【作者】 薛丽；

【导师】 陈玉国；

【作者基本信息】 山东大学 ， 急诊医学， 2012， 博士

【摘要】 背景大量流行病学资料表明,适量饮酒有保护心血管系统的作用,可降低冠状动脉粥样硬化性心脏病(冠心病,coronary artery disease, CAD)的发生率及病死率。早期研究多认为酒中的非乙醇活性成分起到了重要作用,后来研究发现乙醇对心血管系统起主要保护作用。实验室研究发现,低剂量乙醇可通过激活腺苷受体依赖的PI3K-Akt通路增强脐静脉内皮细胞内内皮型一氧化氮合酶(edothelial nitric oxide synthase, eNOS)的表达及活性,并增加一氧化氮(nitric oxide, NO)生成及释放。然而,乙醇作为一种外源性的小分子有机化合物,可通过自由扩散进入细胞内进行代谢,经由酒精代谢系统进行代谢时可伴随产生活性氧(reactive oxygen species, ROS)。活性氧对内皮细胞eNOS有双面作用,并取决于ROS的水平。大量活性氧可通过eNOS脱偶联或者拮抗其上游调节分子来抑制eNOS活性,导致内皮细胞功能紊乱；而少量活性氧可以增强eNOS的活性。而在乙醇干预时的ROS水平取决于抗氧化体系的拮抗作用。近来研究发现,ALDH2不仅代谢乙醛,还可通过氧化丙二醛(malondialdehyde, MDA)和4-羟基壬烯醛(4-hydroxynonenal,4-HNE)等毒性醛类物质来发挥间接抗氧化作用,拮抗ROS生成。多项研究结果表明调控ALDH2表达和活性可以影响机体细胞对氧化应激的应答。ALDH2活性可在转录、转录后调节与修饰、翻译和翻译后修饰不同水平上进行调节。尤其是,ALDH2活性可受乙酰化修饰的调控,其可被线粒体SIRT3直接脱乙酰化而导致酶活性下降。SIRT3具有依赖NAD+的组蛋白脱乙酰基酶活性,位于线粒体基质中,可以通过调控线粒体蛋白乙酰化水平触发一系列细胞适应性改变来应答代谢应激。SIRT3活性对细胞内NAD+/NADH比值变化十分敏感,呈正相关。有意思的是,乙醇代谢会导致NAD+/NADH比值下降,因此很有可能抑制SIRT3活性。因此,我们提出以下假说：乙醇可通过增强ALDH2酶活性来调节细胞内ROS水平,进而影响乙醇增强eNOS活性的作用；且SIRT3依赖的乙酰化修饰作用可能参与乙醇对ALDH2活性的调控过程。依赖于SIRT3的ALDH2乙酰化修饰可能是乙醇保护作用的重要机制,是防治动脉粥样硬化(atherosclerosis, AS)疾病的新靶点。方法1.细胞培养与处理：培养人主动脉内皮细胞(human aortic endothelial cells, HAECs),使用内皮细胞培养基进行培养。待细胞生长融合至80-90%时,用不同浓度的乙醇刺激不同时间；用P13K抑制剂或外源性NAD预处理细胞时,先用试剂预处理细胞24小时后,再用乙醇继续孵育30分钟；2. siRNA和质粒转染：用特异性siRNA对细胞ALDH2和SIRT3进行抑制,用构建好的SIRT3过表达质粒提高细胞内SIRT3表达。首先用脂质体与siRNA/质粒形成复合物,后转染入HAECs。转染后的细胞用／不用ROS清除剂预处理后,再加入乙醇继续孵育；3.实时定量RT-PCR检测：使用Trizol试剂提取细胞RNA,逆转录成cDNA后使用SYBR Green进行实时定量RT-PCR。基因的相对表达量采用2-△△ct计算。4. Western blotting检测：使用细胞裂解液提取细胞总蛋白,使用Western blotting检测方法从蛋白水平检测用于检测HAECs中ALDH2、eNOS、p-eNOS、 Akt、p-Akt、PI3K、SIRT3、4-HNE等蛋白的表达量。5.免疫共沉淀：用裂解液提取细胞总蛋白,加入抗体和蛋白A/G琼脂糖吸附球共同孵育4℃过夜进行免疫沉淀,高速离心将琼脂糖球去除并吸取上清液进行后续Western blotting检测。6.内皮细胞eNOS活性检测：应用eNOS检测试剂盒来分析细胞eNOS的活性,在激发波长495nm,发射波长515nm下进行吸光度测量。eNOS相对活性用实验组与对照组的活性比值来表示。7.线粒体ALDH2酶活性检测：提取细胞线粒体,线粒体ALDH2活性是通过在酶标仪上监测A340nm下NAD+转化为NADH的含量变化所得。ALDH2酶活性单位为nmol/mg/min。8.线粒体SIRT3活性检测：采用SIRT3活性荧光定量检测试剂盒检测,在激发光340nm和发射光440nm检测荧光值。SIRT3相对活性是实验组与对照组的活性比值来表示。9.细胞内ROS水平检测：应用DCFH-DA荧光探针对细胞内活性氧簇进行标记,后在流式细胞仪上进行检测。10.线粒体NAD+/NADH比值检测：细胞收集后提取线粒体,采用NAD+/NADH比值检测试剂盒进行检测,分别在450nm处读取NADt和NADH数值。NAD+/NADH比值计算式为：[NADt-NADH]/NADH。结果1.乙醇对内皮细胞eNOS活性的作用呈剂量依赖性：在不同浓度乙醇处理细胞30min后,随乙醇浓度增加,内皮细胞eNOS活性逐渐升高,在20mM乙醇作用下达到最高峰(1.67倍,P<0.001),而后随乙醇浓度增加eNOS活性逐渐下降,至100mM乙醇作用时回到基线水平。乙醇对eNOS活性调控呈时间依赖性：在乙醇作用5min后,内皮细胞eNOS活性即开始升高,在30min后达最大值,之后逐渐下降,且在乙醇作用120min后,内皮细胞eNOS活性仍然显著高于对照组(P<0.001)；2.20mM乙醇作用30min后可使p-eNOS和p-Akt蛋白表达明显增加,与乙醇对eNOS活性的作用趋势一致,且乙醇的这一作用可被P13K抑制剂拮抗,提示乙醇是通过激活PI3K/Akt通路提高内皮细胞eNOS活性的；3. ALDH2的活性在乙醇刺激下呈时间依赖性改变：乙醇处理后15min,内皮细胞ALDH2活性即开始明显增加,并在30min达到最大值(1.65倍,P<0.001),但随后即迅速下降,但对ALDH2mRNA和蛋白表达没有影响；4. ALDH2siRNA抑制内皮细胞ALDH2表达后,可拮抗乙醇增加Akt和eNOS活性的作用；5.20mM乙醇对细胞内ROS含量没有影响。而在ALDH2siRNA预孵育的细胞中发现,乙醇干预细胞后ROS含量明显升高66%。且ROS清除剂(NAC和DTT)可拮抗ALDH2siRNA对乙醇内皮保护的抑制作用；6.乙酰化ALDH2蛋白表达变化与乙醇刺激呈时间依赖性。乙醇处理后15min,内皮细胞乙酰化ALDH2蛋白表达即开始明显增加,并在30min达到峰值(P<0.001),但随后即明显下降,与ALDH2活性变化趋势一致；7.20mM乙醇处理15min后SIRT3活性即下降,在30min时下降最为明显,然后随时间增加又逐渐恢复到基线水平。且过表达SIRT3可以抵抗乙醇上调ALDH2乙酰化和活性的作用；8. SIRT3过表达的内皮细胞受乙醇刺激后,ROS水平明显增加。且乙醇激活Akt和eNOS的作用也被SIRT3过表达质粒所抑制；9.乙醇处理内皮细胞15min后NAD+/NADH比值显著下降,在30min时下降达最低值。外源性NAD干预下,乙醇抑制SIRT3活性的作用明显减弱,且可抑制乙醇上调ALDH2和eNOS活性的作用。结论1.乙醇通过激活PI3K-Akt通路提高eNOS活性；2.乙醇可上调ALDH2活性；3. ALDH2是一种内源性抗氧化应激因子,使细胞在乙醇处理后保持氧化还原状态稳定,从而使乙醇增强eNOS活性的作用顺利实现；4.乙醇通过降低SIRT3活性,上调ALDH2乙酰化水平和活性；5.乙醇通过降低NAD+/NADH比值抑制SIRT3活性。背景内皮衰老是导致动脉粥样硬化(atherosclerosis, AS)发生发展的重要原因。内皮细胞衰老是正常血管随增龄发生的自然现象,同时伴随端粒缩短和端粒酶活性下降,亦称为复制性衰老,是老年人发生AS的主要原因。适量饮酒可以有效预防老年人AS疾病的发生,降低冠心病发病率、死亡率和全因死亡率,具有延缓衰老,延长人类寿命的作用。因此本实验着重研究饮酒抗衰老的作用机制,来探讨防治AS新的靶分子蛋白。一些研究发现,低剂量乙醇可以提高静息内皮细胞eNOS活性,增加NO释放。既然乙醇可以增强静息内皮细胞eNOS的活性,那么乙醇的这一作用机制很可能也存在于衰老内皮细胞中。且以往一项研究表明,适量乙醇摄入可以抵抗增龄相关的认知功能障碍,提示乙醇可以影响衰老进程。SIRT1是一类调控生物体寿命的重要蛋白,属于第三类组蛋白去乙酰化酶。它可以与染色质、转录因子、转录共调控因子和非组蛋白底物相互作用,通过去酰化作用调节细胞生存、衰老、凋亡等发生发展等一系列生理病理过程。SIRT1作为一个重要的细胞内调控蛋白,其自身的表达和活性不仅受基因表达的调控,还受细胞内NAD+/NADH比值、氧化应激、翻译后修饰和SIRT1核-浆穿梭变化影响。论文I中已证实适量乙醇可快速上调NAD+/NADH比值而影响SIRT3活性,由于SIRT1与SIRT3同源性较高,催化活性域的氨基酸序列同源性可达89%,故乙醇很有可能也会影响SIRT1的生物学功能。我们前期研究发现这乙醇增强静息内皮细胞eNOS活性的作用可被乙醇诱导的高活性乙醛脱氢酶2(aldehyde dehydrogenase2, ALDH2)所调控,ALDH2起到“分子开关”作用。那么,乙醇是否可通过调控ALDH2影响内皮衰老,目前还无相关报道。线粒体ALDH2是一种酒精代谢关键酶,在心血管系统表达丰富。流行病学研究发现高活性的ALDH2可能会预防AS的发生发展。国内外陆续有研究表明,ALDH2具有拮抗衰老的作用。我们前期研究已证实ALDH2是一种内源性抗氧化应激因子。而氧化应激学说是衰老机制理论的重要组成部分,尤其是可影响SIRT1功能,故ALDH2可能是通过抗氧化应激来影响SIRT1功能,进而发挥抑制衰老的作用,是防治AS的新靶点。因此我们提出如下假说：乙醇具有延缓内皮复制性衰老的作用,且SIRT1与ALDH2这两种抗衰老蛋白均可能在此过程中发挥重要介导作用；低剂量乙醇可能通过上调ALDH2活性诱导SIRT1生物学功能增强,进而延缓内皮衰老、拮抗AS发生与发展。方法1.细胞培养与处理：培养人主动脉内皮细胞(human aortic endothelial cells, HAECs),使用内皮细胞培养基在37℃和5%CO2孵育箱内进行培养。2.构建HAECs复制性衰老模型：采用连续传代方法构建内皮细胞复制性衰老模型,每一代细胞传代前均行细胞数目检测,计算群体倍增水平(population doubling level, PDL)。本实验中发现PDL22细胞继续培养后会进入衰老状态,故将PDL22作为本实验的衰老模型。3.乙醇处理衰老细胞：待衰老细胞生长融合至85%左右时,饥饿24h后用不同浓度乙醇(OmM,5mM,20mM,50mM,100mM)处理6天。4.细胞活性检测：采用CCK-8试剂盒进行检测,在450nnm处测定吸光度值。5.p-半乳糖苷酶染色：对细胞进行p-半乳糖苷酶染色,衰老细胞呈现蓝染,并在光学显微镜下计数染色阳性细胞数目。6. BrdU增殖检测：BrdU可代替胸腺嘧啶掺入到新合成的DNA中,用免疫细胞化学法标记BrdU,并在酶标仪上450nm处读取吸光度值来检测细胞增殖能力。7. siRNA和质粒转染：用特异性siRNA对细胞ALDH2和SIRT1进行抑制,用ALDH2表达质粒和特异性定位核／胞质的SIRT1过表达质粒分别提高细胞内ALDH2和核／胞质内SIRT1表达。首先用脂质体与siRNA/质粒形成复合物,后转染入HAECs。转染后的细胞再加入乙醇继续孵育；8.实时定量RT-PCR检测：使用Trizol试剂提取细胞RNA,逆转录成cDNA后使用SYBR Green进行实时定量RT-PCR。基因的相对表达量采用2-△△ct的计算方法。9. Western blotting检测：使用细胞裂解液提取细胞总蛋白,使用Western blotting检测方法从蛋白水平检测用于检测细胞中ALDH2、eNOS、SIRT1等蛋白的表达量。10.内皮细胞eNOS活性检测：应用eNOS检测试剂盒来分析细胞eNOS的活性,在激发波长495nm,发射波长515nm下进行吸光度测量。eNOS相对活性是用实验组与对照组的活性比值来表示的。11.线粒体ALDH2酶活性检测：提取细胞线粒体,线粒体ALDH2活性是通过在酶标仪上监测A340nm下NAD+转化为NADH的含量变化所得。ALDH2酶活性单位为nmol/mg/min。12.免疫荧光染色：细胞培养终止后,用一抗反应液进行孵育,随后用荧光标记的二抗进行处理,最后用含DAPI的防淬灭封片剂封片在免疫共聚焦显微镜下观察。结果1.通过对HAECs细胞株连续传代培养,根据细胞形态学、SA-β-gal衰老染色、BrdU增殖检测、细胞活性等指标变化表明,PDL8细胞属于年轻细胞,而PDL22细胞开始进入衰老状态；2.衰老细胞中eNOS蛋白、mRNA表达和活性均明显下降,表明内皮复制性衰老伴随内皮功能下降；3.通过检测不同浓度(0mM、5mM、20mM、50mM、100mM)乙醇处理6天后细胞细胞衰老表型的变化,结果显示20mM乙醇有延缓内皮衰老的作用,5mM乙醇对内皮衰老表型改变不大,而50Mm和100mM乙醇明显降低细胞数目；4.20mM乙醇可增加衰老内皮细胞内eNOS mRNA和蛋白表达,并增强其活性,进一步证实20mM乙醇有改善衰老内皮细胞功能的作用；5. SIRT1在衰老细胞内的分布发生明显变化：与年轻PDL8细胞相比,衰老细胞内SIRT1在核内蛋白表达明显下降,而在胞质中表达增强,但是SIRT1的总蛋白量没有发生改变,即SIRT1发生了核浆转位；6.通过特异性过表达衰老细胞内核/胞质SIRT1发现,定位核而非胞质的SIRT1具有抵抗内皮细胞复制性衰老的作用；7.20mM乙醇可增强衰老内皮细胞内SIRT1在核中的表达,抑制其在胞浆中的蛋白表达,即乙醇有抑制衰老内皮细胞SIRT1核浆转位的作用；进一步应用SIRT1siRNA特异性抑制其表达后,乙醇延缓内皮衰老的作用明显削弱；8.与年轻细胞相比,衰老细胞内ALDH2蛋白、mRNA表达和活性明显下降,提示在内皮细胞复制性衰老进程中,ALDH2表达和活性明显下降；9.应用ALDH2过表达质粒转染PDL22细胞后6天,结果表明ALDH2过表达可明显延缓HAECs复制性衰老并改善内皮功能；10.20mM乙醇可增加衰老内皮细胞内ALDH2mRNA和蛋白表达,并增强其活性；应用ALDH2特异性siRNA转染PDL22细胞来抑制其表达后,结果发现乙醇延缓内皮衰老的作用明显减弱；11.ALDH2特异性siRNA转染PDL22细胞后再用乙醇孵育,结果发现乙醇抑制SIRT1核浆转位的作用明显削弱。结论1.成功建立HAECs复制性衰老模型；2.乙醇具有延缓HAECs复制性衰老并增强衰老内皮细胞功能的作用；3. HAECs复制性衰老伴随SIRT1核浆转位,乙醇可通过抑制SIRT1核浆转位来延缓HAECs复制性衰老；4. HAECs复制性衰老伴随ALDH2表达和活性下降,乙醇可通过增强ALDH2表达和活性来延缓HAECs复制性衰老；5.乙醇可通过增强ALDH2表达和活性,来抑制SIRT1核浆转位,ALDH2-SIRT1通路可能是乙醇延缓内皮衰老的重要分子机制。更多还原

【Abstract】 BackgroundExtensive epidemiological studies links moderate alcohol consumption with reduced risk of cardiovascular morbidity and mortality. Initially, non-alcoholic components have been thought to play the primary role in the protective effects induced by drinking. However, recent studies found that ethanol may also be involved in the protection. Low-dose ethanol can rapidly activate PI3K/Akt pathway to increase endothelial nitric-oxide synthase (eNOS) activity by an adenosine receptor-dependent mechanism in human umbilical vein endothelial cells (HUVECs).In addition, ethanol, as a small exogenous liposoluble molecule, can easily pass through the cell membrane and be oxidized by metabolic enzymes systems, accompanied by reactive oxygen species (ROS) generation. ROS have detrimental or beneficial effects on eNOS activation depending on its concentrations. During ethanol exposure, ROS level is dependent on the capability of antioxidant systems to antagonize ROS generation.Mitochondrial aldehyde dehydrogenase (ALDH2), a key enzyme in ethanol metabolism, has been found to possess the antioxidant property. In vitro and in vivo studies suggest that regulation of ALDH2activity can have influence on the cellular response to oxidative stress. ALDH2activity can be affected by gene regulation or post-translational modifications. For example, ALDH2can be directly deacetylated by SIRT3resulting the inactivation of ALDH2.SIRT3, a NAD+-dependent class Ⅲ histone deacetylase, is localized in the mitochondrial matrix. It can elicit adaptive responses to metabolic stresses by regulating mitochondrial protein acetylation levels. SIRT3enzymatic activity has been found very sensitive to the NAD+/NADH ratio. Interestingly, enzymes of ethanol metabolism convert NAD+to NADH leading to the reduced NAD+/NADH ratio, which may have the potential to affect SIRT3activity.In present study, we hypothesized that ethanol can activate ALDH2to regulate ROS level, which may be involved in the protective effects of ethanol on endothelial function. SIRT3-dependent acetylation may play an important role in the potential mechanisms involved. SIRT3-dependent ALDH2acetylation may be a therapeutic target in treating cardiovascular diseases.Methods1. Cell culture and treatment:Human aortic endothelial cells (HAECs) were maintained in endothelial cell medium (ECM) in a water-saturated atmosphere of5%CO2and95%air at37℃. When cells were more that80-90%confluence, different doses of ethanol were added into cells for the designated times. Cells were treated with PI3K inhibitors and NAD for24h before incubated with20mM ethanol for another30min.2. Cell transfection:The specific siRNAs were used to inhibit ALDH2and SIRT3expression, whereas SIRT3plasmids were used to elevate SIRT3expression. Cells were transfected with the siRNA duplexes or plasmids by the Lipofectamine2000according to the manufacturer’s instructions. Cells transfected with ALDH2siRNA were then incubated with or without ROS scavengers for30min before ethanol treatment for another30min.3. Real-time PCR:Total RNA were extracted from treated cells by Trizol, and then reverse-transcripted into cDNAs. Real time PCR was performed using SYBR Green detection system. The relative mRNA levels were calculated by2-ΔΔct.4. Western blotting analysis:Total cellular protein was extracted by lysis buffer. The protein expression of ALDH2、eNOS、p-eNOS、Akt、p-Akt、PI3K、SIRT3、4-HNE in HAECs were detected using Western blotting analysis.5. Immunoprecipitation:Cellular total protein was prepared by use of lysis buffer. For immunoprecipitation, the total protein was incubated with appropriate antibody precoupled to protein A/G-agarose beads at4℃overnight. Then agarose beads were removed by high-speed centrigugalization, and then the supernatant were collected to be analyzed by western blotting.6. eNOS activity assay:eNOS activity was measured by the conversion of L-arginine to NO. Then the fluorescent identity was determined by fluoro-spectrophotometer analysis at excitation495nm and emission515nm.7. ALDH2activity assay:The mitochondria ALDH2enzymatic activity was determined by monitoring the reductive reaction of NAD+to NADH at A340nm in a spectrophotometer. Enzyme activity was expressed as nmol/mg/min.8. SIRT3activity assay:The mitochondrial SIRT3activity was assayed using a deacetylase colorimetric activity assay kit according to the manufacture’s instructions.9. Assessment of intracellular ROS levels:Treated cells were stained with2’,7’-dichlorodihydro-fluorescein diacetate, then underwent fluorescent intensity measurement by flow cytometry.10. NAD+/NADH assay:Mitochontrial NAD+/NADH ratio were quantified using an NAD+/NADH assay kit according to the assay instructions. Total NADt and NADH were detected at450nm in a spectrophotometer. NAD+/NADH ratio is calculated as:[NADt-NADH]/NADH.Results1. Ethanol dose-dependently increased then decreased eNOS activity, with peak activity at20mM (1.67-fold, p<0.001). Additionally, time-course study showed that increase of eNOS activity began at5min after ethanol incubation and got the peak at30min (p<0.001);2. Ethanol (20mM) significantly upregulated p-Akt protein expression at30min, which protective effects of ethanol could be blocked by PI3K inhibitors, confirming that ethanol can activate PI3K/Akt pathway to increase eNOS activity;3. ALDH2activity increased significantly after15min of ethanol incubation, and got the peak at30min (1.65-fold,p<0.001), and then decreased rapidly. However, ALDH2mRNA and protein expression were not affected; 4. ALDH2siRNA could inhibit the protective effects of ethanol on activation of Akt and eNOS;5. ROS level had no change after incubation with ethanol. However, ROS concentration was markedly increased by approximately60%in cells expressing ALDH2siRNA after ethanol treatment. Administration of ROS scavengers into cells expressing ALDH2siRNA inhibited ethanol-induced ROS accumulation and increased activation of Akt and eNOS;6. After ethanol treatment, ALDH2acetylation showed time-dependent increase and then decrease, with peak at30min (2.2-fold, p<0.001), consistently with the changes of ALDH2activity;7. After incubation with ethanol, SIRT3activity decreased with time, and got the minimum value at30min. Moreover, SIRT3overexpression could reverse ethanol-elevated ALDH2acetylation and activity;8. ROS level increased significantly in cells overexpressing SIRT3after ethanol incubation. Subsequently, SIRT3overexpression reversed ethanol-induced Akt/eNOS activaton;9. Mitochondrial NAD+/NADH radio decreased by65%at30min after ethanol treatment. NAD treatment reversed the effects of ethanol on SIRT3, ALDH2, Akt and eNOS.Conclusion1. Ethanol can upregulate eNOS activity through activating PI3K-Akt pathway;2. Ethanol can increase ALDH2activity;3. ALDH2is an endogenous anti-oxidant factor, which activation mediates the protective effects of ethanol on eNOS activation through preventing ROS accumulation;4. Ethanol-induced ALDH2activation is positively relevant with acetylation of ALDH2dependent on reduced SIRT3activity;5. Ethanol can inhibit SIRT3activity through reducing NAD+/NADH ratio. BackgroundEndothelial senescence-induced endothelial dysfunction plays an important role in the initiation and the progression of atherosclerosis (AS). Endothelial senescence, as a key characteristic of vascular aging, is companied by reduced telomere length and telomerase activity, which is also called replicative senescence contributing to the increased risk for AS mortality in old-aged population. Moderate alcohol consumption has been found prevent the AS, delay aging and extend lifespan in old persons. Thus detecting the molecular mechanisms involved the inhibitory effects of ethanol on aging may help explore the novel targets for prevention and therapies of AS.Some researchers found that low-dose ethanol can increase eNOS activity and NO release in silent endothelial cells. We presumed that this protective effect of ethanol may also be detected in the senescent endothelial cells. Moreover, a previous study suggests that moderate ethanol ingestion can resist aging-related cognitive dysfunction. Thus, ethanol may prevent aging-related AS through improving endothelial function.SIRT1, a NAD+-dependent class III histone deacetylase, plays an important role in cell survival and replicative senescence. It plays an important role in a wide variety of processes, including apoptosis, senescence, differentiation and aging by interactions with a variety of chromatin-associated proteins and non-histone targes. SIRT1activity can be affected by gene regulation, NAD+/NADH ratio, oxidative stress, post-translational modifications and nucleocytoplasmic shuttling. Our previous studies have found that ethanol can rapidly upregulate NAD+/NADH ratio to affect SIRT3activity. In view of the high degree of homology between SIRT1and SIRT3, SIRT3biological function may also be affected by ethanol.Our previous findings suggest that the protective effects of ethanol on eNOS activity can be mediated by aldehyde dehydrogenase2(ALDH2), which functions as the molecular swich. However, whether ethanol can regulate ALDH2to affect endothelial senescence has no related reports until now. Mitochondrial ALDH2, known as an important enzyme in ethanol metabolism, exists abundantly in cardiovascular system. Epidemiological studies report that ALDH2with high enzymatic activity may prevent the development of AS. Interestingly, ALDH2has been found to regulate aging processes in vitro and in vivo. Furthermore, ALDH2possesses the property to inhibit oxidative stress, which is known as an important contributor for aging especially through affecting SIRT1function. Thus, ALDH2may have an association with SIRT1function to regulate senescence-related diseases, which means ALDH2may be the new target for preventing AS.In present study, we hypothesized that ethanol has the protective effects against endothelial senescence in HAECs, in which progression SIRT1and ALDH2may both be involved. Low-dose ethanol may upregulate ALDH2activity to induce the inhibitory effects of SIRT1on endothelial senescence.Methods1. Cell culture:Human aortic endothelial cells (HAECs) were maintained in endothelial cell medium (ECM; Sciencell, USA) in a water-saturated atmosphere of5%CO2and95%air at37℃.2. Endothelial replicative senescence model:The replicative senescence model of HAECs was constructed by consecutive passage assay. Before each passage, the total cell number were obtained to calculate the population doubling level (PDL). We found that At PDL22, cells exhibited senescence-associated phenotype.3. Cell treatment:When senescent cells were more that80-90%confluence, different doses of ethanol (OmM,5mM,20mM,50mM,100mM)was added into cells for the designated times.4. Cell viability assay:HAECs were seeded in the96-well plates, and the cell viability was detected using the CCK-8assays according to the manufacturer’s instructions. The absorption at450nm was measured spectroscopically.5. Senescence-associated β-galactosidase (SA-β-gal) assay:Cells were stained with β-galactosidase, and the blue-stained cells and the total number of cells were counted under light microscopy.6. BrdU incorporation assay:BrdU incorporates into cellular DNA during cell proliferation, and is detected by immunofluorescent staining. Then the fluorescent identity was determined by fluoro-spectrophotometer analysis at450nm.7. Cell transfection:The specific siRNAs were used to inhibit ALDH2and SIRT1expression, whereas ALDH2and SIRT1plasmids were used to elevate their expression. Cells were transfected with the siRNA duplexes or plasmid by the Lipofectamine2000according to the manufacturer’s instructions. Cells transfected with siRNAs or plasmids were then incubated with ethanol.8. Real-time PCR:Total RNA were extracted from treated cells by Trizol, and then reverse-transcripted into cDNAs. Real time PCR was performed using SYBR Green detection system. The relative mRNA levels were calculated by2-ΔΔct.9. Western blotting analysis:Total cellular protein was extracted by lysis buffer. The protein expression of ALDH2、eNOS、SIRT1in HAECs were detected using Western blotting analysis.10. eNOS activity assay:eNOS activity was measured by the conversion of L-arginine to NO by use of a nitricoxide synthase assay kit. Then the fluorescent identity was determined by fluoro-spectrophotometer analysis at excitation495nm and emission515nm.11. ALDH2activity assay:The mitochondria ALDH2enzymatic activity was determined by monitoring the reductive reaction of NAD+to NADH at A340nm in a spectrophotometer. Enzyme activity was expressed as nmol/mg/min. 12. Immunofluorescence Microscopy:Cells were grown on sterilized glass coverslips and processed for immunofluorescence microscopy by staining eNOS, ALDH2and Sirt1.Results1. After purifying HAECs by consecutive passage, we identified PDL8cells as young cells and PDL22cells as senescent cells according to the cellular changes in morphology, SA-β-gal activity and BrdU incorporation.2. Senescent cells (PDL22) exhibited decreased eNOS protein and mRNA expression, accompanied by reduced activity, indicating that eNOS inactivation can be observed in senescent cells.3.20mM ethanol may delay endothelial cell replicative senescence. However, other doses of ethanol (5,50,100mM) had no beneficial effects on senescent cells.4. Ethanol treatment dose-dependently increased then decreased eNOS protein and mRNA expression, with peak value at20mM. As well, eNOS activity was also increased by20mM ethanol.5. Compared to young cells, in senescent cells, the nuclear expression of SIRT1decreased whereas the cytoplasmic expression increased at the same time. However, the total protein expression of SIRT1had no change in senescent cells, indicating SIRT1translocation from nucleus into cytoplasm in senescent cells.6. In order to determine the association of this alteration in SIRT1subcellular localization with the cellular senescence, SIRT1-EGFP (nuclear SIRT1) and mtNLS-EGFP (cytoplasmic SIRT1) were used in this study. The results suggest that the nuclear but not cytoplasmic SIRT1has impact against replicative senescence.7.20mM ethanol decreased the cytoplasmic SIRT1protein expression and increased the nuclear SIRT1protein expression, indicating20mM ethanol can inhibit SIRT1nuclear export in senescent cells.8. In senescent HAECs, ALDH2activity decreased. Further study found that ALDH2protein and mRNA expression were also decreased in senescent cells.9. ALDH2overexpression could delay endothelial senescence. 10.20mM ethanol could increase ALDH2protein and mRNA expression, accompanied by the increased ALDH2activity in senescent cells. After transfection with ALDH2siRNA to inhibit its expression, the protective effects of ethanol against senescence were attenuated significantly.11. In the presence of ALDH2siRNA, the inhibitory effects of ethanol on SIRT1nuclear export were attenuated.Conclusion1. The replicative senescent model of HAECs can be constructed by consecutive passage assay;2. Ethanol can delay endothelial senescence and improve endothelial function in HAECs;3. SIRT1translocates from nucleus into cytoplasm in senescent HAECs, which can be blocked by ethanol to delay endothelial senescence;4. The expression and activity of ALDH2decrease in senescent HAECs, which can be inhibited by ethanol to delay endothelial senescence;5. Ethanol can upregulate ALDH2expression and activity to inhibit SIRT1nuclear export, which may be the important molecular mechanisms in the protective effects of ethanol against endothelial senescence.更多还原