枳实总酚提取物及柚皮素对血管内皮功能损伤的保护机制研究

【作者】 李辉；

【导师】 刘元艳；

【作者基本信息】 北京中医药大学 ， 中药化学， 2020， 硕士

【摘要】 血管内皮功能损伤是促使动脉粥样硬化发生及发展的首要因素,随后诱发多种心血管疾病的发生,在世界范围内具有高发病率和死亡率,并且在许多国家已经成为沉重的负担,因此迫切需要探索更多能够用于心血管疾病的治疗策略。枳实是中医临床常用中药,来源于芸香科(Rutaceae)橘属(Citrus)植物酸橙Citrus aurantium L.及其栽培变种或甜橙C.sinensis Osbeck的干燥幼果,始载于《神农本草经》。我们前期的研究结果发现,酸橙枳实总酚提取物(total phenolic extracts of Citrus aurantium L.,TPE-CA)含有丰富的与人类健康相关的可食用活性酚类化合物,其中包括黄酮,黄酮醇,黄烷酮及其糖基衍生物,多甲氧基黄酮和香豆素等。另外,许多研究表明活性酚类化合物具有显著的心血管保护作用,并能通过调控体内血脂,血糖的代谢和改善血管内皮功能而发挥治疗作用,继而降低心血管疾病的发病率和死亡率。而由于TPE-CA具有成分多样性与复杂性的特点,使其能够通过作用于体内多个靶点发挥多方面的药理作用,进而达到整体调节体内平衡及稳态作用,但其具体的血管内皮功能保护机制还有待进一步研究。在第一章的研究中,我们首先通过高蛋氨酸饮食建立了大鼠高同型半胱氨酸血症(HHcy)模型以造成血管内皮功能损伤,随后采用多种实验方法评估了 TPE-CA对血管内皮功能损伤大鼠的保护效果。其中包括采用Elisa法对大鼠血浆同型半胱氨酸(Hcy)含量及血管活性介质水平进行了检测。同时,对大鼠胸主动脉进行Masson染色分析以评价其病理损伤程度。实验结果发现,TPE-CA可明显的降低血管内皮功能损伤模型大鼠血浆中的Hcy含量,并调节其血管活性介质如一氧化氮(NO)、内皮素-1(ET-1)、前列环素(PGI2)、血栓素A2(TXA2)和血管紧张素Ⅱ(AngⅡ)的释放。此外,病理分析结果表明,TPE-CA对大鼠的胸主动脉形态损伤有一定的修复作用,可恢复血管正常形态。基于此,我们一方面采用Western blotting对Hcy自身代谢途径相关酶的表达水平进行了深入的分析,另一方面应用网络药理学全面深入地挖掘其发挥血管内皮功能保护作用的潜在机制。依据网络药理学分析结果,我们选择花生四烯酸(AA)代谢的两条途径进行进一步的实验验证。结果发现,TPE-CA治疗HHcy不仅可以通过上调Hcy代谢中转硫途径代谢酶活性有效的降低血浆中Hcy水平,还可以通过激活AA代谢中CYPs表氧化酶途径和抑制CYPs羟化酶途径来恢复HHcy诱导的血管内皮功能损伤。此外,基于上述实验结果及课题组前期研究,柚皮素(Nar)作为TPE-CA中最具生物活性的成分,被筛选出用以进行进一步的机制研究。我们首先建立了人脐静脉内皮细胞(HUVEC)氧化损伤模型,然后给予Nar进行干预,采用荧光标记法对其中细胞质活性氧(Cyto-ROS)、线粒体活性氧(Mito-ROS)及Ca2+的含量进行了详细的测定,以评估Nar的保护效果。随后,为深入揭示Nar发挥保护作用的潜在机制,本研究还采用精准转录组测序技术在转录组水平高通量测序分析,以筛选各组之间差异表达基因,继而提取相关的信号通路。转录组学实验结果提示,AMPKα/Sirt1信号通路可能是Nar缓解内皮细胞氧化应激的潜在机制。因此我们通过Westernblotting对AMPKα,Sirt1及其下游蛋白eNOS的表达水平进行了初步的测定,采用Elisa分别对线粒体细胞色素c及细胞质细胞色素c进行了检测,以评估各组细胞线粒体功能。进一步地,我们设计了两条siRNA(si-Sirt1&si-AMPK)通过细胞瞬时转染精准敲低目标蛋白的表达水平以进一步验证机制的准确性。除此之外,我们还设计了进一步的动物实验,包括采用Masson染色分析大鼠经Nar干预前后的病理情况;应用Elisa测定大鼠体内超氧化物歧化酶(SOD)、丙二醛(MDA)及NO 的含量;利用 Westernblotting 分析上述 AMPKα/Sirt1信号通路的相关蛋白表达水平,以初步探究Nar在体内是否也能发挥改善血管内皮功能损伤的作用。结果发现,AMPKa和Sirt1是Nar发挥内皮细胞保护作用的关键蛋白靶点,Nar对AMPKα/Sirt1信号途径具有显著的调控效力,可有效的改善线粒体功能,对抗由Ca2+超载引发的呼吸链损伤。另一方面,Nar还可发挥抗氧化作用,促使ROS消除,降低机体氧化应激程度,进而阻碍eNOS的解耦联反应,促进其下游产物NO的合成。实现“始端-终端”双调控,共同维系血管内皮功能。综上所述,本研究中通过药效学研究及组织病理学分析全面的评估了 TPE-CA及Nar对血管内皮细胞的保护效果。另外,实验中还采用网络药理学分析及转录组学技术以整体方式深入的揭示了 TPE-CA及Nar潜在的血管内皮功能保护机制,并在进一步的实验中通过细胞瞬时转染、Westernblotting、Elisa、免疫荧光技术、组织切片分析等方法在体内及体外水平均进行了详细的验证。结果表明,多酚类活性成分可通过调节AA代谢途径、改善线粒体功能及对抗氧化应激保护血管内皮细胞,其在由血管内皮损伤引起的心血管疾病中具有开发潜力。同时为其它中药活性成分群的药理机制研究提供了新的研究方法。更多还原

【Abstract】 Endothelial dysfunction is the culprit in accelerating the development of atherosclerosis and subsequently inducing several cardiovascular diseases(CVDs),which possess high morbidity and mortality rates worldwide.CVDshave broughta heavy burden in many countries,thus looking for more therapeutic strategies for CVDs is urgently.The young fruit of Citrus aurantium L.named Zhishi have been officially listed in 2015 edition of the Chinese Pharmacopoeia due to its important therapeutic properties for humans.Base on our previous research,total phenolic extracts of Citrus aurantium L.(TPE-CA)is a naturally obtained phenolic mixture,mainly containing abundant edible bioactive phenolic compounds associated with human health,such as flavones,flavanones and their glycosyl derivatives,flavonols,polymethoxyflavones and coumarins.In addition,accumulating evidences suggested that bioactive phenolic compounds possessed potent cardiovascular protective effects,and exerted therapeutic effect by regulating blood lipid,blood glucose and vascular endothelial function,and subsequently reduced cardiovascular morbidity and mortality.Though TPE-CA is a representative multi-compositions and multi-targeted mixture of traditional Chinese medicine(TCM)that performs its synergistic therapeutic efficacy through regulation of the multiple biological processes in body systems,the underlying protective mechanisms involved in the endothelial dysfunction still need to furtherly study.In the first chapter,we firstly utilized high methionine diet for the hyperhomocysteinemia(HHcy)model establishment so as to induce the injury on endothelial function.After that,the protective effects of TPE-CA on HHcy induced endothelial dysfunction was firstly assessed,including measuring the level of homocysteine(Hcy)and vasoactive mediators in rat plasma.At the same time,the histopathological evaluation of thoracic aortas was conducted so as to evaluate the vascular damage degree.Present results showed that TPE-CA could markedly reduce the Hcy content in endothelial dysfunctional rat plasma and regulate the release of several vasoactive mediators,such as endothelin-1(ET-1),nitric oxide(NO),prostacyclin(PGI2),angiotensin II(Ang II)and thromboxane A2(TXA2).Furthermore,according to the results of histopathological evaluation,we found that thoracic aortas exhibited an obvious repair state in myofiber rupture,collagen proportion and the vascular wall architecture after treated by TPE-CA.In this light,we applied Western blotting for the determination of expression level of metabolic enzymes involved in Hcy self-metabolism pathway,and performed network pharmacology to comprehensively dig the potential protective mechanism of TPE-CA on vascular endothelial dysfunction.Base on the results of network pharmacology,the arachidonic acid(AA)metabolism pathways were screened for further experimental validation.Our data indicated that endothelial dysfunctional rat treated by TPE-CA could not only effectively reduce the Hcy level in plasma through up-regulating transsulfuration pathway,but also restore the HHcy-induced vascular endothelial dysfunction by activating CYPs epoxygenase signal transduction pathway and inhibiting CYPs hydroxylase signal transduction pathway.In addition,combined the aforementioned results and previous study in our lab,the naringenin(Nar)as a most bioactive compound was selected for further mechanism research.We firstly established the human umbilical vein endothelial cell(HUVEC)oxidative injury model,then the protective effects of Nar were assessed after intervened with Nar for 24 h,including employing fluorescence probe for the content detection of cytoplasmic reactive oxygen species(Cyto-ROS),mitochondrial reactive oxygen species(Mito-ROS)and intracellular Ca2+.After that,to deeply uncover the cytoprotective mechanism of Nar,transcriptome sequencing technology was employed for high throughtput sequencing so as to screen the differentially expressed genes(DEGs)between each group.According to the results of transcriptome sequencing,the AMPKα/Sirtl signaling pathway possessed the highest score,and it was the most possible cytoprotective mechanism of Nar.Thus,the expression level of associated proteins,such as AMPKa.Sirtl and downstream protein of eNOS,was initially detected by western blotting.Besides,the mitochondrial function was evaluated via detecting the content of cytochrome c in mitochondria and cytoplasm.To further confirm that the protective effect of Nar against Hcy provoked HUVEC oxidative stress mediated via the AMPKα/Sirtl pathway,two specific targeting siRNAs including siPRKAA1 and siSirtl were applied for AMPKa and Sirtl knockdown in HUVECs,respectively.Moreover,further animal experiments,such as Masson staining,Elisa and western blotting,were designed to initially explore whether Nar ameliorated the endothelial dysfunction in vivo.Experimental results confirmed that AMPKa and Sirtl were the pivotal targets of Nar,Nar activated AMPKα/Sirtl signaling pathway could not only rescue the Ca2+disrupted mitochondrial function consequently reduce the ROS production,but also upregulate the activity of downstream protein of eNOS subsequently increase the production of NO,ameliorate the endothelial dysfunction in“origin-end”double regulation manner.In summary,current research employed pharmacodynamics study and histopathology to global assess the potential protective effects of TPE-CA and Nar on vascular endothelial cell.Moreover,network pharmacology and transcriptome sequencing technology were conducted to comprehensively reveal the potential protective mechanism of TPE-CA and Nar on vascular endothelial function,and performed related experimental validation.Present reaserch confirmed that phenolic compounds can improve the vascular endothelial function through regulating the AA metabolism pathway,ameliorating the mitochondrial function and countering the oxidative stress.Also,Current methods may provide a new direction for pharmacological mechanism exploration of other TCM active ingredient groups,and provide certain theoretical basis on the application and development potential of polyphenols active ingredients in CVDs.更多还原