摘要
背景
心力衰竭是各种心血管疾病发展的终末阶段,具有较高的发病率和死亡率。近年来各种药物治疗使心力衰竭患者的生活质量大大提高,但其生存率仍无明显改善。究其原因,主要是其发病机制还不十分清楚。自噬是广泛存在于真核细胞内的一种溶酶体依赖性的降解途径,细胞通过单层或双层膜包裹待降解物形成自噬小体,然后运送到溶酶体形成自噬溶酶体并进行多种酶的消化及降解,以实现细胞自身的代谢和细胞器的更新,自噬对维持细胞的自身稳态具有重要意义。最近的研究表明自噬在心血管疾病的发生、发展过程中发挥着重要作用。因此阐明自噬在心衰中的确切作用,有助于进一步了解心衰的发病机制,为心衰的有效防治提供新的思路和策略。然而,目前自噬在心衰中的具体作用尚不明了,自噬在心衰中的时序性表达变化和对心肌的产生有利或有害作用值得进一步探讨。
目的
建立小鼠主动脉弓缩窄(Transverse aortic arch constriction, TAC)模型,观察TAC小鼠心衰进展过程中自噬功能的时序性变化,并探讨心衰过程中自噬功能紊乱的可能机制。
实验Ⅰ
方法
1.实验分组
170只雄性C57BL/6小鼠随机分为正常组(50只),假手术组(50只)和心衰组(70只)。各组分别于0天、3天、7天、14天、28天处死8-11只小鼠。
2.动物模型的建立
小鼠腹腔注射0.08%戊巴比妥钠麻醉、固定后,在大体显微镜下行TAC术。在颈部中心位置作一纵行10mm切口,钝性分离左、右颈总动脉,于双侧颈总动脉分支处盲穿并结扎主动脉弓,27号针头与主动脉弓一同结扎后,抽出针头,以控制主动脉弓狭窄率(60±5%)。假手术组的手术步骤同上,但不结扎主动脉弓。
3.存活率分析
保留存活记录并绘制曲线图。
4.超声心动图检测
所有小鼠于处死前行经胸壁超声心动图检测,计算左室射血分数(Ejection fraction, EF%)、左室重量(Left ventricular mass weight, LVMW)。
5.组织病理学及免疫组织化学染色检查
留取小鼠心脏标本,行苏木素-伊红染色(Haematoxylin eosin, HE)、Masson染色,观察心脏大体形态学改变；行免疫组织化学染色检测心肌内各指标的表达：
(1)自噬指标：微管相关蛋白1轻链3(Microtubule-associate protein1light chain3, LC3)、Bcl-2相互作用蛋白(Bcl-2interacting coiled-coil protein-1, Beclin-1)、Beclin-1相互作用蛋白(RUN domain protein as Beclin1-interacting and cysteine-rich containing, Rubicon)、P62(Sequestosome1/SQSTM1);
(2)炎症指标：单核细胞趋化蛋白-1(Monocyte chemoattractant protein-1, MCP-1).细胞间粘附分子-1(Intercellular adhesion molecule-1, ICAM-1)、Toll样受体4(Toll-like receptor4, TLR4)、髓样分化因子88(Myeloid differentiation factor88, Myd88)、基质金属蛋白酶-9(Matrix metalloproteinase-9, MMP-9)、基质金属蛋白酶抑制剂-1(Tissue inhibitor of matrix metalloprotease-1, TIMP-1);
(3)氧化应激指标：4-羟基壬烯醛(4-hydroxynonenal,4-HNE)、8-羟基-2’-脱氧鸟苷(8-hydroxy-2'-deoxyguanosine,80HdG)。
6.实时定量聚合酶链反应(Real-time quantitative polymerase chain reaction, Real-time PCR):检测心肌内LC3a、LC3b、自噬相关基因(Autophagy-related gene, Atg) Atg5、Atg7、Rubicon、Beclin-1的表达。
7.统计学分析
统计学处理计量资料以均数±标准差(x±s)表示,所有数据用SPSS11.5软件进行统计学分析。两组数据间比较采用两独立样本t检验,多组间均数比较采用包含Bonferroni post-hoc检验的单因素或双因素方差分析。多组间的两两比较采用费希尔的LSD法。P<0.05为差异有统计学意义。
结果
1.实验动物的一般情况
本研究共120只小鼠进行了手术,50只为假手术组,只挂线不结扎主动脉弓,1只死于麻醉意外,其余全部存活；70只为心衰组,TAC手术当天存活小鼠56只,手术成功率为80%。
2.心脏超声学检测结果
心脏超声图像显示术后28天心衰组小鼠左心室室壁变薄,心腔明显扩大,心肌收缩力减退。与正常组和假手术组相比,心衰组小鼠术后EF%明显下降,术后第3天由术前的73.4±3.5%降至41±10.4%(P<0.01)；术后7天至28天EF%下降较缓慢,术后28天小鼠EF%为36.2±5.7%(P<0.01)。心衰组小鼠术后LVMW较正常组和假手术组显著增高(P<0.05),术后3天LVMW由42.2±10.2增至52.5±12.7,术后28天增至58.2±11.0。
3.病理学检测
(1)病理学染色
HE染色和Masson染色均显示随着心衰时间的延长,心衰组小鼠心肌细胞直径增大,心脏横径和左心室腔不断增大。术后28天心肌纤维化明显,与正常组和假手术组比较具有显著性差异(P<0.05或P<0.01)。
(2)自噬因子表达变化
与正常组和假手术组相比,心衰组小鼠心肌LC3表达明显增高,呈双峰分布,在3天和7天表达持续升高,在7天达到第一个峰值(P<0.01)；在14天略有降低(P<0.05或P<0.01)；在28天表达明显增强,达到第二个峰值(P<0.01)。
与正常组和假手术组相比,心衰组小鼠心肌Beclin-1表达在术后3天和7天,明显升高(P<0.01)。
与正常组和假手术组相比,心衰组小鼠心肌P62表达在7天和28天显著升高(P<0.05或P<0.01)。
与正常组和假手术组相,心衰组小鼠心肌Rubicon的表达在术后3天、7天、14天、28天均显著升高(P<0.05或P<0.01)。
(3)炎症因子表达变化
与正常组和假手术组相比,心衰组小鼠心肌MCP-1表达明显升高,呈双峰分布,在3天表达较高(P<0.01),在7天表达略有减低(P<0.05或P<0.01),在14天和28天又持续升高(P<0.01)。
与正常组和假手术组相比,心衰组小鼠心肌ICAM-1表达在各时间点均显著升高,在7天、28天表达较高(P<0.01),3天、14天表达略低(P<0.05)。
与正常组和假手术组相比,心衰组小鼠心肌MMP-9表达在14天、28天表达略有升高(P<0.05)。
与正常组和假手术组相比,心衰组TIMP-1表达在28天显著升高(P<0.05或P<0.01),余无显著性差异。
与正常组和假手术组相比,心衰组TLR4表达在各时间点均显著升高,并随时间不断增强,与正常组和假手术组比较差异具有统计学意义(P<0.05或P<0.01)。
与正常组和假手术组相比,心衰组小鼠Myd88表达在14天显著升高(P<0.05),余无显著性差异。
(4)氧化应激因子表达变化
心衰组小鼠心肌80HdG的表达明显增强,术后3天阳性率为24.18±6.63%,较假手术组(10.40±4.54%)和正常组(3.13±1.33%)显著增强(P<0.05或P<0.01)；术后7天略下降至11.58±3.05%；术后14天、28天又持续上升(分别为28.56±7.31%,P<0.05：30.79±7.47%,P<0.01)。
心衰组小鼠心肌4-HNE表达也显著增强,在术后3天升高至正常组的6-7倍,7天略下降至3-4倍,14天、28天又升高至4-6倍,与正常组和假手术组比较具有统计学意义(P<0.01)。
4. Real-time PCR结果
与正常组和假手术组比较,心衰组小鼠心肌LC3a mRNA的水平随时间呈单峰分布,在7天达到最高(P<0.01),余时间点无显著性差异；
与正常组和假手术组比较,心衰组LC3b的mRNA表达水平也呈单峰分布,在3天、7天表达最高(P<0.05),28天恢复到正常水平；
与正常组和假手术组比较,心衰组小鼠心肌Atg5mRNA表达水平在3天、7天、14天明显增高(P<0.01),在28天基本降至基础水平；
与正常组和假手术组比较,心衰组小鼠心肌Atg7mRNA表达水平在3天明显升高(P<0.01),达到高峰,在28天基本降至基础水平,余时间点差异无统计学意义(P>0.05)。
与正常组和假手术组比较,心衰组小鼠心肌Beclin-1mRNA表达水平表现为在第3天显著升高(P<0.01),在7天、14天迅速降至基础水平,在28天略有升高,但无统计学意义(P>0.05)。
与正常组和假手术组比较,心衰组小鼠心肌Rubicon的mRNA表达水平随时间呈双峰分布,在7天、28天达到最高值(P<0.01),14天略有降低(P<0.05)。
实验Ⅱ
方法
1.实验动物
180只雄性C57BL/6小鼠随机分为正常组(50只),假手术组(50只)和心衰组(80只)。以上各组组内又随机分为3个亚组：雷帕霉素组、雷帕霉素加氯喹组和生理盐水组,共9个亚组。各亚组分别于TAC术后7天、28天处死8-10只小鼠,小鼠处死前4h分别给予腹腔注射雷帕霉素(30mg/kg)或(和)氯喹(2mg/kg)或生理盐水(0.2ml)。
2.动物模型的建立
动物模型的建立方法同实验Ⅰ。
3.病理学检测
LC3免疫组化检测各组心肌LC3的蛋白表达水平。
4. Real-time PCR检测
检测各组LC3a、LC3b的mRNA表达水平。
结果
1.病理学染色
在不应用雷帕霉素和氯喹干预情况下,术后7天心衰组小鼠心肌LC3表达阳性率为21.43±2.9%,术后28天达28.20±3.24%,与正常组和假手术组比较均具有显著性差异(P<0.01)。
术后7天,雷帕霉素均可以引起正常组、假手术组和心衰组小鼠心肌LC3表达水平的升高(P<0.05)；雷帕霉素和氯喹联合应用可以引起正常组、假手术组小鼠心肌LC3表达的进一步升高(P<0.01)。在心衰组中,雷帕霉素和氯喹组的LC3表达与雷帕霉素组相比无显著性差异(P>0.05)。
术后28天,在正常组和假手术组中,雷帕霉素可以引起LC3蛋白表达的升高(P<0.05或P<0.01),雷帕霉素和氯喹联合应用可以引起LC3蛋白表达的进一步升高(P<0.05或P<0.01)。在心衰组中,雷帕霉素组LC3表达较生理盐水组略有升高,但无统计学意义(P>0.05)；雷帕霉素和氯喹组LC3表达与雷帕霉素组相比显著下降(P>0.05)。
2. Real-time PCR结果
与正常组和假手术组相比,心衰组小鼠心肌LC3a的mRNA水平在术后7天明显升高,达正常组的100-120倍(P<0.01)；术后28天降至接近正常水平(P>0.05)。
TAC术后7天,雷帕霉素可以引起正常组和假手术组LC3a的mRNA水平的升高(P<0.05),心衰组LC3a mRNA水平略有升高,但无显著性差异(P>0.05)。雷帕霉素和氯喹联合应用均可以进一步升高各组LC3a的mRNA水平。(P<0.05或P<0.01)。
TAC术后28天,雷帕霉素可以引起正常组、假手术组和心衰组的LC3a的mRNA水平的升高(P<0.05或P<0.01),雷帕霉素和氯喹联合应用可以进一步升高正常组和假手术组LC3a的mRNA水平(P<0.05或P<0.01)。但在心衰组中,雷帕霉素和氯喹组与雷帕霉素组相比,LC3a的mRNA水平没有进一步升高,反而迅速降低(P<0.01)。
与正常组和假手术组相比,心衰组小鼠心肌LC3b的mRNA水平在术后7天明显升高,达正常组的3-5倍(P<0.05)；在术后28天略有升高,无显著性差异(P>0.05)。
TAC术后7天,雷帕霉素均可诱导正常组、假手术组和心衰组LC3b的mRNA水平的升高(P<0.05或P<0.01)；雷帕霉素与氯喹联合应用均引起各组LC3b的mRNA水平的进一步升高(P<0.05或P<0.0])。
TAC术后28天,雷帕霉素均可诱导正常组、假手术组和心衰组LC3b的mRNA表达(P<0.05或P<0.01)；雷帕霉素与氯喹联合应用可进一步诱导正常组、假手术组LC3b的mRNA表达(P<0.01),但在心衰组中,雷帕霉素和氯喹组LC3b表达与雷帕霉素组相比有所升高,但无显著性差异(P>0.05)。
3.自噬流的改变
免疫组化及PCR结果均显示：心衰组小鼠在TAC术后7天,心肌自噬功能正常；与正常组和假手术组比较,心衰组雷帕霉素诱导的自噬流无显著性差异(P>0.05)。术后28天,心衰组小鼠心肌自噬功能异常,与正常组和假手术组比,心衰组雷帕霉素诱导的自噬流明显减少甚至消失(P<0.01)。
结论
1.在TAC小鼠模型中,心脏压力超负荷的早期,心功能代偿,雷帕霉素诱导的自噬流正常,自噬功能正常；在心脏压力超负荷晚期,心功能失代偿,雷帕霉素诱导的自噬流减弱甚至消失,自噬小体清除阶段的自噬功能发生紊乱。
2.在小鼠TAC模型中,在心衰的不同时期自噬基因的发生动态变化,自噬、炎症、氧化应激三种机制相互影响,共同促进TAC小鼠心衰的进展和心功能的降低。
背景
心力衰竭是严重危害人类健康的常见心血管疾病之一。随着人口老龄化日益加重,心衰的发病率逐年提高。有效防治心衰已成为医学界亟待解决的重要问题。自噬是细胞内物质代谢的重要途径,是降解再循环系统的重要组成部分。研究发现,自噬参与了心血管疾病的发生、发展过程。大量基础与临床研究已证实芪苈强心胶囊具有强心、利尿、扩血管、抑制神经内分泌激活等作用,能有效改善心衰症状,缓解心肌损伤和心功能的恶化。但其对心衰发生发展过程中自噬功能的作用尚不明了。
目的
1.明确芪苈强心胶囊改善心衰小鼠心功能代偿期和失代偿期的作用。
2.探讨芪苈强心胶囊对心衰小鼠心功能代偿期和失代偿期自噬功能的影响及其相关机制。
方法：
1.实验动物
300只雄性C57BL/6小鼠随机分为假手术组(60只)和横向主动脉弓缩窄术(Transverse aortic arch constriction, TAC)组(240只)。假手术组给予生理盐水灌胃；TAC组又随机分为芪苈强心组,培哚普利组和生理盐水组(心衰组),分别给予芪苈强心、培哚普利、生理盐水灌胃。
假手术组、芪苈强心组、培哚普利组和心衰组组内又随机分为生理盐水组、雷帕霉素组、雷帕霉素加氯喹组3个亚组,处死前4h分别给予腹腔注射生理盐水、雷帕霉素、雷帕霉素加氯喹,共12个亚组,分别于14天,28天处死各亚组内8-14只小鼠。
2.模型的建立
模型建立方法同论文Ⅰ。
3.存活率分析
保留存活记录并绘制图表。
4.超声心动图检测
所有小鼠于处死前行超声心动图检测左室射血分数(Ejection fraction,EF%),左室重量(Left ventricular mass weight, LVMW)。
5.组织病理学及免疫组织化学染色检查
留取小鼠心脏标本,行苏木素-伊红染色(Haematoxylin eosin, HE)、Masson染色心脏大体形态学改变；行免疫组织化学染色检测雷帕霉素和雷帕霉素加氯喹干预组的小鼠心肌内的微管相关蛋白1轻链3(Microtubule-associate protein1light chain3, LC3)的表达,检测内未经雷帕霉素或氯喹干预的小鼠心肌内LC3和如下各指标的表达：
(1)自噬指标：Bcl-2相互作用蛋白(Bcl-2interacting coiled-coil protein-1, Beclin-1)、Beclin-1相互作用蛋白(RUN domain protein as Beclin1-interacting and cysteine-rich containing, Rubicon)、P62(Sequestosome1/SQSTM1);
(2)炎症指标：基质金属蛋白酶-9(Matrix metalloproteinase-9, MMP-9)、基质金属蛋白酶抑制剂-1(Tissue inhibitor of matrix metalloprotease-1, TIMP-1);
(3)氧化应激指标：4-羟基壬烯醛(4-hydroxynonenal,4-HNE)、8-羟基-2’-脱氧鸟苷(8-hydroxy-2'-deoxyguanosine,8OHdG)。
6.实时定量聚合酶链反应(Real-time quantitative polymerase chain reaction, Real-time PCR)
检测雷帕霉素组、雷帕霉素加氯喹干预组的小鼠心肌内的LC3a、LC3b的mRNA表达。检测未经雷帕霉素或氯喹干预组小鼠心肌内自噬相关基因(Autophagy-related gene, Atg)Atg5、Atg7、LC3a、LC3b、Rubicon Beclin-1的表达。
7.统计学分析
统计学处理计量资料以均数±标准差(x±s)表示,所有数据用SPSS11.5软件进行统计学分析。两组数据间比较采用两独立样本t检验,其他数据多组间均数比较采用包含Bonferroni post-hoc检验的单因素或双因素方差分析。多组间的两两比较采用费希尔的LSD法。P<0.05为差异有统计学意义。
结果：
1.动物的一般情况
与假手术组比较,心衰组小鼠生存率显著降低(P<0.01),培哚普利和芪苈强心胶囊均能有效改善心衰小鼠的生存率(P<0.05)。
2.心脏超声学检测
在术后14天心功能代偿期,心衰组小鼠EF%明显下降,LVMW明显增大,与假手术相比具有统计学意义(P<0.01)。培哚普利和芪苈强心胶囊均能显著提高心衰小鼠的EF%(P<0.05或P<0.01),缓解LVMW的增高(P<0.05或P<0.01)。
在术后28天心功能失代偿期,心衰组小鼠EF%进一步降低,而LVMW进一步升高,与假手术组相比具有统计学意义(P<0.01)。与心衰组相比,培哚普利组和芪苈强心组的EF%显著增高(P<0.01),LVMW显著降低(P<0.05)；两药物组之间EF%和LVMW无显著性差异。
3.病理学和免疫组织化学检测
(1)病理学染色
HE染色显示：术后14天、28天,心衰组小鼠心肌细胞直径明显增大,心脏横径明显增大,左心室腔显著增大,以28天更为显著；培哚普利组和芪苈强心组小鼠心肌肥大及左心室腔扩大程度较心衰组显著降低。
Masson染色显示：术后14天各组心肌胶原含量无显著性差异。术后28天,与假手术组相比,心衰组、培哚普利组和芪苈强心组小鼠心肌胶原含量明显增多(P<0.01)；与心衰组相比,培哚普利组和芪苈强心组小鼠心肌胶原含量显著降低(P<0.01)。
(2)自噬功能检测
在不使用雷帕霉素或氯喹干预情况下,术后14天、28天,与假手术组相比,心衰组、培哚普利组、芪苈强心组小鼠心肌LC3表达阳性率明显增高(P<0.01)；与心衰组相比,培哚普利组和芪苈强心组小鼠心肌LC3表达降低(P<0.05或P<0.01)。
术后14天,雷帕霉素均可以引起各组的LC3表达的升高(P<0.05或P<0.01)；雷帕霉素和氯喹联合应用后,假手术组、芪苈强心组、培哚普利组LC3表达进一步升高(P<0.05或P<0.01)；在心衰组中,雷帕霉素和氯喹联合应用后LC3表达略有升高,但与应用雷帕霉素相比没有显著性差异(P>0.05)。
术后28天,在除心衰组外的各组中,雷帕霉素可以诱导LC3蛋白表达升高(P<0.05或P<0.01),雷帕霉素和氯喹联合应用可引起LC3蛋白表达的进一步升高(P<0.05或P<0.01)。在心衰组中,应用雷帕霉素后,LC3表达稍有升高,但无统计学意义(P>0.05)。联合应用雷帕霉素和氯喹后,与单独应用雷帕霉素相比,LC3表达无显著性差异(P>0.05)。
(3)自噬基因的表达
术后14天,各组P62蛋白表达水平较低,无显著性差异(P>0.05)。术后28天,与假手术组相比,心衰组P62蛋白表达水平显著升高(P<0.01),芪苈强心组、培哚普利组P62蛋白表达水平无显著性差异(P>0.05)。
术后14天、28天,心衰组、芪苈强心组、培哚普利组Beclin-1表达与假手术组相比均显著增高(P<0.01)。术后14天,芪苈强心组、培哚普利组Beclin-1表达较心衰组显著增高(P<0.05或P<0.01),术后28天,芪苈强心组、培哚普利组Beclin-1表达较心衰组显著降低(P<0.05)。
术后14天、28天,与假手术组相比,心衰组、芪苈强心组、培哚普利组Rubicon表达均显著增高(P<0.01)；与心衰组相比,芪苈强心组、培哚普利组Rubicon表达均显著降低(P<0.05或P<0.01)。
(4)氧化应激
术后14天、28天,与假手术组比较,心衰组、培哚普利组和芪苈强心组小鼠心肌的4-HNE、8OHdG表达进一步增强(P<0.01)；与心衰组相比,培哚普利组和芪苈强心组小鼠心肌的4-HNE、8OHdG表达显著降低(P<0.05或P<0.01)。
(5) MMPs/TIMPs表达
术后14天,与假手术组和培哚普利组相比,心衰组和芪苈强心组小鼠心肌MMP-9表达增多(P<0.01)。术后28天,与假手术组相比,心衰组、芪苈强心组、培哚普利组小鼠心肌的MMP-9表达显著增多(P<0.01)；与心衰组相比,芪苈强心组和培哚普利组MMP-9表达显著降低(P<0.05)。
术后14天,各组小鼠心肌TIMP-1表达无显著性差异(P>0.05)。术后28天,与假手术组相比,心衰组、芪苈强心组、培哚普利组小鼠心肌TIMP-1表达较显著增多(P<0.01)；与心衰组相比,培哚普利组和芪苈强心组TIMP-1表达显著增多(P<0.01)。
4. Real-time PCR结果
(1)LC3的表达
TAC术后14天,与假手术组相比,心衰组、培哚普利组和芪苈强心组小鼠LC3a mRNA水平均升高(P<0.05或P<0.01)。与心衰组相比,培哚普利组和芪苈强心组LC3a mRNA水平明显升高(P<0.01)。术后28天,培哚普利组和芪苈强心组LC3a mRNA水平较假手术组和心衰组显著升高(P<0.05),而心衰组LC3amRNA水平与假手术比较差异无统计学意义。
TAC术后14天,培哚普利组和芪苈强心组小鼠心肌LC3b mRNA表达较假手术组和心衰组升高(P<0.01),心衰组与假手术组LC3b mRNA表达水平无显著性差异(P>0.05)。术后28天,培哚普利组和芪苈强心组小鼠心肌LC3b的mRNA水平较假手术组升高(P<0.05或P<0.01),培哚普利组较心衰组显著升高(P<0.05),其余各组间无显著性差异。
(2)自噬流的检测
TAC术后14天,雷帕霉素可以显著引起各组LC3a mRNA水平的升高(P<0.01),雷帕霉素和氯喹联合应用引起LC3a mRNA水平的进一步升高(P<0.01)。芪苈强心组和培哚普利组LC3a升高水平显著大于心衰组和假手术组(P<0.01)。
TAC术后28天,雷帕霉素可显著引起各组LC3a的mRNA水平的升高(P<0.01)。与雷帕霉素组相比,雷帕霉素和氯喹联合应用均可以进一步升高培哚普利组、芪苈强心组和假手术组LC3a的mRNA水平(P<0.01),而心衰组LC3a的mRNA水平不升反降,呈低水平表达(P<0.01)。
TAC术后14天,雷帕霉素可以显著引起各组LC3b mRNA水平的升高(P<0.01),雷帕霉素和氯喹联合应用可促进LC3b mRNA水平的进一步升高(P<0.01)。TAC组雷帕霉素诱导的自噬流较其余各组略有降低,但无显著性差异(P>0.05),其余各组间无显著性差异(P>0.05)。
TAC术后28天,雷帕霉素均可引起各组LC3b的mRNA水平升高(P<0.05或P<0.01)。在培哚普利组、芪苈强心组和假手术组中,与雷帕霉素组相比,雷帕霉素加氯喹组LC3b的mRNA水平显著升高(P<0.01)；在心衰组中,与雷帕霉素组相比,雷帕霉素加氯喹组LC3b的mRNA水平无显著性变化(P>0.05)。与假手术组相比,芪苈强心组和培哚普利组雷帕霉素诱导的自噬流显著降低(P<0.05),心衰组雷帕霉素诱导的自噬流消失,自噬功能紊乱。
(3)自噬基因的表达
TAC术后14天,与假手术组相比,心衰组、培哚普利组和芪苈强心组小鼠心肌Atg5mRNA表达显著增高(P<0.01),培哚普利组较心衰组显著降低(P<0.05),其余各组间无显著性差异。术后28天,芪苈强心组和培哚普利组较假手术组和心衰组Atg5mRNA表达显著增高(P<0.01)。
TAC术后14天、28天,芪苈强心组和培哚普利组Atg7mRNA表达水平显著高于假手术组和心衰组(P<0.05或P<0.01)。
TAC术后14天,芪苈强心组和培哚普利组小鼠心肌Beclin-1mRNA表达水平较假手术组和心衰组显著升高(P<0.05或P<0.01)。术后28天心衰组Beclin-1mRNA表达水平较其余各组显著升高(P<0.01)。
TAC术后14天、28天,心衰组、培哚普利组和芪苈强心组小鼠心肌Rubicon的1mRNA表达较假手术组显著升高(P<0.01)。术后14天,培哚普利组和芪苈强心组Rubicon的mRNA表达较心衰组显著升高(P<0.01)；术后28天,培哚普利组和芪苈强心组Rubicon的mRNA表达较心衰组显著降低(P<0.01)。
结论
1.芪苈强心胶囊能够改善TAC小鼠心衰代偿期、失代偿期心功能,有效改善心肌重构,且与培哚普利疗效相当。
2.芪苈强心胶囊能够抑制心肌氧化应激、改善心肌自噬功能是其改善心衰的重要机制。
背景
在心衰患者中普遍存在抑郁、焦虑等心理应激症状,心理应激不仅会引起患者的精神和行为障碍,而且可以影响患者的神经内分泌系统和机体功能,进而加重心衰。此外,心衰在其发生发展过程中也可引起或加重心理应激。两者互为因果,形成恶性循环,最终影响预后。由此可见,心理应激在心衰的发生、发展中发挥着重要作用。然而,目前对于心理应激与心衰的研究多集中于临床观察性研究,对于心理应激对心衰的具体作用机制尚不清楚。
抑郁、焦虑等心理应激可促进肾素-血管紧张素系统(renin-angiotensin system, RAS)的激活和血管紧张素Ⅱ(AngiotensinⅡ, AngⅡ)的生成。而AngⅡ参与了心衰的发生、发展过程。最近的研究表明,AngⅡ参与了心肌细胞的自噬。自噬是是广泛存在于大部分真核细胞中的一种生命现象。自噬作用主要是清除和降解细胞内受损伤的细胞结构、衰老的细胞器、生物大分子等,同时为细胞内细胞器的构建提供原料,即细胞结构的再循环。自噬在正常情况下呈低水平表达,而自噬功能的缺陷可导致心功能的异常和心衰的发生。自噬功能异常是心衰发生、发展的重要机制。然而,心理应激对心衰过程中自噬功能的研究未见报道。
目的
1.观察心理应激对心衰早期自噬功能的影响；
2.探讨心理应激对心衰自噬功能的作用机制。
实验一
方法
1.实验动物
雄性C57BL/6小鼠随机分为假手术组(30只)和横向主动脉弓缩窄术(Transverse aortic arch constriction, TAC)组(120只)。TAC组内又随机分为应激组(40只)、应激加卡托普利组(卡托普利组,40只)、心衰对照组(心衰组,40只)。各组于TAC术后3天,分别给予心理应激和药物干预7天,为排除灌胃影响,除卡托普利组外各组分别给予生理盐水(0.2ml/day)。术后11天处死小鼠,为了检测自噬流,各组于处死前4h分别随机分为三个亚组并给予腹腔注射雷帕霉素(30mg/kg)或氯喹(2mg/kg)或生理盐水(0.2m1)。
2.动物模型的建立：同论文Ⅰ。
3.心理应激方法
TAC术后3天,对应激组和卡托普利组进行应激,应激时间为1周。心理应激方法采用幽闭应激和噪音应激。将小鼠放入管壁带孔的50ml针管中2个h,同时进行噪音刺激(110DB、间隔3min、持续5s)。刺激周期为7天。
4.自我修饰行为检测
将10%的蔗糖水喷洒在小鼠背部,进行飞溅测试,观察并记录小鼠自我修饰行为。
5存活率分析：记录小鼠生存状况并绘制曲线图。
6.血流动力学及心脏超声检测
在TAC术后第3天和第10天,应用小动物血压测定仪检测小鼠舒张压(Diastolic blood pressure, DBP)、收缩压(Systolic blood pressure, SBP)和心率(Heart rate, HR),心脏超声检测左室射血分数(Ejection fraction, EF%)和左室重量(Left ventricular muscle weight, LVMW)。
7.血清学检测
心理应激7天后,所有小鼠处死,并收集血清,酶联免疫吸附试验(Enzyme-linked immunosorbent assay, ELISA)检测血清皮质醇水平和Ang Ⅱ含量。
8.病理学和免疫组化染色
留取小鼠心脏标本,行苏木素-伊红染色(Hematoxylin and eosin, HE)、Masson染色；行免疫组织化学染色检测未经氯喹和雷帕霉素干预组的小鼠心肌内的自噬指标：微管相关蛋白1轻链3(Microtubule-associate protein1light chain3, LC3)、Bcl-2相互作用蛋白(Bcl-2interacting coiled-coil protein-1, Beclin-1)、Beclin-1相互作用蛋白(RUN domain protein as Beclin1-interacting and cysteine-rich containing, Rubicon)、P62(Sequestosome1/SQSTM1)和氧化应激指标：4-羟基壬烯醛(4-hydroxynonenal,4-HNE)、8-羟基-2’-脱氧鸟苷(8-hydroxy-2'-deoxyguanosine,8OHdG)的表达。检测雷帕霉素和氯喹干预组小鼠心肌内LC3的表达。
9.实时定量聚合酶链反应(Real-time quantitative polymerase chain reaction, Real-time PCR)
Real-time PCR检测未经雷帕霉素和氯喹干预组的LC3a、LC3b、Beclin-1、 Rubicon、自噬相关基因(Autophagy-related gene, Atg) Atg5和Atg7的mRNA表达。检测雷帕霉素和氯喹干预组的LC3a、LC3b的mRNA表达。
10.统计学分析：统计学处理计量资料以均数±标准差(x±s)表示,所有数据用SPSS11.5软件进行统计学分析。Kaplan-Meier法制作生存率曲线,生存率比较采用log rank检验。两组数据间比较采用t检验,其他数据多组间均数比较采用包含Bonferroni post-hoc检验的单因素或双因素方差分析。多组间的两两比较采用费希尔的LSD法。P<0.05为差异有统计学意义。
结果
1.自我修饰行为
应激组和卡托普利组小鼠自我修饰行为较心衰组和假手术组显著较低(P<0.01)。
2.生存率
应激组小鼠生存率(75%)较心衰组(91%)明显降低(P<0.05)；卡托普利组生存率(89%)较应激组有所升高,但无统计学意义。
3.血清学检测
应激组和卡托普利组小鼠血清皮质醇水平较心衰组和假手术组明显升高5-6倍(P<0.01)。应激组小鼠血清AngⅡ水平比心衰组和卡托普利组小鼠明显升高(P<0.01),心衰组与卡托普利组比较差异无统计学意义(P>0.05)。与假手术组相比,卡托普利组小鼠血清AngⅡ水平升高(P<0.05)。
4.血流动力学和心脏超声学检测
应激显著增加了应激组和卡托普利组小鼠的SBP、DBP和HR(P<0.01),卡托普利组的SBP、DBP和HR较应激组降低(P<0.01)。与心衰组相比,应激组小鼠心肌肥厚明显,LVMW (P<.01)和HW/BW (P<0.01)显著增加,EF%明显下降(P<0.01)。卡托普利明显改善了应激引起的心肌肥厚(P<0.01),卡托普利组小鼠LVMW和心脏重量指数(Heart weight/body weight, HW/BW)较应激组显著降低(P<0.01),EF%显著增高(P<0.01)。
5.病理学染色
HE和Masson染色显示应激组心肌肥厚,与心衰组相比,心肌胶原纤维含量增多(P<0.05),卡托普利组心肌胶原含量较应激组有所降低,但无统计学意义(P>0.05)。
在不应用雷帕霉素或氯喹干预情况下,与假手术组相比,其余各组小鼠心肌LC3b表达明显增高(P<0.01)；与心衰组相比,应激组和卡托普利组LC3b表达明显升高(P<0.05或P<0.01)；与应激组相比,卡托普利组LC3b表达明显降低(P<0.01)。
雷帕霉素可以显著诱导假手术组、心衰组、卡托普利组和应激组LC3b表达的升高(P<0.05或P<0.01)。氯喹可以显著诱导假手术组、心衰组、卡托普利组LC3b表达的升高(P<0.01)。但在应激组,氯喹仅引起LC3b表达的轻微升高,无统计学意义(P>0.05)。
在假手术组心肌上有极少量Beclin-1和Rubicon的阳性表达,与假手术组相比,其余各组表达明显增强(P<0.01)；与应激组相比,心衰组和卡托普利组Beclin-1和Rubicon表达明显减弱(P<0.01)。
在假手术组小鼠心肌基本没有4-HNE、OHdG的阳性表达,其余各组表达比假手术组明显增强(P<0.01)；与应激组相比,心衰组和卡托普利组4-HNE和80HdG表达明显减弱(P<0.01),心衰组和卡托普利组4-HNE、8OHdG表达相当,差异无统计学意义(P>0.05)。
6. Real-time PCR结果
在不应用雷帕霉素或氯喹干预情况下,假手术组LC3a mRNA呈低水平表达,其余各组小鼠心肌LC3a mRNA表达较假手术组明显增高(P<0.01)；与心衰组相比,卡托普利组LC3a mRNA表达明显升高(P<0.05),应激组LC3a mRNA表达升高,但无统计学意义(P>0.05)。与假手术组相比,其余各组LC3b mRNA表达明显增高(P<0.01)；应激组LC3b mRNA表达水平较心衰组和卡托普利组明显增高(P<0.05或P<0.01)。
雷帕霉素可以显著诱导各组LC3b mRNA表达水平的升高。雷帕霉素干预后,应激组LC3b mRNA表达较假手术组升高(P<0.05),其余各组间无显著差异(P>0.05)。氯喹可以显著诱导假手术组、心衰组、卡托普利组LC3b mRNA表达的升高(P<0.01),但仅引起应激组LC3b mRNA表达的轻微升高,无统计学意义(P>0.05)。氯喹干预后,卡托普利组LC3b mRNA表达较假手术组(P<0.01)和应激组(P<0.05)明显升高。
在不应用雷帕霉素和氯喹干预的情况下,假手术组小鼠心肌Beclin-1、 Rubicon、Atg5、Atg7mRNA呈低水平表达,其余各组Beclin-1、Rubicon、Atg5、 Atg7mRNA表达较假手术组明显升高(P<0.01)；与心衰组相比,应激组Beclin-1、 Rubicon、Atg5、Atg7mRNA表达显著增高(P<0.01)；与应激组相比,卡托普利组Beclin-1、Rubicon mRNA表达均显著降低(P<0.01)。
实验二
方法
1.刺激细胞
对细胞进行AngⅡ刺激,刺激浓度分别为0μM和0.1μM,置于37℃细胞培养箱孵育22h,然后在各孔中分别加入雷帕霉素(1μM)或巴弗洛霉素(50nM)继续孵育2h,进行下一步检测。为检测氧化应激在AngⅡ通路中的作用,对细胞进行AngⅡ刺激,刺激浓度分别为0μM和0.1μM,同时加入或不加入N-乙酰半胱氨酸(N-acetylcysteine, NAC)50μM,置于37℃细胞培养箱孵育22h。然后在各孔中分别加入雷帕霉素(1μM)或巴弗洛霉素(50nM)继续孵育2h。
2.免疫荧光
免疫荧光检测H9c2细胞LC3b和4-HNE表达水平。
3. Western blot检测
通过细胞蛋白的提取、蛋白浓度测定、凝胶电泳、转膜、标记一抗和二抗、曝光等步骤检测LC3b、Rubicon、Beclin-1、β-action的表达水平。
4. Real-time PCR
通过提取细胞RNA, Real-time PCR检测LC3a、LC3b、Atg5、Atg7、Beclin-1、 Rubicon表达变化。
结果
1. AngⅡ对H9c2细胞自噬的作用
对细胞进行AngⅡ (0.1μM)刺激24h,免疫荧光染色检测LC3b的表达,显示AngⅡ刺激后,LC3b的表达明显升高(P<0.01)；雷帕霉素和巴弗洛霉素均可进一步诱导LC3b的表达(P<0.01)。
2.NAC能够抑制AngⅡ引起的自噬和氧化应激的升高
在AngⅡ (0.1μM)刺激24h的同时,加入氧化应激的抑制剂NAC,免疫荧光染色发现：Ang Ⅱ能够显著升高H9c2细胞LC3b的蛋白表达(P<0.01)；与AngⅡ组相比,NAC能够显著抑制H9c2细胞LC3b的蛋白表达(P<0.01)。PCR结果显示：AngⅡ能够显著升高H9c2细胞的LC3a、LC3b mRNA表达水平(P<0.05或P<0.01)；与AngⅡ组相比AngⅡ和NAC共培养能够显著降低LC3b mRNA表达水平(P<0.05)。Western blot对LC3b的检测也进一步证实了该结果。同时,与AngⅡ组相比,AngⅡ和NAC共培养后,雷帕霉素或巴佛洛霉素诱导的LC3b水平显著降低(P<0.01)。
免疫荧光染色发现：Ang Ⅱ能够显著升高H9c2细胞4-HNE的蛋白表达(P<0.01)；与AngⅡ组相比,AngⅡ和NAC共培养能够显著抑制H9c2细胞4-HNE的蛋白表达(P<0.01)。
3.NAC对AngⅡ诱导的自噬基因表达的影响
在AngⅡ (0.1μM)刺激24h的同时,加入氧化应激的抑制剂NAC, PCR检测Atg5、Atg7、Beclin-1、Rubicon mRNA表达水平的改变；Western blot检测Beclin-1、Rubicon的蛋白表达。结果显示AngⅡ可以引起Atg5、Atg7、Beclin-1、 Rubicon mRNA表达水平的升高(P<0.01)；与AngⅡ组相比,AngⅡ和NAC共培养能够显著抑制H9c2细胞Atg5、Beclin-1、Rubicon mRNA表达水平(P<0.05或P<0.01)。Western blot显示AngⅡ可以引起Beclin-1、Rubicon蛋白表达水平升高(P<0.01)；与AngⅡ组相比,AngⅡ和NAC共培养能够显著降低H9c2细胞Beclin-1、Rubicon蛋白表达水平(P<0.05或P<0.01)。
结论
1.心理应激可以导致心衰小鼠自噬小体清除阶段的功能紊乱；
2. AngⅡ诱导的氧化应激通路的激活是心理应激导致的自噬功能紊乱的重要机制。
Background
Heart failure (HF) is a chronic, progressive illness that is highly prevalent in the worldwide. Despite its place among the leading causes of morbidity, pharmacological and mechanic remedies have only been able to slow the progression of the disease. There is a critical need to further understand its underlying mechanisms. Autophagy is a major catabolic pathway by which mammalian cells degrade and recycle macromolecules and organelles. It plays a critical role in removing protein aggregates, as well as damaged or excess organelles, to maintain intracellular homeostasis and to keep the cell healthy. Recnetly, Autophagic vacuoles are found in cardiomyocytes in ischemic hearts, and in human and hamster cardiomyopathic failing hearts. However, the precise role of autophagy in the HF is still unclear and remains to be elucidated.
Objectives
Establish the transverse aortic arch constriction (TAC) model, investigate the autophagic flux and autophagic gene expression in the progression of HF, and thus discuss the underlying mechanisms.
Experiment I
Methods
1. Animals
170male C57BL/6mice were divided into three groups:the normal group (n=50), the sham group (n=50) and the TAC group (n=70).8-11mice were sacrificed at0,3,7,14or28days after surgery in each group.
2. Pressure-overload HF
The use of a horizontal incision at the level of the suprasternal notch allows direct visualization of the transverse aorta without entering the pleural space and thus obviates the need for mechanical ventilation. The transverse aorta was banded between the right innominate and left carotid arteries to the diameter of a27-gauge needle using a7-0silk suture. Sham operations were served as a control for the TAC group.
3. Echocardiography Analysis
Left ventricular mass weight (LVMW) and Ejection fraction (EF%) were detected to evaluate cardiac function.
4. Histological and Immunochemical Analysis
Haematoxylin eosin (HE) and Masson staining were preformed to investigate cardiac hypertrophy myocardial fibrosis. Specific immunohistochemical staining was also performed with antibodies against microtubule-associated protein light chain3(LC3), Bcl-2interacting coiled-coil protein-1(Beclin-1), RUN domain protein as Beclin1-interacting and cysteine-rich containing (Rubicon), monocyte chemotactic protein-1(MCP-1), intercellular adhesion molecule (ICAM-1), toll-like receptor4(TLR4), myeloid differentiation factor88(Myd88), matrix metallo proteinase-9(MMP-9), tissue inhibitor of metalloproteinase-1(TIMP-1),4-hydroxy-2-Nonenal (4-HNE),8-hydroxydeoxyguanosine (8OHdG).
5. Real-time quantitative polymerase chain reaction (Real-time PCR)
Real-time PCR was used to analyze the mRNA levels of LC3a, LC3b, Rubicon, Beclin-1, Autophagy5(Atg5) and Atg7.a-Actin was a normalization control.
6. Statistical analysis
Statistical analysis involved use of SPSS11.5for Windows. Normally distributed data were analyzed by one-way ANOVA and non-normally distributed data by Kruskal-Wallis one-way ANOVA. Student t test was used to compare data for two groups. Data are expressed as mean±SD. P<0.05was considered statistically significant.
Results
1. Survival rate
Survival probability in the TAC group is lower than the normal group and the sham group (P<0.01).
2. Echocardiography analysis
Compared to the sham group, the TAC group showed enhanced cardiac hypertrophy, EF%was significantly decreased from73.4±3.5%to41±10.4%on the3rd day (P<0.01) and further decreased to36.2±5.7%on the28th day (P<0.01); LVMW was significantly increased, it increased from42.2±10.2to52.5±12.7on the3rd day after surgery (P<0.05) and further increased to58.2±11.0on the28th day (P<0.05).
3. Histological and Immunochemical Analysis
Histology staining:HE and Masson staining revealed increased myocardium cross-sectional area and more cardiac fibrosis in hearts of the TAC group than the normal group and the sham group especially on the28th day (P<0.05or P<0.01).
Autophagosomal marker assay:We analyzed the expression of autophagosomal markers such as LC3, P62, Beclin-1, Rubicon, Compared with the normal group and the sham group, the protein expression levels of LC3, P62significantly increased and peaked on the7th day and the28th day (P<0.05or P<0.01); The expression level of Beclin-1was increased on the3rd day and the7th day (P<0.01) and Rubicon expression level markedly increase on the3rd day, the7th day, the14th day and the28th day(P<0.05or P<0.01).
Inflammation assay:The protein expression level of inflammation markers such as MCP-1, ICAM-1, TLR4, Myd88, MMP-9, TIMP-1were significantly increased in the TAC group at different time point after TAC surgery (P<0.05or P<0.01).
Oxidative stress assay
Both4-HNE and8OHdG protein levels were increased in TAC mice on the3rd day, the14th day and the28th day (P<0.05or P<0.01).
4. Real-time PCR
Compared to the normal group and the sham group, the mRNA level of LC3a was markedly increased in TAC mice on the7th day (P<0.01), it showed no significant difference at other time points. The mRNA level of LC3b was increased on the3rd day (P<0.01) and the7th day (P<0.05) compared to the normal group and the sham group. Compared to the normal group and sham group, the mRNA level of Atg5were significantly increased on the3rd day, the7th day and the14th day while the expression of Atg7and Beclin-1increased on the3rd day (P<0.01). The mRNA level of Rubicon was higher at the7rd,14th and28th day than the normal group and the sham group (P<0.05or P<0.01). There's no significant different between the normal group and the sham group (P>0.05).
Experiment Ⅱ
Methods
1. Animal
180male C57BL/6mice were divided into three groups:the normal group (n=50), the sham group (n=50) and the TAC group (n=80). To investigate autophagic flux, mice in each group were randomly assigned to treatment with physiological saline or chloroquine or rapamycin by intraperitoneal injection4h before being killed.8-10mice were killed at the7th day and the28th day after surgery in each subgroup.
2. Pressure-overload HF
TAC model was produced by the method in experiment Ⅰ.
3. Autophagic flux assay
To detect autophagic flux in the heart, we measured expression of LC3by immunohistochemistry and Real-time PCR.
Results
1. Histological and Immunochemical Analysis
The protein level of LC3was markedly increased in TAC mice on the7th day (21.43±2.9%, P<0.01) and the28th day (28.2±03.24%, P<0.01) compared with the normal group and the sham group. Rapamycin significantly increased the expression of LC3in all conditions (P<0.05), and combination with rapamycin and chloroquine further increased the expression LC3except the TAC group on the7th day (P<0.01). Rapamycin significantly increased the expression of LC3(P<0.05or P<0.01), and combination with rapamycin and chloroquine further increased the expression LC3in the normal group and the sham group on the28th day (P<0.05or P<0.01). Rapamycin slightly increased the expression of LC3(P>0.05) and combination with rapamycin and chloroquine couldn't further increase the expression LC3in TAC mice on the28th day (P>0.05).
2. Real-time PCR
The mRNA expression of LC3a and LC3b was significant increased at the end of the7th day compared with the normal group and the sham group (P<0.05or P<0.01).
On the7th day, rapamycin significantly increased the mRNA expression of LC3a except the TAC group (P<0.05), and increased the mRNA expression of LC3b in every group (P<0.05or P<0.01); combination with rapamycin and chloroquine further increased the expression LC3a and LC3b in every group (P<0.05or P<0.01). On the28th day, rapamycin significantly increased the mRNA expression of LC3a and LC3b in every group (P<0.05or P<0.01). Combination with rapamycin and chloroquine further increased the expression of LC3a and LC3b in every group except the TAC group.
3. Autophagic flux assay
Autophagic flux induced by rapamycin made no significant difference in every groups on the7th day. But it significantly decreased on the28th day in the TAC group compared with the normal group and the sham group.
Conclusions
1. Autophagy was compensatory enhanced at early stage of pathological pressure overload and showd normal autophagic flux induced by rapamycin in the TAC group. At the late stage, it showed autophagy dysfunciton and lower autophagic flux induced by rapamycin.
2. Autophagy genes change dynamically and play an important role in the progression of cardiac remodeling in TAC mice.
3. Autophagy and inflammation/oxidative stress are closely related in the progression of cardiac remodeling in TAC mice, and TLR4-Myd88pathway may be involved in the underlying mechanisms.
Background
Heart failure (HF) is the leading cause of morbidity and mortality worldwide. There is a critical need to explore new therapeutic approaches in heart failure. Autophagy is a cellular response to starvation as well as a quality-control system that can deliver damaged organelles and long-lived proteins from the cytoplasm to lysosomes for clearance. Autophagy helps clear intracellular protozoa, bacteria and viruses and functions in antigen presentation. Growing evidence suggested that autophagy took part in the development and progression of heart diseases. Traditional Chinese drug has been used in the treatment of HF. Basic and clinical research showed that Qiliqiangxin could up-regulation the contents of ATP, improve the hemodynamics, inhibit neurohormonal activation, thus effectively improve cardiac function and the prognosis in patients. But the role of Qiliqiangxin in the autophagic mechanisms is still unclear in the progression of HF.
Objectives
1. To test the hypotheses that the traditional Chinese medicine Qiliqiangxin can improve cardiac function in the compensated stage and the decompensated stage of HF in transverse aortic arch constriction (TAC) mice.
2. To elucidate the autophagic mechanism of the traditional Chinese medicine Qiliqiangxin in the compensated stage and the decompensated stage of HF in TAC mice.
Methods
1. Animal
C57BL/6mice underwent TAC surgery (n=300), a model of pressure-overload heart failure, or sham surgery (n=60). We randomly divided the TAC mice into3groups for treatment (n=80each):the perindopril group, the Qiliqiangxin group and the TAC group. Mice were killed on the14th and28th day, to investigate autophagic flux, mice in each group were randomly assigned to treatment with physiological saline (0.2ml), chloroquine (30mg/kg) or rapamycin (2mg/kg) by intraperitoneal injection4h before being killed.
2. Pressure-overload HF:TAC model was produced by the method in Part I
3. Echocardiography Analysis
Left ventricular mass weight (LVMW) and LV ejection fraction (EF%) were detected to evaluate cardiac function.
4.Histological and Immunochemical Analysis
Haematoxylin eosin (HE) and Masson were preformed to investigate cardiac hypertrophy myocardial fibrosis. Specific immunohistochemical staining was also performed to detect the expression of microtubule-associated protein light chain3(LC3) in every subgroups, and detect the expression of Bcl-2interacting coiled-coil protein-1(Beclin-1), RUN domain protein as Beclin1-interacting and cysteine-rich containing (Rubicon), matrix metallo proteinase-9(MMP-9), tissue inhibitor of metalloproteinase-1(TIMP-1),4-hydroxy-2-Nonenal (4-HNE),8-hydroxy-2'-deoxyguanosine (8OHdG) in mice that were injection with physiological saline.
5. Real-time quantitative polymerase chain reaction (Real-time PCR)
Real-time PCR was used to analyze the mRNA levels of LC3a, LC3b in every subgroups, and the levels of Rubicon, Beclin-1, Autophagy5(Atg5), and Atg7in in mice that were injection with physiological saline. a-Actin was a normalization control.
6. Statistical analysis
Statistical analysis involved use of SPSS11.5for Windows. Normally distributed data were analyzed by one-way ANOVA and non-normally distributed data by Kruskal-Wallis one-way ANOVA. Student t test was used to compare data for2groups. Data are expressed as mean±SD. P<0.05was considered statistically significant.
Results
1. Echocardiography Analysis
On the14th day after surgery, acute pressure overload significantly decreased EF%and increased LVMW compared to the sham group (P<0.01). Both the perindopril group and the Qiliqiangxin group showed higher EF%and lower LVMW compared to the TAC group (P<0.05or P<0.01).
On the28th day after surgery, chronic pressure overload further decreased EF%and increased LVMW in the TAC group compared to the sham group (P<0.01). Either perindopril or Qiliqiangxin could improve EF%(P<0.01) and LVMW (P<0.05) in the TAC mice. There is no significant difference between the perindopril group and the Qiliqiangxin group.
2. Histological and Immunochemical Analysis
(1) Histology staining
HE and Masson staining revealed increased myocyte cross-sectional area and more cardiac fibrosis in hearts of TAC mice than sham mice especially on the28th day (P<0.01). Either perindopril or Qiliqiangxin could help relieve such pathological change both on the14th day and the28th day (P<0.05).
(2) Autophagic function
On the14th and the28th day, the expression of LC3was significantly higher in the TAC group, the perindopril group and the Qiliqiangxin group in comparasion with the sham group (P<0.01). Compared with the TAC group, the perindopril group and the Qiliqiangxin group showed lower LC3positive rate (P<0.05or P<0.01).
On the14th day, rapamycin significantly increased the expression of LC3in every group (P<0.05or P<0.01); combination with rapamycin and chloroquine further increased the expression LC3except the TAC group on the14th day (P<0.05or P<0.01).
On the28th day, rapamycin significantly increased the expression of LC3(P<0.05or P<0.01) and combination with rapamycin and chloroquine further increased the expression LC3in every group (P<0.05or P<0.01)except the TAC group on the28th day.
(3) Autophagic gene
On the28th day, the p62protein expression level in the TAC group was significantly increased compared with the perindopril group, the Qiliqiangxin group and the sham group (P<0.01).
On the14th day and the28th day, the expression of Beclin-1in the sham group was significantly lower than the other groups (P<0.01).It was much higher in the perindopril group and the Qiliqiangxin group than the TAC group on the14th day (P<0.05or P<0.01).On the28th day, it was higher in the TAC group than the perindopril group and the Qiliqiangxin group (P<0.05).
On the14th and the28th day, the expression of Rubicon in the sham group was significantly lower than the other groups (P<0.01), and it was much lower in the perindopril group and the Qiliqiangxin group than the TAC group (P<0.05or P<0.01).
(4) Oxidative stress markers
On the14th day and the28th day, the expression of4-HNE and8OHdG were much higher in the TAC group, the perindopril group and the Qiliqiangxin group than the sham group (P<0.01). The4-HNE and8OHdG protein expression levels were much higher in the TAC group than the perindopril group and Qiliqiangxin group (P<0.05or P<0.01) on the28th day.
(5) MMPs/TIMPs
On the14th day, the expression of MMP-9were increased in the TAC group and the Qiliqiangxin group compared with the sham group and the perindopril group (P<0.01). On the28th day, the expression level of MMP-9was significantly increased in the TAC group, the perindopril group and the Qiliqiangxin group than the sham group (P<0.01), and it was lower in the perindopril group and Qiliqiangxin group than the TAC group (P<0.05).
On the14th day, there is no significant difference in every groups,on the28th day, the expression of TIMP-1were much higher in the TAC group, the perindopril group and the Qiliqiangxin group than the sham group (P<0.01). It was much higher in the perindopril group and the Qiliqiangxin group than the TAC group on the28th day (P<0.01).
3. Real-time PCR
(1) LC3expression
On the14th day, the mRNA expression of LC3a in the TAC group, the perindopril group and the Qiliqiangxin group were significantly increased in comparison with the sham group (P<0.05or P<0.01). Compared with the TAC group, the mRNA expression of LC3a was significantly higher (P<0.01). On the28th day, the mRNA expression of LC3a was significantly higher in the perindopril group and the Qiliqiangxin group compared with the sham group and the TAC group (P<0.01).
The mRNA expression of LC3b was significantly increased in the perindopril group and the Qiliqiangxin group compared with the TAC group and the sham group on the14th day (P<0.01). The mRNA expression of LC3b in the TAC group, the perindopril group and the Qiliqiangxin group were significantly increased in comparison with the sham group on the28th day (P<0.05or P<0.01). Furthermore, the mRNA expression of LC3b in the perindopril group were significantly higher in comparison with the TAC group on the28th day (P<0.05)..
(2) Autophagic flux
On the14th day, rapamycin significantly increased the mRNA expression of LC3a in every group (P<0.01), combination with rapamycin and chloroquine further increased the expression LC3a in every group (P<0.01).
On the28th day, rapamycin significantly increased the mRNA expression of LC3a in every group (P<0.01); combination with rapamycin and chloroquine further increased the expression LC3a in every groups except the TAC group (P<0.01).
On the14th day, rapamycin significantly increased the mRNA expression of LC3b in every group, combination with rapamycin and chloroquine further increased the expression LC3b in every group, P<0.01).
On the28th day, rapamycin significantly increased the mRNA expression of LC3b in every group (P<0.05or P<0.01); combination with rapamycin and chloroquine further increased the expression LC3b in every groups except the TAC group (P<0.01).
Autophagic flux was much lower in the TAC group but made no significant differedce on the14th day. On the28th day, the autophagic flux was further decreased in the TAC group compared to the other groups (P<0.01). And it was lower in the perindopril group and the Qiliqiangxin group compared to the sham group (P<0.05).
(3) Autophagic gene
On the14th day, the expression of Atg5were much higher in the TAC group, the perindopril group and the Qiliqiangxin group than the sham group (P<0.01). It was much lower in the perindopril group than the TAC group (P<0.05). On the28th day, compared to the sham group and the TAC group, the mRNA level of Atg5was significantly increased in the perindopril group and the Qiliqiangxin group (P<0.01).
On the14th day and the28th day, the expression of Atg7were higher in the perindopril group and the Qiliqiangxin group than the TAC group and the sham group (P<0.05or P<0.01).
The mRNA level of Beclin-1in the perindopril group and the Qiliqiangxin group were significantly higher than the other groups on the14th day (P<0.05or P<0.01), and it was higher in the TAC group than the other groups on the28th day (P<0.01).
On the14th and the28th day, the mRNA expression of Rubicon were much higher in the TAC group, the perindopril group and the Qiliqiangxin group than the sham group (P<0.01). It was much higher in the perindopril group and the Qiliqiangxin group than the TAC group on the14th day (P<0.01), and lower in the perindopril group and the Qiliqiangxin group than the TAC group on the28th day (P<0.01).
Conclusions
1. A reversing effect of Qiliqiangxin on the cardic remodeling in TAC mice was observed in the present study.
2. Qiliqiangxin could anti-oxidative stress and improve autophagic function, and has the same effect as perindopril.
Background
Heart failure (HF) is a major public health concern associated with significant mortality and morbidity. Symptoms of psychological stress, such as anxiety and depression, are common among patients with HF. Furthermore, the presence of these disorders significantly leads to increased symptoms of HF, poor quality of life and an opposite effect on prognosis. Thus, psychological stress may have an important role in the development of HF. However, previous studies were prospective observational studies, so mechanisms relating psychological stress to HF are still not totally understood.
Depression and anxiety disorders were linked to dysfunction of the renin-angiotensin system (RAS) and generation of Angiotensin Ⅱ(AngⅡ). AngⅡ contributes to the progression of HF, and recent study showed that AngⅡ takes part in cardiomyocyte autophagy.Autophagy is a highly conserved cellular mechanism that plays a key role in the turnover of long-lived proteins, RNA, and other macromolecules. In the heart, autophagy occurs at low levels under normal conditions, then defects in the process cause cardiac dysfunction and HF. Autophagy may be an important mechanism underlying the progression of HF. Whether psychological stress induces autophagic dysfunction in HF is not totally understood. Psychological stress may affect cardiac autophagy through AngⅡ, thus adversely affecting cardiac remodeling.
The angiotensin-converting enzyme inhibitor (ACEI) captopril can reduce the activity of the renin-angiotensin system (RAS), blocking the conversion of AngI to Ang Ⅱ.We observed autophagy in mice with HF induced by transverse aortic constriction (TAC) and investigated whether psychological stress influences autophagy in HF and the role of captopril in this progression, thus discuss the underlying mechanism.
Objectives
1. To observe autophagy in early stage of HF in TAC mice, investigated whether psychological stress influence the autophagy in HF.
2. To discusse the role of Ang Ⅱ in this progression, thus studied the underlying mechanisms.
Experiment I
Methods
1. Experimental Protocol
150male C57BL/6mice (7-8weeks old) were underwent TAC surgery (n=120), a model of pressure-overload HF, or sham surgery (n=30). On the3rd day after surgery, we randomly divided the TAC mice into3groups for treatment (n=40):the combined acoustic and restraint stress group (stress), the stress plus captopril group (captopril) and the TAC control group (TAC). To investigate autophagic flux, mice in each group were randomly assigned to treatment with physiological saline(0.2ml), chloroquine (30mg/kg) or rapamycin (2mg/kg) by intraperitoneal injection4h before being killed.
2. Combined acoustic and restraint stress
Stressed mice were exposed to combined acoustic and restraint stress for7days. They were placed in50-ml conical centrifuge tubes with multiple ventilation holes without pinning the tail for2h. Meanwhile, acoustic stress was produced by an alarm fixed on the top of the stimulator and introduced every3min at110dB lasting for5s.
3. Splash test
The splash test was performed at the beginning and end of the stress procedure. An amount of10%sucrose solution was squirted on the dorsal coat of mice that were in their home cage. Grooming behaviour was recorded as total frequency.
4. Echocardiography and hemodynamic analysis
Echocardiography and hemodynamic analysis was performed at the beginning and end of1-week stress treatment. Systolic blood pressure (SBP), diastolic blood pressure (DBP) and heart rate (HR) were measured by the tail-cuff method. Echocardiography was performed and left ventricular mass weight (LVMW) and ejection fraction (EF%) were detected to evaluate cardiac function.
5. Survival analysis
Survival rate of every group was record.
6. Blood collection and biochemical measurements
After1-week stress, all animals were killed, and blood was collected. Serum samples were prepared. Enzyme-linked immunosorbent assay (ELISA) kits were used to assay serum concentration of corticosterone and Ang Ⅱ.
7. Histology and immunohistochemical staining
Tissues were embedded in paraffin, and coronal sections (5μm) underwent general histological staining with haematoxylin&eosin (HE) and Masson. Specific immunohistochemical staining was also performed with antibody against microtubule-associated protein light chain3b (LC3b) in every group and performed with antibodies against Bcl-2interacting coiled-coil protein-1(Beclin-1), RUN domain protein as Beclin1-interacting and cysteine-rich containing (Rubicon),4-hydroxy-2-Nonenal (4-HNE) and8-hydroxydeoxyguanosine (8OHdG,) in groups injected with physiological saline.
8. Real-time quantitative polymerase chain reaction (Real-time PCR)
Real-time PCR was used to analyze the mRNA levels of LC3a, LC3b in every group and Beclin-1, Rubicon, Autophagy5(Atg5), and Atg7in groups injected with physiological saline.
9. Statistical analysis
Statistical analysis involved use of SPSS11.5for Windows. Survival was assessed by the Kaplan-Meier method with chi-square analysis. Normally distributed data were analyzed by one-way ANOVA and non-normally distributed data by Kruskal-Wallis one-way ANOVA. Student t test was used to compare data for2groups. Data are expressed as mean±SD. P<0.05was considered statistically significant.
Results
1. Grooming frequency
Stressed mice groomed significantly less than did unstressed mice (P<0.01).
2. Survival rate
Kaplan-Meier survival analysis showed cumulative survival probability significantly shorter for stressed mice (75%,27/36) than TAC mice (91%,33/36, P<0.05).
3. Bichemical studies
Seven-day stress increased serum corticosterone levels in the stress group and the captopril group (6-7fold, P<0.01) and compared with the sham group and the TAC group.
The serum level of Ang II was significantly higher in the stressed than the TAC and captopril mice (P<0.01), with no difference in Ang Ⅱ level between the captopril and the TAC mice (P>0.05). Moreover, the serum level of Ang Ⅱ was higher but not significantly in the TAC than the sham mice (P<0.05).
4. Hemodynamic analysis and echocardiography
Stress significantly elevated SBP, DBP and HR in the stress and the captopril groups (P<0.01). The levels were lower with captopril than with stress alone. Compared to the TAC group, the stressed group showed enhanced cardiac hypertrophy, including increased LVMW (P<0.01) and ratio of heart weight to body weight (HW/BW, P<0.01). Most importantly, EF%was significantly lower in stressed than TAC mice after1week of stress (P<0.01). The captopril group showed higher EF%, lower LVMW and HW/BW compared to the stress group (P<0.01).
5. Histology staining for cardiac fibrosis
Masson staining revealed more cardiac fibrosis in hearts of stressed than TAC mice (P<0.05). Masson staining revealed less cardiac fibrosis exists in hearts of captopril mice, with no significant difference (P>0.05).
6. Autophagic flux assay
To determine whether stress induces autophagic dysfunction in the heart, we measured expression of LC3b by immunohistochemistry and Real-time PCR. The level of LC3b was markedly increased in TAC mice (P<0.01). Furthermore, stress further upregulated the expression of LC3b (P<0.01). Administration of captopril decreased expression of LC3b compared to stress alone (P<0.01).
Autophagic flux was markedly increased in TAC mice as compared with the sham group but significantly decreased in the stressed group as compared with the TAC group. The captopril group showed upregulated autophagic flux as compared with stress alone. Rapamycin significantly increased the expression of LC3b in all conditions (P<0.05or P<0.01), which indicates no autophagosome formation dysfunction under stress. Therefore, decreased clearance occurred after stress, and such autophagic responses could be reduced by captopril.
7. Autophagosomal marker assay
Stress significantly increased the mRNA levels of Beclin-1, Rubicon, Atg5and Atg7(P<0.01). The expression of Rubicon and Beclin-1were lower with captopril than stress alone (P<0.01). Immunohistochemical staining results were consistent with Real-time PCR results.
8. Cardiac oxidative stress assay
To investigate oxidative stress in the heart, we examined the expression of4-HNE and8OHdG. Both4-HNE and8OHdG levels were increased in TAC mice (P<0.01) and further exaggerated in stress mice (P<0.01). Moreover, levels were lower with captopril than stress alone (P<0.01).
Experiment II
Method
1. Cell Stimulation
In some experiments, cells were pretreated with Ang II (0.1μM), N-acetylcysteine (NAC,50μM), rapamycin (1μM) or bafilomycin A1(Baf-A1,50nM).
2. Immunofluorescence
The protein levels of LC3b and4-HNE were measured by immunofluorescence.
3. Western blot
The protein levels of LC3b, Rubicon, Beclin-1, β-action were measured by Western blot.
4. Real-time PCR
LC3a, LC3b, Atg5, Atg7, Beclin-1, Rubicon mRNA levels were measured by Real-time PCR after RNA extraction and reverse transcription.
Result
1. Ang Ⅱ promotes autophagy
To further confirm the role of Ang Ⅱ in autophagy, H9c2cells were incubated with vehicle (control) or AngⅡ (0.1μM) for22h, then rapamycin or bafilomycin Al were added to the medium and co-incubated for2h., finally underwent immunofluorescence. Ang Ⅱ promoted the expression of LC3b (P<0.01). Ang Ⅱ enhanced autophagic flux. Rapamycin or Baf-A1enhanced AngⅡ-induced increased ofLC3b.
2. Role of oxidative stress in induction of autophagy
To evaluate the role of oxidative stress in Ang Ⅱ-induced autophagy induction, cells were treated with vehicle (control) or Ang Ⅱ with or without NAC (50μM), then underwent immunofluorescence and western blot analysis. Ang Ⅱ enhanced LC3b and4-HNE expression (P<0.01), which was partly inhibited by NAC (P<0.01). Furthermore, Rapamycin or Baf-Al enhanced Ang Ⅱ-induced increased of LC3b which was partly inhibited by NAC (P<0.01).
3. Expression of Autophagy gene
AngⅡ enhanced Atg5, Atg7, Beclin-1, Rubicon mRNA expression and Beclin-1(P<0.05or P<0.01)., Rubicon protein expression, which was partly inhibited by NAC (P<0.05or P<0.01).
Conclusions
1. Stress may induce autophagic flux dysfunction in clearing autophagosomes and adversely affects cardiac hypertrophy and HF in mice with TAC.
2. Endogenous Ang Ⅱ plays a key role in autophagic dysfunction via the oxidative stress response to stress.
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