【作者】 高峰；

【导师】 祝善俊；

【作者基本信息】 第三军医大学 ， 内科学， 2012， 博士

【摘要】 背景和目的：近年来心力衰竭的治疗取得了一定的进展，但其发病率和死亡率在全世界范围内仍然居高不下。因此，探讨好的治疗方法及研制新的药物是医学界亟待解决的问题。心力衰竭是一组临床上极为常见的心血管综合征，冠心病、高血压、心肌梗塞、心律失常、瓣膜异常都可引起心力衰竭。心衰时对心脏构和功能的研究经历了相当长的时期，直到20试剂90年代才意识到心衰发生发展的基础是心室重构，心室重构是心衰患者发病率和死亡率的决定因素。心室重构是指心脏受到损伤后基因表达、分子、细胞和心肌间质的变化，临床表现为心脏大小、形状和功能的改变。心室重构的调节主要包括肾素-血管紧张素-醛固酮系统、炎性细胞因子系统、机械应激、氧化应激、高盐、高糖、转化生长因子等都能在一定程度上影响心室重构的进行。心脏通过能量代谢将储存于脂肪酸或葡萄糖中的化学能转化为机械能，为心脏收缩和舒张提供能量。代谢底物从脂肪酸到葡萄糖利用的适应性转换在心肌重构的病理生理过程中起到关键性作用。糖脂代谢异常可通过影响心肌能量代谢，促使心肌代谢重构。盐是高血压、心肌肥厚重要的环境因素，盐代谢异常可通过增加氧化应激，促使心肌重构。本研究从脂质，高盐两个方面对心肌重构进行探讨。过氧化物酶增殖体激活受体γ共活化因子-1(PGC-1α)在高代谢率的组织表达较高，例如心脏，肌肉和棕色脂肪组织。PGC-1α和核受体家族或是非核受体家族相互作用，来调控不同的细胞能量代谢通路，在心肌肥厚和心衰的进展中PGC-1α起着重要的作用。例如，PGC-1α和雌激素相关受体作用，来调节丙酮酸脱氢酶4（PDK4），通过对PDK4的调控，从而完成对葡萄糖氧化的调节。文献报道，在心肌肥厚的发展中，心肌对葡萄糖的利用增加，PGC-1α和PDK4的蛋白表达受到抑制，都是其特征性的表现。UCP2在心肌中大量表达，减少ROS产生和减轻氧化应激反应，iNOS表达上调可增加心肌纤维化、心肌硝基化和启动心肌重构等。PPARδ可激活UCP2、减少iNOS表达，减轻氧化应激，改善心肌重构。他汀类药物（HMG-CoA还原酶抑制剂）是临床上广泛应用的降脂药物，人们发现他汀类药物产生的获益远大于降脂作用本身善内皮功能现已发现和证实的作用包括改善血管内皮功能、抗炎、抗氧化、稳定斑块、改善凝血功能、减少神经内分泌激活、刺激内皮祖细胞分化、逆转心肌重构、减少骨质吸收及抗肿瘤等多效性。盐是高血压，心肌肥厚重要的环境因素，盐的摄入与高血压患病率呈明显的量效关系，我国心血管病的发病率率呈北高男低的特点。而我国南北方饮食有显著差异，北方喜食高盐，云、贵、川居民喜食辣椒食品，已报道辣椒素能调节高盐诱导血压升高。基于以上背景，我们提出如下科学假说：阿托伐他汀是否能够通过对PGC-1α的调节，而改善炎性状态下的心肌的糖脂代谢紊乱，逆转心肌重构？辣椒素激活TRPV1调节高盐诱导的血压的同时，是否能改善高盐诱导心肌重构？材料与方法：第一部分：胚胎来源的大鼠心肌细胞系H9c2，在DMEM培养基（10%的胎牛血清）中生长，种植的密度维持在5,000cells/cm2，培养基每两天更换一次，细胞在5%CO2and95%O2条件下生长。待近于融合时，更换成无血清的培养基，继续培养24小时。细胞分组有H9c2cells, H9c2+TNF-α, H9c2+TNF-α+Atorvastatin, siPGC-1α分别进行了研究。第二部分：采用离体实验和在体实验。在体实验C57BL/6J野生型(WT)和TRPV1基因敲除(TRPV1–/–)小鼠作为研究对象进行饮食干预，离体实验以辣椒素刺激心肌细胞为研究对象。WT和TRPV1–/–小鼠随机分为普食组(NS组)，高盐组(HS)，高盐加辣椒素组(HS+Cap)干预1年，从2月龄开始。1.心肌细胞H9c2培养，用TNF-α刺激，同时用阿托伐他汀干预细胞我们检测以上各组的葡萄糖氧化率。2.心肌细胞H9c2培养，用TNF-α刺激，同时用不同浓度的阿托伐他汀干预细胞1-25μmol/L持续24小时，后收集细胞提蛋白行western检测PGC-1α的抗体。同时给予甲羟戊酸，金合欢醇，尨牛儿基尨牛儿醇等观察阿托伐他汀对所起的作用和这几种物质的关系。3.细胞分组有H9c2cells, H9c2+TNF-α, H9c2+TNF-α+Atorvastatin，干预结束后，提蛋白行western检测PDK4和CPT14.用RNA干扰技术，敲减PGC-1α后检测心肌细胞中PDK4和CPT1的蛋白表达情况，5.心肌细胞分组control, GFP+TNF-α, siPGC-1α+TNF-α，然后检测各组的葡萄糖的氧化率。6.用蛋白免疫印迹检测TRPV1蛋白的表达；用免疫荧光染色观察TRPV1在H9C2心肌细胞的具体分布。7.比率荧光成像系统检测辣椒素刺激下H9C2心肌细胞[Ca2+]i的变化，观察TRPV1特异性拮抗剂iRTX对钙信号变化的影响。8.用超声评价各组小鼠的心功能。9.检测各组小鼠的心脏重量/体重，应用Masson染色检测胶原沉积，DHE染色检测超氧阴离子水平，组织切片HE染色观察肥厚指标。10.检测心脏组织的蛋白表达PPAR-δ, inos, ucp2。11.检测心肌细胞PPAR-δ蛋白表达。结果：1. TNF-α诱导心肌细胞葡萄糖氧化率显著增加，而用阿托伐他汀共培养后显著抑制了这种增加。2.阿托伐他汀对抗TNF-α诱导的心肌细胞PGC-1α的下调，并呈计量依赖性的，但这种下调和甲羟戊酸，金合欢醇有关，而与尨牛儿基尨牛儿醇无关。3. TNF-α刺激心肌细胞后，会使PGC-1α，PDK4，CPT1显著下调，而阿托伐他汀能逆转TNF-α的这种效果。4.在心肌细胞中敲减PGC-1α，而PGC-1α的靶基因PDK4，CPT1显著下调。5.在心肌细胞中敲减PGC-1α，使得葡萄糖氧化率显著升高。6. TRPV1在H9C2心肌细胞和小鼠心肌组织均有表达。7.辣椒素刺激H9C2心肌细胞引起浓度依赖性的[Ca2+]i升高， TRPV1特异性拮抗剂iRTX预处理可抑制这种细胞内钙信号的变化。8.高盐饮食诱导使心功能恶化，辣椒素能改善高盐诱导的心功能。而在TRPV1-/-小鼠没有观察到这种效果。9.长期高盐饮食诱导使心肌肥厚和纤维化，而辣椒素能改善高盐诱导的肥厚，纤维化。10.辣椒素能上调高盐诱导后心肌组织中的PPARδ, UCP2蛋白表达，而减少iNOS的表达。11.辣椒素能上调心肌细胞中PPARδ的蛋白表达。结论：1.TNF-α诱导心肌细胞葡萄糖氧化率显著增加，而阿托伐他汀对抗了这种增加。而且相应的蛋白表达发生了改变，TNF-α刺激导致了PGC-1α, PDK4and CPT1的蛋白表达显著下调，阿托伐他汀同时也逆转了这些蛋白的下调，同时阿托伐他汀对抗PGC-1α的下调呈浓度依赖性的。2.在心肌细胞中我们应用满病毒系统，敲减PGC-1α，同时检测了PGC-1α相关的蛋白PDK4, CPT1显著下调。而心肌细胞的葡萄糖的氧化率也在PGC-1α敲减后，显著的增加。3.阿托伐他汀对抗炎症状态下心肌细胞葡萄糖氧化率的增加和PGC-1α上调有关，这些变化可能和类异戊烯的代谢物有关。4.辣椒素激活TRPV1能减轻高盐摄入诱导的氧化应激，减少超氧阴离子产生，改善高盐膳食导致的心功能的损伤，减轻高盐摄入导致的心肌肥厚。5.长期膳食辣椒素激活TRPV1，能上调高盐诱导后PPARδ, UCP2蛋白表达，减少iNOS的产量，体外细胞实验进一步证实了这种观点。6.长期膳食辣椒素激活TRPV1可能在高盐摄入导致的血压升高和心脏肥大中具有潜在的预防作用，可能成为人群中生活方式预防高盐膳食导致的高血压及心肌肥厚等的一种简单、有效的方法。更多还原

【Abstract】 Background and objective:Despite the prominent progress in treatment of chronic heart failure, heart failure isstill a major cause of morbidity and mortality worldwide. Look for better drug andtreatment method remains a challenge in cardiology.Heart failure is a very common clinicalcardiovascular syndrome. Hypertension, coronary heart disease, myocardial infarction,arrhythmia, valvular abnormalities can all lead to heart failure.The study of cardiacstructure and function, has experienced quite a long period of time in heart failure. Until the1990s, people realized that the basis for the development of heart failure is ventricularremodeling. Ventricular remodeling is the determinants of morbidity and mortality inpatients with heart failure. Ventricular remodeling refers to gene expression,changes inquality between the molecules, cells and stormal cells when the heart damaged. Clinicalrealization of the changes in heart size, shape and function.The regulation of ventricularremodeling include RAAS, immunity,inflammation, mechanical stress, oxidative stress,high salt, high sugar, and so on. They can influence ventricular remodeling to a certainextent.Heart energy metabolism can be transformed into mechanical energy stored in the fattyacid and glucose in the chemical to provide energy for contraction and relaxation of theheart. Metabolic substrate from fatty acid to glucose utilization of the adaptive conversionplays an important role in the pathological process of myocardial remodeling. Abnormalglucose and lipid metabolism by affecting myocardial energy metabolism, and promotemyocardial remodeling. Salt is important environmental factors of hypertension, cardiachypertrophy. Salt metabolic abnormalities may contribute to myocardial remodeling byincreased oxidative stress.This study to explore the two aspects of the lipid and high salt onmyocardial remodeling.The peroxisomal proliferator-activator receptor co-activator1α (PGC-1α) is highly expressed in tissues with high metabolic rates, such as heart, muscle and brown adiposetissue. PGC-1α is indispensable for the heart to match the increased demand for ATP andwork output in response to various physiological stimuli, including stress and pressureoverload. Except for peroxisomal proliferator-activator receptorγ, PGC-1α also could reactwith other members of the nuclear receptor transcription factor superfamily and nonnucleartranscription factors to control diverse cellular energy metabolic pathways. For example,PGC-1α interacts with ERRα and activates the expression of pyruvate dehydrogenasekinase4(PDK4), which is a negative regulator of glucose oxidation. It has been reportedthat under the development of cardiac hypertrophy, which is characteristic by increasedutilization of glucose, the expression of PGC-1α and PDK4are repressed. UCP2is highlyexpressed in the myocardium and reduce the production of ROS and reduce oxidative stress.iNOS expression raised to increase myocardial fibrosis, myocardial nitration, and startmyocardial remodeling. PPARdelta can activation of UCP2to reduce the expression ofiNOS, reduce oxidative stress, and improve myocardial remodeling.The statins are widely used lipid-lowering drugs in the clinical, it was discovered thebenefit of statins is much larger than lipid-lowering effect itself. In the present,it has beendiscovered and confirmed the role include improved endothelial function,anti-inflammatory, antioxidant, the stable plaque, improve coagulation, to reduceneurohormonal activation, stimulate endothelial progenitor cell differentiation and reversalof myocardial remodeling, reduce bone resorption,anti-tumorpleiotropic and so on.Salt isan important environmental factors of hypertension, cardiac hypertrophy. Salt intake andthe prevalence of hypertension was a significant dose-effect relationship. The incidence ofhypertension in China was from north to south, may be around the eating habits are closelyrelated. Northern residents eating high-salt, Yunnan, Guizhou, Sichuan and other residentsin addition to eating salty, and hi hot meals. Capsaicin has been reported to be able to adjustthe high-salt-induced high blood pressure.Based on the above background, we propose the following scientific hypotheses:Atorvastatin through the regulation of PGC-1a, to improve the inflammatory status ofmyocardial glucose and lipid metabolism disorders, and thus reverse myocardialremodeling? Capsaicin activation of TRPV1to adjust the high-salt-induced blood pressure,while improve cardiac remodeling induced by high salt? Materials and Methods:Part one: The embryonic rat-heart-derived H9c2cells (Cell Bank, Chinese Academy ofSciences, Shanghai, China) were maintained in growth medium composed of DMEMsupplemented with10%fetal bovine serum. H9c2cells were plated at a density of5,000cells/cm2and allowed to proliferate in growth medium. Medium was changed every2days.After incubation at37°C in humid air (5%CO2and95%O2) for near confluence, the H9c2cells were then deprived of serum and incubated for another24h before TNF-α (100ng/mL)treatment. The present study is involved in vitro experiments. In vivo models include H9c2cells, H9c2+TNF-α, H9c2+TNF-α+Atorvastatin, siPGC-1α were also observed. Part two:The present study includes in vivo and in vitro experiments. In vivo models were C57BL/6Jwild-type (WT) mice and TRPV1-null mutant (TRPV1–/–) mice fed with interventionaldiet. In vitro models include cultured H9c2cells exposured to capsaicin. WT mice andTRPV1–/–were randomly grouped and fed with a normal salt (0.5%sodium, NS) diet, ahigh salt (8%sodium, HS) diet and a high salt plus capsaicin (8%sodium+0.01%capsaicin,HS+Cap) diet for1year beginning at8weeks of age.1. H9c2cardiomyocytes were incubated with tumor necrosis factor-α (TNF-α,100ng/mL) in the presence or absence of atorvastatin. Then, we calculated the glucoseoxidation of each group.2.Immunoblottings of PGC-1α were performed in H9c2cardiomyocytes stimulatedwith TNF-α (100ng/mL) in the presence or absence of different concentration atorvastatin(ATV,1-25μmol/L) for24h. H9c2cardiomyocytes were co-incubated with atorvastain andmevalonate, farnesol, geranylgeraniol in the presence of TNF-α, then investigate the proteinexpression of PGC-1α.3. Protein expression of PDK4and CPT1were conducted in TNF-α-stimulatedcardiomyocytes in the presence or absence of atorvastatin.4. Selective silencing of PGC-1α by RNA interference in H9c2cardiomyocytes wasperformed using a lentiviral system. Furthermore, Protein expression of PDK4and CPT1were conducted in siPGC-1α cardiomyocytes.5. Glucose oxidation was conducted in various groups, including control, GFP+TNF-α,siPGC-1α+TNF-α. 6. Expressions of TRPV1protein in H9c2cells and myocardium were detected byimmunoblotting. Distribution of TRPV1in H9c2cells was shown by immunofluorescence.7. Capsaicin induced [Ca2+]i change was examined by PTI systems. Capsaicininduced a concentration-dependent [Ca2+]i increase in H9c2cells, which was inhibited byiRTX pretreatment.8. Cardiac function was evaluated by echocardiogram.9HW/BW%and Collagen deposition were detected, and expression of nitrotyrosinewere determined by Masson trichrome, Superoxide anion in heart was detected bydihydroethidium (DHE).Cardiac hypertrophy was evaluated by hematoxylin/eosin stainsof different group histological sections.10Protein expressions of PPAR-δ, inos, ucp2in heart were examined by western blot.11. Protein expressions of PPAR-δ in H9c2cells was examined by western blot.Results：1. TNF-α induced a significant increase in glucose oxidation rate with respect tountreated H9c2cells.Whereas, co-incubation of TNF-α-stimulated H9c2cells withatorvastatin abolished this increase.2. Atorvastatin counteracts TNF-α-induced PGC-1αsuppression in a dose-dependentmanner in cardiomyocytes. The effect of atorvastatin on PGC-1α was almost abolished bymevalonate and partially by farnesol, but not by geranylgeraniol.3. The protein levels showed that TNF-α stimulation led to a significant reduction ofPGC-1α target genes, PDK4and CPT1, whereas these changes of metabolic genes weremarkedly inhibited in the presence of atorvastatin.4. Silence of PGC-1α suppressed PDK4and CPT1protein expressions incardiomyocytes.5. Silence of PGC-1α increases the glucose oxidation in TNF-α-stimulatedcardiomyocytes.6. TRPV1existed in both H9c2cells and mice myocardium.7. Capsaicin induced a concentration-dependent [Ca2+]i increase in H9c2cells, whichwas inhibited by iRTX pretreatment.8. Dietary capsaicin improves cardiac function in WT mice on long-term high-salt dietthrough TRPV1activation. However, capsaicin had no effects on TRPV1-/-mice. 9. Activation of TRPV1by dietary capsaicin attenuates cardiac hypertrophy andfibrosis on long-term high salt diet. But, capsaicin had no effects on TRPV1-/-mice.10. Activation of TRPV1by dietary capsaicin upregulates PPARδ, UCP2proteinexpression and decreases iNOS production in mice on long-term high-salt diet.11.TRPV1activation by capsaicin increases PPARδ expression in cardiomyocytes.Conclusions：1. TNF-α induced a significant increase in glucose oxidation rate with respect tountreated H9c2cells.Whereas, co-incubation of TNF-α-stimulated H9c2cells withatorvastatin abolished this increase. Furthermore, the protein levels showed that TNF-αstimulation led to a significant reduction of PGC-1α target genes, PDK4and CPT1,whereas these changes of metabolic genes were markedly inhibited in the presence ofatorvastatin.2. We employ the siRNA to knockdown of PGC-1α in H9c2cells. And investigate theprotein expression of PDK4, CPT1and the glucose oxidation rate in inflammatory status. Itshowed that PGC-1α silence significantly suppressed the protein expressions of PDK4andCPT1in cardiomyocytes. The glucose oxidation rate was markedly increased by PGC-1αsilence in TNF-α-stimulated H9c2cells.3. Atorvastatin inhibits TNF-α-induced glucose oxidation through PGC-1αup-regulation in cardiomyocytes, which might be associated with the regulation ofisoprenoid metabolites.4. Activation of TRPV1by capsaicin attenuates high-salt intake-induced oxidativestress, reduces production superoxide anion, and improves high-salt intake-inducedimpairment of cardiac function, alleviate high-salt intake-induced cardiac hypertrophy.5. Activation of TRPV1by dietary capsaicin upregulates PPARδ, UCP2proteinexpression and decreases iNOS production in mice on long-term high-salt diet. but not inthose of TRPV1-null mice. Vitro studies further support that capsaicin upregulates PPARδthrough TRPV1activation.6. Activation of TRPV1by long-term dietary capsaicin might play a potential role inthe prevention of the development of high-salt intake-induced cardiac hypertrophy. Chronicdietary capsaicin may represent a simple and effective lifestyle intervention in populationswith high-salt diet-induced hypertension and cardiac hypertrophy.更多还原