七氟醚调控 C/EBPβ-δ 分泌酶通路介导 AD 模型中的 Tau 异常磷酸化

目的：探讨吸入麻醉药七氟醚是否会通过C/EBPβ-δ secretase途径，促进AD模型的Tau蛋白异常磷酸化。同时研究七氟醚暴露是否会对AD模型小鼠的认知功能和记忆功能造成影响，产生AD的相关行为学变化。

方法：实验分为离体实验和在体实验。离体实验中以小鼠N₂A细胞系作为研究对象，验证七氟醚暴露下神经元细胞的C/EBPβ-δ secretase通路的表达情况。将野生型N₂A细胞（Wild Type，WT）和转入APP基因的N₂A细胞分为空白组（con组）和七氟醚组（Sevo组）共四组。七氟醚组放入4%七氟醚中暴露6h，之后分别提取四组的细胞总蛋白，检测C/EBP β-δ secretase通路与Tau N368、AT8蛋白的表达情况。在体实验则使用C57与APP/PS1小鼠，分为Con组和Sevo组，Sevo组在3%七氟醚暴露6h。进行旷场和水迷宫实验，检测小鼠的运动能力和认知功能。提取小鼠海马蛋白，检测C/EBPβ-δ secretase通路蛋白与Tau N 368、AT8的蛋白表达情况。

结果：和空白组相比，七氟醚组的N₂A  APP细胞中的C/EBPβ，δ secretase，Tau N368，AT8的含量都显著上升（P＜0.05），但WT型N₂A细胞的空白组与七氟醚组无明显差异（P＞0.05）。经过七氟醚暴露后的APP/PS1小鼠，运动能力不受影响（P＞0.05），但逃避潜伏期与平台探索时间显著减少（P＜0.05），C57小鼠经过暴露后无明显差异（P＞0.05）。七氟醚暴露的APP/PS1小鼠海马C/EBPβ，δ secretase，Tau N368，AT8的含量都显著上升（P＜0.05），但是C57小鼠海马蛋白在七氟醚暴露前后无明显差异（P＞0.05）。对于转入AD风险基因更多的5×FAD小鼠模型，四月龄进行七氟醚暴露后，就可以观察到C/EBPβ，δ secretase，Tau N368，AT8蛋白含量的显著上升（P＜0.05）。

结论：七氟醚不会导致N2A WT细胞和C57小鼠的C/EBPβ-δ secretase通路上调以及Tau蛋白异常磷酸化，也不会导致C57小鼠的认知功能障碍。但对于转入了AD风险基因的细胞和小鼠，会导致其C/EBPβ-δ secretase通路的上调，并且促进Tau N368和AT 8等异常磷酸化蛋白的表达。

[1] COLLABORATORS G B D D F. Estimation of the global prevalence of dementia in 2019 and forecasted prevalence in 2050: an analysis for the Global Burden of Disease Study 2019[J]. Lancet Public Health, 2022, 7(2): e105-e125.
[2] 任汝静, 殷鹏, 王志会, 等. 中国阿尔茨海默病报告2021[J]. 诊断学理论与实践, 2021, 20(04): 317-337.
[3] XIE J, VAN HOECKE L, VANDENBROUCKE R E. The Impact of Systemic Inflammation on Alzheimer's Disease Pathology[J]. Front Immunol, 2021, 12: 796867.
[4] FUKUI K, TAKATSU H, SHINKAI T, et al. Appearance of amyloid beta-like substances and delayed-type apoptosis in rat hippocampus CA1 region through aging and oxidative stress[J]. J Alzheimers Dis, 2005, 8(3): 299-309.
[5] YANG T, ZHANG Y, CHEN L, et al. The potential roles of ATF family in the treatment of Alzheimer's disease[J]. Biomed Pharmacother, 2023, 161: 114544.
[6] TAN M S, TAN L, JIANG T, et al. Amyloid-beta induces NLRP1-dependent neuronal pyroptosis in models of Alzheimer's disease[J]. Cell Death Dis, 2014, 5(8): e1382.
[7] QUINN J P, CORBETT N J, KELLETT K A B, et al. Tau Proteolysis in the Pathogenesis of Tauopathies: Neurotoxic Fragments and Novel Biomarkers[J]. J Alzheimers Dis, 2018, 63(1): 13-33.
[8] ZHANG Z, SONG M, LIU X, et al. Cleavage of tau by asparagine endopeptidase mediates the neurofibrillary pathology in Alzheimer's disease[J]. Nat Med, 2014, 20(11): 1254-62.
[9] BALLATORE C, LEE V M, TROJANOWSKI J Q. Tau-mediated neurodegeneration in Alzheimer's disease and related disorders[J]. Nat Rev Neurosci, 2007, 8(9): 663-72.
[10] WANG L, BENZINGER T L, SU Y, et al. Evaluation of Tau Imaging in Staging Alzheimer Disease and Revealing Interactions Between beta-Amyloid and Tauopathy[J]. JAMA Neurol, 2016, 73(9): 1070-7.
[11] WISCHIK C M, HARRINGTON C R, STOREY J M. Tau-aggregation inhibitor therapy for Alzheimer's disease[J]. Biochem Pharmacol, 2014, 88(4): 529-39.
[12] CHALERMPALANUPAP T, KINKEAD B, HU W T, et al. Targeting norepinephrine in mild cognitive impairment and Alzheimer's disease[J]. Alzheimers Res Ther, 2013, 5(2): 21.
[13] HERRMANN N, LANCTOT K L, KHAN L R. The role of norepinephrine in the behavioral and psychological symptoms of dementia[J]. J Neuropsychiatry Clin Neurosci, 2004, 16(3): 261-76.
[14] BRAAK H, DEL TREDICI K. Where, when, and in what form does sporadic Alzheimer's disease begin?[J]. Curr Opin Neurol, 2012, 25(6): 708-14.
[15] WEINSHENKER D. Long Road to Ruin: Noradrenergic Dysfunction in Neurodegenerative Disease[J]. Trends Neurosci, 2018, 41(4): 211-223.
[16] BURKE W J, LI S W, WILLIAMS E A, et al. 3,4-Dihydroxyphenylacetaldehyde is the toxic dopamine metabolite in vivo: implications for Parkinson's disease pathogenesis[J]. Brain Res, 2003, 989(2): 205-13.
[17] LI S W, LIN T S, MINTEER S, et al. 3,4-Dihydroxyphenylacetaldehyde and hydrogen peroxide generate a hydroxyl radical: possible role in Parkinson's disease pathogenesis[J]. Brain Res Mol Brain Res, 2001, 93(1): 1-7.
[18] KANG S S, ZHANG Z, LIU X, et al. TrkB neurotrophic activities are blocked by alpha-synuclein, triggering dopaminergic cell death in Parkinson's disease[J]. Proc Natl Acad Sci U S A, 2017, 114(40): 10773-10778.
[19] XIONG J, ZHANG Z, YE K. C/EBPbeta/AEP Signaling Drives Alzheimer's Disease Pathogenesis[J]. Neurosci Bull, 2023.
[20] WANG H, CHEN G, AHN E H, et al. C/EBPbeta/AEP is age-dependently activated in Parkinson's disease and mediates alpha-synuclein in the gut and brain[J]. NPJ Parkinsons Dis, 2023, 9(1): 1.
[21] VALENTE T, MANCERA P, TUSELL J M, et al. C/EBPbeta expression in activated microglia in amyotrophic lateral sclerosis[J]. Neurobiol Aging, 2012, 33(9): 2186-99.
[22] CHEN J M, DANDO P M, STEVENS R A, et al. Cloning and expression of mouse legumain, a lysosomal endopeptidase[J]. Biochem J, 1998, 335 ( Pt 1)(Pt 1): 111-7.
[23] ZHANG Z, TIAN Y, YE K. delta-secretase in neurodegenerative diseases: mechanisms, regulators and therapeutic opportunities[J]. Transl Neurodegener, 2020, 9: 1.
[24] XIA Y, WANG Z H, ZHANG Z, et al. Delta- and beta- secretases crosstalk amplifies the amyloidogenic pathway in Alzheimer's disease[J]. Prog Neurobiol, 2021, 204: 102113.
[25] XIONG J, KANG S S, WANG Z, et al. FSH blockade improves cognition in mice with Alzheimer's disease[J]. Nature, 2022, 603(7901): 470-476.
[26] CHEN C, ZHOU Y, WANG H, et al. Gut inflammation triggers C/EBPbeta/delta-secretase-dependent gut-to-brain propagation of Abeta and Tau fibrils in Alzheimer's disease[J]. EMBO J, 2021, 40(17): e106320.
[27] WU Z, CHEN C, KANG S S, et al. Neurotrophic signaling deficiency exacerbates environmental risks for Alzheimer's disease pathogenesis[J]. Proc Natl Acad Sci U S A, 2021, 118(25).
[28] XIA Y, WANG Z H, ZHANG J, et al. C/EBPbeta is a key transcription factor for APOE and preferentially mediates ApoE4 expression in Alzheimer's disease[J]. Mol Psychiatry, 2021, 26(10): 6002-6022.
[29] HOLMES C. Review: systemic inflammation and Alzheimer's disease[J]. Neuropathol Appl Neurobiol, 2013, 39(1): 51-68.
[30] YANG C W, FUH J L. Exposure to general anesthesia and the risk of dementia[J]. J Pain Res, 2015, 8: 711-8.
[31] PEREZ ORTIZ J M, SWERDLOW R H. Mitochondrial dysfunction in Alzheimer's disease: Role in pathogenesis and novel therapeutic opportunities[J]. Br J Pharmacol, 2019, 176(18): 3489-3507.
[32] BALDO B A. Toxicities of opioid analgesics: respiratory depression, histamine release, hemodynamic changes, hypersensitivity, serotonin toxicity[J]. Arch Toxicol, 2021, 95(8): 2627-2642.
[33] ANTHONY I C, NORRBY K E, DINGWALL T, et al. Predisposition to accelerated Alzheimer-related changes in the brains of human immunodeficiency virus negative opiate abusers[J]. Brain, 2010, 133(Pt 12): 3685-98.
[34] TIAN D, XING Y, GAO W, et al. Sevoflurane Aggravates the Progress of Alzheimer's Disease Through NLRP3/Caspase-1/Gasdermin D Pathway[J]. Front Cell Dev Biol, 2021, 9: 801422.
[35] YU Y, YANG M, ZHUANG X, et al. Neurotoxic 18-kDa apolipoprotein E fragment production contributes to anesthetic sevoflurane-induced tau phosphorylation and neuroinflammation in vitro[J]. Hum Exp Toxicol, 2022, 41: 9603271221102519.
[36] HENEKA M T, GOLENBOCK D T, LATZ E. Innate immunity in Alzheimer's disease[J]. Nat Immunol, 2015, 16(3): 229-36.
[37] LI N, MA Y, LI C, et al. Dexmedetomidine alleviates sevoflurane-induced neuroinflammation and neurocognitive disorders by suppressing the P2X4R/NLRP3 pathway in aged mice[J]. Int J Neurosci, 2022: 1-11.
[38] WU Z, LIU X, CHENG L, et al. Delta-secretase triggers Alzheimer's disease pathologies in wild-type hAPP/hMAPT double transgenic mice[J]. Cell Death Dis, 2020, 11(12): 1058.
[39] WU Y, NIU X, LI P, et al. Lactobacillaceae improve cognitive dysfunction via regulating gut microbiota and suppressing Abeta deposits and neuroinflammation in APP/PS1 mice[J]. Arch Microbiol, 2023, 205(4): 118.
[40] HOU X, JIANG H, LIU T, et al. Depletion of gut microbiota resistance in 5xFAD mice enhances the therapeutic effect of mesenchymal stem cell-derived exosomes[J]. Biomed Pharmacother, 2023, 161: 114455.
[41] ZHANG T, HAN Y, WANG J, et al. Comparative Epidemiological Investigation of Alzheimer's Disease and Colorectal Cancer: The Possible Role of Gastrointestinal Conditions in the Pathogenesis of AD[J]. Front Aging Neurosci, 2018, 10: 176.
[42] CHEN C, LIAO J, XIA Y, et al. Gut microbiota regulate Alzheimer's disease pathologies and cognitive disorders via PUFA-associated neuroinflammation[J]. Gut, 2022, 71(11): 2233-2252.
[43] LIU M, SONG S, CHEN Q, et al. Gut microbiota mediates cognitive impairment in young mice after multiple neonatal exposures to sevoflurane[J]. Aging (Albany NY), 2021, 13(12): 16733-16748.
[44] HUANG X, YING J, YANG D, et al. The Mechanisms of Sevoflurane-Induced Neuroinflammation[J]. Front Aging Neurosci, 2021, 13: 717745.
[45] LUCA M, DI MAURO M, DI MAURO M, et al. Gut Microbiota in Alzheimer's Disease, Depression, and Type 2 Diabetes Mellitus: The Role of Oxidative Stress[J]. Oxid Med Cell Longev, 2019, 2019: 4730539.
[46] SOLA E, MOYANO P, FLORES A, et al. Cadmium-promoted thyroid hormones disruption mediates ROS, inflammation, Abeta and Tau proteins production, gliosis, spongiosis and neurodegeneration in rat basal forebrain[J]. Chem Biol Interact, 2023, 375: 110428.
[47] AHN E H, LEI K, KANG S S, et al. Mitochondrial dysfunction triggers the pathogenesis of Parkinson's disease in neuronal C/EBPbeta transgenic mice[J]. Mol Psychiatry, 2021, 26(12): 7838-7850.
[48] ZHOU Y, ZHANG Y, WANG H, et al. Microglial pyroptosis in hippocampus mediates sevolfurane-induced cognitive impairment in aged mice via ROS-NLRP3 inflammasome pathway[J]. Int Immunopharmacol, 2023, 116: 109725.
[49] BARTOLETTI A, CANCEDDA L, REID S W, et al. Heterozygous knock-out mice for brain-derived neurotrophic factor show a pathway-specific impairment of long-term potentiation but normal critical period for monocular deprivation[J]. J Neurosci, 2002, 22(23): 10072-7.
[50] PHILLIPS H S, HAINS J M, ARMANINI M, et al. BDNF mRNA is decreased in the hippocampus of individuals with Alzheimer's disease[J]. Neuron, 1991, 7(5): 695-702.
[51] MURER M G, BOISSIERE F, YAN Q, et al. An immunohistochemical study of the distribution of brain-derived neurotrophic factor in the adult human brain, with particular reference to Alzheimer's disease[J]. Neuroscience, 1999, 88(4): 1015-32.
[52] KANG S S, AHN E H, YE K. Delta-secretase cleavage of Tau mediates its pathology and propagation in Alzheimer's disease[J]. Exp Mol Med, 2020, 52(8): 1275-1287.
[53] ZHAO J, REN J, LIU S, et al. Repeated exposure to sevoflurane in neonatal rats impairs cognition in adulthood via the PKA-CREB-BDNF signaling pathway[J]. Exp Ther Med, 2021, 22(6): 1442.
[54] DALL E, BRANDSTETTER H. Structure and function of legumain in health and disease[J]. Biochimie, 2016, 122: 126-50.
[55] BASURTO-ISLAS G, GRUNDKE-IQBAL I, TUNG Y C, et al. Activation of asparaginyl endopeptidase leads to Tau hyperphosphorylation in Alzheimer disease[J]. J Biol Chem, 2013, 288(24): 17495-507.
[56] SCHLEGEL K, AWWAD K, HEYM R G, et al. N368-Tau fragments generated by legumain are detected only in trace amount in the insoluble Tau aggregates isolated from AD brain[J]. Acta Neuropathol Commun, 2019, 7(1): 177.
[57] 刘硕, 曹云鹏. 阿尔茨海默病脑TAU蛋白磷酸化位点综述[J]. 中风与神经疾病杂志, 2022, 39(09): 857-860.
[58] ALONSO A D, GRUNDKE-IQBAL I, BARRA H S, et al. Abnormal phosphorylation of tau and the mechanism of Alzheimer neurofibrillary degeneration: sequestration of microtubule-associated proteins 1 and 2 and the disassembly of microtubules by the abnormal tau[J]. Proc Natl Acad Sci U S A, 1997, 94(1): 298-303.
[59] WEGMANN S, EFTEKHARZADEH B, TEPPER K, et al. Tau protein liquid-liquid phase separation can initiate tau aggregation[J]. EMBO J, 2018, 37(7).
[60] DI PATRE P L, READ S L, CUMMINGS J L, et al. Progression of clinical deterioration and pathological changes in patients with Alzheimer disease evaluated at biopsy and autopsy[J]. Arch Neurol, 1999, 56(10): 1254-61.
[61] SPRUNG J, KRUTHIVENTI S C, WARNER D O, et al. Exposure to surgery under general anaesthesia and brain magnetic resonance imaging changes in older adults[J]. Br J Anaesth, 2019, 123(6): 808-817.
[62] LIANG T Y, PENG S Y, MA M, et al. Protective effects of sevoflurane in cerebral ischemia reperfusion injury: a narrative review[J]. Med Gas Res, 2021, 11(4): 152-154.
[63] SHANG S, SUN F, ZHU Y, et al. Sevoflurane preconditioning improves neuroinflammation in cerebral ischemia/reperfusion induced rats through ROS-NLRP3 pathway[J]. Neurosci Lett, 2023, 801: 137164.
[64] FU Z, WU X, ZHENG F, et al. Activation of the AMPK-ULK1 pathway mediated protective autophagy by sevoflurane anesthesia restrains LPS-induced acute lung injury (ALI)[J]. Int Immunopharmacol, 2022, 108: 108869.
[65] LI G, WANG Y, CAO F, et al. Sevoflurane Promotes Neurodegeneration Through Inflammasome Formation in APP/PS1 Mice[J]. Front Neurosci, 2021, 15: 647136.
[66] YAO Y, KANG S S, XIA Y, et al. A delta-secretase-truncated APP fragment activates CEBPB, mediating Alzheimer's disease pathologies[J]. Brain, 2021, 144(6): 1833-1852.