【作者】 江端；

【导师】 唐其柱；

【作者基本信息】 武汉大学 ， 内科学， 2014， 博士

【摘要】 背景：心脏重构是心脏衰竭的关键环节,常继发于高血压等疾病引起的长期心脏负荷过重,心脏重构在初始阶段具有代偿功能,但心脏重构后期的心肌纤维化等会引起心脏功能障碍,它的特点是心脏肥大、纤维化、氧化应激和心肌细胞凋亡。目前已有报道Akt、PKC等多种信号通路参与了心脏重构各个过程,临床上有多种药物治疗心衰过程中的心脏重构,这些药物针对于心脏重构各环节。橙皮素,它属于柑橘类黄酮,主要包含黄酮类化合物,具有抗炎、抗肿瘤等多种药理性能。研究表明橙皮素具有一定的抗氧化和抗凋亡的功能,然而,橙皮素在心脏重构中的作用尚无报道,因此我们采用主动脉缩窄术(Aortic binding, AB)研究橙皮素对心脏重构过程中心肌肥大,纤维化、氧化应激和心肌细胞凋亡的影响。实验方法：将野生型C57BL/6小鼠(雄性8～10周龄)随机分为对照-假手术组,橙皮素-假手术组,对照-手术组和橙皮素-手术组,采用主动脉缩窄术建立实验动物模型,手术成功后根据分组给予不同处理7周。主要通过以下指标检测心脏重构各过程和相关分子机制情况：一、心肌肥厚：1.采用心动超声仪检测不同组小鼠的心率和IVSd、LVDd、LVPWd、IVSs、 LVDs、LVPWs;2.采用血流动力学监测各组小鼠LVEF、LVFS、ESP和dP/dT max;3.采用解剖研究各组小鼠心脏重量(HW)、肺脏重量(LW)、心脏体重比(HW/BW)和心脏重量肋骨直径比(HW/TL);4.采用组织切片WGA染色和HE染色研究各组小鼠心脏体积和心肌细胞横截面积；5.采用实时定量PCR研究ANP、BNP、α-肌球蛋白重链(a-MHC)等增生性标记物的表达情况；6.采用western blot研究各组小鼠心脏中PKC-Akt和MAPK信号通路信号分子的表达情况；二、心肌纤维化：1.采用PSR染色研究各组小鼠心肌纤维化情况；2.采用实时定量PCR检测小鼠心肌纤维化介质的表达情况2.采用western blot研究Smad蛋白级联相关蛋白的表达情况；三、氧化应激采用实时定量PCR检测小鼠心肌氧化酶亚基mRNA的表达情况；四、细胞凋亡1.采用TUNEL法检测各组小鼠心肌细胞凋亡情况2.采用western blot研究Smad蛋白级联和裂解PARP、BAX、BCL-2等凋亡相关蛋白的表达情况。实验结果：第一部分：心肌肥厚1.主动脉缩窄术引起小鼠的心率明显增加,假手术组心率为518士13.34beats/min,而AB小鼠心率增加到522.38±11.90beats/min,橙皮素处理组与假手术组无显著性差别；2.AB术后LVEF、LVFS显著降低,3.HE切片显示AB术后小鼠心脏体积和心肌细胞横截面积增大,而橙皮素能明显降低AB术引起的心脏体积和心肌细胞横截面积的增大；4.实时定量PCR发现AB术后ANP等心肌增生标记物mRNA表达显著增加,橙皮素能显著降低这些心肌增生标记物；5. western blot结果表明慢性压力负荷引起小鼠心肌PKCα/βⅡ-AKT和JNK信号通路的激活,橙皮素能降低这两个信号通路的激活.第二部分：心肌纤维化1.PSR染色结果表明AB术诱导的压力负荷引起左心室纤维化区域增大,胶原体积和含量增加,橙皮素明显降低这种现象；2.实时定量PCR结果显示慢性压力负荷引起心肌组织中的TGF-β1等纤维化介质mRNA表达增加,橙皮素能显著降低这些纤维化介质的表达；3. western blot结果表明压力负荷引起磷酸化的Smad2和Smad3表达显著增加,而与AB手术组相比,橙皮素显著降低Smad2和Smad3蛋白磷酸化水平。第三部分：一、氧化应激实时定量PCR结果显示慢性压力负荷增加NADPH氧化酶亚单位mRNA表达,降低SODl和SOD2mRNA的表达,而橙皮素能降低NADPH氧化酶亚单位mRNA的表达、增加SOD1和SOD2mRNA的表达。二、细胞凋亡1.TUNEL法结果表明慢性压力负荷引起心肌细胞凋亡增加,橙皮素能减低心肌细胞的凋亡；2. western blot实验显示慢性超压力负荷明显增加裂解PARP、BAX、BCL-2、 BCL-XL、Bak和裂解的caspase-3蛋白表达,橙皮素能抑制裂解PARP、Bax/Bcl-2、Bak和裂解的caspase-3等蛋白的表达。结论：1.主动脉缩窄术所致的慢性压力负荷引起小鼠心肌肥厚、心肌纤维化、氧化应激增加和心肌细胞凋亡；2.橙皮素降低慢性压力负荷引起的心肌肥厚,该作用是通过PKCα/βⅡ-AKT和JNK信号通路实现的；3.橙皮素通过Smad级联蛋白信号通路降低慢性压力负荷引起的心肌纤维化；4.橙皮素降低压力负荷引起的心肌氧化应激；5.橙皮素降低心肌细胞凋亡,并降低凋亡相关蛋白的表达；这些结果表明,橙皮素是心脏重构和心脏衰竭的潜在治疗药物。更多还原

【Abstract】 Background:Cardiac remodeling, which is caused by long-term cardiac overload caused by hypertension and other diseases, is a key factor of heart failure. In the initial stages, cardiac remodeling has compensatory function, but myocardial fibrosis may lead to cardiac dysfunction, which is characterized by cardiac hypertrophy, fibrosis, oxidative stress and apoptosis of myocardial cells. It has been reported that Akt, PKC and other signaling pathways were involved in various processes of cardiac remodeling. There are a variety of drugs in clinical treatment of heart failure in the processes of cardiac remodeling. Hesperetin, which belongs to citrus flavonoids, is mainly expressed in flavonoids. It has several pharmacological properties, such as anti-inflammatory, anti-tumor and so on. Hesperetin have some antioxidant and anti-apoptotic function, however, the role of hesperetin in cardiac remodeling remains elusive. So we study the effect of hesperetin on the process of cardiac remodeling, including myocardial hypertrophy, fibrosis, oxidative stress and cardiac myocyte apoptosis, by aortic banding (AB).Methods: Wild-type C57BL/6mice (8-10weeks old male) were randomly were randomly assigned to four groups: vehicle-sham (n=10), hesperetin-sham (n=9), vehicle-AB (n=10) and hesperetin-AB (n=10). Aortic banding (AB) was performed as described previously. Then,1week after AB or surgery, the animals were treated with30mg/kg/day of hesperetin or vehicle for7weeks. We mainly study cardiac remodeling process and molecular mechanisms underlying the case through the following indicators:A. cardiac hypertrophy:1. detected different groups of mice heart rate and IVSd, LVDd, LVPWd, IVSs, LVDs, LVPWs by echocardiography;2. monitored mice in each group LVEF, LVFS, ESP and dP/dT max by hemodynamic apparatus;3. investigated mice heart weight (HW), lung weight (LW), heart weight ratio (HW/BW) and heart weight rib diameter ratio (HW/TL) by anatomical studies;4. studied cardiac volume and cardiac myocyte cross-sectional area by using WGA staining and HE staining;5. studied mRNA expression of ANP, BNP, a-myosin heavy chain (a-MHC) and other proliferative marker by using real-time quantitative PCR6. investigated the protein expression of mice heart PKC-Akt and MAPK signaling pathways signaling molecules by western blot studiesB. myocardial fibrosis:1. studied myocardial fibrosis mice in each group situations using PSR staining;2. detected the expression of cardiac fibrosis media by real-time quantitative PCR3. detected expression of Smad proteins cascade related protein using western blotC. Oxidative StressDetected mRNA expression mouse myocardium oxidase subunit by real-time quantitative PCRD. Apoptosis1.detected myocyte apoptosis using TUNEL assay2.detected the expression of Smad proteins cascade and cleavaged PARP, BAX, BCL-2and other apoptosis-related protein by western blot;Results:Part I:cardiac hypertrophy1. Aorta banding significantly increased heart rate. The heart rate of vehicle-sham group is518±13.34, while heart rate increased to522.38±11.90in vehicle-AB group. There is no significant difference between hesperetin-AB group and vehicle-sham group;2. LVEF, LVFS significantly reduced in vehicle-AB mice;3. HE sections showed that heart volume and cross-sectional area of myocardial cells increased in vehicle-AB group, and hespertin can significantly reduce increasement of the volume and cardiac myocyte cross-sectional area, which is caused by AB surgery;4. Real-time quantitative PCR indicated that myocardial proliferation markers,such as ANP mRNA, was significantly increased after AB surgery, hesperetin can significantly reduce cardiac markers proliferation;5. Western blot results showed that in mice with chronic pressure overload induced the activation of myocardial PKCa/β Ⅱ-AKT and JNK signaling pathway, and hesperetin can reduce the activation of these two signaling pathways. Part II:Myocardial fibrosis1. PSR staining showed that AB surgery-induced left ventricular pressure overload increased areas of fibrosis, collagen volume and content. Hesperetin significantly reduce this phenomenon;2. Real-time quantitative PCR results showed that chronic pressure overload increased mRNA expression of myocardial tissue fibrosis media such as TGF-β1. Hespertin can significantly reduce the expression of these fibrosis medium;3. Western blot results showed that pressure overload significantly increased phosphorylation of Smad2and Smad3expression. While compared with the AB, hesperetin significantly reduced phosphorylation levels of Smad2and Smad3.Part III:First,Oxidative stressReal-time quantitative PCR results showed that chronic pressure overload increased mRNA expression of NADPH oxidase subunit, reduced mRNA expression of SOD1and SOD2, whereas hesperetin can reduce the mRNA expression of NADPH oxidase subunit increased mRNA expression of SOD1and SOD2.Second, Apoptosis1. TUNEL results showed that chronic pressure overload increased myocardial apoptosis and hespertin can reduce cardiac myocyte apoptosis;2. Western blot experiments showed that chronic pressure overload significant increased protein expression of cleavaged PARP, BAX, BCL-2, BCL-XL, Bak and cleaved caspase-3, and hesperetin can inhibit the protein expression of cleavaged PARP, Bax/Bcl-2, Bak and cleaved caspase-3, etc.Conclusions:We found that orange flavonoid hesperetin protected against cardiac hypertrophy, fibrosis, apoptosis and dysfunction induced by aortic banding. These findings possible exploit a potential therapeutic drug to cardiac remodeling and heart failure.更多还原