey are used significantly less frequently compared with the otherguideline-recommended medications.
In recent years, low dose of aldosterone receptor antagonists has beenused in increasing number of experimental studies. It is generally acceptedthat “low-dose” refers to a non-pressure-lowering dose, although the doses ofaldosterone receptor antagonists vary with different reports. It was found thatthe low-dose spironolactone (1mg/kg) had no effect on systolic blood pressurebut improved diastolic dysfunction comparable to a conventional dose (30mg/kg) in a transgenic rat model of RAAS over-expression. A subpressordose of eplerenone markedly ameliorated vascular endothelial dysfunction,cardiac inflammation and fibrosis in salt-sensitive hypertensive rats to asimilar degree as an antihypertensive dose of amlodipine. These resultssuggest that the low-dose of aldosterone receptor antagonists exert organprotective effect independent of hemodynamic changes. The clinical study hasshown that low-dose of spironolactone significantly reduce the risk ofhyperkalemia. Thus, we hypothesize that low doses of aldosterone antagonists,added to ACEI or ARB therapy potentiates the cardioprotective effects, andwith little potential of hyperkalemia.
To verify our hypothesis, this study was designed to evaluate theeffectiveness of "low-dose" and conventional-dose aldosterone antagonistspironolactone in combination with ARB irbesartan on cardiac hypertrophicremodeling induced by abdominal aorta coarctation (PartialAbdominalAortic Constriction, PAAC) in rats. Plasma K level was measured to assesshyperkalemia risk. The effects of low-dose spironolactone in combinationwith irbesartan on myocardial oxidative stress were investigated in order tounderstand the pharmacological mechanisms underlying the potentiationeffects of the two-drug combination. To further explore the mechanism ofaldosterone induced myocardial hypertrophy, the possibility thatNF-E2-related factor (nuclear factor erythroid2-related factor2, Nrf2) wasinvolved in the regulation of cardiac hypertrophy induced by exogenousaldosterone was tested by using Nrf2knockout mice. The results will providesome new insights into the clinical application of aldosterone antagonists totreat cardiac hypertrophy and heart failure.
Section1The effects of low-dose spironolactone added to irbesartan onmyocardial remodelling induced by pressure overload inrats
Objective: To evaluate the effectiveness of "low-dose" andconventional-dose aldosterone antagonist spironolactone in combination withARB irbesartan on cardiac hypertrophic remodeling induced by PAAC in rats. Plasma K level was measured to assess hyperkalemia risk.
Methods: Male SD rats were chosen, the rats were prepared the model ofmyocardial hypertrophy by PAAC. At4weeks after surgery, cardiachypertrophy was confirmed by echocardiographic measurements. Then, PAACrats were divided into4groups: model, irbesartan (Ir,15mg/kg daily),conventional dose of spironolactone (20mg/kg daily) added toirbesartan(CSp+Ir) and low dose spironolactone (1mg/kg daily) added toirbesartan(LSp+Ir). At the end of8weeks, Left ventricular mass index(LVW/BW) was assessed by morphology measurement. The thicknesses ofleft ventricle walls and cardiac function were measured by transthoracicechocardiography. Left ventricular pressure and function were assessed byhemodynamic examination. Cardiomyocyte hypertrophy and collagenaccumulation in cardiac tissue were measured by HE and Masson stainingrespectively. The mRNA expression of ANP, β-MHC, Procollagen I andTGF-β were measured with RT-PCR.
Results:
1Effects on cardiac hypertrophy:1) Echocardiography showed that allrats subjected to PAAC developed cardiac hypertrophy at4weeks aftersurgery. Left ventricular posterior wall thickness (LVPW), interventricularseptum thickness (IVs) were increased, while left ventricular end diastolicdimension (LVd) was decreased in PAAC compared with age-matched shamrats. Which indicated that PAAC in this experimental condition result in hearthypertrophy and the model is successful. There was no significant differenceamong the experimental groups before therapy.2) At the end of drugtreatment, body weight was similar among the experimental groups. The ratioof LV weight to body weight (LVW/BW), an index of LV hypertrophy, was34%greater in model rats than in sham rats, and the overload-induced increasein this parameter was not significantly reduced by monotherapy with Ir, but itwas comparably attenuated by low-dose and conventional Sp added to Ir.3)Echocardiography revealed that LVPW, IVs and LVd were greater in modelrats than in sham rats at8weeks. Monotherapy with Ir didn’t affect these parameters. Combination therapy led to significant decrease in theseparameters compared with monotherapy with Ir, whereas there was nodifference in treatment with low-dose and conventional Sp added to Ir.4)Histological analysis revealed that hemodynamic overload had increased thecross-sectional area of cardiac myocytes in model rats by45%compared withthat apparent in sham rats. The extent of load induced cardiomyocytehypertrophy was reduced by treatment with low-dose and conventional Spadded to Ir (p<0.01). Whereas there was no difference in treatment withlow-dose and conventional Sp added to Ir.5) Hemodynamic overload resultedin upregulation of the expression of ANP and β-MHC, molecular markers ofhypertrophy, in the heart of model rats, and this effect was inhibited bylow-dose and conventional Sp added to Ir. Whereas there was no difference intreatment with low-dose and conventional Sp added to Ir. The results indicatedthat low dose Sp added to Ir can reduce PAAC induced cardiac hypertrophy,furthermore, the effect of low dose Sp is comparable with conventional Sp.
2Effects on cardiac fibrosis: Marked interstitial fibrosis was also detectedin the left ventricle of model rats as compared with matched Sham; thisincrease in cardiac fibrosis was also significantly reduced by low-dose andconventional Sp added to Ir. There was no difference in treatment withlow-dose and conventional Sp added to Ir. The rise in collagen type I andTGF-β1gene expression in the myocardium from model rats was attenuatedby low-dose and conventional Sp added to Ir. Whereas there was no differencein treatment with low-dose and conventional Sp added to Ir. The resultsindicated that low dose Sp added to Ir can reduce PAAC induced cardiacfibrosis, and the effect of low dose Sp is comparable with conventional Sp.
3Effects on cardiac Function: Echocardiography revealed left ventricularejection fraction(LVEF), left ventricular fractional shortening (LVSF) werenot different among the experimental groups (p>0.05), which indicatedsystolic function remained unaltered. Haemodynamic values obtained at theend of the treatment period. PAAC increase systolic blood pressure(SBP) anddiastolic blood pressure(DBP). Ir alone had no significant effect on SBP and DBP (p>0.05), but SBP and DBP was significantly reduced by low-dose andconventional Sp added to Ir. There was diastolic dysfunction in model rats ascompared with Sham as illustrated by increased LVEDP and decreasedDP/dtmin/LVSP (each p <0.05), but dP/dtmaxwere not different from Sham,which indicated systolic function remained unaltered in CMC-PAACrats,which is consistent with the result of echocardiography. Both low-doseand conventional Sp added to Ir reduced LVEDP and increased DP/dt min/LVSP(each p <0.05). The results indicated that low dose Sp added to Ir can reducePAAC induced diastolic dysfunction, and the effect of low dose Sp iscomparable with conventional Sp.
4Effects on the concentration of plasma K+and Na+: There was nodifference in the plasma concentration of Na+among all groups of rats.However, conventional dose of Sp added to Ir significantly elevated potassiumlevel in plasma, whereas, low-dose Sp did not affect the plasma K+concentration. The results indicated that low dose Sp added to Ir do not affectthe plasma K+concentration, which is was superior to conventional Sp.
Conclusion: Low doses of aldosterone antagonists spironolactone,added to ARB therapy potentiates the cardioprotective effects, and with littlepotential of hyperkalemia.
Section2The effects of combined low dose of spironolactone andirbesartan on myocardial oxidative stress induced bypressure overload in rats
Objective: To examine the effects of low dose of spironolactone,irbesartan and their combination on myocardial oxidative stress, and explorethe probable mechanism of the additive beneficial effects on cardiachypertrophy and diastolic dysfunction.
Methods: The levels of malondialdehyde (MDA) were measured asindirect oxidative marker. NADPH oxidase activity of left ventricular tissuehomogenate was measured by lucigenin chemiluminescence. SOD activitywas evaluated by nitro-blue tetrazolium methold. Western blot was used toevaluate the protein expression of both NADPH oxidase subunits including NOX2，NOX4and SOD subunits including SOD-1and SOD-2.
Results:
1Effects on MDA level: We evaluate the level of oxidative stress bydeterminating malondialdehyde (Maleic Dialdehyde, MDA) content.Compared with the sham operation group, model group of myocardial MDAcontent increased significantly (p<0.01), which demonstrate thatPAAC induced myocardial oxidative stress injury. Ir monotherapy, but notlow dose Sp, significantly reduced myocardial MDA level (p<0.05). Thecombination of Ir and low dose Sp further reduce the myocardialMDA level (p<0.05). These results suggest that Ir can significantly improvethe oxidative stress in myocardium, but Ir and low dose spironolactonecombined with better results.
2Effects on cardiac NADPH oxidase activity: NADPH oxidase is oneof the main enzyme to generating active oxygen in myocardialtissue, we observed the changes of NADPH oxidase activity in myocardium.Compared with the sham operation group, model group of myocardialNADPH oxidase activity was significantly enhanced (p<0.01).LSp group, Ir+LSp group, Ir decreased the activity of NADPH oxidase, butthere is significant difference only in Ir+LSp group(p<0.05). Compared withLSp group, combined treatment further reduced the activity of NADPHoxidase (p<0.05) in myocardium.
3Effects on cardiac NADPH oxidase subunits expression: In order tofurther illustrate the change mechanism of NADPH oxidase activity, weused immunoblotting to examine myocardial NADPH oxidasesubunit NOX2, NOX4protein expression. PAAC significantly increased theexpression of myocardial NADPH oxidase isoforms of NOX4protein(p<0.01), but no significant changes in NOX2isoforms. Monotherapy with Iror low dose Sp tended to reduce the expression of NOX4protein, but thedifference did not reach statistical significance. However, the combination ofIr and low dose Sp significantly attenuated the increases in the expression ofNOX4protein (p<0.05). These results suggest a better inhibitory effect of Ir combined with low dose of Sp on the expression of NOX4protein.
4Effects on cardiac SOD activity：Compared with the sham operationgroup, model group of myocardial SOD activity decreased significantly(p<0.05)，and it tended to be reduced by monotherapy with Ir or low dose Sp,but the difference did not reach statistical significance. However, thecombination of Ir and low dose Sp significantly attenuated the decreases inSOD activity (p<0.05).
5Effects on cardiac SOD subunits expression: In order to explainthe mechanism of SOD change, we used immunoblotting to examinemyocardial SOD subtype: SOD-1, SOD-2. Compared with the shamoperation group, model group of myocardial SOD-2proteinexpression increased significantly (p<0.01), but SOD-1had noobvious change. Monotherapy with Ir or low dose Sp tended to increase theexpression of SOD-2protein, but the difference did not reach statisticalsignificance. However, the combination of Ir and low dose Sp significantlyattenuated the decreases in the expression of SOD-2.
Conclusions:
1Abdominal aortic constriction caused the increase of MDA content inrat myocardial tissue, enhanced NADPH oxidase activity and the expressionof subunit NOX4, and decreased the activity of SOD and the expression ofSOD-2subunit, suggesting that oxidative stress induced by NADPH oxidaseand SOD is involved in the process of myocardial hypertrophy and diastolicdysfunction in PAAC rats.
2Low dose Sp combined with irbesartan can reduce PAAC inducedmyocardial oxidative stress injury, which may play a role by inhibiting theNADPH oxidase activity and enhancement of SOD activity.
Section3Regulation effect of Nrf2transcription factor on aldosteroneinduced cardiac hypertrophy in mice
Objective: To explore the role of Nrf2in regulation of myocardialhypertrophy induced by external aldosterone, further explorethe mechanism of aldosterone induced myocardial hypertrophy.
Methods: The Nrf2-null mice backcrossed into a ICR background weregifted by academician Li Chunyan of Department of Internal Medicine,Second Affiliated Hospital of Hebei Medical University. The genotypesof the animal were identified from the DNA， mRNA and proteinlevel. Aldosterone-treated wild type(WT) and Nrf2-null mice were implantedwith osmotic mini-pumps for subcutaneous infusion for4wk.(1mg/kg/·day)for4wk. Heart mass index (HW/BW) was assessed by morphologymeasurement. The thicknesses of left ventricle walls and cardiac function weremeasured by transthoracic echocardiography. Blood aldosterone concentrationwere measured using [125I] aldosterone radioimmunoassay. Myocardialcells were digested by trypsin method, and loaded DCFH-DA dye to observethe level of ROS. Western blot was used to evaluate the protein expression ofNrf2. The mRNA expression of Nrf2, HO-1, NQO-1were measured withRT-PCR.
Results:
1Nrf2knockout mice identification: PCR and agarose electrophoresis oftail DNA show that amplified bands of Nrf2-null mice is in400bp position, and the WT mice is in700bp. we confirmed that the Nrf2ispresent in the WT mouse hearts The protein (Western blot) and RNAexpression (RT-PCR) analysis confirmed disruption of Nrf2in the KO mouseheart.
2Effects on plasma aldosterone level: Plasma aldosterone concentrationwere significantly higher than corresponding control group (p<0.05),suggesting that osmotic pump with constant speed can release of aldosteroneinto mice, which significantly increased aldosterone level in the body.
3Echocardiography: Echocardiography revealed left ventricular ejectionfraction(LVEF), left ventricular fractional shortening (LVSF) were notdifferent among the experimental groups (p>0.05), which indicated systolicfunction remained unaltered. The Nrf2-null in baseline have a norma lleftVentricular anterior wall thickness(LVID), left ventricular posterior wallthickness(LVPW), LVEF, LVFS. These results indicate that there is no evidence for cardiac dysfunction due to disruption of Nrf2. Aldosteroneinfusion led to an increase in LVID and LVPW, however, the aldosterone–induced increase in LVID and LVPW were exaggerated in Nrf2-null mice.These results suggest that Nrf2deficiency exaggerates aldosterone-induced thechange of echocardiography parameters.
4Effects on HW/BW: Aldosterone infusion led to an increase in heartweight to body weight ratio (HW/BW), however, the aldosterone–inducedincrease in HW/BW was exaggerated in Nrf2-null mice. These resultssuggest that Nrf2deficiency exaggerates aldosterone-induced the change ofHW/BW.
5Effects on expression of cardiac hypertrophy gene ANP, β-MHC:Aldosterone infusion led to an upregulation of cardiac hypertrophy geneANP, β–MHC. However, the aldosterone-induced molecular markers ofhypertrophy changes were exaggerated in Nrf2-null mice.
6Effects on ROS level: In view of the anti-oxidative defence role of Nrf2in the heart, we investigated the role of Nrf2in regulating aldosterone-inducedmyocardial oxidative stress. Aldosterone infusion increased myocardial levelsof H2-DCFDA, a marker of ROS, indicating elevated levels of oxidative stressin the heart. The aldosterone-induced oxidative stress levels in the heart weremarkedly enhanced as a result of Nrf2deficiency.
7Effects on expression of Nrf2and antioxidative enzymes, HO-1、NQO-1: The chronic aldosterone infusion-enhanced the mRNA and proteinexpression Nrf2in WT myocardium. In response to the sustained aldosteronestimulation, several Nrf2downstream genes, including HO-1, NQO-1, in WTbut not in Nrf2-null hearts were up-regulated at mRNA level.
Conclusion: Our present study demonstrates that Nrf2is a criticalregulator for maintaining structural and functional integrity of the heart in thesetting of sustained aldosterone stimulation. Mechanistically, itis likely thatNrf2protects against maladaptive cardiac remodelling and dysfunction byrepressing ROS production and up-regulating antioxidative enzymes.
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