叶酸与动脉粥样硬化的研究进展

叶酸是同型半胱氨酸（homocysteine,Hcy）代谢过程中的重要辅助因子,叶酸缺乏可引起血清Hcy浓度增加,Hcy水平升高又是心血管疾病发生的独立危险因素。叶酸通过合成S-腺苷蛋氨酸（也称S-腺苷甲硫氨酸,SAM）参与机体的DNA甲基化反应。动脉粥样硬化（atherosclerosis,AS）是一种多因素相关的疾病,包括营养、遗传和环境因素。     

同型半胱氨酸对Aβ经毒性促进作用

目的 研究同型半胱氨酸是否促进β淀粉样肽(β-amyloid,Aβ)对体外培养海马细胞的神经毒性.方法 用同型半胱氨酸和/或Aβ42处理体外培养的海马细胞,观察神经细胞的凋亡情况以及神经细胞内的游离钙浓度的改变、DNA损伤和氧化损伤.结果 同型半胱氨酸(250μmol/L)和Aβ42(10μmol/L)同时作用海马细胞的凋亡率显著高于Hcy或Aβ42单独作用,甚至高于两者单独作用之和.Hcy没有促进Aβ42对DNA的作用;在神经兴奋毒和氧化损伤作用方面,同型半胱氨酸(250 μmol/L)和Aβ42 (10 μmol/L)产生了协同作用,同型半胱氨酸可促进Aβ42引发海马细胞内游离钙浓度升高和MDA的产生增多.结论 同型半胱氨酸可通过神经兴奋毒作用、氧化损伤和引发凋亡等机制促进Aβ42对体外培养海马细胞的神经毒性作用.     

向饮食要开心

<正>有研究表明,同型半胱氨酸水平过高会使抑郁症的患病几率提高2倍!而橙汁、菠菜和谷类食物中的叶酸,恰好能抑制同型半胱氨酸的这种作用。叶酸通过将同型半胱氨酸转化为S-腺苷甲硫氨酸(一种抵抗抑郁症的化学成分),能够振奋精神,改变人的抑郁状     

槲皮素对高同型半胱氨酸血症大鼠甲基代谢相关酶活性的影响

目的研究槲皮素对高同型半胱氨酸血症(HHcy)大鼠甲基代谢相关酶活性的影响。方法 48只Wistar大鼠,按照血清Hcy水平将动物分成4组,分别为对照组(AIN-93饲料)、1%蛋氨酸组(1%Met)、0.5%槲皮素组(0.5%Q)、1%蛋氨酸+0.5%槲皮素组(1%Met+0.5%Q),每组12只,雌雄各半,单笼饲养。用相应饲料喂养6周后,乙醚麻醉,球后静脉丛采血,离心取血清,处死后取肝脏。检测血清同型半胱氨酸(Hcy)、S-腺苷蛋氨酸(SAM)、S-腺苷同型半胱氨酸(SAH)、血清和肝脏槲皮素及其甲基化产物含量以及肝脏儿茶酚-O-甲基转移酶(COMT)、蛋氨酸合成酶(MS)、5,10-亚甲基四氢叶酸还原酶(MTHFR)、丝氨酸羟甲基转移酶(SHMT)、二氢叶酸还原酶(DHFR)活性。采用RT-PCR法检测肝脏COMT mRNA的表达情况。结果 1%Met组血清Hcy水平显著升高;0.5%Q组血清SAM水平显著升高,肝脏COMT活性和mRNA表达显著降低;1%Met+0.5%Q组血清Hcy水平与对照组无统计学差异,MS、MTHFR和SHMT活性显著增强。结论槲皮素可增强HHcy大鼠肝脏MTHFR和SHMT的活性,这可能是槲皮素降低Hcy的重要靶点。     

SAH抑制内皮细胞增殖和ER-α表达作用

目的 探讨胞内S-腺苷同型半胱氨酸(SAH)升高抑制血管内皮细胞增殖和雌激素受体-α(ER-α)表达变化的关系.方法 以永生化的人脐静脉内皮细胞(HUVEC)为研究对象,经不同浓度的S-腺苷同型半胱氨酸水解酶(SAHH)强效抑制剂3-deazaadenosine(DZA)处理24,48,72 h.利用细胞计数法、流式细胞仪和脱氧核糖核酸转移酶缺口末端标记法(TUNEL)检测细胞的增殖凋亡能力,蛋白印迹法检测ER-α蛋白的表达,荧光定量-PCR(qRT-PCR)检测其mRNA表达,甲基化特异PCR法(MSP)检测ER-α基因启动子甲基化状态.结果 经DZA处理后,HUVEC增殖能力降低(主要受阻于G1/G0期),细胞凋亡率增加(P<0.05),ER-α mRNA和蛋白的相对表达量降低(P<0.05),但其启动子甲基化不发生改变.结论 胞内SAH升高抑制HUVEC的增殖能力与ER-α的表达变化有关,但这种作用不是通过改变ER-α基因启动子的甲基化而实现.     

丝氨酸对大鼠高同型半胱氨酸血症的干预作用

目的探讨不同蛋白水平下补充丝氨酸对大鼠高同型半胱氨酸血症的干预效果。方法将48只健康Wistar大鼠随机分为低蛋白组〔10%酪蛋白(10C),10C+0.75%蛋氨酸(10CM),10CM+2.5%丝氨酸〕和高蛋白组〔40%酪蛋白(40C),40C+0.75%蛋氨酸(40CM),40CM+2.5%丝氨酸〕。实验喂养14 d后处死动物,采集血液、肝脏样品,用于测定血液生化指标及肝脏蛋氨酸中间代谢物和酶学指标。结果丝氨酸的添加显著抑制了低蛋白、高蛋白饲料中蛋氨酸诱导的同型半胱氨酸浓度的升高,同时降低了肝脏S-腺苷甲硫氨酸浓度。丝氨酸的补充恢复了由低蛋白饲料中添加蛋氨酸导致的肝脏丝氨酸浓度的降低。结论丝氨酸对低蛋白饲料诱导的高同型半胱氨酸血症的干预效果显著强于高蛋白组,显著抑制了由蛋氨酸添加诱导的高同型半胱氨酸血症的发生。     

砷对Keap1抑制HaCaT细胞凋亡及抗氧化水平的影响

目的 基于抑制Kelch样环氧氯丙烷相关蛋白1（Kelch-like ECH-assoicated protein 1,Keap1）基因,研究砷对人永生化角质形成细胞凋亡及抗氧化水平的影响。方法 培养细胞72 h,分为5组:空白对照组、阴性对照（Keap1基因抑制未染砷）、Keap1基因抑制+低砷（2.9 μmol/L）、Keap1基因抑制+中砷（5.8 μmol/L）和Keap1基因抑制+高砷（29.0 μmol/L）组;用流式细胞仪检测细胞凋亡率;用实时荧光定量聚合酶链式反应检测核因子E2相关因子2（nuclear factor erythroid 2-related factor 2,Nrf2）、Keap1 mRNA水平;采用酶联免疫吸附试验检测谷胱甘肽、血红素氧化酶、环氧化酶、S-腺苷甲硫氨酸与同型半胱氨酸蛋白水平。结果 Nrf2和Keap1 mRNA表达不全相等（均有P<0.05）;与阴性对照组相比,低砷组S-腺苷甲硫氨酸和同型半胱氨酸蛋白表达上调（均有P<0.05）,血红素氧化酶蛋白在低、中、高砷组均上调（均有P<0.05）,环氧化酶蛋白在中、高砷组上调（均有P<0.05）,谷胱甘肽蛋白在高砷组上调（F=7.24,P=0.001）。结论 基于Keap1基因抑制,砷暴露上调抗氧化酶活性,提高抗氧化水平,细胞凋亡下降;随着砷剂量增加,Nrf2表达下调,抗氧化水平失衡,细胞凋亡增加。     

腹腔内注射同型半胱氨酸对大鼠学习记忆的影响及其神经生理机制

目的通过在体实验观察高同型半胱氨酸血症动物学习记忆功能及海马长时程增强(LTP)的变化,探讨补充叶酸是否能够改善高同型半胱氨酸血症所导致的学习记忆改变。方法以腹腔注射同型半胱氨酸(Hcy)的方法建立高同型半胱氨酸血症动物模型,动物分为对照组、Hcy处理组、Hcy处理补充叶酸组和补充叶酸组。结果动物经过四周腹腔内注射Hcy,血浆Hcy水平异常升高,动物的生长受到明显抑制。实验动物在注射Hcy后对外界环境的变化恐惧心理增强,自发探究行为减少,动物出现明显的学习记忆障碍。腹腔注射Hcy大鼠海马突触传递效率显著降低。补充叶酸大鼠的学习记忆能力及LTP与对照组无显著差别。结论 Hcy可使动物自发探究行为减少,出现明显的学习记忆障碍,LTP的改变可能是机制之一。补充叶酸可以显著改善高同型半胱氨酸血症所导致的学习记忆功能改变。     

营养

不同补锌剂量对大鼠海马nNOS蛋白表达影响;β,β-胡萝卜素改善H2O2对成骨细胞损伤的影响;叶酸、VB6、VB12体外对同型半胱氨酸致主动脉内皮细胞损伤的保护作用;维生素C对磁场损伤胚胎神经细胞保护作用;番茄红素对人胃癌细胞生长的抑制作用     

叶酸对大鼠蛋氨酸诱发高同型半胱氨酸水平的影响

目的探讨叶酸对高同型半胱氨酸(HCY)血症血浆HCY水平及SOD、M DA、血脂的影响。方法采用高蛋氨酸饮食复制高HCY模型,大鼠30只,雄性,平均体重(150±50)g。随机分为三组:正常对照组,不作任何处理;蛋氨酸组与叶酸处理组,共同喂养6周后,心脏取血。分别测定各组血浆HCY、SOD、M DA、血脂的水平。结果蛋氨酸组、叶酸处理组与对照组相比,HCY、M DA含量均显著升高,SOD水平降低,差异有极显著性(P<0.01);叶酸处理组与蛋氨酸组相比,HCY、M DA含量明显下降,SOD水平升高,差异也有显著性(P<0.05)。而TC、TG、HDL在蛋氨酸组、叶酸处理组和对照组间均无显著性差异(P>0.05)。结论高同型半胱氨酸能增强脂质过氧化的作用,叶酸可抵抗高同型半胱氨酸引起的脂质过氧化作用及对其引起血管损伤可能有预防作用。     

Neurotoxic mechanism of homocysteine in hippocampal neurons

Abstract

Homocysteine (Hcy) is a neurotoxic amino acid that accumulates in several neurodegenerative disorders. We examined the consequences of treatment of cultured rat hippocampal neurons with Hcy and the effects of folate, S-adenosyl methionine (SAM) and MK-801 on Hcy-induced neuron apoptosis and the related molecular mechanisms were also observed. Primary hippocampal neurons were treated with 250 μM Hcy for 4 h resulted in apoptosis time-dependently. 100 μM S-adenosyl methionine (SAM) and 10 μM folate significantly repressed, although 1 μM MK-801, an NMDA receptor inhibitor did not, Hcy-induced neuron apoptosis. SAM and folate significantly repressed Hcy-induced neuron DNA damage. Hcy induced neuron calcium overload through activation of NMDA receptors. Hcy treatment also induced a significant increase in MDA level, but did not affect the neurons' total antioxidant capacity (T-AOC). Hcy (250 μM) significantly increased the expression of p53 and Bax while decreased the expression of Bcl-2. SAM and folate inhibited the changes of apoptosis-related protein expression induced by Hcy. These findings indicate that Hcy compromises neuronal homeostasis by not only DNA damage, but also neuron exitotoxicity, oxidative injury, apoptosis, and expression changes of apoptosis-related proteins.     

Depletion of intracellular zinc induced apoptosis in cultured hippocampal neurons through Raf/MEK/ERK pathways

An experiment was performed to observe the changes in Raf-1 kinase/mitogen-activated protein kinase ERK (MEK)/extracellular signal-regulated kinase (ERK) signaling pathways in cultured hippocampal neurons and its correlation with neurons apoptosis induced by intracellular zinc depletion. Cultured hippocampal neurons were exposed to a cell membrane-permeant zinc chelator TPEN (2 μM), and to TPEN plus zinc sulfate (5 μM) for 24 h. Cultures were then processed to detect neuronal viability by the methyl thiazolyl tetrazolium assay, while apoptosis rate was simultaneously observed by the flow cytometric analysis. Caspase-3, Raf-1, pMEK, pERK1/2, and pCREB protein levels were examined by Western blot assays. The viability in TPEN-incubated neurons was notably decreased, apoptosis rate and expression of caspase-3 significantly increased compared to untreated controls. The significant down-regulation of Raf/MEK/ERK signaling pathway and expression of pCREB were decreased in TPEN-treated neurons. Co-addition of zinc almost completely reversed TPEN-induced alterations described. The results demonstrated zinc-modulated apoptosis and the expression of Raf/MEK/ERK at the protein level in hippocampal neurons. It is possible that zinc depletion-induced apoptosis in cultured hippocampal neurons may be relevant to the changes of Raf/MEK/ERK signaling pathway.     

心理应激对大鼠叶酸和同型半胱氨酸的影响及补充叶酸的调节作用

目的明确应激状态下机体血浆和脑脊液中同型半胱氨酸(Hcy)浓度及组织叶酸含量的变化,探讨叶酸在应激状态下对Hcy水平的调节作用。方法以束缚作为应激原建立大鼠心理应激模型,动物分为对照组、束缚3w组,束缚3w同时补充叶酸组。认识心理应激对机体Hcy和叶酸水平的影响,及应激状态下补充叶酸对机体Hcy水平的调节作用。结果心理应激动物叶酸代谢发生了显著的变化,血浆叶酸水平显著低于对照组,小肠粘膜上皮、肝脏、皮层和海马组织中叶酸的含量也明显降低。补充叶酸可以显著升高心理应激后机体的叶酸水平,降低心理应激所致的血浆同型半胱氨酸水平升高。结论应激状态下机体叶酸含量的降低可能是同型半胱氨酸水平升高的重要原因之一。     

长链非编码RNA核富含丰富的转录本1对癫痫模型海马神经元凋亡的影响和作用机制

目的 探讨长链非编码RNA(lncRNA)核富含丰富的转录本1(NEAT1)对癫痫细胞模型海马神经元凋亡的影响及作用机制,以期为NEAT1成为癫痫治疗的新靶点提供依据。方法 于2019年8月—2020年10月期间,体外培养大鼠海马神经元细胞,无镁诱导制备癫痫海马神经元模型,实验分为:对照组(正常细胞外液)、模型组(无镁细胞外液)、转染对照组(转染非特异性siRNA+无镁细胞外液)和转染组(转染NEAT1特异性siRNA+无镁细胞外液)。实时荧光定量PCR(qPCR)检测NEAT1和miR-29b-3p的表达变化,双荧光素酶报告基因实验检测NEAT1是否靶向调控miR-29b-3p,酶联免疫吸附法(ELISA)检测白细胞介素-1(IL-1)、白细胞介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)的含量,AnnexinV-FITC/PI双染色法检测海马神经元细胞凋亡情况,蛋白免疫印迹法(Western blot)检测各组细胞中B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、Toll样受体4(TLR4)和核因子-κB(NF-κB)的表达水平。结果 与对照组相比,模型组海马神经元凋亡率及NEAT1、Bax、TLR4和NF-κB的表达水平升高(F＝50.980、73.668、65.635、13.203、10.292,P＜0.05),IL-1、IL-6和TNF-α的含量增多(F＝33.107、33.857、51.129,P＜0.05),miR-29b-3p和Bcl-2的表达水平降低(F＝145.023、67.655,P＜0.05);而抑制NEAT1的表达能够降低神经元凋亡,抑制Bax、TLR4和NF-κB的表达,促进miR-29b-3p和Bcl-2的表达,减少IL-1、IL-6和TNF-α的分泌。双荧光素酶报告基因实验证实NEAT1和miR-29b-3p的靶向关系。结论 lncRNA NEAT1靶向下调miR-29b-3p的表达阻断TLR4/NF-κB信号通路来抑制癫痫模型海马神经元的凋亡。     

腺相关病毒载体介导的人类HIF-1α基因对海马神经细胞凋亡的影响

目的构建人类缺氧诱导因子-1α(human hypoxia inducible factor,HIF-1α)的腺病毒相关载体(rAAV-HIF-1α),并观察其对β-淀粉样蛋白25-35(Aβ25-35)诱导原代培养海马神经细胞凋亡及细胞内游离钙离子浓度的影响。方法构建含人类HIF-1α基因的AVV载体rAAV-HIF-1α转染BHK21细胞并选择培养获得的G418抗性细胞,即AVV载体细胞株。以重组单纯疱疹病毒感染此细胞株获得rAAV-HIF-1α,PCR技术鉴定病毒,地高辛标记的CMV探针点杂交方法测定重组病毒滴度电泳测定病毒纯度,SDS-PAGE电泳测定病毒纯度,Aβ25-35旅导原代培养海马神经细胞凋亡。Western印迹检测目的蛋白的表达。流式细胞术分析细胞凋亡百分率,激光共聚焦显微镜测定细胞内游离钙离子浓度。结果 PCR证实rAAV-HIF-1α内含有人HIF-1α基因序列,病毒滴度为1×1012/ml,纯度大于98%,感染原代培养海马神经细胞后能够表达目的蛋白,同时可显著降低Aβ25-35诱导海马神经细胞凋亡百分率及细胞内游离子浓度上调(P0.05)。结论缺氧诱导因子-1α基因转染可能通过抑制细胞内游离钙离子浓度上调使海马神经细胞对Aβ25-35产生抗凋亡作用。     

Effects of intracellular zinc depletion on the expression of VDAC in cultured hippocampal neurons

An experiment was conducted to investigate whether intracellular zinc depletion can actually change expression of voltage-dependent anion channel 1 (VDAC1) and VPAC2 in cultured hippocampal neurons as well as their significance. Hippocampal neurons were obtained by primary culture from hippocampus of newborn Wistar rats. Cultured hippocampal neurons were exposed to a cell membrane-permeable zinc chelator N,N,N',N'-tetrakis (2-pyridyl methyl) ethylenediamine (TPEN) (2 μM), and to TPEN plus zinc sulfate (5 μM) for 1 or 24 hours. Cultures were then processed to detect neuronal injury by lactate dehydrogenase (LDH) assay, intracellular Ca(2+) with the fluorescent probe fluo-3/AM, reactive oxygen species (ROS) generation using 2',7'-dichlorofluorescein diacetate (DCFH-DA) assay, nuclear morphology by Hoechst 33342, VDAC1, and VDAC2 protein levels by western blot, and VDAC1 and VDAC2 mRNA levels by RT-PCR. The results demonstrated that exposure of hippocampal neurons to TPEN (2 μM) for 24 hours induced notably neuronal injury, significantly increased the number of apoptotic nuclei, up-regulated the expression of VDAC1 protein level and down-regulated the expression of VDAC2 protein level. Significant down-regulation of mRNA levels for VDAC1 and VDAC2 were observed in TPEN-treated neurons. Co-addition of zinc almost completely reversed TPEN-induced neuronal injury and above alterations in VDAC1 and VDAC2 protein levels and mRNA levels. Present results implicate a possibility that up-regulation of VDAC1 and down-regulation of VDAC2 may participate in hippocampal neuron injury induced by zinc deficiency.     

叶酸、VB6、VB12对血管细胞粘附分子-1表达影响

目的 观察叶酸(FA)、维生素B6(W6)、维生素B12(VB12)对同型半胱氨酸(Hcy)上调内皮细胞血管细胞粘附分子-1(VCAM-1)表达抑制作用.方法 将对数生长期大鼠主动脉内皮细胞随机分为5组,空白对照组、1 mmol/L Hcy组、抑制组I(1 mmol/L Hcy+5 μmol/L FA)、抑制组Ⅱ(1 mmol/L Hcy+5 μmol/L FA+5 nmol/LVB12)、抑制组HI(1 mmol/L Hcy+5 μmol/L FA+0.1 μmol/L VB6+5 nmol/L VB12)、逆转录聚合酶链反应(RT-PCR)检测血管细胞粘附分子VCAM-1的表达.结果 与空白对照组比较,1 mmol/L Hcy明显上调核转录因子Kb(NF-Kb)和VCAM-1 mRNA的表达,内皮-单核细胞粘附率由6.03%增加到59.04%(P<0.05);与1 mmol/LHcy组比较,3个抑制组NF-Kb(F=11.547,P=0.000)、VCAM-1 mRNA(F=94.014,P=0.000)的表达均明显下调,抑制组Ⅰ、Ⅱ、Ⅲ的内皮-单核细胞的粘附率分别为44.98%,27.23%,10.83%,均明显低于1 mmol/L Hcy组(P<0.05).结论 叶酸、VB6、VB12均可抑制NF-Kb、VCAM-1 mRNA的表达,减少内皮-单核细胞的粘附,缓解Hcy对血管内皮细胞的损伤作用,且三者同时使用效果更好.     

营养素防护同型半胱氨酸致ECV304细胞损伤和凋亡

目的: 探讨叶酸、维生素E(VE)和硒(Se)对同型半胱氨酸(homocysteine, Hcy)诱导ECV304细胞损伤和凋亡的防护作用及其机制。方法: 采用倒置显微镜观察和MTT实验检测各种营养素对Hcy细胞损伤作用的防护,采用琼脂糖凝胶DNA电泳和流式细胞仪检测各种营养素对Hcy诱导细胞凋亡作用的防护。并通过测定细胞内脂质过氧化水平、细胞内抗氧化酶活力及对细胞内活性氧产生的改变研究其保护机制。结果: 叶酸、VE和Se都可防护Hcy的细胞增殖抑制和诱导细胞凋亡作用,并都可明显减少细胞内MDA含量。叶酸可以明显增加细胞内SOD活力,叶酸和Se可以明显增加细胞内GSH-Px活力。VE对Hcy诱导的细胞内活性氧产生具有明显抑制作用,而Se和叶酸抑制作用不明显。结论: 叶酸、VE和Se都可以防护Hcy导致的细胞增殖抑制,并可以抑制Hcy诱导的细胞凋亡,但其作用机制不同。     

Effects of intracellular zinc depletion on the expression of VDAC in cultured hippocampal neurons

Abstract

An experiment was conducted to investigate whether intracellular zinc depletion can actually change expression of voltage-dependent anion channel 1 (VDAC1) and VPAC2 in cultured hippocampal neurons as well as their significance. Hippocampal neurons were obtained by primary culture from hippocampus of newborn Wistar rats. Cultured hippocampal neurons were exposed to a cell membrane-permeable zinc chelator N,N,N′,N′-tetrakis (2-pyridyl methyl) ethylenediamine (TPEN) (2 µM), and to TPEN plus zinc sulfate (5 µM) for 1 or 24 hours. Cultures were then processed to detect neuronal injury by lactate dehydrogenase (LDH) assay, intracellular Ca²⁺ with the fluorescent probe fluo-3/AM, reactive oxygen species (ROS) generation using 2′,7′-dichlorofluorescein diacetate (DCFH-DA) assay, nuclear morphology by Hoechst 33342, VDAC1, and VDAC2 protein levels by western blot, and VDAC1 and VDAC2 mRNA levels by RT–PCR. The results demonstrated that exposure of hippocampal neurons to TPEN (2 µM) for 24 hours induced notably neuronal injury, significantly increased the number of apoptotic nuclei, up-regulated the expression of VDAC1 protein level and down-regulated the expression of VDAC2 protein level. Significant down-regulation of mRNA levels for VDAC1 and VDAC2 were observed in TPEN-treated neurons. Co-addition of zinc almost completely reversed TPEN-induced neuronal injury and above alterations in VDAC1 and VDAC2 protein levels and mRNA levels. Present results implicate a possibility that up-regulation of VDAC1 and down-regulation of VDAC2 may participate in hippocampal neuron injury induced by zinc deficiency.