胎球蛋白降低局灶性脑缺血大鼠脑损伤及与炎症的关联

摘要/Abstract

摘要： 目的：研究胎球蛋白（Fetuin）在大鼠局灶性脑缺血中的作用及与炎症的关系；对氧糖剥夺神经元损伤的保护作用。方法：大鼠大脑中动脉闭塞（middle cerebral artery occlusion，MCAO）60 min制备局灶性脑缺血再灌注(cerebral ischemia reperfusion)模型（MCAO/R）。经神经行为学特征、氯代三苯基四氮唑(2,3,5-triphenyltetrazolium chloride，TTC)染色、免疫组织化学、Western印迹杂交及定量PCR方法分析：①MCAO/R不同时间段Fetuin在损伤梗塞区域的表达；②MCAO 30 min后Fetuin（500，50，25，5 mg·kg-1）静脉注射(i.v.)，24 h后大脑梗塞体积及神经行为学特征；③MCAO后不同时间点（15，30，60，120 min）Fetuin（50 mg·kg-1）i.v.，24 h后脑梗塞体积的变化；④MCAO 30 min后Fetuin（50 mg，i.v.），24 h后脑缺血区域巨噬细胞（macrophages，MØ）、小胶质细胞（microglia，MG）及肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）的变化；⑤Fetuin对体外氧糖剥夺/复氧（oxygen glucose deprivation/reoxygenation，OGD/R）神经元损伤的影响。结果：①大鼠脑缺血2 h损伤区域Fetuin mRNA及蛋白表达增强，48h达高峰；②Fetuin改善神经行为学特征，缩小脑梗塞体积，作用呈量-效依赖关系；③MCAO后15，30及60 min给予Fetuin均缩小脑梗塞体积，作用强弱依次为15＞30＞60 min。120 min则无明显效果；④Fetuin减少MØ/MG在脑损伤区域的聚集，抑制TNF-α的表达；⑤Fetuin（1~100 µmol·L-1）减少OGD/R神经元乳酸脱氢酶（lactate dehydrogenase，LDH）释放（与Fetuin浓度呈反向关系）。结论：MCAO/R大鼠缺血性脑损伤组织自身表达Fetuin；外源性Fetuin改善脑缺血大鼠神经行为学特征，缩小缺血性脑梗塞体积，对OGD/R导致的神经元损伤有保护作用；提示Fetuin参与对神经元的保护而产生对脑缺血有益作用，其作用与抑制MØ/MG在病灶区域的聚集和TNF-α表达有关。

关键词: Fetuin, 局灶性脑缺血, 巨噬细胞（MØ, ）, 小胶质细胞（MG）, 肿瘤坏死因子-&, alpha, （TNF-&, alpha, ）

Abstract: Objective: To investigate whether the Fetuin is inducible in ischemic region and has beneficial efficacy to improve ischemic injuries in focal reversible cerebral ischemic rat model, as well as the possible mechanisms of its action. Methods: Adult male Sprague-Dawley (SD) rats with ischemia via 60 min middle cerebral artery occlusion (MCAO) and subsequent 24 h reperfusion were performed. ① The expression of endogenous Fetuin was detected by Western and real-time RT-PCR analysis at the different times of MCAO/R. ② For dose response, Fetuin (500, 50, 25 and 5 mg·kg-1) was intravenously administered 30 min after the initiation of MCAO and the effect of Fetuin against acute ischemic injury was evaluated by neurological deficit score (NDS), and 2,3,5-triphenyltetrazolium chloride (TTC) staining as well; ③ In time cause study, Fetuin was also intravenously administered (50 mg·kg-1 only) 15, 30, 60 and 120 min respectively after initiation of MCAO to evaluate the potential efficacy of Fetuin for improving ischemic damage by TTC staining; ④ CD68 and TNF-α in ischemic regions were detected by real-time RT-PCR and Western blotting/immunohistochemistry to further investigate accumulation of Fetuin and its anti-inflammatory property; ⑤ The neuronal viability assay was performed by measurement of lactate dehydrogenase (LDH) in the medium to determine the anti-hydrogen peroxide injury of Fetuin in vitro model of oxygen-glucose deprivation/reoxygenation (OGD/R). Results: ① Endogenous Fetuin in response to cerebral ischemia is accumulated at 2 h after initiation of MCAO in damaged areas both in mRNA and protein levels, which remained elevation and reached a peak at 48 h; ② Treatment with Fetuin (500, 50, 25, 5 mg·kg-1) dramatically diminished infarct volumes and improved neurological deficit in a dose-dependent mann; ③ In time course study, animals treated with Fetuin (50 mg·kg-1) had significantly smaller infarct size time-dependently at 15, 30 and 60 min after initiation of MCAO but no protection at 120 min; ④ Fetuin decreased macrophage (MØ)/microglia (MG) accumulation, and down-regulated tumor necrosis factor α (TNF-α) expression both in mRNA and protein levels in damaged regions; ⑤ Treatment with Fetuin from 1-100 μmol·L-1 in vitro while OGD/R (24 h) gradually reduced LDH in the medium. Conclusion: Our data indicate that Fetuin, after MCAO/R, is upregulated in the injured area of brain, as well as plays a critical role in neuronal survival and protection/therapy of cerebral ischemia-reperfusion injury by, at least in part, inhibition of inflammatory cell MØ/MG accumulation and down-regulation of cytokine TNF-α expression in ischemic regions. These results also suggest that Fetuin may be a candidate protein for the treatment of cerebral ischemia and OGD-induced neurodegenerative diseases.

Key words: Fetuin, focal reversible cerebral ischemia, macrophage（MØ, ）, microglia（MG）, tumor necrosis factor &, alpha, (TNF-&, alpha, ）

车建途，图娅，王孔江. 胎球蛋白降低局灶性脑缺血大鼠脑损伤及与炎症的关联[J]. 神经药理学报, 2012, 2(6): 18-27.

CHE Jian-tu, TU Ya, WANG Kong-jiang. Fetuin Attenuates Cerebral lschemic Injury in Rats Associated with Decreases in Macrophage/Microglia and Tumor Necrosis Factor α[J]. Acta Neuropharmacologica, 2012, 2(6): 18-27.

参考文献

[1] Becker K J. Inflammation and acute stroke [J]. Curr Opin Neurol, 1998, 11(1):45-49.

[2] 张健颖, 张颖冬. 缺血性脑损害病理生理过程的炎症反应[J]. 中华脑血管病杂志:电子版, 2011, 5(2): 24-31.

[3] Frank C Barone, Arvin B, White R F, et al. Tumor necrosis factor-alpha. a mediator of focal ischemic brain injury [J]. Stroke, 1997, 28(6):1233-1244.

[4] Ryota Tanaka, Komine-Kobayashi M, Mochizuki H, et al. Migration of enhanced green fluorescent protein expressing bone marrow-derived microglia/macrophage into the mouse brain following permanent focal ischemia [J]. Neuroscience, 2003 117(3):531-539.

[5] Tomimoto H, Ichiro Akiguchi, Hideaki Wakita, et al. Glial expression of cytokines in the brains of cerebrovascular disease patients [J]. Acta Neuropathol, 1996, 92(3):281-287.

[6] Hedley C A Emsley, Pippa J Tyrrell. Inflammation and infection in clinical stroke[J]. J Cereb Blood Flow Metab, 2002, 22(12):1399-1419.

[7] L Creed Pettigrew, Mark S Kindy, Stephen Scheff, et al. Focal cerebral ischemia in the TNFalpha-transgenic rat [J]. J Neuroinflammation, 2008, 5:47.

[8] Michael Ombrellino, Wang Hai-chao, Yang Huan, et al. Fetuin, a negative acute phase protein, attenuates TNF synthesis and the innate inflammatory response to carrageenan [J]. Shock, 2001, 15(3):181-185.

[9] Pascal Lim, Jean-Phillipe Collet, Stephane Moutereau, et al. Fetuin-A is an independent predictor of death after ST-elevation myocardial infarction [J]. Clin Chem, 2007, 53(10):1835-1840.

[10] Marc W Merx, Cora Schafer, Ralf Westenfeld, et al. Myocardial stiffness, cardiac remodeling, and diastolic dysfunction in calcification-prone fetuin-A-deficient mice [J]. J Am Soc Nephrol, 2005, 16(11):3357-3364.

[11] Peter Stenvinkel, Wang Kai, Abdul Rashid Qureshi, et al. Low fetuin-A levels are associated with cardiovascular death: Impact of variations in the gene encoding Fetuin [J]. Kidney Int, 2005, 67(6):2383-2392.

[12] Longa E Z, Philip R Weinstein, Carlson S, et al. Reversible middle cerebral artery occlusion without craniectomy in rats [J]. Stroke, 1989, 20(1):84-91.

[13] Bederson J B, Pitts L H, Germano S M, et al. Evaluation of 2,3,5-triphenyltetrazolium chloride as a stain for detection and quantification of experimental cerebral infarction in rats [J]. Stroke, 1986, 17(6): 1304-1308.

[14] Susan X Jiang, Jittiwud Lertvorachon, Sheng T Hou, et al. Chlortetracycline and demeclocycline inhibit calpains and protect mouse neurons against glutamate toxicity and cerebral ischemia [J]. J Biol Chem, 2005, 280(40):33811-33818.

[15] John P MacManus, Jian Ming, Edward Preston, et al. Absence of the transcription factor E2F1 attenuates brain injury and improves behavior after focal ischemia in mice [J]. J Cereb Blood Flow Metab, 2003, 23(9):1020-1028.

[16] Brewer G J. Serum-free B27/neurobasal medium supports differentiated growth of neurons from the striatum, substantia nigra, septum, cerebral cortex, cerebellum, and dentate gyrus [J]. J Neurosci Res, 1995, 42(5):674-683.

[17] Markus Ketteler, Philipp Bongartz, Ralf Westenfeld, et al. Association of low fetuin-A (AHSG) concentrations in serum with cardiovascular mortality in patients on dialysis: a cross-sectional study [J]. Lancet, 2003, 361(9360):827-833.

[18] Armin Schneider, Rainer Wysocki, Claudia Pitzer, et al. An extended window of opportunity for G-CSF treatment in cerebral ischemia [J]. BMC, 2006, 4:36.

[19] Von Bulow F A, M Stagaard Janas, O B F Terkelsen, et al. Human fetuin/alpha 2 HS glycoprotein in colloid and parenchymal cells in human fetal pituitary gland [J]. Histochemistry, 1993, 99(1):13-22.

[20] Iadecola C, Alexander M. Cerebral ischemia and inflammation [J]. Curr Opin Neurol, 2001, 14(1):89-94.

[21] Paula M Hughes, Peter R Allegrini, Markus Rudin, et al. Monocyte chemoattractant protein-1 deficiency is protective in a murine stroke model [J]. J Cereb Blood Flow Metab, 2002, 22(3):308-317.

[22] Jin Rong, Yang Guo-jun, Li Guo-hong. Inflammatory mechanisms in ischemic stroke: role of inflammatory cells [J]. J Leukoc Biol, 2010, 87(5):779-89.

[23] Zheng Zhen, Midori A Yenari. Post-ischemic inflammation: molecular mechanisms and therapeutic implications [J]. Neurol Res, 2004, 26(8):884-892.

[24] Zhang M, Caragine T, Wang H, et al. Spermine inhibits proinflammatory cytokine synthesis in human mononuclear cells: a counterregulatory mechanism that restrains the immune response [J]. J Exp Med, 1997, 185(10):1759-1768.

[25] Gregersen R, Lambertsen K, Finsen B. Microglia and macrophages are the major source of tumor necrosis factor in permanent middle cerebral artery occlusion in mice [J]. J Cereb Blood Flow Metab, 2000, 20(1):53-65.

[26] Sean D Lavine, Florence M Hofman, Berislav V Zlokovic. Circulating antibody against tumor necrosis factor-alpha protects rat brain from reperfusion injury [J]. J Cereb Blood Flow Metab, 1998, 18(1):52-58.

[27] Annadora J Bruce, Warren Boling, Mark S Kindy, et al. Altered neuronal and microglial responses to excitotoxic and ischemic brain injury in mice lacking TNF receptors [J]. Nat Med, 1996, 2(7):788-794.

[28] Hiroshi Nawashiro, Kaoru Tasaki, Christl A Ruetzler, et al. TNF-alpha pretreatment induces protective effects against focal cerebral ischemia in mice [J]. J Cereb Blood Flow Metab, 1997, 17(5):483-490.