簡易檢索 / 詳目顯示

研究生: 黃景怡
Huang, Chimg-Yi
論文名稱: 利用初級細胞培養以及小鼠模式確認對阿茲海默氏症具治療潛力之中草藥
Identification of potential Chinese herbs for Alzheimer’s disease using primary culture and mouse models
指導教授: 謝秀梅
Hsieh, Hsiu-Mei
學位類別: 碩士
Master
系所名稱: 生命科學系
Department of Life Science
論文出版年: 2016
畢業學年度: 104
語文別: 中文
論文頁數: 101
中文關鍵詞: NH037神經保護寡聚體Aβ認知焦慮
英文關鍵詞: NH037, neuroprotection, oligomeric Aβ, cognition, anxiety
DOI URL: https://doi.org/10.6345/NTNU202204367
論文種類: 學術論文
相關次數: 點閱:94下載:4
分享至:
查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報
  • 阿茲海默氏症(Alzheimer’s disease; AD)是目前最常見的漸進式神經退化性疾病,主要因海馬迴的神經元受損而導致認知功能障礙。據估計,全球有超過3500萬人罹患AD。AD起始的主要關鍵為類澱粉胜肽(beta-amyloid peptides; Aβ)堆積和tau蛋白質的過度磷酸化。近期有愈來越多的證據顯示,對於多因性之疾病AD而言,使用多目標特性的傳統中草藥治療效果可能優於單靶藥物。因此,近年來中草藥於AD的研究和治療越來越受到重視。在此研究中,我們建立了以寡聚體Aβ25-35和Aβ42處理的初級海馬迴神經細胞平台進行中草藥的篩選,希望藉此篩選出具有神經保護功效的中草藥。從此篩藥平台中,我們發現NH037中草藥能有效的增加神經元數目、神經突起長度、神經突起分支數目,並伴隨提高無活性GSK3β的量及減少Aβ堆積與脂質氧化壓力等神經保護效果。更進一步,於兩側海馬迴CA1區域注射寡聚體Aβ25-35的C57BL/6 小鼠動物實驗中,我們也發現NH037中草藥可以減少小鼠的焦慮及認知功能異常的問題,而在病理分析中我們可以看到NH037前處理減少Aβ類澱粉蛋白質堆積、tau蛋白質磷酸化、神經發炎反應與增加突觸相關蛋白質表現量及正腎上腺素神經元與血清素神經元數目以對抗寡聚體Aβ25-35所造成的細胞毒性。因此, NH037可能有潛力避免認知、非認知損傷以及誘發AD相關的病因特性。

    Alzheimer’s disease (AD) is the most common neurodegenerative disease associated with progressive damage in hippocampal neurons and cognitive dysfunctions. It is estimated that over 35 million people worldwide suffered from the disease. Both the accumulation of beta-amyloid peptides (Aβ) and tau protein phosphorylation are regarded as crucial events in the initiation of AD. Recently, more evidences show that the multi-target characteristics of traditional Chinese Herb Medicine (CHM) may be advantageous over single-target drugs against the multifactorial nature of AD. These drugs have therefore attracted much attention in the research and treatment in AD. In the present study, we established mouse primary hippocampal neuronal culture treated with oligomeric Aβ25-35 and Aβ42 as the screening platform of CHM. From the in vitro screening results , we found that CHM NH037 can prevent the decrease of neuronal number, neuritic length, branch number, lipid oxidation, amyloid deposition , and NH037 increasing the level of inactived GSK3β under neurotoxicity of oligomeric Aβ. Furthermore, the administration of NH037 also reduced anxiety and the cognitive impairment in the C57BL/6J mice treated with bilateral intrahippocampal CA1 injection of oligomeric Aβ25-35. From pathological analysis, we further found that the pretreatment of NH037 decreased the levels of Aβ deposition, tau protein phosphorylation, neuroinflammation, and increased the levels of synapse-related protein expression, noradrenergic and serotonergic neurons against the toxicity of oligomer Aβ25-35. Therefore, using CHM NH037 is a potential therapeutic strategy to prevent the cognitive, noncognitive dysfunction, and related pathogenic characterizations of AD.

    目錄 1.英文摘要 5 2.中文摘要 7 3. 縮寫表 9 4. 前言 11 4.1阿茲海默氏症 (Alzheimer’s disease) 11 4.3初級海馬迴神經元培養 13 4.4類澱粉蛋白質(Aβ) 13 4.6神經發炎反應 15 4.7 動物模式的建立 16 4.8中草藥做為治療AD之選擇 16 4.9中草藥葛根 (NH037) 18 5. 研究材料及方法 19 5.1動物 19 5.2中草藥的製備 19 5.3小鼠海馬迴初級神經元培養及藥物處理 19 5.4 Aβ42寡聚體之製備 20 5.5 Aβ25-35寡聚體的製備 21 5.6細胞免疫螢光染色分析(Immumocytochemical, ICC) 21 5.7乳酸脫氫酶分析(LDH assay) 22 5.8動物實驗與藥物處理 22 5.9動物行為分析(Behavioral analysis) 23 5.10免疫組織化學染色(Immunohistochemistry) 25 5.11西方墨點轉漬法(Western blot) 27 5.12統計分析 (Statistical analysis) 28 6. 結果 29 6.1單獨給予不同中草藥不影響初級海馬迴細胞之培養 29 6.2 NH021與NH037中草藥可以有效對抗Aβ42寡聚體所造成的細胞損傷 29 6.3 NH014,NH021與NH037中草藥可以有效對抗Aβ25-35寡聚體所造成 的細胞損傷 31 6.4 NH037在Aβ42寡聚體體外平台對神經細胞保護的分子機制分析 32 6.5 NH037在Aβ25-35寡聚體體外平台對神經細胞保護的分子機制分析 33 6.6 NH037緩解Aβ25-35寡聚體所誘發的焦慮行為但不影響自主運動(locomotor activity) 34 6.7 NH037減緩Aβ25-35寡聚體所誘發的短期記憶能力受損 35 6.8 NH037緩解Aβ25-35寡聚體誘導的認知功能受損 35 6.9 NH037可以有效緩解Aβ25-35寡聚體所造成的神經細胞tau蛋白質過度磷酸化路徑的活化情形 36 6.10 NH037能降低Aβ的堆積 38 6.11 NH037能有效避免Aβ25-35寡聚體所誘導的發炎反應情形 39 6.12 NH037能緩解因Aβ25-35寡聚體所誘發的突觸相關蛋白下降的情形 40 6.13 NH037可以有效緩解海馬迴CA1注射Aβ25-35寡聚體所造成之正腎上腺素及血清素神經元之傷害 40 6.14 NH037能夠顯著提升CAMKII的表現量 41 6.15 NH037不影響神經滋養因子(BDNF)的表現量 41 7. 討論 42 8. 參考資料46 9. 圖表 54

    Ali T, Kim MO (2015) Melatonin ameliorates amyloid beta-induced memory deficits, tau hyperphosphorylation and neurodegeneration via PI3/Akt/GSk3beta pathway in the mouse hippocampus. J Pineal Res 59:47-59.
    Braak H, Braak E, Strothjohann M (1994) Abnormally phosphorylated tau protein related to the formation of neurofibrillary tangles and neuropil threads in the cerebral cortex of sheep and goat. Neuroscience letters 171:1-4.
    Brahmbhatt S, Gupta A, Sharma AC (2005) Bigendothelin-1 (1-21) fragment during early sepsis modulates tau, p38-MAPK phosphorylation and nitric oxide synthase activation. Mol Cell Biochem 271:225-237.
    Cavallini A, Brewerton S, Bell A, Sargent S, Glover S, Hardy C, Moore R, Calley J, Ramachandran D, Poidinger M, Karran E, Davies P, Hutton M, Szekeres P, Bose S (2013) An unbiased approach to identifying tau kinases that phosphorylate tau at sites associated with Alzheimer disease. J Biol Chem 288:23331-23347.
    Chen PT, Chen CL, Lin LT, Lo CH, Hu CJ, Chen RP, Wang SS (2016) Design of Peptide Substrate for Sensitively and Specifically Detecting Two Abeta-Degrading Enzymes: Neprilysin and Angiotensin-Converting Enzyme. PLoS One 11:e0153360.
    Cheng Y, Feng Z, Zhang QZ, Zhang JT (2006) Beneficial effects of melatonin in experimental models of Alzheimer disease. Acta pharmacologica Sinica 27:129-139.
    Chintamaneni M, Bhaskar M (2012) Biomarkers in Alzheimer's disease: a review. ISRN pharmacology 2012:984786.
    Davis M (1992) The role of the amygdala in fear and anxiety. Annu Rev Neurosci 15:353-375.
    De Felice FG (2013) Alzheimer's disease and insulin resistance: translating basic science into clinical applications. The Journal of clinical investigation 123:531-539.
    Deininger MH, Fimmen BA, Thal DR, Schluesener HJ, Meyermann R (2002) Aberrant neuronal and paracellular deposition of endostatin in brains of patients with Alzheimer's disease. The Journal of neuroscience : the official journal of the Society for Neuroscience 22:10621-10626.
    Del Tredici K, Braak H (2012) Lewy pathology and neurodegeneration in premotor Parkinson's disease. Mov Disord 27:597-607.
    Elias MF, Beiser A, Wolf PA, Au R, White RF, D'Agostino RB (2000) The preclinical phase of alzheimer disease: A 22-year prospective study of the Framingham Cohort. Archives of neurology 57:808-813.
    Eroglu C, Barres BA (2010) Regulation of synaptic connectivity by glia. Nature 468:223-231.
    Fan LL, Sun LH, Li J, Yue XH, Yu HX, Wang SY (1992) The protective effect of puerarin against myocardial reperfusion injury. Study on cardiac function. Chin Med J (Engl) 105:11-17.
    Fiorani M, Guidarelli A, Blasa M, Azzolini C, Candiracci M, Piatti E, Cantoni O (2010) Mitochondria accumulate large amounts of quercetin: prevention of mitochondrial damage and release upon oxidation of the extramitochondrial fraction of the flavonoid. The Journal of nutritional biochemistry 21:397-404.
    Gao C, Liu Y, Jiang Y, Ding J, Li L (2014) Geniposide ameliorates learning memory deficits, reduces tau phosphorylation and decreases apoptosis via GSK3beta pathway in streptozotocin-induced alzheimer rat model. Brain Pathol 24:261-269.
    Garcia-Mediavilla V, Crespo I, Collado PS, Esteller A, Sanchez-Campos S, Tunon MJ, Gonzalez-Gallego J (2007) The anti-inflammatory flavones quercetin and kaempferol cause inhibition of inducible nitric oxide synthase, cyclooxygenase-2 and reactive C-protein, and down-regulation of the nuclear factor kappaB pathway in Chang Liver cells. European journal of pharmacology 557:221-229.
    Gebicke-Haerter PJ (2001) Microglia in neurodegeneration: molecular aspects. Microsc Res Tech 54:47-58.
    Gerhard A, Trender-Gerhard I, Turkheimer F, Quinn NP, Bhatia KP, Brooks DJ (2006) In vivo imaging of microglial activation with [11C](R)-PK11195 PET in progressive supranuclear palsy. Movement disorders : official journal of the Movement Disorder Society 21:89-93.
    Grimes CA, Jope RS (2001) The multifaceted roles of glycogen synthase kinase 3beta in cellular signaling. Progress in neurobiology 65:391-426.
    Hampel H, Ewers M, Burger K, Annas P, Mortberg A, Bogstedt A, Frolich L, Schroder J, Schonknecht P, Riepe MW, Kraft I, Gasser T, Leyhe T, Moller HJ, Kurz A, Basun H (2009) Lithium trial in Alzheimer's disease: a randomized, single-blind, placebo-controlled, multicenter 10-week study. The Journal of clinical psychiatry 70:922-931.
    Harkany T, O'Mahony S, Keijser J, Kelly JP, Konya C, Borostyankoi ZA, Gorcs TJ, Zarandi M, Penke B, Leonard BE, Luiten PG (2001) Beta-amyloid(1-42)-induced cholinergic lesions in rat nucleus basalis bidirectionally modulate serotonergic innervation of the basal forebrain and cerebral cortex. Neurobiol Dis 8:667-678.
    Hoppe JB, Frozza RL, Horn AP, Comiran RA, Bernardi A, Campos MM, Battastini AM, Salbego C (2010) Amyloid-beta neurotoxicity in organotypic culture is attenuated by melatonin: involvement of GSK-3beta, tau and neuroinflammation. J Pineal Res 48:230-238.
    Hu J, Huang HZ, Wang X, Xie AJ, Liu D, Wang JZ, Zhu LQ (2015) Activation of Glycogen Synthase Kinase-3 Mediates the Olfactory Deficit-Induced Hippocampal Impairments. Mol Neurobiol 52:1601-1617.
    Iqbal K, Grundke-Iqbal I, Zaidi T, Merz PA, Wen GY, Shaikh SS, Wisniewski HM, Alafuzoff I, Winblad B (1986) Defective brain microtubule assembly in Alzheimer's disease. Lancet 2:421-426.
    Iqbal K, Wang GP, Grundke-Iqbal I, Wisniewski HM (1989) Laboratory diagnostic tests for Alzheimer's disease. Progress in clinical and biological research 317:679-687.
    Ishizawa K, Dickson DW (2001) Microglial activation parallels system degeneration in progressive supranuclear palsy and corticobasal degeneration. Journal of neuropathology and experimental neurology 60:647-657.
    Isla AG, Vazquez-Cuevas FG, Pena-Ortega F (2016) Exercise Prevents Amyloid-beta-Induced Hippocampal Network Disruption by Inhibiting GSK3beta Activation. J Alzheimers Dis 52:333-343.
    Kanter M, Unsal C, Aktas C, Erboga M (2013) Neuroprotective effect of quercetin against oxidative damage and neuronal apoptosis caused by cadmium in hippocampus. Toxicology and industrial health.
    Katzman R (1989) Alzheimer's disease is a degenerative disorder. Neurobiology of aging 10:581-582; discussion 588-590.
    Kayed R, Head E, Thompson JL, McIntire TM, Milton SC, Cotman CW, Glabe CG (2003) Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis. Science 300:486-489.
    Kitazawa M, Oddo S, Yamasaki TR, Green KN, LaFerla FM (2005) Lipopolysaccharide-induced inflammation exacerbates tau pathology by a cyclin-dependent kinase 5-mediated pathway in a transgenic model of Alzheimer's disease. The Journal of neuroscience : the official journal of the Society for Neuroscience 25:8843-8853.
    Latimer DA, Gallo JM, Lovestone S, Miller CC, Reynolds CH, Marquardt B, Stabel S, Woodgett JR, Anderton BH (1995) Stimulation of MAP kinase by v-raf transformation of fibroblasts fails to induce hyperphosphorylation of transfected tau. FEBS letters 365:42-46.
    Lee J, Kim MS (2007) The role of GSK3 in glucose homeostasis and the development of insulin resistance. Diabetes research and clinical practice 77 Suppl 1:S49-57.
    Lee VK (1991) Language changes and Alzheimer's disease: a literature review. Journal of gerontological nursing 17:16-20.
    Li J, Ding X, Zhang R, Jiang W, Sun X, Xia Z, Wang X, Wu E, Zhang Y, Hu Y (2015) Harpagoside ameliorates the amyloid-beta-induced cognitive impairment in rats via up-regulating BDNF expression and MAPK/PI3K pathways. Neuroscience 303:103-114.
    Li J, Wang G, Liu J, Zhou L, Dong M, Wang R, Li X, Li X, Lin C, Niu Y (2010) Puerarin attenuates amyloid-beta-induced cognitive impairment through suppression of apoptosis in rat hippocampus in vivo. Eur J Pharmacol 649:195-201.
    Lin F, Xie B, Cai F, Wu G (2012) Protective effect of Puerarin on beta-amyloid-induced neurotoxicity in rat hippocampal neurons. Arzneimittelforschung 62:187-193.
    Liu J, Chi N, Chen H, Zhang J, Bian Y, Cui G, Xiu C (2013) Resistin protection against endogenous Abeta neuronal cytotoxicity from mitochondrial pathway. Brain Res 1523:77-84.
    Liu L, Drouet V, Wu JW, Witter MP, Small SA, Clelland C, Duff K (2012) Trans-synaptic spread of tau pathology in vivo. PloS one 7:e31302.
    Lovestone S, Reynolds CH, Latimer D, Davis DR, Anderton BH, Gallo JM, Hanger D, Mulot S, Marquardt B, Stabel S, et al. (1994) Alzheimer's disease-like phosphorylation of the microtubule-associated protein tau by glycogen synthase kinase-3 in transfected mammalian cells. Current biology : CB 4:1077-1086.
    Lucas JJ, Hernandez F, Gomez-Ramos P, Moran MA, Hen R, Avila J (2001) Decreased nuclear beta-catenin, tau hyperphosphorylation and neurodegeneration in GSK-3beta conditional transgenic mice. The EMBO journal 20:27-39.
    Maphis N, Xu G, Kokiko-Cochran ON, Jiang S, Cardona A, Ransohoff RM, Lamb BT, Bhaskar K (2015) Reactive microglia drive tau pathology and contribute to the spreading of pathological tau in the brain. Brain 138:1738-1755.
    McGaugh JL (2002) Memory consolidation and the amygdala: a systems perspective. Trends Neurosci 25:456.
    Morris JC, Selkoe DJ (2011) Recommendations for the incorporation of biomarkers into Alzheimer clinical trials: an overview. Neurobiology of aging 32 Suppl 1:S1-3.
    Nyakas C, Granic I, Halmy LG, Banerjee P, Luiten PG (2011) The basal forebrain cholinergic system in aging and dementia. Rescuing cholinergic neurons from neurotoxic amyloid-beta42 with memantine. Behav Brain Res 221:594-603.
    Price DL, Cork LC, Struble RG, Whitehouse PJ, Kitt CA, Walker LC (1985) The functional organization of the basal forebrain cholinergic system in primates and the role of this system in Alzheimer's disease. Ann N Y Acad Sci 444:287-295.
    Qi L, Ke L, Liu X, Liao L, Ke S, Wang Y, Lin X, Zhou Y, Wu L, Chen Z, Liu L (2016) Subcutaneous administration of liraglutide ameliorates learning and memory impairment by modulating tau hyperphosphorylation via the glycogen synthase kinase-3beta pathway in an amyloid beta protein induced alzheimer disease mouse model. Eur J Pharmacol 783:23-32.
    Qicheng F (1980) Some current study and research approaches relating to the use of plants in the traditional Chinese medicine. J Ethnopharmacol 2:57-63.
    Rama Rao KV, Curtis KM, Johnstone JT, Norenberg MD (2013) Amyloid-beta inhibits thrombospondin 1 release from cultured astrocytes: effects on synaptic protein expression. J Neuropathol Exp Neurol 72:735-744.
    Raskind MA, Peskind ER (1994) Neurobiologic bases of noncognitive behavioral problems in Alzheimer disease. Alzheimer Dis Assoc Disord 8 Suppl 3:54-60.
    Reinikainen KJ, Soininen H, Riekkinen PJ (1990) Neurotransmitter changes in Alzheimer's disease: implications to diagnostics and therapy. J Neurosci Res 27:576-586.
    Rodriguez-Carballo E, Gamez B, Ventura F (2016) p38 MAPK Signaling in Osteoblast Differentiation. Front Cell Dev Biol 4:40.
    Rodriguez JJ, Noristani HN, Verkhratsky A (2012) The serotonergic system in ageing and Alzheimer's disease. Prog Neurobiol 99:15-41.
    Sabogal-Guaqueta AM, Munoz-Manco JI, Ramirez-Pineda JR, Lamprea-Rodriguez M, Osorio E, Cardona-Gomez GP (2015) The flavonoid quercetin ameliorates Alzheimer's disease pathology and protects cognitive and emotional function in aged triple transgenic Alzheimer's disease model mice. Neuropharmacology 93:134-145.
    Seibenhener ML, Wooten MW (2012) Isolation and culture of hippocampal neurons from prenatal mice. J Vis Exp.
    Shen ZX (2004) Brain cholinesterases: II. The molecular and cellular basis of Alzheimer's disease. Medical hypotheses 63:308-321.
    Small SA, Wu EX, Bartsch D, Perera GM, Lacefield CO, DeLaPaz R, Mayeux R, Stern Y, Kandel ER (2000) Imaging physiologic dysfunction of individual hippocampal subregions in humans and genetically modified mice. Neuron 28:653-664.
    Stepanichev MY, Zdobnova IM, Zarubenko, II, Lazareva NA, Gulyaeva NV (2006) Studies of the effects of central administration of beta-amyloid peptide (25-35): pathomorphological changes in the Hippocampus and impairment of spatial memory. Neuroscience and behavioral physiology 36:101-106.
    Tan Y, Liu M, Wu B (2008) Puerarin for acute ischaemic stroke. Cochrane Database Syst Rev CD004955.
    Tell V, Hilgeroth A (2013) Recent developments of protein kinase inhibitors as potential AD therapeutics. Front Cell Neurosci 7:189.
    Tolosa E, Litvan I, Hoglinger GU, Burn D, Lees A, Andres MV, Gomez-Carrillo B, Leon T, Del Ser T, Investigators T (2014) A phase 2 trial of the GSK-3 inhibitor tideglusib in progressive supranuclear palsy. Movement disorders : official journal of the Movement Disorder Society 29:470-478.
    Wang Q, Wu T, Chen X, Ni J, Duan X, Zheng J, Qiao J, Zhou L, Wei J (2006) Puerarin injection for unstable angina pectoris. Cochrane Database Syst Rev CD004196.
    Waring SC, Rosenberg RN (2008) Genome-wide association studies in Alzheimer disease. Archives of neurology 65:329-334.
    Wenk GL (2003) Neuropathologic changes in Alzheimer's disease. The Journal of clinical psychiatry 64 Suppl 9:7-10.
    Williams RJ, Spencer JP, Rice-Evans C (2004) Flavonoids: antioxidants or signalling molecules? Free radical biology & medicine 36:838-849.
    Xu L, Zheng N, He Q, Li R, Zhang K, Liang T (2013) Puerarin, isolated from Pueraria lobata (Willd.), protects against hepatotoxicity via specific inhibition of the TGF-beta1/Smad signaling pathway, thereby leading to anti-fibrotic effect. Phytomedicine 20:1172-1179.
    Yamada K, Nabeshima T (2003) Brain-derived neurotrophic factor/TrkB signaling in memory processes. J Pharmacol Sci 91:267-270.
    Yamauchi T (2005) Neuronal Ca2+/calmodulin-dependent protein kinase II--discovery, progress in a quarter of a century, and perspective: implication for learning and memory. Biol Pharm Bull 28:1342-1354.
    Yang HJ, Hwang JT, Kwon DY, Kim MJ, Kang S, Moon NR, Park S (2013) Yuzu extract prevents cognitive decline and impaired glucose homeostasis in beta-amyloid-infused rats. J Nutr 143:1093-1099.
    Yankner BA, Duffy LK, Kirschner DA (1990) Neurotrophic and neurotoxic effects of amyloid beta protein: reversal by tachykinin neuropeptides. Science 250:279-282.
    Yao RQ, Qi DS, Yu HL, Liu J, Yang LH, Wu XX (2012) Quercetin attenuates cell apoptosis in focal cerebral ischemia rat brain via activation of BDNF-TrkB-PI3K/Akt signaling pathway. Neurochemical research 37:2777-2786.
    Yoshiyama Y, Higuchi M, Zhang B, Huang SM, Iwata N, Saido TC, Maeda J, Suhara T, Trojanowski JQ, Lee VM (2007) Synapse loss and microglial activation precede tangles in a P301S tauopathy mouse model. Neuron 53:337-351.
    Youdim KA, Shukitt-Hale B, Joseph JA (2004) Flavonoids and the brain: interactions at the blood-brain barrier and their physiological effects on the central nervous system. Free radical biology & medicine 37:1683-1693.
    Zhang HY, Liu YH, Wang HQ, Xu JH, Hu HT (2008) Puerarin protects PC12 cells against beta-amyloid-induced cell injury. Cell Biol Int 32:1230-1237.
    Zhang Z, Zhao R, Qi J, Wen S, Tang Y, Wang D (2011) Inhibition of glycogen synthase kinase-3beta by Angelica sinensis extract decreases beta-amyloid-induced neurotoxicity and tau phosphorylation in cultured cortical neurons. J Neurosci Res 89:437-447.
    Zou Y, Hong B, Fan L, Zhou L, Liu Y, Wu Q, Zhang X, Dong M (2013) Protective effect of puerarin against beta-amyloid-induced oxidative stress in neuronal cultures from rat hippocampus: involvement of the GSK-3beta/Nrf2 signaling pathway. Free Radic Res 47:55-63.

    下載圖示
    QR CODE