Icariin, a pharmacologically active component isolated from the Chinese herb Epimedium, has been shown to improve spatial learning and memory abilities in Alzheimer’s disease (AD) rats through inhibition of Aβ production and tau protein hyperphosphorylation. However, the potential mechanism of icariin-induced protective effects against mitochondrial dysfunctions in AD still remains unclear. In the present study, we investigated the effect of icariin on the modulation of mitochondrial transport and distribution in primary hippocampal cultures from triple-transgenic (3× Tg) AD mice. The results showed that icariin enhanced mitochondrial motility and increased mitochondrial index and mitochondrial length and size in the diseased neurons. Additionally, the expression of the key mitochondrial enzyme, pyruvate dehydrogenase-E1α (PDHE1α), and the post synaptic density protein 95 (PSD95), was preserved in AD neurons after icariin treatment, accompanied by a downregulation of Aβ and phosphorylated tau expression in the corresponding areas. Further study showed that icariin treatment resulted in a decrease in mitochondrial fission protein dynamin-related protein 1 (Drp1) and an increase in fusion protein Mitofusin 2 (Mfn2). These data indicate that icariin can promote mitochondrial transport, protect mitochondria against fragmentation and preserve the expression of mitochondrial and synaptic functional proteins in AD neurons. Thus, icariin may be a potential therapeutic complement for AD and other mitochondrial malfunction-related neuronal degenerative diseases.

目的 探讨阿尔茨海默病(AD)病变与肝Ⅰ相药酶基因表达的相关性,为早期和长期药物干预AD病程提供实验参考.方法 制备B6.129-PS1 M146V/APPSwe/TauP301L三重转基因AD模型小鼠(3×Tg AD小鼠)及同系非转基因正常小鼠(NTg小鼠)肝组织总RNA和总蛋白质样品,逆转录PCR/实时荧光定量PCR技术检测肝核因子4(HNF4)等7种核受体和细胞色素氧化酶P4501a2(Cyp1a2)等7种CYP基因mRNA相对表达量,比较基因转录谱差异;Western蛋白印迹法检测技术检测差异显著的目的基因蛋白质水平.结果 与NTg小鼠相比,3×Tg AD小鼠HNF4、雌激素受体(ERα)及组成型雄甾烷受体均下调近66％(P＜0.01),孕烷X受体和芳香烃受体分别上调1.84和1.64倍(P＜0.05),糖皮质激素受体及受氧化应激激活的核因子E2相关因子未见显著变化;主要药物代谢酶Cyp2b10及Cyp2a5 mRNA分别降低至正常小鼠水平的3％(即下降97％,P＜0.01)和30％(P＜0.05),Cyp2e1、Cyp2j9、Cyp3a11基因的转录水平则分别上调了2.26倍、2.42倍和3.66倍(P＜0.05);Cyp1a2和Cyp2d22基因转录在两种小鼠中无明显差异.Western蛋白印迹分析证实,3×Tg AD小鼠ERα和CYP2a5明显下降(P＜0.05)而Cyp3a11表达则明显上升(P＜0.05),蛋白质表达变化的趋势与其相应mRNA水平改变一致.结论 3×Tg AD小鼠肝内核受体基因及CYP酶基因转录谱发生显著改变,提示AD病变可能干扰机体肝代谢功能,对机体内源性及外源性化合物生物转运产生影响.

This study investigated the neuroprotective properties of icariin (an effective component of traditional Chinese herbal medicine Epimedium) on neuronal function and brain energy metabolism maintenance in a triple-transgenic mouse model of Alzheimer's disease (3 × Tg-AD).3 × Tg-AD mice as well as primary neurons were subjected to icariin treatment. Morris water maze assay, magnetic resonance spectroscopy (MRS), Western blotting, ELISA, and immunohistochemistry analysis were used to evaluate the effects of icariin administration.Icariin significantly improved spatial learning and memory retention in 3 × Tg-AD mice, promoted neuronal cell activity as identified by the enhancement of brain metabolite N-acetylaspartate level and ATP production in AD mice, preserved the expressions of mitochondrial key enzymes COX IV, PDHE1α, and synaptic protein PSD95, reduced Aβ plaque deposition in the cortex and hippocampus of AD mice, and inhibited β-site APP cleavage enzyme 1 (BACE1) expression. Icariin treatment also decreased the levels of extracellular and intracellular Aβ1-42 in 3 × Tg-AD primary neurons, modulated the distribution of Aβ along the neurites, and protected against mitochondrial fragmentation in 3 × Tg-AD neurons.Icariin shows neuroprotective effects in 3 × Tg-AD mice and may be a promising multitarget drug in the prevention/protection against AD.

OBJECTIVE To develop a CYP2A6 gene transcriptional high-capacity screening system and to evaluate the regulatory effects of four environmental estrogenic compounds and three phytoestrogens on CYP2A6 gene transcription. METHODS Three luciferase reporter plasmids containing the CYP2A6gene- 2460 ~ + 1 5'-flanking region( pGL3-2A6 UP),the potential enhancer region host estrogen response element( ERE)-like tandem in the 5' of promoter( pGL3-2A6 EP),and three copies of this ERE-like tandem in the 5' of promoter( pGL3-2A6 3EP) were constructed. Each reporter plasmid DNA was co-transfected into HepG2 cells with receptor pcDNA3. 0-ERα and pRL-TK. Dual-luciferase assay was performed after 48 h treatment by different estrogenic compounds,including four environmental pollutants 2,2',4,4'-tetrabromodiphenyl ether( BDE-47),bisphenol( ABPA),fenvalerate( FEN),perfluorooctanoic acid( PFOA) and three phytoestogens icariin( ICA),daidzein( DAI) and quercetin( QUE),as well as 0. 1% DMSO( negative control) and 10 nmol·L- 1estradiol( positive control). RESULTS pGL3-2A6 3EP reporter construct represented the strongest concentration-dependent induction response to the estradiol treatment. CYP2A6 transcription activity was significantly up-regulated by all the seven estrogenic compounds. Among these chemicals,BDE-47 was of particular interest with remarkable induction fold changes from 1. 45 ± 0. 13 to 3. 30 ± 0. 13( P 0. 01) corresponding to concentrations ranging from 1. 0 to 100 nmo·lL- 1. Phytoestrogens ICA,DAI and QUE represented a similar concentrationinduction pattern. 241% to 283 % increases compared with DMSO control in CYP2A6 promoter activity were observed under phytoestrogens 10 μmo·lL- 1treatment. CONCLUSION CYP2A6 promoter activity is induced by estrogen and estrogenic compounds in an estrogen receptor-dependent manner,implying the biological effects of common environmental endocrine disrupting the chemicals' regulation of CYP2A6 gene transcription in the human body. Furthermore,this mechanism can also explore individual nicotine metabolism and carcinogenic susceptibility under hazardous and estrogen-rich environments.

Mitochondrial dynamics refers to the morphological changes of mitochondria undergoing continuous fission and fusion.Dynamin related GTPase 1,Fis1,mitochondrial fission factor,mitofusin 1/2,and optic atrophy protein 1 have been found to play key roles in this process.They are subject to upstream regulations including phosphorylation,S-nitrosylation,ubiquitination,sumoylation and proteolysis.Normal mitochondrial functioning is an indispensible part of the maintenance of synapses morphology and function.Neurotoxicants may affect mitochondrial dynamics and then result in the dysfunction of oxidative phosphorylation and increas reactive oxygen species production and neuron apoptosis.More and more researches suggest that the imbalance of mitochondrial dynamics is a key pathological event in the early stage of Alzheimer disease.

评述了阿尔茨海默病(Alzheimer's disease,AD)的多种病因学说,包括胆碱能学说、β-淀粉样级联学说、ABC学说(aging,beta-amyloid,channel)、tau蛋白过度磷酸化学说、氧化应激学说、神经细胞凋亡学说和基因突变学说等.指出AD是多因素引起的病变,其发病机制也必然有多种.由于该病与人体衰老过程密切相关,发病机理复杂,且神经细胞丢失后不具再生能力,这就决定了研究治疗AD有效药物的艰巨性.还指出AD防治工作应重视早期预报和早期干预,发展早期诊断方法包括正电子发射计算机断层扫描、磁共振成像,及血液和尿液中寻找灵敏的生物标志物等技术方法.目前有关AD防治药物包括一线治疗药物、疫苗、尚在研究阶段的药物、辅助性治疗药物、中药以及医疗保健品.前3类药物因疗效或研究结果长期未取得突破性进展,人们开始关注后3类药物对延缓AD的作用及机理.课题组重点开展后3种药物防治AD的作用和机理研究,发现硒代蛋氨酸、辅酶Q-10和中药组分淫羊藿甙对防治AD有好的疗效.综述了目前抗AD药物的研究现状和面临的挑战,提出AD的防治策略是:摒弃针对单一致病因素的治疗思路,从多因素及内在相互联系的系统生物学角度,开展AD防治机制及新药研发.

目的 探讨人体尼古丁主要代谢酶细胞色素P450 (CYP)2A6及其同族成员CYP2A13肽链结构中,影响其尼古丁5′-羟化代谢活性的关键性氨基酸残基.方法 使用前期制备的CYP2A6和CYP2A13系列氨基酸互换突变体: CYP2A6V117A, CYP2A6G164H, CYP2A6I208S, CYP2A6R372H和CYP2A6S465P以及CYP2A13A117V, CYP2A13H164G, CYP2A13S208I,CYP2A13H372R和CYP2A13P465S,比较其与相应野生蛋白酶的尼古丁5′-羟化催化反应的动力学参数.结果 各突变体对2个CYP2A蛋白酶的尼古丁代谢活性影响不同.对于CYP2A6,I208S突变对酶活性的影响显著,导致表观反应常数Km及最大反应速度Vmax由野生型62.25 μmol·L~(-1)和6.53 mol·min~(-1)·moL~(-1)变化为345 μmol·L~(-1)和2.19 mol·min~(-1)·moL~(-1),但该位点对CYP2A13酶活性无显著影响;对于CYP2A13,H372R突变对酶活性的影响最为显著,导致Km 及Vmax由野生型的26.01 μmol·L~(-1)和24.51 mol·min~(-1)·moL~(-1)变为148.7 μmol·L~(-1)和6.11 mol·min~(-1)·moL~(-1), 此位点对CYP2A6无显著影响. 其他位点突变对酶活性影响较小或不显著.结论 CYP2A家族蛋白中,I208与H372分别是影响CYP2A6和CYP2A13对尼古丁代谢的关键残基.对于同家族蛋白酶而言,关键性氨基酸的作用并不总是一一对应.

【Objective】To compare the catalytic kinetic characteristics of major human nicotine metabolic enzymes—the wild-type CYP2A6 and CYP2A13 and the enzyme protein of their mutants in coumarin 7-hydrocylation, to identify the key amino acid residues that influence their catalytic activities, and to support fundamental data to develop biomarkers for the assessment of CYP2A gene polymorphism, smoking behavior, and cancer susceptibility. 【Methods】 A set of mutants with reciprocal substitution of amino acid residues at 300, 301 and 369 positions of CYP2A6 and CYP2A13 were generated by point mutation and the insect baculovirus protein expression system. The catalytic reaction activities of all enzyme proteins were determined at different coumarin concentrations and kinetic parameters were obtained to compare with that of wild-type enzyme proteins. 【Results】 Compared with that of wild-type CYP2A6, the catalytic efficiency (Kcat =Vmax / Km) of Ile300→Phe mutant decreased significantly, while that of Gly301 → Ala mutant increased significantly (Kcat of 1.64 and 8.02 vs. 3.56 of the wild-type CYP2A6, P0.01).Correspondently, the catalytic efficiency of CYP2A13 Phe300 → Ile mutant increased, while that of CYP2A13 Ala301 → Gly mutant decreased remarkably (Kcat of 1.03 and 0.06 vs.0.27 of the wild-type CYP2A13, P 0.05). The changes of the catalytic characteristics were due to the changes of Vmax. Amino acid residues Ser or Gly at position 369 in either CYP2A6 or CYP2A13 did not show significant influence on the enzyme catalytic efficiency in coumarin 7-hydroxlation. 【Conclusions】 Amino acid residues at position 300 and 301 are the key residues in both CYP2A6 and CYP2A13 that affect the catalytic activities of coumarin 7-hydroxylation. However, the amino acid residues at position 369 do not affect the catalytic activities of coumarin metabolism.