摘要
现代社会中,锰元素被广泛用于冶金、电池以及杀虫剂等工农业生产中,特别是作为新一代的汽油防爆剂使用,使得环境中锰的含量逐渐升高,锰的毒性危害也日益受到人们的重视。研究表明,长期接触锰元素能够引起基底神经节和锥体外系统的神经毒性,并且能够增加帕金森疾病的发病几率。
目前为止,锰对神经系统的毒性作用机制尚不十分清楚。以往对锰神经毒性的研究,主要关注锰对神经元的直接损伤和对星形胶质细胞功能的影响上,而中枢神经系统中的小胶质细胞的作用却一直被忽视。小胶质细胞是脑内具有巨噬细胞类似功能的非特异性免疫细胞,在维持中枢神经系统的正常功能中发挥重要作用。过度激活的小胶质细胞释放大量的神经毒性因子,如一氧化氮(NO)、活性氧簇(ROS)、肿瘤坏死因子α(TNF-α)、白介素1β(IL-1β)和白介素6(IL-6),以及谷氨酸(Glu)等兴奋性氨基酸,可诱发或参与多种神经退行性疾病的发生。因此,本论文以NO、Glu和胞内ROS为检测指标,考察了氯化锰(MnCl2)单独作用和与细菌脂多糖(LPS)联合作用对小胶质细胞活化的影响,并进行相关的机制探讨;还考察了米诺环素等四种单体化合物对MnCl2诱导活化的小胶质细胞NO释放的影响,为研究锰神经毒性的机制和临床药物干预提供理论依据。
首先,MTT的结果发现,MnCl2(300、1000μM)影响N9小胶质细胞的存活率。实验选用MnCl2(10、30、100μM)进行后期实验。接着,运用Griess法检测MnCl2对N9细胞NO释放的影响,结果显示单独MnCl2不能引起N9细胞NO的释放,但是能显著增加LPS诱导的N9细胞NO释放。说明MnCl2对小胶质细胞NO释放的增加,需要小胶质细胞的活化为前提。10 ng/mL的LPS对N9细胞NO的释放没有影响,但与MnCl2合用后,能够显著刺激N9细胞释放NO,说明MnCl2能够使阈下浓度的LPS诱导N9细胞释放NO。实验还发现,提前给予LPS 3 h不能使单独的MnCl2具有刺激N9细胞释放NO的能力;但是提前给予MnCl23 h,LPS对N9细胞的NO释放比单独LPS刺激的NO释放量高。
采用原代培养的大鼠小胶质细胞验证以上实验结果。结果显示,MnCl2单独作用,不能增加大鼠原代小胶质细胞NO释放,但是能显著增强LPS诱导的小胶质细胞NO的释放。这些结果都与MnCl2作用于N9小胶质细胞系的结果一致。
采用高效液相结合荧光检测器的方法研究了MnCl2和MnCl2+LPS两种模型活化N9细胞释放谷氨酸(Glu)水平的变化。首次发现了MnCl2能够刺激N9细胞释放谷氨酸,此作用可被谷氨酸/胱氨酸反向转运体(Xc转运体)的抑制剂L-a-aminoadipic acid (AAA)显著抑制。并且,MnCl2和LPS联合作用对N9细胞谷氨酸的释放有协同增加作用。本文进一步研究了MnCl2促进N9细胞谷氨酸释放的机制,发现MnCl2作用N9细胞早期,能够显著增加细胞内ROS的含量以及降低细胞内GSH水平。抗氧化剂NAC不但能够抑制细胞内ROS的增加,提高细胞内GSH水平,而且能够明显抑制MnCl2刺激的谷氨酸释放。RT-PCR的结果也证实,MnCl2能够显著增加小胶质细胞Xc转运体mRNA的表达。以上结果说明,小胶质细胞的氧化应激和Xc转运体的激活可能均参与了氯化锰诱导的N9细胞谷氨酸的释放。
最后,本文考察了药物对MnCl2+LPS和LPS刺激N9细胞NO释放的抑制作用。结果表明,米诺环素,姜黄素以及白藜芦醇对两种模型都具有显著的抑制作用,但对两种模型的抑制率存在差异,可能是因为药物抑制小胶质细胞活化的主要分子靶点不同决定的。
综上所述,MnCl2对小胶质细胞NO的释放需要小胶质细胞的活化为前提。本文首次发现氧化应激和Xc转运体的激活参与了MnCl2刺激的小胶质细胞谷氨酸的释放。提示小胶质细胞释放的谷氨酸可能也参与了锰诱导的神经系统兴奋性毒性。此外,米诺环素、白藜芦醇和姜黄素能够抑制MnCl2与LPS联合诱导的N9小胶质细胞的活化。以上结果丰富了锰的神经毒性机制研究,并为寻找相应的防治药物提供了一定的理论依据。
Manganese,(Mn), used in many industries, is becoming a serious environmental contaminant. It has been established that neurons related to motor function can be damaged by Mn accumulation in the globus pallidus and striatum. There are increasing evidences indicating that exposure to excessive levels of Mn participates in numerous disorder diseases of human central nervous system (CNS) such as hyperactivity, chronic Parkinson's disease and manganism.
However, the underlying mechanism of Mn-induced neurotoxicity remains poorly understood. In past, the study on the toxicity of manganese used to focus on the neuron and astrocyte injury, while the microglial effects on the nerve toxicity by manganes were always neglected. Microglia are resident monocytes in the CNS, functionally similar to macrophages. They play a major role in homeostatic and reparative functions but unregulated response or over-activation of microglia can have disastrous neurotoxic consequences. This might be mediated by the microglial release of NO, ROS, TNF-a, IL-1β, IL-6 and glutamate (Glu), which is associated with several neurodegenerative diseases. The present dissertation evaluated the NO、ROS and Glu production in MnCl2-activated microglia, and studied on the mechanism of MnCl2-activated microglia. Then, we evaluated the effects of Curcumin; Resveratrol; Pseudoginsenoside-PF11 and Minocycline on the models above.
Firstly, the effect of MnCl2 on microglial cells viability was assessed using MTT assays. The result showed that 300 and 1000μM MnCl2 groups can significantly reduce the viability of N9 microglia cells, so the final MnCl2 concentration was 0-100μM for next experiments. Our results showed MnCl2 can potentiate NO production in LPS-activated microglia, which indicated that manganese could significantly increase NO production by activated microglial cells. Furthermore, MnCl2 with LPS in subthreshold concentration can induce the NO production in microglia. Exposure to LPS 3 h before MnCl2 activation, No significant nitrite production was observed, but exposure to MnCl2 3 h before LPS activation, compared to LPS lonely activation, the nitrite production was significant higher.
Besides the N9 microglial cell line, we next determined the effect of MnCl2 on the release of NO in primary rat microglia. Although, MnCl2 did not result in significant production of NO as measure, it can potentiate the NO production in LPS-activated microglia, which is similar to observation for the N9 microglial cell line.
The release of glutamate from MnCl2-stimulated N9 cells was examined by HPLC. we reported, for the first time that manganese chloride (MnCl2) significantly increases Glu release in a time-and concentration-dependent manner in N9 cells, which may be mediated by intracelluar ROS, mainly via xCT.
Finally, the effects of Curcumin, Resveratrol, Pseudoginsenoside-PF11 and Minocycline on the NO production in LPS/MnCl2+LPS-activated microglial cells were assessed by Griess reaction. It was found that the inhibition of Curcumin, Resveratrol and Minocycline were different in two models, which could be due to the different molecular pharmacological targets.
In conclusion, we first demonstrate that MnCl2 can induce sustained release of Glu in microglia which may be mediated by intracelluar ROS, mainly via xCT. Curcumin, Resveratrol and Minocycline could inhibit the activation of MnCl2-induced microglia. They may have therapeutic potential in the treatment of neurodegenerative diseases accompanied by microglial activation. These results may provide a novel insight into the potential mechanism of action for manganese-induced neurotoxicity.
引文
[1]Wedler FC, Denman RB, Roby WG. Glutamine synthetase from ovine brain is a manganese (Ⅱ) enzyme. Biochem.1982; 21(6):63-89.
[2]Gerber GB, Leonard A, Hantson P. Mutagenicityand teratogenicity of manganese compounds. Crit. Rev. Oncol. Hematol.2002; 42(9):25-34.
[3]孟宪忠。锰的生物化学及缺乏和中毒,国外医学·卫生学分册,1986;(1):26-29.
[4]Liliana Quintanar. Manganese neurotoxicity:A bioinorganic chemist's perspective,Inorganica. Chimica. Acta.2008; 4 (2):875-884
[5]Erikson KM, Suber RL, Aschner M. Glutamate/aspartate transporter (GLAST), taurine transporter and metallothionein mRNA levels are differentially altered in astrocytes exposed to manganese chloride, manganese phosphate or manganese sulfate. Neurotoxicol.2002; 23 (15):281-288.
[6]Dorman DC, Struve MF, Marshall MW, Parkinson CU, James RA, Wong BA. Tissue manganese concentrations in young male rhesus monkeys following Subchronic manganese sulfate inhalation. Toxicol. Sci.2006; 92 (1):201-210.
[7]Misselwitz B, Muhler A, Weinmann HJ. A toxicologic risk for using manganese complexes:A literature survey of existing data through several medical specialties. Invest. Radiol.1995; 30 (17):611-620.
[8]Rovetta F, Catalani S, Steimberg N, Boniotti J,Gilberti ME, Mariggio MA. Mazzoleni G. Organ-specific manganese toxicity:a comparative in vitro study on five cellular models exposed to MnCl2. Toxicol. in Vitro.2007; 21 (11):284-292.
[9]杜艳芬,李新,张哲成,王纪佐。慢性锰中毒的锥体外系表现四例报告,中华神经科杂志。 2004;37(5):126-128.
[10]Olanow CW. Manganese-induced parkinsonism and Parkinson's disease. Ann NY Acad Sci.2004; 1012 (56):209-212.
[11]Racette BA, McGeer ML, Moerlein SM, Mink JW, Videen TO, Perlmutter JS. Welding-related parkinsonism:clinical features, treatment, and pathophysiology. Neuro.2001; 56 (13):8-13
[12]McGeer PL, Itagaki S, Boyes BE, McGeer EG. Reactive microglia are positive for HLA-DR in the substantia nigra of Parkinson's and Alzheimer's disease brains. Neurol.1988; 38 (8):1285-91.
[13]Bernard W. Economic implications of manganese neurotoxicity. Neurotoxicol.2006; 27 (3):362-368.
[14]张志红,徐明。慢性锰中毒的神经毒理机制,职业与健康。2003;21(1):6-7.
[15]Oberdorster G, Sharp Z, Atudorei V, Elder A, Gelein R, Kreyling W. Translocation of inhaled ultrafine particles to the brain. Inhal Toxicol.2004; 16 (7):437-45.
[16]Davalos D, Grutzendler J, Yang G, Kim JV, Zuo Y, Jung S, Littman DR, Dustin ML, Gan WB. ATP mediates rapid microglial response to local brain injury in vivo. Nat Neurosci.2005; 8 (5):752-758.
[17]Mergler D, Baldwin M, Belanger S, Larribe F, Beuter A, Bowler R. Manganese neurotoxicity, a continuum of dysfunction:results from a community based study. Neurotoxicol.1999:20 (8):327-42.
[18]Hochberg F, Miller G, Valenzuela R, McNelis S, Crump KS, Covington T. Late motor deficits of Chilean manganese miners:a blinded control study. Neurol.1996; 47 (9):788-95.
[19]Crossgrove JS, Allen DD, Bukaveckas BL, Rhineheimer SS, Yokel RA. Manganese distribution across the blood-brain barrier:evidence for carrier-mediated influx of managanese citrate as well as manganese and manganese transferrin. Neurotoxicol.2003; 24 (1):3-13.
[20]Andersen ME, Gearhart JM, Clewell HJ. Pharmacokinetic data needs to support risk assessments for inhaled and ingested manganese. Neurotoxicol.1999; 20 (12):161-71.
[21]Vitarella D, Wong BA, Moss OR, Dorman DC. Pharmacokinetics of inhaled manganese phosphate in male Sprague-Dawley rats following subacute (14-day) exposure. Toxicol. Appl. Pharmacol.2000; 163 (17):279-85.
[22]Spadoni F, Stefani A, Morello M, Lavaroni F, Giacomini P, Sancesario G. Selective vulnerability of pallidal neurons in the early phases of manganese intoxication.Exp. Brain Res.2000; 135 (30):544-551.
[23]Donaldson J, McGregor D, LaBella F. Manganese neurotoxicity:A model for free radical mediated neurodegeneration. Can. J. Physiol. Pharmacol.1982; 60 (11):1398-1405.
[24]Dukhande VV, Malthankar-Phatak GH, Hugus JJ, Daniels CK,Lai JC. Manganese-induced neurotoxicity is differentially enhanced by glutathione depletion in astrocytoma and neuroblastoma cells.Neurochem. Res. 2006:311 (16):349-1345.
[25]Crompton M, Costi A. A heart mitochondrial Ca2+-dependent pore of possible relevance to re-perfusion-induced injury.Evidence that ADP facilitates pore interconversion between the closed and open states. J. Biochem.1990; 266(1):33-9.
[26]Gavin CE, Gunter KK, Gunter TE. Manganese and calcium transport in mitochondria:implicationsfor manganese toxicity. Neurotoxicol.1999; 20 (23):445-53.
[27]Takahashi H, Yamaguchi M. Regulatory effect of regu-calcinon(Ca2+,Mg2+)-ATPase in rat liver plasma membranes comparison with the activation by Mn2+ and Co2+. Mol Cell Biochem.1993; 124(2):169-174.
[28]Vig PJ, Nath R, Desaiah D. Metal inhibition of calmodulin activity in monkey brain. J. Appl. Toxicol. 1989; 9 (5):313-316.
[29]Block ML,Hong JS. Microglia and inflammation-mediated neurodegeneration:multiple triggers with a common mechanism. Prog. Neurobiol.2005; 76(2):77-98.
[30]Liu B, Hong JS. Role of microglia in inflammation-mediated neurodegenerative disease:mechanisms and strategies for therapeutic intervention. J. Pharmacol. Exp. Ther.2003:304 (12):1-7.
[31]Nimmerjahn A, Kirchhoff F, Helmchen F. Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. Sci.2005; 308 (2):1314-1318.
[32]Morgan SC, Taylor DL, Pocock JM. Microglia release activators of neuronal proliferation mediated by activation of mitogen-activated protein kinase, phosphatidylinositol-3-kinase/Akt and delta-Notch signalling cascades. J. Neurochem.2004; 90 (4):089-101.
[33]Mirza B, Hadberg H, Thomsen P, Moos T. The absence of reactive astrocytosis is indicative of a unique inflammatory process in Parkinson's disease. Neurosci.2000; 95 (2):425-432.
[34]Loeffler DA, DeMaggio AJ, Juneau PL, Havaich MK, LeWitt PA. Effects of enhanced striatal dopamine turn over in vivo on glutathione oxidation. Clin. Neuropharmacol.1994; 17 (1):370-379.
[35]Kim Y. High signal intensities on Tl-weighted MRI as a biomarker of exposure to manganese. In. Health. 2004; 42 (4):111-115.
[36]Kim YS, Choi DH, Block ML, Lorenzl S, Yang L, Kim YJ, Sugama S, Cho BP, Hwang O, Browne SE, Kim SY, Hong JS, Beal MF, Joh TH. A pivotal role of matrix metalloproteinase-3 activity in dopaminergic neuronal degeneration via microglial activation. J. FASEB.2007; 21 (1):179-187.
[37]Wilms H, Rosenstiel P, Sievers J, Deuschl G, Zecca L, Lucius R. Activation of microglia by human neuromelanin is NF-kappaB dependent and involves p38 mitogen-activated protein kinase:implications for Parkinson's disease. J. FASEB.2003; 17 (2):500-502.
[38]Takeuchi H, Wang J, Kawanokuchi J, Mitsuma N, Mizuno T, Suzumura A. Interferon-y induces microglial-activation-induced cell death:a hypothetical mechanism of relapse and remission in multiple sclerosis. Neurobiol. Dis.2006; 22 (3):33-39.
[39]Schofield E, Kersaitis C, Shepherd CE, Kril JJ, Halliday GM. Severity of gliosis in Pick's disease and frontotemporal lobar degeneration:tau-positive glia differentiate these disorders. Brain.2003; 126 (23): 827-840.
[40]Zheng Z, Yenari MA. Post-ischemic inflammation:molecular mechanisms and therapeutic implications. Neurol. Res.2004; 26 (4):884-892.
[41]Barger SW, Goodwin ME, Porter MM, Beggs ML. Glutamate release from activated microglia requires the oxidative burst and lipid peroxidation. J. Neurochem.2007; 101 (5):1205-13.
[42]Bal-Price A, Brown GC. Inflammatory neurodegeneration mediated by nitric oxide from activated glia-inhibiting neuronal respiration, causing glutamate release and excitotoxicity. J. Neurosci.2001; 21 (17): 6480-6491.
[43]O'Shea RD, Lau CL, Farso MC, Diwakarla S, Zagami CJ, Svendsen BB, Feeney SJ, Callaway JK, Jones NM, Pow DV, Danbolt NC, Jarrott B, Beart PM. Effects of lipopolysaccharide on glial phenotype and activity of glutamate transporters:Evidence for delayed up-regulation and redistribution of GLT-1. Int. Neurochem.2006; 48 (6-7):604-10.
[44]Kato S, Negishi K, Mawatari K, Kuo CH. A mechanism for glutamate toxicity in the C6 glioma cells involving inhibition of cystine uptake leading to glutathione depletion. Neurosci.1992; 48 (4):903-914.
[45]Murphy TH, Miyamoto M, Sastre A, Schnaar RL, Coyle JT. Glutamate toxicity in a neuronal cell line involves inhibition of cystine transport leading to oxidative stress. Neu.1989; 2 (6):1547-1558.
[46]Simonian NA, Coyle JT. Oxidative stress in neurodegenerative diseases. Annu. Rev. Pharmacol. Toxicol. 1996; 36 (2):083-106.
[47]Chang JY, Liu LZ. Manganese potentiates nitric oxide production by microglia. Mol. Bra. Res.1999; 68 (2):22-28.
[48]Zhang P, Wong TA, Lokuta KM, Turner DE, Vujisic K, Liu B. Microglia enhance manganese chloride-induced dopaminergic neurodegeneration:role of free radical generation, Exp. Neuro.2009; 217 (3):219-230.
[49]杨剑文,胡治平,米诺环素在神经系统疾病中的保护作用,药学与临床研究。2007;15(6):433-436.
[50]Yrjanheikki J, Keinanen R, Pellikka M, Hokfelt T, Koistinaho J. Tetracyclines inhibit microglial activation and are neuroprotective in global brain ischemia. Proc. Nat. Acad. Sci.1998; 95 (26): 15769-15774.
[51]Tikka TM, Koistinaho JE. Minocycline provides neuroprotection against N-methyl-D-aspartate neurotoxicity by inhibiting microglia. J. Immunol.2001; 166 (12):7527-3753.
[52]Suk K. Minocycline suppresses hypoxic activation of rodent microglia in culture. Neurosci Lett.2004; 366(2):167-171.
[53]Bureau G, Longpre F, Martinoli MG. Resveratrol and quercetin, two natural polyphenols, reduce apoptotic neuronal cell death induced by neuroinflammation. J. Neurosci. Res.2008; 86 (2):403-410.
[54]Bi XL, Yang JY, Dong YX, Wang JM, Cui YH, Ikeshima T, Zhao YQ, Wu CF. Resveratrol inhibits nitric oxide and TNF-alpha production by lipopolysaccharide-activated microglia. Int. Immunopharmacol. 2005; 5(1):185-193.
[55]Chang JY, Chavis JA, Liu LZ, Drew PD. Cholesterol oxides induce programmed cell death in microglial cells. Biochem. Biophy. Res. Commun.1998; 249 (23):817-821.
[56]庞战军, 周玫。自由基医学研究方法。人民卫生出版社。2000;34(12):224-226.
[57]Gill K, Menez JF, Lucas D, Deitrich RA. Enzymatic pruduction of acetaldehyde from ethanol in rat brain tissue. Alc. Clin. Exp. Res.1992; 16 (5):910-915.
[58]Qin L, Liu Y, Wang T, Wei SJ, Block ML, Wilson B, Liu B, Hong JS. NADPH oxidase mediates lipopolysaccharide-induced neurotoxicity and proinflammatory gene expression in activated microglia. J. Biol. Chem.2004; 279 (5):1415-1421.
[59]Crittenden PL, Filipov NM. Manganese-induced potentiation of in vitro proinflammatory cytokine production by activated microglial cells is associated with persistent activation of p38 MAPK, Toxicol. In. Vitro.2008; 22 (24):18-27.
[60]McGeer PL, Schwab C, Parent A, Doudet D. Presence of reactive microglia in monkey substantia nigra years after 1-methyl-4-phenyl-dropyridine administration. Ann. Neurol.2003; 54 (5):599-604.
[61]Takeuchi H, Mizuno T, Zhang G, Wang J, Kawanokuchi J, Kuno R, Suzumura A. Neuritic beading induced by activated microglia is an early feature of neuronal dysfunction toward neuronal death by inhibition of mitochondrial respiration and axonal transport. J. Biol. Chem.2005; 280 (11):10444-10454.
[62]Qin S, Colin C, Hinners I, Gervais A, Cheret C, Mallat M. System XC-and apolipoprotein E expressed by microglia have opposite effects on the neurotoxicity of amyloid-peptide 1-40. J. Neurosci.2006; 26 (12): 3345-3356.
[63]Zhang P, Wong TA, Lokuta KM, Turner DE, Vujisic K, Liu B. Microglia enhance manganese chloride-induced dopaminergic neurodegeneration:role of free radical generation, Exp. Neurol.2009; 217 (3):219-230.
[64]Garthwaite J, Garthwaite G, Hajos F. Amino acid neurotoxicity:relationship to neuronal depolarization in rat cerebellar slices. Neurosci.1986; 18 (2):449-60.