激活TRPV1上调UCP2防治糖脂代谢紊乱致动脉粥样病变的机制研究
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摘要
背景和目的：
动脉粥样硬化(Atherosclerosis, AS)仍然是心源性猝死和脑血管意外等致残、致死疾病的主要病因。目前的治疗手段，如药物、介入手术和外科干预并不能有效地遏止我国心脑血管病的高发趋势。多项研究显示某些膳食因子如DASH (Dietary Approach toStop Hypertension)饮食，地中海（Mediterranean）饮食，Prudent饮食等可有效地降低心脑血管病的发病率和病死率。我国流行病学调查研究结果表明，我国南方地区冠心病的发病率和死亡率均显著低于北方，该现象除了跟北方地区气候因素及人群盐的摄入量要显著高于南方以外，还可能跟南方，尤其是西南地区人群膳食喜辣的生活方式有密切的联系。辣椒素是辣椒中辣味的主要成分，其作用靶点是辣椒素受体，亦称瞬时受体电位通道香草醛亚家族1(Transient Receptor potential vanilloid1, TRPV1)，当辣椒素与TRPV1结合后可促进细胞内Ca2+浓度的增加，从而介导一系列的病理生理反应。前期研究表明，膳食辣椒素通过激活TRPV1具有显著的心血管代谢保护作用：激活血管内皮细胞上的TRPV1受体可增加细胞内Ca2+的浓度，通过cAMP/PKA/eNOS信号通路从而促进一氧化氮(Nitric Oxide, NO)的释放，改善血管内皮依赖性舒张功能，降低自发性高血压大鼠(Spontanously Hypertensive Rats, SHR)的血压；激活脂肪细胞上的TRPV1可显著抑制3T3-L1前脂肪细胞的分化成熟，防止脂质的形成，从而具有防治肥胖的作用。课题组成员近期研究还发现，膳食辣椒素可通过激活血管平滑肌细胞(Vascularsmooth muscle cells, VSMCs)上的TRPV1受体，减少VSMCs对脂质的摄取，防止VSMCs泡沫化并减少脂质在ApoE敲除小鼠血管的沉积；膳食辣椒素还可防止高盐环境对血管功能的氧化应激性损害从而导致的血压升高，该研究提示激活TRPV1具有显著的对抗氧化应激导致的血管功能损害。脂质代谢紊乱，长期的慢性炎症和机体内氧化应激水平的升高是AS发生、发展的重要机制。前期研究揭示了激活TRPV1对脂质代谢的调节作用从而防治AS斑块形成的机制，但关于激活TRPV1对AS病理状态下氧化应激导致的心血管损害的影响及机制值得进一步深入研究。
在心血管危险因素如血脂异常，血糖升高的影响下，活性氧(Reactive oxide species,ROS)产生增加，使血管内皮一氧化氮合酶(eNOS)脱偶联，经精氨酸途径生成的NO与超氧阴离子结合生成具有极强破坏力的硝基化产物，进一步破坏细胞结构和相关的酶系，还可使血管平滑肌NO/cGMP信号通路功能下降从而导致动脉粥样硬化相关的血管功能损害。近年发现，存在于线粒体膜上的解偶联蛋白2(uncoupling protein2, UCP2)通过介导质子漏降低线粒体膜电位，使呼吸链氧化磷酸化解偶联，具有调节能量代谢和ROS生成的作用。UCP2敲除小鼠由于体内抗氧化能力下降，在高脂饮食的诱导下血管内可产生显著的AS斑块。另一方面，既往研究提示膳食辣椒素可通过激活肝脏细胞中的TRPV1上调UCP2的表达，从而具有防治非酒精性脂肪肝的作用。为此，在本研究中我们提出如下理论假设，膳食辣椒素通过激活血管内皮细胞TRPV1可上调UCP2蛋白的表达，从而减少由于糖脂代谢紊乱导致的ROS生成增加，改善心血管系统的结构与功能。本研究的开展将进一步阐明TRPV1在动脉粥样硬化血管的结构与功能损害过程中的重要作用，为AS相关的血管功能障碍的防治提供新的干预靶点，并为膳食因子干预AS尤其是冠状动脉粥样硬化性心脏病提供实验依据。
材料与方法：
ApoE敲除(Apolipoprotein E，ApoE-/-)小鼠是目前公认的研究AS较为理想的模型，ApoE-/-小鼠能自发形成高脂血症和AS。db/db小鼠是瘦素受体缺陷型小鼠，具有高胰岛素血症，胰岛素抵抗和血糖升高等类似人类2型糖尿病的特征，是目前研究2型糖尿病及相关的血管损伤的重要动物模型。为证实我们的研究假设，在考察辣椒素对脂质代谢紊乱致动脉粥样病变血管损伤影响的实验研究中，我们利用已有的TRPV1-/-，ApoE-/-（C57BL/6J背景）和UCP2-/-（C57BL/6J背景）小鼠杂交培育出双基因敲除小鼠ApoE-/-/TRPV1-/-，ApoE-/-/UCP2-/-结合ApoE-/-小鼠和相应的野生型(Wild type, WT)小鼠开展相关的实验研究。而在考察辣椒素对糖代谢紊乱致血管损伤影响的实验研究中我们则利用TRPV1-/-（C57BL/6J背景）及相应的野生型小鼠，db/db（C57BL/KsJ背景）及相应的野生型小鼠开展相关的研究。
1.首先观察膳食辣椒素对AS小鼠冠脉功能和形态的作用。(1)采用微血管张力测定仪分析长期膳食辣椒素对AS小鼠冠状动脉血管功能的影响。雄性8周龄ApoE-/-，ApoE-/-/TRPV1-/-，ApoE-/-/UCP2-/-小鼠给予普通饮食，高脂饮食和高脂+辣椒素(含0.01%辣椒素)干预24周后取冠状动脉观察血管对辣椒素诱导的血管舒张功能变化，乙酰胆碱诱导的血管内皮依赖性舒张功能，硝酸甘油诱导的血管非内皮依赖性舒张功能变化的影响。(2) H&E染色，油红O染色和Masson染色观察长期膳食辣椒素对冠状动脉狭窄程度的影响。(3)利用生物电放大器观察小鼠体表心电图的改变及辣椒素的干预作用；以异丙肾上腺素腹腔注射负荷后利用动态心电技术观察缺血性ST段改变的情况以评估膳食辣椒素干预后小鼠的抗心肌缺血的能力。
2.观察膳食辣椒素对AS小鼠心脏重构和功能以及远期生存的影响。(1)利用小动物超声技术观察膳食辣椒素干预后高脂喂养的AS小鼠心脏的结构与功能的改变，观察的主要指标包括心脏室间隔的厚度，收缩功能，舒张功能，主动脉血流速度，冠脉血流速度等。(2)观察长期膳食辣椒素对小鼠远期生存的影响，并描绘生存曲线，计算小鼠平均生存时间。
3.观察激活TRPV1防治脂质紊乱致动脉粥样血管损害的机制。(1)利用氧化修饰的低密度脂蛋白（ox-LDL）干预血管内皮细胞，观察辣椒素对ROS生成水平的影响，免疫印迹法（Western blotting）观察辣椒素对UCP2，PKA的调节作用；(2) Western blotting观察长期膳食辣椒素对AS小鼠主动脉组织TRPV1，eNOS, p-eNOS，PKA，p-PKA，UCP2，Nitrotyrosine等分子表达的影响。(3)荧光染色和western blotting观察长期膳食辣椒素对小鼠血管组织ROS，NO和硝基化水平的影响。
4.膳食辣椒素对糖代谢紊乱及相关血管损害的影响及机制。(1)利用动态血糖技术观察膳食辣椒素对TRPV1-/-和相应的野生型小鼠血糖水平的影响；(2)观察膳食辣椒素对db/db小鼠体重、血糖、血压和血管功能的影响；(3) H&E染色及荧光染色观察膳食辣椒素对db/db小鼠胰岛的形态与功能的影响；(4) Western blotting观察长期膳食辣椒素对db/db小鼠主动脉组织TRPV1，eNOS, p-eNOS，PKA，p-PKA，UCP2和P22phox等分子的表达水平的影响；(5)分别利用DHE与DAF-2DA荧光探针染色观察血管组织中ROS及NO水平。
结果：
1.膳食辣椒素对AS小鼠冠脉功能和形态的影响：(1)高脂饮食使AS小鼠辣椒素诱导的血管舒张功能和乙酰胆碱诱导的血管内皮依赖性舒张功能显著减退，膳食辣椒素可有效地防止高脂饮食导致的ApoE-/-小鼠冠脉对辣椒素和乙酰胆碱诱导的血管舒张的减退，但对硝酸甘油诱导的非内皮依赖性舒张反应无显著的作用；膳食辣椒素对高脂饮食诱导的ApoE-/-/TRPV1-/-和ApoE-/-/UCP2-/-小鼠辣椒素诱导的舒张反应，乙酰胆碱和硝酸甘油诱导的舒张反应无显著影响。(2)长期膳食辣椒素可显著减轻高脂喂养ApoE-/-小鼠的冠状动脉狭窄程度。(3)体表心电图和动态心电图结果显示，高脂饮食使AS小鼠出现显著的ST段压低，而长期膳食辣椒素可显著改善ApoE-/-小鼠ST段压低情况，增强ApoE-/-小鼠对抗异丙肾上腺素诱导的心肌缺血能力；膳食辣椒素对ApoE-/-/TRPV1-/-,ApoE-/-/UCP2-/-的ST段压低和异丙肾上腺素的负荷后的心肌缺血无显著的改善作用。
2.膳食辣椒素对AS小鼠心脏结构和功能的影响以及远期生存的观察结果：(1)高脂饮食使AS小鼠心脏增大、左心室重量、心脏/体重比值增加；(2)超声结果提示，膳食辣椒素可显著防止高脂饮食导致的ApoE-/-小鼠的左心室重构，改善左室的舒张功能，但对收缩功能无显著的影响。(3)长期膳食辣椒素可显著延长高脂饮食喂养ApoE-/-小鼠的生存时间，对ApoE-/-/TRPV1-/-, ApoE-/-/UCP2-/-小鼠的生存时间无显著影响。
3. TRPV1/PKA/UCP2通路在防治脂质代谢紊乱致动脉粥样病变中的作用：(1)辣椒素和eNOS的抑制剂(L-NAME，100μM)可显著对抗ox-LDL导致的血管内皮细胞ROS水平的上升，而TRPV1阻断剂、PKA拮抗剂和UCP2的抑制剂可显著减弱辣椒素的作用。(2) Western blotting结果提示辣椒素可呈浓度依赖性的上调UCP2的表达，并可被TRPV1和PKA的拮抗剂所拮抗。(3)长期膳食辣椒素显著上调高脂喂养ApoE-/-小鼠主动脉组织中TRPV1，p-PKA，UCP2，p-eNOS的表达水平，降低硝基酪氨酸水平；DHE染色表明膳食辣椒素还可显著降低ApoE-/-小鼠血管内膜超氧阴离子水平，增加NO的水平；膳食辣椒素对ApoE-/-/TRPV1-/-, ApoE-/-/UCP2-/-小鼠血管组织中TRPV1，p-PKA，UCP2，p-eNOS和硝基酪氨酸的表达水平，血管内膜的超氧阴离子和NO水平无显著的影响。
4.膳食辣椒素对糖代谢紊乱及相关的血管损害的作用：(1)膳食辣椒素可显著降低WT小鼠空腹血糖水平，改善糖耐量，降低24h平均动态血糖水平，但对TRPV1-/-小鼠的血糖水平无显著的影响。14周膳食辣椒素的干预可显著降低db/db小鼠的体重和空腹血糖，改善小鼠对胰岛素的敏感性，改善胰岛形态与功能。(2)膳食辣椒素可显著改善db/db小鼠的乙酰胆碱诱导的血管内皮功能依赖性舒张功能，但对硝酸甘油诱导的非内皮依赖性舒张功能无显著的影响；(3) Western blotting提示与相应的WT小鼠比较，TRPV1， p-PKA在db/db小鼠的主动脉和肠系膜动脉组织中的表达显著下降，NADPH-P22phox，UCP2的表达上调，而膳食辣椒素可修复db/db小鼠血管组织中TRPV1，p-PKA的表达，增加p-eNOS，UCP2的水平降低NADPH-P22phox的水平。
结论：
1.辣椒素激活TRPV1通过上调血管内皮细胞中PKA/UCP2信号通路，可显著减少ox-LDL导致的血管内皮细胞ROS水平的升高。
2.膳食辣椒素通过激活TRPV1以UCP2依赖的方式显著防止高脂饮食导致的AS小鼠冠脉功能的减退，从而减轻冠脉的狭窄程度，防止心肌重构和舒张功能的减退，并最终延长AS小鼠的生存时间，其机制涉及长期膳食辣椒素通过激活TRPV1上调PKA/UCP2信号通路，从而显著降低血管组织中超氧阴离子和硝基酪氨酸的水平，增加血管内皮NO的水平。
3.膳食辣椒素通过激活TRPV1改善葡萄糖代谢紊乱及相关的血管功能损害，其机制与辣椒素可修复TRPV1与p-PKA在糖代谢紊乱血管中的表达，上调UCP2的功能降低P22phox的活性有关。
4.膳食辣椒素通过激活TRPV1显著改善糖脂代谢紊乱致动脉粥样病变，其机制涉及激活TRPV1可上调血管内皮细胞中的PKA/UCP2通路，从而减少ROS的生成，增加NO的水平。
Back Ground and Objectives:
Atherosclerosis (AS) is still the leading cause of the cardiogenic sudden death and strokein many countries. Nowadays, therapeutic methods such as drugs, interventional therapies andsurgeries can not prevent the increase morbility and mortality of cardiovascular andcerebrovascular diseases. Several studies indicated dietary factor such as DASH diet,Mediterranean diet and Prudent diet can decreased the morbility and mortality ofcardiovascular and cerebrovascular diseases effectively. Epidemiology studies data from ourcountry showed that, the prevalence of morbility of cardiovascular and cerebrovasculardiseases in the south of China is much lower than in the north of China. Besides for the highersalt intake of the north China people, the cause of this difference may be also due to the spicyfood prevalence in south especially in the southwestern of China. Capsaicin is the majorgradient in Hot pepper, its target is Transient Receptor potential vanilloid1(TRPV1), whencapsaicin binding to the TRPV1, it can lead to rise calcium ion (Ca2+) level intracellular, thusmediate a series of pathologic physiology reaction. Our previous studies implicated thatdietary capsaicin exist cardiometabolic protection through TRPV1activation. Activation ofTRPV1in endotheliual cells (ECs) promote the Ca2+influx to the intracellular, then activatesthe cAMP/PKA/eNOS signal pathway thus increase the nitric oxide (NO) release fromvascular endothelium, improves the endothelium-dependent relaxation, hence lower the bloodpressure in Spontanously Hypertensive Rats (SHR). Activation of TRPV1in preadipocyte(3T3-L1) cells, inhibited the maturity of the3T3-L1thus prevents lipid formation and obesity.Recently, we found that activation of TRPV1by the specific agonists, capsaicin andresiniferatoxin (RTX), can dose-dependently increased cytosolic calcium and significantlyreduced the accumulation of lipids in VSMC from Wild type (WT) mice but not TRPV1knock out (TRPV1-/-) mice. Long-term activation of TRPV1significantly reduced lipidstorage and atherosclerotic lesions in the aortic sinus and in the thoracoabdominal aorta from AS mice treated with high-fat diet. These findings indicated that TRPV1activationameliorates high-fat diet-induced atherosclerosis. Chronic administration of capsaicin reducedthe high-salt diet-induced endothelial dysfunction and nocturnal hypertension in part bypreventing the generation of superoxide anions and NO reduction of mesenteric arteriesthrough vascular TRPV1activation. The above studies suggested that activation of TRPV1has protected effects against vascular dysfunction cause by oxidative stress. Lipid metabolismdisorders, chronic inflammation and oxidative stress are the major mechanisms of AS. Ourrecently study found that TRPV1activation by dietary capsaicin improves lipids metabolismthus prevents lipids accumulation. However, the effects of TRPV1activation on oxidativestress under the pathological state of AS is need further investigated.
Under the cardiovascular risk factors, such as high cholesterol diet and high glucoselevel, reactive oxide species (ROS) generates from mitochondrial, excessive ROS can damagethe mitochondrial DNA, lead to abnormal mitochondrial membrane pore channels open,decrease mitochondrial membrane potential hence lead to the mitochondrial respiratory chaindysfunction and promote cell apoptosis. More important, excessive generates of ROS alsolead to endothelial nitric oxide synthase (eNOS) uncoupling. NO, which generate from L-arginine will bond to superoxide anion then generation more destructive products,peroxynitrite. Damage cells structure and relevant enzymes as well as impaired the NO/cGMPsignal pathway thus lead to the vascular dysfunction in AS.Recent years, researchers discoveried that uncoupling protein2(UCP2), a protein existed inthe mitochondrial membrane, can modulates the mitochondrial membrane potential, thusreducing the mitochondrial ROS generation. Some other studies found UCP2knock out(UCP2-/-) mice fed with high fat diet can develop AS significantly. Our previous study showedthat dietary capsaicin prevents non-alcoholic fatty liver through up-regulates UCP2expression in liver via TRPV1activation. Therefore, we propose the hypothesis that,activation of artery endothelial cells TRPV1by dietary capsaicin decreases the ROSproduction induced by the metabolism disorder of lipids and glucose, improves vasculardysfunction.
This study will further clarify the important role of TRPV1in the process of vasculardysfunction in atherosclerosis, provide new therapy target for AS vascular dysfunction therapy,and also, provide theoretical basis on dietary factors intervention AS especially coronary atherosclerosis heart disease.
Material and Methods:
ApoE knock out (ApoE-/-) mice is a well model of AS, mice can formation ofhyperlipidemia and AS spontaneously. Leptin receptor deficient db/db mice existedhyperinsulinism, insulin resistant, high glucose levels and obesity that were similar to humantype2diabetes. It is also an important animal model for type2diabetes and related vasculardysfunction study. In order to verify our hypothesis, we use the TRPV1-/-，ApoE-/-and UCP2-/-mice generates the ApoE-/-/TRPV1-/-, ApoE-/-/UCP2-/-mice and then we use these mice forhigh fat induced atherosclerosis vascular dysfunction study. On the other hand, we useTRPV1-/-and WT mice, purchase the db/db mice and the matched WT mice to explore theeffects of dietary capsaicin on high glucose induced vascular dysfunction.
We explore the effects of dietary capsaicin on AS mice coronary function by wiremyograph. Eight weeks old male ApoE-/-, ApoE-/-/TRPV1-/-and ApoE-/-/TRPV1-/-mice werefed with normal chow (ND), high fat diet (HF) and high fat diet plus capsaicin (HC,0.01%capsaicin inside). At the end of24weeks, the vasorelaxation of coronary induced by capsaicin,acetylcholine (Ach) and nitroglycerin (NTG) were studied. H&E stain, oil red stain andMasson stain were used to explore the effects of dietary capsaicin on the degree of coronaryartery stenosis. Using bioelectric amplifier, we observe the change of electrocardiogram (ECG)in mice. After intraperitoneal injection of isoprenaline (1.5mg/kg), we using dynamicelectrocardiogram to observe the ischemic ECG changes and the ST segement duration timein mice in order to assess the effects of dietary capsaicin to resist myocardial ischemia inmice.
We also use small animal ultrasound to observe the effects of dietary intervention on thecardiac structure and function in AS mice, including cardiac ventricular septal thickness andsystolic function, diastolic function in left heart and aortic blood flow, coronary blood flowvelocity, etc. Fluorescence staining and western blotting to analysis the effects of long-termdietary capsaicin on ROS and Nitrotyrosine levels of aortic from AS mice. Observe theinfluence of long-term dietary on survival time of AS mice, and make the survival curve,calculating the average survival time.
In vitro study, we cultured the pig iliac endothelial cells (PIEC), and treated withox-LDL, then using dihydroethidium (DHE) probe and immunobloting to observe the effects of capsaicin in ox-LDL induced ROS level and nitrotyrosine increase. Finally, we explore thechanges of TRPV1, eNOS, p-eNOS, PKA, p-PKA, UCP2in AS mice aortic tissue by westernblot and the effects of dietary capsaicin on these changes.
In high glucose level induced vascular dysfunction, we first observe the effects ofTRPV1activation on blood glucose level. TRPV1-/-and WT mice were treat with dietarycapsaicin (0.01%) for24weeks, body weight, fasting blood glucose level, IntraperitonealGlucose Tolerance Testing (IPGTT), dynamic blood glucose levels were observed. Then weobserve the effects of dietary capsaicin on db/db mice body weight, fasting blood glucoselevels, intraperitoneal insulin-tolerance test (IPITT) and islet fuction and islet morphologywere observed by immunofluorescence and H&E stain. Myograph analysis the mesenteric andaortic arteries vasorelaxation in db/db mice and the WT mice treated with capsaicin or not.Use immunofluorescence to study the ROS and NO levels in mice mesenteric arteries. We useimmunoblotting analysis the expressions of TRPV1, eNOS, p-eNOS, PKA, p-PKA, UCP2inmice arteries tissues.
Results:
Capsaicin induced vasorelaxation in coronary arteries was impaired in high fat diettreated AS mice, and chronic dietary capsaicin improves the capsaicin induced vasorelaxationin ApoE-/-mice coronary arteries but not in TRPV1or UCP2knock out AS mice coronaryarteries. We also found Ach induced vasorelaxation was impaired in high fat diet treated mice,and chronic dietary capsaicin improves the vasorelaxation induced by Ach also in TRPV1andUCP2dependent manner. ECG studies found that there existed ST segment depression in highfat diet treated AS mice and24weeks dietary capsaicin improves the ST depression inTRPV1and UCP2dependent manner.
Ultrasonic results indicated that, high fat diet caused the cardiac remodeling in AS micetreated with high fat diet. Dietary capsaicin decreased the interventricular septal thickness,improves the heart diastolic function but not the systolic function in TRPV1and UCP2dependent manner. TRPV1or UCP2knock out decreased the survival time of AS mice treatedwith high fat diet, and long time dietary capsaicin can prolong the span life time in ApoE-/-mice treated with high fat diet.
Ox-LDL induced a significant increase superoxide anion and nitrotyrosine levels inPIECs, and capsaicin decrease the ROS and peroxynitrite level in a dose dependet manner, but this effects was blocked by TRPV1antagnist, PKA antagonist and UCP2inhibitorrespectively.
In animal study, dietary capsaicin decreased the ROS production and increased the NOlevels in AS mice aortic treated with high fat diet. Western blot results indicated that, TRPV1expression decreased in high fat diet treat ApoE-/-mice aortic, and dietary capsaicin restorethe TRPV1expression in UCP2dependent manner. Dietary capsaicin also increased thep-eNOS, UCP2levels and decreased the nitrotyrosine levels in ApoE-/-mice vascular treatedwith high fat diet.
TRPV1activation decreased the blood glucose level both in db/db mice and the WTmice. Dietary capsaicin also improves db/db mice insulin sensitivity and restores isletfunction.14weeks dietary capsaicin improves db/db mice arteries endothelium-dependentvasorelaxation but not the endothelium-independent vasorelaxation. Western blottingindicated dietary capsaicin increases TRPV1, p-eNOS, p-PKA and UCP2expression in db/dbmice arteries, decreases P22phoxin mice arteries.
Conclusions:
Dietary capsaicin restores capsaicin induced vasorelaxation, improves coronary arteriesendothelium-dependent vasorelaxation and decreases artherosclerosis stenosis hence improvesthe ischemic ECG changes in high fat treat AS mice in TRPV1and UCP2dependent manner.Long term dietary capsaicin improves cardiac remodeling and vascular velocity in AS micethus prolong the life span in AS mice also in UCP2dependent manner through TRPV1activation.
Capsaicin prevents ox-LDL induced ROS and nitrotyrosine increase via PKA/UCP2pathway through TRPV1activation. Chronic dietary capsaicin increase TRPV1, p-eNOS,UCP2and p-PKA expression and decreased nitrotyrosine in AS mice.
TRPV1activation decrease blood glucose levels, improves insulin sensitivity and restoreislet morphology in db/db mcie. Chornic dietary capsaicin decreases the ROS production andincreases NO level thus improves db/db mice endothelium-dependent vasorelaxation, thismay be due to the increases expression of TRPV1, p-eNOS, PKA and UCP2, decreases theexpression of P22phoxin mice arteries.
动脉粥样硬化(Atherosclerosis, AS)仍然是心源性猝死和脑血管意外等致残、致死疾病的主要病因。目前的治疗手段，如药物、介入手术和外科干预并不能有效地遏止我国心脑血管病的高发趋势。多项研究显示某些膳食因子如DASH (Dietary Approach toStop Hypertension)饮食，地中海（Mediterranean）饮食，Prudent饮食等可有效地降低心脑血管病的发病率和病死率。我国流行病学调查研究结果表明，我国南方地区冠心病的发病率和死亡率均显著低于北方，该现象除了跟北方地区气候因素及人群盐的摄入量要显著高于南方以外，还可能跟南方，尤其是西南地区人群膳食喜辣的生活方式有密切的联系。辣椒素是辣椒中辣味的主要成分，其作用靶点是辣椒素受体，亦称瞬时受体电位通道香草醛亚家族1(Transient Receptor potential vanilloid1, TRPV1)，当辣椒素与TRPV1结合后可促进细胞内Ca2+浓度的增加，从而介导一系列的病理生理反应。前期研究表明，膳食辣椒素通过激活TRPV1具有显著的心血管代谢保护作用：激活血管内皮细胞上的TRPV1受体可增加细胞内Ca2+的浓度，通过cAMP/PKA/eNOS信号通路从而促进一氧化氮(Nitric Oxide, NO)的释放，改善血管内皮依赖性舒张功能，降低自发性高血压大鼠(Spontanously Hypertensive Rats, SHR)的血压；激活脂肪细胞上的TRPV1可显著抑制3T3-L1前脂肪细胞的分化成熟，防止脂质的形成，从而具有防治肥胖的作用。课题组成员近期研究还发现，膳食辣椒素可通过激活血管平滑肌细胞(Vascularsmooth muscle cells, VSMCs)上的TRPV1受体，减少VSMCs对脂质的摄取，防止VSMCs泡沫化并减少脂质在ApoE敲除小鼠血管的沉积；膳食辣椒素还可防止高盐环境对血管功能的氧化应激性损害从而导致的血压升高，该研究提示激活TRPV1具有显著的对抗氧化应激导致的血管功能损害。脂质代谢紊乱，长期的慢性炎症和机体内氧化应激水平的升高是AS发生、发展的重要机制。前期研究揭示了激活TRPV1对脂质代谢的调节作用从而防治AS斑块形成的机制，但关于激活TRPV1对AS病理状态下氧化应激导致的心血管损害的影响及机制值得进一步深入研究。
在心血管危险因素如血脂异常，血糖升高的影响下，活性氧(Reactive oxide species,ROS)产生增加，使血管内皮一氧化氮合酶(eNOS)脱偶联，经精氨酸途径生成的NO与超氧阴离子结合生成具有极强破坏力的硝基化产物，进一步破坏细胞结构和相关的酶系，还可使血管平滑肌NO/cGMP信号通路功能下降从而导致动脉粥样硬化相关的血管功能损害。近年发现，存在于线粒体膜上的解偶联蛋白2(uncoupling protein2, UCP2)通过介导质子漏降低线粒体膜电位，使呼吸链氧化磷酸化解偶联，具有调节能量代谢和ROS生成的作用。UCP2敲除小鼠由于体内抗氧化能力下降，在高脂饮食的诱导下血管内可产生显著的AS斑块。另一方面，既往研究提示膳食辣椒素可通过激活肝脏细胞中的TRPV1上调UCP2的表达，从而具有防治非酒精性脂肪肝的作用。为此，在本研究中我们提出如下理论假设，膳食辣椒素通过激活血管内皮细胞TRPV1可上调UCP2蛋白的表达，从而减少由于糖脂代谢紊乱导致的ROS生成增加，改善心血管系统的结构与功能。本研究的开展将进一步阐明TRPV1在动脉粥样硬化血管的结构与功能损害过程中的重要作用，为AS相关的血管功能障碍的防治提供新的干预靶点，并为膳食因子干预AS尤其是冠状动脉粥样硬化性心脏病提供实验依据。
材料与方法：
ApoE敲除(Apolipoprotein E，ApoE-/-)小鼠是目前公认的研究AS较为理想的模型，ApoE-/-小鼠能自发形成高脂血症和AS。db/db小鼠是瘦素受体缺陷型小鼠，具有高胰岛素血症，胰岛素抵抗和血糖升高等类似人类2型糖尿病的特征，是目前研究2型糖尿病及相关的血管损伤的重要动物模型。为证实我们的研究假设，在考察辣椒素对脂质代谢紊乱致动脉粥样病变血管损伤影响的实验研究中，我们利用已有的TRPV1-/-，ApoE-/-（C57BL/6J背景）和UCP2-/-（C57BL/6J背景）小鼠杂交培育出双基因敲除小鼠ApoE-/-/TRPV1-/-，ApoE-/-/UCP2-/-结合ApoE-/-小鼠和相应的野生型(Wild type, WT)小鼠开展相关的实验研究。而在考察辣椒素对糖代谢紊乱致血管损伤影响的实验研究中我们则利用TRPV1-/-（C57BL/6J背景）及相应的野生型小鼠，db/db（C57BL/KsJ背景）及相应的野生型小鼠开展相关的研究。
1.首先观察膳食辣椒素对AS小鼠冠脉功能和形态的作用。(1)采用微血管张力测定仪分析长期膳食辣椒素对AS小鼠冠状动脉血管功能的影响。雄性8周龄ApoE-/-，ApoE-/-/TRPV1-/-，ApoE-/-/UCP2-/-小鼠给予普通饮食，高脂饮食和高脂+辣椒素(含0.01%辣椒素)干预24周后取冠状动脉观察血管对辣椒素诱导的血管舒张功能变化，乙酰胆碱诱导的血管内皮依赖性舒张功能，硝酸甘油诱导的血管非内皮依赖性舒张功能变化的影响。(2) H&E染色，油红O染色和Masson染色观察长期膳食辣椒素对冠状动脉狭窄程度的影响。(3)利用生物电放大器观察小鼠体表心电图的改变及辣椒素的干预作用；以异丙肾上腺素腹腔注射负荷后利用动态心电技术观察缺血性ST段改变的情况以评估膳食辣椒素干预后小鼠的抗心肌缺血的能力。
2.观察膳食辣椒素对AS小鼠心脏重构和功能以及远期生存的影响。(1)利用小动物超声技术观察膳食辣椒素干预后高脂喂养的AS小鼠心脏的结构与功能的改变，观察的主要指标包括心脏室间隔的厚度，收缩功能，舒张功能，主动脉血流速度，冠脉血流速度等。(2)观察长期膳食辣椒素对小鼠远期生存的影响，并描绘生存曲线，计算小鼠平均生存时间。
3.观察激活TRPV1防治脂质紊乱致动脉粥样血管损害的机制。(1)利用氧化修饰的低密度脂蛋白（ox-LDL）干预血管内皮细胞，观察辣椒素对ROS生成水平的影响，免疫印迹法（Western blotting）观察辣椒素对UCP2，PKA的调节作用；(2) Western blotting观察长期膳食辣椒素对AS小鼠主动脉组织TRPV1，eNOS, p-eNOS，PKA，p-PKA，UCP2，Nitrotyrosine等分子表达的影响。(3)荧光染色和western blotting观察长期膳食辣椒素对小鼠血管组织ROS，NO和硝基化水平的影响。
4.膳食辣椒素对糖代谢紊乱及相关血管损害的影响及机制。(1)利用动态血糖技术观察膳食辣椒素对TRPV1-/-和相应的野生型小鼠血糖水平的影响；(2)观察膳食辣椒素对db/db小鼠体重、血糖、血压和血管功能的影响；(3) H&E染色及荧光染色观察膳食辣椒素对db/db小鼠胰岛的形态与功能的影响；(4) Western blotting观察长期膳食辣椒素对db/db小鼠主动脉组织TRPV1，eNOS, p-eNOS，PKA，p-PKA，UCP2和P22phox等分子的表达水平的影响；(5)分别利用DHE与DAF-2DA荧光探针染色观察血管组织中ROS及NO水平。
结果：
1.膳食辣椒素对AS小鼠冠脉功能和形态的影响：(1)高脂饮食使AS小鼠辣椒素诱导的血管舒张功能和乙酰胆碱诱导的血管内皮依赖性舒张功能显著减退，膳食辣椒素可有效地防止高脂饮食导致的ApoE-/-小鼠冠脉对辣椒素和乙酰胆碱诱导的血管舒张的减退，但对硝酸甘油诱导的非内皮依赖性舒张反应无显著的作用；膳食辣椒素对高脂饮食诱导的ApoE-/-/TRPV1-/-和ApoE-/-/UCP2-/-小鼠辣椒素诱导的舒张反应，乙酰胆碱和硝酸甘油诱导的舒张反应无显著影响。(2)长期膳食辣椒素可显著减轻高脂喂养ApoE-/-小鼠的冠状动脉狭窄程度。(3)体表心电图和动态心电图结果显示，高脂饮食使AS小鼠出现显著的ST段压低，而长期膳食辣椒素可显著改善ApoE-/-小鼠ST段压低情况，增强ApoE-/-小鼠对抗异丙肾上腺素诱导的心肌缺血能力；膳食辣椒素对ApoE-/-/TRPV1-/-,ApoE-/-/UCP2-/-的ST段压低和异丙肾上腺素的负荷后的心肌缺血无显著的改善作用。
2.膳食辣椒素对AS小鼠心脏结构和功能的影响以及远期生存的观察结果：(1)高脂饮食使AS小鼠心脏增大、左心室重量、心脏/体重比值增加；(2)超声结果提示，膳食辣椒素可显著防止高脂饮食导致的ApoE-/-小鼠的左心室重构，改善左室的舒张功能，但对收缩功能无显著的影响。(3)长期膳食辣椒素可显著延长高脂饮食喂养ApoE-/-小鼠的生存时间，对ApoE-/-/TRPV1-/-, ApoE-/-/UCP2-/-小鼠的生存时间无显著影响。
3. TRPV1/PKA/UCP2通路在防治脂质代谢紊乱致动脉粥样病变中的作用：(1)辣椒素和eNOS的抑制剂(L-NAME，100μM)可显著对抗ox-LDL导致的血管内皮细胞ROS水平的上升，而TRPV1阻断剂、PKA拮抗剂和UCP2的抑制剂可显著减弱辣椒素的作用。(2) Western blotting结果提示辣椒素可呈浓度依赖性的上调UCP2的表达，并可被TRPV1和PKA的拮抗剂所拮抗。(3)长期膳食辣椒素显著上调高脂喂养ApoE-/-小鼠主动脉组织中TRPV1，p-PKA，UCP2，p-eNOS的表达水平，降低硝基酪氨酸水平；DHE染色表明膳食辣椒素还可显著降低ApoE-/-小鼠血管内膜超氧阴离子水平，增加NO的水平；膳食辣椒素对ApoE-/-/TRPV1-/-, ApoE-/-/UCP2-/-小鼠血管组织中TRPV1，p-PKA，UCP2，p-eNOS和硝基酪氨酸的表达水平，血管内膜的超氧阴离子和NO水平无显著的影响。
4.膳食辣椒素对糖代谢紊乱及相关的血管损害的作用：(1)膳食辣椒素可显著降低WT小鼠空腹血糖水平，改善糖耐量，降低24h平均动态血糖水平，但对TRPV1-/-小鼠的血糖水平无显著的影响。14周膳食辣椒素的干预可显著降低db/db小鼠的体重和空腹血糖，改善小鼠对胰岛素的敏感性，改善胰岛形态与功能。(2)膳食辣椒素可显著改善db/db小鼠的乙酰胆碱诱导的血管内皮功能依赖性舒张功能，但对硝酸甘油诱导的非内皮依赖性舒张功能无显著的影响；(3) Western blotting提示与相应的WT小鼠比较，TRPV1， p-PKA在db/db小鼠的主动脉和肠系膜动脉组织中的表达显著下降，NADPH-P22phox，UCP2的表达上调，而膳食辣椒素可修复db/db小鼠血管组织中TRPV1，p-PKA的表达，增加p-eNOS，UCP2的水平降低NADPH-P22phox的水平。
结论：
1.辣椒素激活TRPV1通过上调血管内皮细胞中PKA/UCP2信号通路，可显著减少ox-LDL导致的血管内皮细胞ROS水平的升高。
2.膳食辣椒素通过激活TRPV1以UCP2依赖的方式显著防止高脂饮食导致的AS小鼠冠脉功能的减退，从而减轻冠脉的狭窄程度，防止心肌重构和舒张功能的减退，并最终延长AS小鼠的生存时间，其机制涉及长期膳食辣椒素通过激活TRPV1上调PKA/UCP2信号通路，从而显著降低血管组织中超氧阴离子和硝基酪氨酸的水平，增加血管内皮NO的水平。
3.膳食辣椒素通过激活TRPV1改善葡萄糖代谢紊乱及相关的血管功能损害，其机制与辣椒素可修复TRPV1与p-PKA在糖代谢紊乱血管中的表达，上调UCP2的功能降低P22phox的活性有关。
4.膳食辣椒素通过激活TRPV1显著改善糖脂代谢紊乱致动脉粥样病变，其机制涉及激活TRPV1可上调血管内皮细胞中的PKA/UCP2通路，从而减少ROS的生成，增加NO的水平。
Back Ground and Objectives:
Atherosclerosis (AS) is still the leading cause of the cardiogenic sudden death and strokein many countries. Nowadays, therapeutic methods such as drugs, interventional therapies andsurgeries can not prevent the increase morbility and mortality of cardiovascular andcerebrovascular diseases. Several studies indicated dietary factor such as DASH diet,Mediterranean diet and Prudent diet can decreased the morbility and mortality ofcardiovascular and cerebrovascular diseases effectively. Epidemiology studies data from ourcountry showed that, the prevalence of morbility of cardiovascular and cerebrovasculardiseases in the south of China is much lower than in the north of China. Besides for the highersalt intake of the north China people, the cause of this difference may be also due to the spicyfood prevalence in south especially in the southwestern of China. Capsaicin is the majorgradient in Hot pepper, its target is Transient Receptor potential vanilloid1(TRPV1), whencapsaicin binding to the TRPV1, it can lead to rise calcium ion (Ca2+) level intracellular, thusmediate a series of pathologic physiology reaction. Our previous studies implicated thatdietary capsaicin exist cardiometabolic protection through TRPV1activation. Activation ofTRPV1in endotheliual cells (ECs) promote the Ca2+influx to the intracellular, then activatesthe cAMP/PKA/eNOS signal pathway thus increase the nitric oxide (NO) release fromvascular endothelium, improves the endothelium-dependent relaxation, hence lower the bloodpressure in Spontanously Hypertensive Rats (SHR). Activation of TRPV1in preadipocyte(3T3-L1) cells, inhibited the maturity of the3T3-L1thus prevents lipid formation and obesity.Recently, we found that activation of TRPV1by the specific agonists, capsaicin andresiniferatoxin (RTX), can dose-dependently increased cytosolic calcium and significantlyreduced the accumulation of lipids in VSMC from Wild type (WT) mice but not TRPV1knock out (TRPV1-/-) mice. Long-term activation of TRPV1significantly reduced lipidstorage and atherosclerotic lesions in the aortic sinus and in the thoracoabdominal aorta from AS mice treated with high-fat diet. These findings indicated that TRPV1activationameliorates high-fat diet-induced atherosclerosis. Chronic administration of capsaicin reducedthe high-salt diet-induced endothelial dysfunction and nocturnal hypertension in part bypreventing the generation of superoxide anions and NO reduction of mesenteric arteriesthrough vascular TRPV1activation. The above studies suggested that activation of TRPV1has protected effects against vascular dysfunction cause by oxidative stress. Lipid metabolismdisorders, chronic inflammation and oxidative stress are the major mechanisms of AS. Ourrecently study found that TRPV1activation by dietary capsaicin improves lipids metabolismthus prevents lipids accumulation. However, the effects of TRPV1activation on oxidativestress under the pathological state of AS is need further investigated.
Under the cardiovascular risk factors, such as high cholesterol diet and high glucoselevel, reactive oxide species (ROS) generates from mitochondrial, excessive ROS can damagethe mitochondrial DNA, lead to abnormal mitochondrial membrane pore channels open,decrease mitochondrial membrane potential hence lead to the mitochondrial respiratory chaindysfunction and promote cell apoptosis. More important, excessive generates of ROS alsolead to endothelial nitric oxide synthase (eNOS) uncoupling. NO, which generate from L-arginine will bond to superoxide anion then generation more destructive products,peroxynitrite. Damage cells structure and relevant enzymes as well as impaired the NO/cGMPsignal pathway thus lead to the vascular dysfunction in AS.Recent years, researchers discoveried that uncoupling protein2(UCP2), a protein existed inthe mitochondrial membrane, can modulates the mitochondrial membrane potential, thusreducing the mitochondrial ROS generation. Some other studies found UCP2knock out(UCP2-/-) mice fed with high fat diet can develop AS significantly. Our previous study showedthat dietary capsaicin prevents non-alcoholic fatty liver through up-regulates UCP2expression in liver via TRPV1activation. Therefore, we propose the hypothesis that,activation of artery endothelial cells TRPV1by dietary capsaicin decreases the ROSproduction induced by the metabolism disorder of lipids and glucose, improves vasculardysfunction.
This study will further clarify the important role of TRPV1in the process of vasculardysfunction in atherosclerosis, provide new therapy target for AS vascular dysfunction therapy,and also, provide theoretical basis on dietary factors intervention AS especially coronary atherosclerosis heart disease.
Material and Methods:
ApoE knock out (ApoE-/-) mice is a well model of AS, mice can formation ofhyperlipidemia and AS spontaneously. Leptin receptor deficient db/db mice existedhyperinsulinism, insulin resistant, high glucose levels and obesity that were similar to humantype2diabetes. It is also an important animal model for type2diabetes and related vasculardysfunction study. In order to verify our hypothesis, we use the TRPV1-/-，ApoE-/-and UCP2-/-mice generates the ApoE-/-/TRPV1-/-, ApoE-/-/UCP2-/-mice and then we use these mice forhigh fat induced atherosclerosis vascular dysfunction study. On the other hand, we useTRPV1-/-and WT mice, purchase the db/db mice and the matched WT mice to explore theeffects of dietary capsaicin on high glucose induced vascular dysfunction.
We explore the effects of dietary capsaicin on AS mice coronary function by wiremyograph. Eight weeks old male ApoE-/-, ApoE-/-/TRPV1-/-and ApoE-/-/TRPV1-/-mice werefed with normal chow (ND), high fat diet (HF) and high fat diet plus capsaicin (HC,0.01%capsaicin inside). At the end of24weeks, the vasorelaxation of coronary induced by capsaicin,acetylcholine (Ach) and nitroglycerin (NTG) were studied. H&E stain, oil red stain andMasson stain were used to explore the effects of dietary capsaicin on the degree of coronaryartery stenosis. Using bioelectric amplifier, we observe the change of electrocardiogram (ECG)in mice. After intraperitoneal injection of isoprenaline (1.5mg/kg), we using dynamicelectrocardiogram to observe the ischemic ECG changes and the ST segement duration timein mice in order to assess the effects of dietary capsaicin to resist myocardial ischemia inmice.
We also use small animal ultrasound to observe the effects of dietary intervention on thecardiac structure and function in AS mice, including cardiac ventricular septal thickness andsystolic function, diastolic function in left heart and aortic blood flow, coronary blood flowvelocity, etc. Fluorescence staining and western blotting to analysis the effects of long-termdietary capsaicin on ROS and Nitrotyrosine levels of aortic from AS mice. Observe theinfluence of long-term dietary on survival time of AS mice, and make the survival curve,calculating the average survival time.
In vitro study, we cultured the pig iliac endothelial cells (PIEC), and treated withox-LDL, then using dihydroethidium (DHE) probe and immunobloting to observe the effects of capsaicin in ox-LDL induced ROS level and nitrotyrosine increase. Finally, we explore thechanges of TRPV1, eNOS, p-eNOS, PKA, p-PKA, UCP2in AS mice aortic tissue by westernblot and the effects of dietary capsaicin on these changes.
In high glucose level induced vascular dysfunction, we first observe the effects ofTRPV1activation on blood glucose level. TRPV1-/-and WT mice were treat with dietarycapsaicin (0.01%) for24weeks, body weight, fasting blood glucose level, IntraperitonealGlucose Tolerance Testing (IPGTT), dynamic blood glucose levels were observed. Then weobserve the effects of dietary capsaicin on db/db mice body weight, fasting blood glucoselevels, intraperitoneal insulin-tolerance test (IPITT) and islet fuction and islet morphologywere observed by immunofluorescence and H&E stain. Myograph analysis the mesenteric andaortic arteries vasorelaxation in db/db mice and the WT mice treated with capsaicin or not.Use immunofluorescence to study the ROS and NO levels in mice mesenteric arteries. We useimmunoblotting analysis the expressions of TRPV1, eNOS, p-eNOS, PKA, p-PKA, UCP2inmice arteries tissues.
Results:
Capsaicin induced vasorelaxation in coronary arteries was impaired in high fat diettreated AS mice, and chronic dietary capsaicin improves the capsaicin induced vasorelaxationin ApoE-/-mice coronary arteries but not in TRPV1or UCP2knock out AS mice coronaryarteries. We also found Ach induced vasorelaxation was impaired in high fat diet treated mice,and chronic dietary capsaicin improves the vasorelaxation induced by Ach also in TRPV1andUCP2dependent manner. ECG studies found that there existed ST segment depression in highfat diet treated AS mice and24weeks dietary capsaicin improves the ST depression inTRPV1and UCP2dependent manner.
Ultrasonic results indicated that, high fat diet caused the cardiac remodeling in AS micetreated with high fat diet. Dietary capsaicin decreased the interventricular septal thickness,improves the heart diastolic function but not the systolic function in TRPV1and UCP2dependent manner. TRPV1or UCP2knock out decreased the survival time of AS mice treatedwith high fat diet, and long time dietary capsaicin can prolong the span life time in ApoE-/-mice treated with high fat diet.
Ox-LDL induced a significant increase superoxide anion and nitrotyrosine levels inPIECs, and capsaicin decrease the ROS and peroxynitrite level in a dose dependet manner, but this effects was blocked by TRPV1antagnist, PKA antagonist and UCP2inhibitorrespectively.
In animal study, dietary capsaicin decreased the ROS production and increased the NOlevels in AS mice aortic treated with high fat diet. Western blot results indicated that, TRPV1expression decreased in high fat diet treat ApoE-/-mice aortic, and dietary capsaicin restorethe TRPV1expression in UCP2dependent manner. Dietary capsaicin also increased thep-eNOS, UCP2levels and decreased the nitrotyrosine levels in ApoE-/-mice vascular treatedwith high fat diet.
TRPV1activation decreased the blood glucose level both in db/db mice and the WTmice. Dietary capsaicin also improves db/db mice insulin sensitivity and restores isletfunction.14weeks dietary capsaicin improves db/db mice arteries endothelium-dependentvasorelaxation but not the endothelium-independent vasorelaxation. Western blottingindicated dietary capsaicin increases TRPV1, p-eNOS, p-PKA and UCP2expression in db/dbmice arteries, decreases P22phoxin mice arteries.
Conclusions:
Dietary capsaicin restores capsaicin induced vasorelaxation, improves coronary arteriesendothelium-dependent vasorelaxation and decreases artherosclerosis stenosis hence improvesthe ischemic ECG changes in high fat treat AS mice in TRPV1and UCP2dependent manner.Long term dietary capsaicin improves cardiac remodeling and vascular velocity in AS micethus prolong the life span in AS mice also in UCP2dependent manner through TRPV1activation.
Capsaicin prevents ox-LDL induced ROS and nitrotyrosine increase via PKA/UCP2pathway through TRPV1activation. Chronic dietary capsaicin increase TRPV1, p-eNOS,UCP2and p-PKA expression and decreased nitrotyrosine in AS mice.
TRPV1activation decrease blood glucose levels, improves insulin sensitivity and restoreislet morphology in db/db mcie. Chornic dietary capsaicin decreases the ROS production andincreases NO level thus improves db/db mice endothelium-dependent vasorelaxation, thismay be due to the increases expression of TRPV1, p-eNOS, PKA and UCP2, decreases theexpression of P22phoxin mice arteries.
引文
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