【作者】 贾素洁；

【导师】 邓汉武；

【作者基本信息】 中南大学 ， 心血管药理， 2007， 博士

【摘要】 动脉粥样硬化（AS）是冠心病发病的主要原因，目前研究认为血管内皮受损在AS发生发展中起关键作用。一氧化氮（NO）主要由NO合酶（NOS）催化L-精氨酸转化而来，在维持血管结构和功能中起重要作用，被认为是一个关键的内源性抗AS分子。近期研究发现，体内存在内源性NOS抑制物非对称二甲基精氨酸（ADMA），能减少NO合成，进而损伤内皮功能。ADMA还能诱导氧化应激和炎症因子生成，促进内皮损伤等，可能是一个新的内源性致AS分子。二甲基精氨酸-二甲胺水解酶（DDAH）是ADMA的代谢酶，其活性下降是体内ADMA累积的主要原因。抗氧化药维生素E、普罗布考等的抗AS作用与增加DDAH活性，降低ADMA水平有关，提示DDAH／ADMA通路可能是AS防治的新靶点。本论文在细胞和动物水平系统探讨了ADMA致内皮功能不全的作用机制、DDAH／ADMA通路在AS形成和防治中的作用及抗氧化药物（口山）酮的抗AS作用。第一章ADMA促动脉粥样硬化作用的研究第一节ADMA促动脉粥样硬化形成的作用研究背景研究显示，间隙连接通讯（GJIC）功能改变在AS发病机制中具有一定意义。GJ由细胞质膜上的蛋白质亚单位—间隙连接蛋白（Cxs）相互衔接而成。血管内皮的自稳态及生理功能的维持，都依赖于内皮GJIC介导的信息交换和协同反应。研究表明，Cx43的表达、空间分布及结构变化以及其引起的GJIC功能变化可能在AS发病机制中具有一定意义。对内皮细胞GJIC功能和Cx43表达的影响是多种AS危险因素致内皮功能不全的机制。大量研究表明，多种因素均可通过增加细胞内氧自由基生成，氧化Cx或者改变其所在膜环境诱发Cx构象改变，导致GJIC功能障碍。基于氧化应激是导致GJIC功能障碍的重要原因以及内源性ADMA能诱发氧化应激，我们推测ADMA可能通过致氧化作用氧化Cx43导致GJIC功能障碍，进而促进AS发生发展。方法实验设4组（n=8）：①野生型C57BL／6J小鼠组；②野生型C57BL／6J小鼠外源性ADMA处理组；③ApoE基因缺陷(ApoE^-/-)小鼠组；④ApoE^-/-小鼠外源性ADMA处理组。ADMA（5 mg／kg）每日皮下注射。4周后，眼眶取血，离心，分离血浆，检测血浆对氧磷酶（PON1）活性、ADMA（HPLC法）浓度；开胸，迅速分离主动脉，观察动脉粥样损伤（苏丹红染色法）及主动脉内皮Cx43蛋白表达（免疫组化法）。结果ApoE^-/-小鼠动脉粥样斑块明显，同时伴有血浆ADMA水平升高；给予外源性ADMA（5 mg／kg，4w）可进一步增加ApoE^-/-小鼠及野生型小鼠血浆ADMA水平，同时显著增加ApoE^-/-小鼠动脉粥样斑块面积及促进野生型小鼠动脉粥样损伤形成；ApoE^-/-小鼠血浆PON1活性显著降低，外源性给予ADMA可明显抑制野生型小鼠和ApoE^-/-小鼠血浆PON1活性；同时，外源性ADMA可以显著降低野生型和ApoE^-/-小鼠主动脉内皮Cx43表达。结论ADMA是AS形成的重要促进物，其作用可能与下调内皮细胞Cx43表达，进而抑制间隙连接细胞间通讯功能有关。第二节NADPH氧化酶介导溶血性磷脂酰胆碱诱发的ADMA升高研究背景内皮功能紊乱与氧化应激密切相关。活性氧（ROS）在氧化应激中起重要作用。NADPH氧化酶途径是内皮细胞活性氧的重要来源。研究表明，内皮细胞增殖及凋亡，缺氧／复氧损伤和NO介导的内皮舒张功能障碍均涉及NADPH氧化酶活性变化。ADMA与血管内皮功能密切相关。多种刺激因素包括氧化型低密度脂蛋白（ox-LDL）均可诱导ADMA生成。已知溶血性磷脂酰胆碱（LPC）是ox-LDL的主要成分，能诱导ADMA生成，并能激活NADPH氧化酶诱发氧化应激。因此，本实验探讨了LPC诱导的ADMA升高是否通过NADPH氧化酶途径诱发氧化应激，进而影响ADMA代谢酶活性而发挥作用。方法将人脐静脉内皮细胞（HUVEC-12）分为：正常对照组；LPC（10mg／L）损伤组；LPC+NADPH氧化酶抑制剂DPI（100和300μM）组；LPC+黄嘌呤氧化酶抑制剂别嘌呤醇（100μM）组。LPC处理前1 h加入药物，细胞培养相应时间后，收集细胞，提取蛋白，检测PRMT I表达（Western blot）、DDAH活性（HPLC法）及细胞内ROS水平（荧光法）；收集细胞培养液，测定NO（Griess法）和ADMA（HPLC法）浓度；MTT法检测细胞活性。结果LPC（10 mg／L）孵育内皮细胞可显著增加细胞内ROS含量，降低内皮细胞活性；降低细胞培养液中NO水平和升高ADMA水平；上调PRMT蛋白表达和降低DDAH活性。预先给予DPI（100和300μM）能显著抑制LPC诱导的上述作用。而别嘌呤醇（100μM）对LPC的作用没有显著影响。药物本身对上述指标没有影响。结论LPC诱发ADMA水平增高与其升高NADPH氧化酶活性，进而上调PRMT表达及降低DDAH活性有关。第三节ADMA对细胞通讯功能的影响研究背景内皮细胞功能完整性的维持需要各个细胞间活动的高度协调，GJIC在其中发挥重要作用。在人脐静脉内皮细胞主要表达Cx43。研究发现，AS发生发展与内皮细胞间GJIC功能缺陷密切相关，Cx43的表达、空间分布以及结构变化对AS具有重要调控作用。LDL是促进AS斑块发生的主要脂质成分，可诱发内皮细胞功能和结构的改变。LDL所诱发的内皮功能不全中，ADMA起重要介导作用。研究发现，LDL所致内皮细胞间GJIC功能改变可能是其致AS的重要机制之一。本实验在细胞水平观察了LPC对内皮细胞GJIC功能和Cx43表达的影响。体内90％以上ADMA经组织细胞中DDAH代谢。DDAH存在DDAH1和DDAH2两种亚型，其中DDAH2主要分布于表达内皮型NOS和诱导型NOS的血管组织，是心血管系统ADMA的主要代谢酶。我们利用基因转染技术，建立了过表达DDAH2的内皮细胞，进一步探讨了DDAH／ADMA通路在LPC所致GJIC功能和Cx43表达改变中的作用。方法（1） LPC对人脐静脉内皮细胞GJIC功能的影响及DDAH／ADMA通路的作用在正常细胞、空载体转染细胞及DDAH2过表达细胞，分别设立空白对照组；LPC处理组，加入LPC（10 mg／L）孵育内皮细胞24 h；LPC+NADPH氧化酶抑制剂diphenyliodioum（DPI，100μM）组；LPC+黄嘌呤氧化酶抑制剂别嘌呤醇（100μM）组。（2） ADMA调节GJIC及Cx43表达的机制用不同浓度ADMA处理内皮细胞不同时间，观察Cx43表达变化。①NADPH氧化酶在ADMA调节Cx43表达中的作用空白对照组；ADMA处理组，ADMA（10μM，4 h）孵育内皮细胞；ADMA+DPI（100μM）组；ADMA+PDTC（10μM）组；ADMA+别嘌呤醇（100μM）组。加入药物30 min后，再加入ADMA（10μM）孵育4 h。②p38MAPK在ADMA调节Cx43表达中的作用空白对照组；ADMA处理组，ADMA（10μM，20 min）孵育内皮细胞；ADMA+DPI（100μM）组；ADMA+p38MAPK抑制剂SB203580（20μM）组；ADMA+PDTC（10μM）组。加入药物30 min后，再加入ADMA（10μM）孵育20 min。③PKC在ADMA调节Cx43表达中的作用空白对照组；ADMA处理组，ADMA（10μM，24 h）孵育内皮细胞；ADMA+DPI（100μM）组；ADMA+PDTC（10μM）组；ADMA+PKC阻断剂H7（10μM）组。加入以上药物30 min后，再加入ADMA（10μM）孵育24 h。PKC激动剂12-O-tetradecanoylpforbal 13-acetate（TPA）组，加入终浓度为100 nM的TPA孵育内皮细胞1 h。结果1．转染DDAH2基因的HUVECs DDAH2表达较正常和空载体转染细胞均有不同程度的升高。2．与正常对照组和空载体转染组相比，过表达DDAH2可显著提高人脐静脉内皮细胞细胞活性、降低细胞内活性氧水平和ADMA浓度。3．过表达DDAH2的人脐静脉内皮细胞GJIC功能显著升高，同时伴有Cx43表达上调。4．LPC可抑制内皮细胞间GJIC并降低Cx43表达，此种作用与激活NADPH氧化酶进而促进活性氧生成有关；过表达DDAH2可显著抑制LPC的作用。5．ADMA可以剂量和时间依赖性抑制内皮细胞间GJIC及降低Cx43表达，NADPH氧化酶抑制剂、p38MAPK抑制剂及蛋白激酶C阻断剂可以抑制ADMA的作用。结论DDAH／ADMA通路介导了LPC对内皮细胞GJIC及Cx43的调节作用，其机制可能涉及活性氧—p38MAPK途径。第二章（口山）酮抗动脉粥样硬化作用及其机制研究研究背景LDL所致内皮细胞间GJIC功能改变可能是其致AS的重要机制。LDL及其氧化产物可以剂量依赖性抑制人脐静脉内皮细胞及血管平滑肌细胞GJIC功能。近年实验表明，AS等多种心血管疾病血管功能不全与ADMA水平升高有关。LDL及ox-LDL是AS时ADMA浓度升高的主要原因，并且ADMA浓度升高也是LDL致内皮功能不全的重要介导因素。多种药物通过降低ADMA浓度可拮抗LDL及ox-LDL的作用。（口山）酮是普遍存在于植物中的一类多酚类化合物，具有强效的抗炎、抗氧化及保护血管内皮作用。此外，多酚类某些化合物具有降血脂作用。3，4，5，6-四羟基（口山）酮是我院药物化学系合成的新型（口山）酮单体化合物。我室以前工作证明，3，4，5，6四羟基（口山）酮对血管内皮细胞与缺血心肌具有保护作用。本实验在在体动物和离体细胞水平，系统研究了（口山）酮对血脂和AS进展的影响及其与内皮细胞GJIC的关系。实验分两个部分：1)在高胆固醇血症大鼠，观察（口山）酮对高胆固醇血症的防治作用；2)在ApoE^-/-小鼠，观察3，4，5，6-四羟基（口山）酮对AS进展的影响及其与内皮细胞GJIC的关系；在培养的内皮细胞进一步探讨3，4，5，6-四羟基（口山）酮对LPC作用的影响及机制。第一节（口山）酮对高胆固醇血症大鼠血脂的影响方法实验分预防性给药和治疗性给药两部分。在预防性给药实验，提前两天给予大鼠三个剂量（口山）酮（10、30和90 mg／kg），然后再灌以高胆固醇乳剂，连续十天；在治疗性给药实验，给予大鼠高脂饮食一个月后，再按血脂水平将高胆固醇大鼠随机分组，给予不同剂量（口山）酮(10、30和90 mg／1（g），连续3周。末次给药后禁食12 h，取血检测血脂及丙二醛水平。结果预防性或治疗性给予（口山）酮均能降低高胆固醇血症大鼠血清总胆固醇和低密度脂蛋白水平。治疗性给药实验中，（口山）酮能显著降低血清丙二醛水平。结论（口山）酮可降低高胆固醇血症大鼠血脂水平，此作用可能与其抗氧化作用有关。第二节（口山）酮对Cx43表达的影响及机制方法1动物实验实验分4组（n=8）：①野生型C57BL／6J小鼠组；②ApoE^-／-小鼠组；③低剂量（口山）酮组，给予ApoE^-／-小鼠（口山）酮（10 mg／kg）；④高剂量（口山）酮组，给予ApoE^-／-小鼠（口山）酮（20 mg／kg）。（口山）酮每日灌胃给药。4周后，眼眶取血，分离血浆，检测血浆PON1活性、血脂和ADMA（HPLC法）浓度；分离主动脉，观察动脉粥样损伤（苏丹红染色法）及Cx43表达（免疫组化）。2细胞实验实验分组：空白对照组；LPC处理组：LPC（10 mg／L）孵育内皮细胞相应时间；LPC+不同剂量3，4，5，6-四羟基（口山）酮处理组，加入终浓度为1，3，10μM（口山）酮30 min后，再加入LPC（10 mg／L）孵育相应时间。MTT法检测细胞活性，荧光法检测细胞内活性氧水平，RT-PCR和Western blot分别检测Cx43 mRNA及蛋白水平。结果在ApoE^-／-小鼠，（口山）酮可显著提高血浆PON1活性，降低血浆ADMA水平，上调Cx43表达；在培养的内皮细胞，（口山）酮可显著抑制LPC诱导的细胞活性降低，活性氧生成增多及Cx43表达下调。结论3，4，5，6-四羟基（口山）酮可抑制AS发展，其机制与降低ADMA水平，改善内皮细胞GJIC功能有关。更多还原

【Abstract】 Atherosclerosis （AS） represents the major cause of death anddisability in human adult populations. The loss of functional or structuralintegrity of vascular endothelium is closely related to the initiation of AS.Nitric oxide （NO） is synthesized from L-arginine under catalysis of NOsynthase （NOS） and is believed to be a crucial endogenous anti-atherosclerotic factor.It was reported that an endogenous NOS inhibitor, asymmetricdimethylarginine （ADMA） can competitively inhibit NOS activity andreduce NO production and consequently damage the function of bloodvessels. In addition, ADMA can induce oxidative stress, production ofpro-inflammatory factors and endothelial dysfunction. ADMA is acceptedas a new endogenous atherogenic factor. Dimethylargininedimethylaminohydrolase （DDAH） is responsible for the degradation ofADMA and the reduced DDAH activity is believed to be the main causeof elevated ADMA levels. Some drugs exert protective effects onendothelial dysfunction via improving DDAH activity and decreasingaccumulation of ADMA. The DDAH/ADMA pathway is believed to be anew target for prevention and treatment of AS. The thesis systematicallystudied the role of DDAH/ADMA pathway in the progression andprevention of AS through the mechanism of ADMA induced endothelialdysfunction, the role of DDAH/ADMA pathway in AS, and the effects ofxanthone.ChapterⅠEffects of ADMA on the development of atherosclerosisPart One Effects of ADMA on the development of atherosclerosisBACKGROUNDIt is suggested that gap junction intercellular communication （GJIC）is involved in the progression of AS. GJIC has been demonstrated to beinvolved in the regulation of a variety of endothelial activities. And thealteration of GJIC activity in endothelial cells could lead to a disorder ofhomeostasis. The alternation in expression, distribution, and the structure of Cx43 is of great significance in AS. Recent studies imply thatendothelial gap junctions are involved in atherogenesis and atherogenicfactors are potential regulators of endothelial gap junctions.It is reported that various stimuli oxidize or change the structure ofCxs and then lead to GJIC dysfunction. Based on the facts that oxidativestress results in GJIC dysfunction and ADMA can induce oxidative stress,we suppose that ADMA might cause oxidization of Cx43 via inducingoxidative stress, which contributes to the development of AS.METHODSMice were divided randomly into 4 groups （n=8）: （1） WT group:C57BL/6J mice treated with saline, （2） exogenous ADMA-treatedC57BL/6J mice group, （3） ApoE^-/-mice group: apoE^-/-mice treated withsaline, and （4） exogenous ADMA-treated ApoE^-/-mice group. ADMA （5mg/kg, s.c） was given for 4 weeks. After treatment, mice were sacrificedand serum was colleted to detect paraoxonase （PON1） activity andADMA concentration （HPLC）. The aortas were excised to assay theatherosclerotic lesion area （SudanⅣstaining） and Cx43 expression（immunohistochemistry）.RESULTSIn ApoE^-/- mice, atherosclerotic lesions were more obviouscompared with C57BL/6J mice, concomitantly with the elevation ofADMA. Exogenous ADMA-treatment induced or aggravatedatherosclerotic lesions in WT and ApoE^-/-mice; Plasma PON1 activitywas significantly reduced in ApoE^-/-mice and exogenous ADMAtreatment further decreased PON1 activity; Exogenous ADMA-treatmentsignificantly decreased Cx43 expression in both ApoE^-/- and WT mice.CONCLUSIONADMA plays a key role in facilitating the development of AS, whichmay be related to the down-regulation of Cx43 and inhibition of GJIC. Part Two: Lysophosphatidylcholine-induced elevation of asymmetricdimethylarginine level by the NADPH oxidase pathwayBACKGROUNDEndothelial dysfunction is closely related to oxidative stress. Recentstudies have suggested that endothelium is a major source of reactiveoxygen species （ROS） and the main source was via NADPH oxidasepathway. NADPH oxidase is involved in multiple pathophysiologicalprocesses such as cell proliferation and apoptosis, NO-mediatedvasorelaxation and anoxia/reoxygenation injury in endothelial cells.Much evidence showed that ADMA was closely related toendothelial dysfunction. Various stimuli including ox-LDL can induceADMA accumulation. Lysophosphatidylcholine （LPC）, a majorcomponent of ox-LDL, can induce ADMA elevation and enhance theactivity of NADPH oxidase. In present study, we tested whether theincreased level of ADMA induced by LPC is due to reduction of DDAHactivity and up-regulation of PRMT expression by the NADPH oxidasepathway.METHODSIn LPC-treated endothelial cells （HUVEC-12）, ROS production（fluorescence）, cell viability （MTT）, ADMA （HPLC） and NO levels（Griess）, activity of DDAH （HPLC） and expression of PRMTⅠ（westernblot） were detected.RESULTSTreatment with LPC （10 mg/L） markedly increased intracellularROS production, expression of PRMTⅠ, levels of ADMA, decreased theconcentration of NO and activity of DDAH. These effects wereattenuated by diphenyliodonium, the NADPH oxidase inhibitor.CONCLUSIONLPC-induced elevation of ADMA was due to reduction of DDAH activity and the up-regulation of PRMT expression by stimulation of ROSproduction via NADPH oxidase pathway.Part Three: Effects of ADMA on intercellular communicationBACKGROUNDThe functional integrity of endothelium requires highly coordinationbetween cells, among which GJIC plays an important role. It has beenreported that dysfunction of GJIC is closely related to initiation anddevelopment of AS. Cx43 is the main isotype expressed in culturedendothelial cells. The alternation in expression, distribution, and thestructure of Cx43 is of great significance in AS.LDL is the main component of serum lipids and can inducefunctional and structural changes of endothelial cells. The effects of LDLare partly mediated by ADMA. Recent studies suggest that inhibition ofGJIC induced by LDL is partly responsible for LDL-induced endothelialdysfunction. In the present study, we further observed the effects of LPCon GJIC activity and Cx43 expression.Over 90% percent of ADMA was metabolized by DDAH. As aresult, the level of ADMA depends on DDAH activity and expression. Bygene transfection, we established DDAH2 overexpressed HUVECs andobserved the role of DDAH/ADMA pathway in LPC-induced GJICinhibition in DDAH2 overexpressed endothelial cells.METHODS（1） Effects of LPC on GJIC and the role of DDAH/ADMA pathwayUntransfected cells, DDAH2 transfected cells and empty vectortransfected cells were treated with LPC （10 mg/L） for 24 h. GJICfunction （Lucifer yellow） and Cx43 expression （RT-PCR, western blot）were determined.（2） The mechanism of ADMA on GJIC function and Cx43expression in HUVEC-12 ①dose and time effects of ADMA on Cx43 expressionCells were incubated with different concentrations of ADMA fordifferent time courses, and then the expression of Cx43 was detected.②the mechanism of ADMA on Cx43 expression1) role of NADPH oxidaseControl group; ADMA treated group: cells were treated with ADMA（10μM） for 4 h; +DPI （100μM） group; +PDTC （10μM） group; + allo（100μM） group; Vehicle group. After treatment with drugs for half anhour, then added ADMA for 4 h.2) role of p38MAPKControl group; ADMA treated group: cells were treated with ADMA（10μM） for 20 min; +DPI （100μM） group; +specific p38MAPKinhibitor SB203580 （20μM） group; +PDTC （10μM group）; + allo （100μM） group; Vehicle group. After treatment with drugs for30 min, thenadded ADMA for 20 min.3) role of PKCControl group; ADMA treated group: cells were treated with ADMA（10μM） for 24 h; +DPI （100μM） group; +PDTC （10μM） group;+specific PKC inhibitor H7 （10μM） group. After treatment with drugsfor 30 min, then added ADMA for 20 min. PKC activator12-O-tetradecanoylpforbal 13-acetate （TPA） group: cells were treatedwith TPA （100 nM） for 1 h.RESULTS1. The expression of DDAH2 was increased in DDAH2 transfected cellscompared with empty vector transfected and untransfected cells.2. DDAH2 overexpression can enhance cell viability, decreaseintracellular ROS production and ADMA concentration.3. DDAH2 overexpression can enhance GJIC function, concomitantlywith up-regulation of Cx43.4. LPC inhibited GJIC and down-regulated expression of Cx43, which isrelated to the excessive production of ROS via activation of NADPHoxidase. DDAH2 overexpression can reverse such effects of LPC.5. ADMA dose- and time-dependently down-regulated Cx43 expression.NADPH oxidase inhibitor, p38MAPK inhibitor and PKC inhibitor can inhibit the effects of ADMA.CONCLUSIONLPC induced inhibition of GJIC was mediated by DDAH/ADMApathway, which may involve ROS-p38MAPK signal pathway.ChapterⅡThe suppression of xanthone on atherogenesis and itsmechanismsBACKGROUNDRecent studies suggested that the alternation of GJIC function playsan important role in LDL induced AS. LDL can dose dependently inhibitGJIC function in smooth muscle cells and endothelial cells. LDL andox-LDL are the main causes of elevated ADMA in AS and the elevationof ADMA also mediates the effects of LDL. Some drugs can inhibit theeffects of LDL via decreasing ADMA levels.Xanthones are polyphenol compounds existing in many plants whichhave potent anti-inflammation, anti-oxidation and endothelial protectiveeffects. Some polyphenol compounds have cholesterol-lowing effects.3,4,5,6-tetrahydroxyxanthone is synthesized by the department ofchemical pharmacy. The previous work of our lab has shown that3,4,5,6-tetrahydroxyxanthone has protective effects on endothelium andmyocardium. In this study, we systematically studied the effects ofxanthone on the progression of AS and its relationship to GJIC both invivo and in vitro.There are two parts: 1) to test the effects of xanthone on serum lipidand oxidative state of hypercholesterolemic rats; 2) to observe the effectsof 3,4,5,6-tetrahydroxyxanthone on the progression of AS in ApoEdeficient mice and its relationship to the alternation of GJIC; and furtherexplored the mechanism of 3,4,5,6-tetrahydroxyxanthone on LPCinduced inhibition of GJIC. Part One Effects of xanthone on serum lipid profiles inhypercholesterolemic ratsMETHODSThe experiments were divided into two parts: in the preventiveexperiment, the rats were treated with xanthone （10, 30 or 90 mg/kg, i.g）for 2 days and then treated with cholesterol emulsion for 10 days in thepresence of xanthone; In the therapeutic experiment, after being fed withfoods rich in cholesterol for 4 weeks, the rats were treated with xanthonefor 3 weeks （i.g.）. Serum lipids and malondialdohyde were measured.RESULTSXanthone, given preventively or therapeutically, markedly decreasedtotal cholesterol and LDL level. Moreover, in the therapeutic experimentxanthone also decreased malondialdohyde content.CONCLUSIONXanthone decreased total cholesterol and LDL level inhypercholesterolemic rats, which may be related to its anti-oxidativeeffects.Part Two Effects of xanthone on expression of Cx43 and itsmechanismMETHODS1 animal studyThe experiment was divided into 4 groups （n=8）:①Wild typeC57BL/6J mice;②ApoE^-/- mice group;③low dose xanthone treatmentgroup, ApoE^-/- mice were given xanthone （10 mg/kg, i.g.）④high dosexanthone treatment group, ApoE^-/- mice were given xanthone （20 mg/kg,i.g.）. After treatment, serum was colleted to detect PON1 activity andADMA concentration （HPLC）; aortas were excised to assay atherosclerotic lesion area （SudanⅣstaining） and Cx43 expression（immunohistochemistry）.2 cell cultureThe experiment was divided into 4 groups: control group; LPCtreated group: cells were treated with LPC （10 mg/L） for different time;different concentration of 3,4,5,6-tetrahydroxyxanthone group: cells weretreated with 3,4,5,6-tetrahydroxyxanthone （1, 3, 10μM） for 30 min, thentreated with LPC （10 mg/L） for different time. ROS production（fluorescence）, cell viability （MTT）, ADMA （HPLC） level and expressionof Cx43 （western blot and RT-PCR） were detected.RESULTSIn ApoE^-/- mice, 3,4,5,6-tetrahydroxyxanthone significantlydiminished atherosclerotic lesion area, increased serum PON1 activity,decreased ADMA and up-regulated Cx43 expression. In cultured cells,3,4,5,6-tetrahydroxyxanthone significantly inhibited LPC induceddecreased cell viability, increased ROS production and down-regulatedCx43 expression.CONCLUSION3,4,5,6-tetrahydroxyxanthone has anti-atherosclerotic effects whichis related to the reduction of ADMA level and consequently improvementof GJIC function.更多还原