霉酚酸酯对实验性自身免疫性脑脊髓炎大鼠的治疗作用及其机制研究
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摘要
【目的】本研究应用霉酚酸酯(mycophenolate mofetil, MMF)对多发性硬化(multiple sclerosis,MS)的动物模型—实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis ,EAE)大鼠进行治疗,通过观察实验动物的临床表现及中枢神经系统(central nervous system,CNS)病理改变,检测脾脏淋巴细胞增殖能力及中枢神经系统中细胞间粘附分子-1(intercellular adhesion molecule-1,ICAM-1)、血管细胞粘附分子-1(vascular cell adhesion molecule-1,VCAM-1)和基质金属蛋白酶-9(matrix metalloproteinase-9, MMP-9)的表达,探讨霉酚酸酯对EAE的治疗作用及其作用机制,为临床治疗多发性硬化提供实验依据。
【方法】(1)采用豚鼠全脊髓匀浆和完全弗氏佐剂(complete freund's adjuvant,CFA)制成的混合乳剂作为抗原,辅以同时皮内注射百日咳疫苗,复制Wistar大鼠急性EAE。(2)将实验动物随机分为3组,正常对照组、EAE模型组和MMF治疗组,每组12只。自免疫后第10天起,MMF治疗组以霉酚酸酯30mg·kg~(-1)·d~(-1)的剂量连续灌胃治疗1周,EAE模型组用等量生理盐水代替。(3)免疫后每日观察实验动物的体重变化并进行临床症状评分;免疫后第16天,3组各取部分大鼠处死。HE染色、Luxol Fast Blue-HE髓鞘染色、Bielschowsky轴突银染色及电镜检查观察中枢神经系统的病理改变;CCK-8法检测脾脏淋巴细胞增殖能力;免疫组织化学方法检测中枢神经系统中ICAM-1、VCAM-1和MMP-9蛋白的表达。各组其余大鼠观察至免疫后第30天处死,每天仍进行体重称量及临床症状评分,以了解整个病程变化。
【结果】(1)建模大鼠出现的临床表现、病理改变均符合急性EAE的病变特点。(2)EAE模型组大鼠临床症状高峰评分为(2.83±1.27),体重下降最高值为(34.58±13.47)g,病程持续(16.33±1.15)d。MMF治疗组大鼠临床症状减轻,症状高峰评分为(1.75±1.12),体重下降最高值(24.67±8.42)g,病程持续(11.67±1.53)d,与EAE组相比差异均有统计学意义(P<0.05)。HE染色显示,EAE组大鼠脑、小脑、脊髓的炎症细胞浸润评分分别为(2.50±0.55)、(3.50±0.84)、(3.83±0.41),经MMF治疗后相应部位评分分别为(1.5±0.55)、(2.00±0.63)、(2.17±0.98),两组比较差异有统计学意义(P<0.05)。Luxol Fast Blue-HE髓鞘染色显示,EAE组大鼠脑、小脑、脊髓的髓鞘脱失评分分别为(1.33±0.52)、(2.00±0.63)、(2.17±0.75),MMF治疗组相应部位评分分别为(0.83±0.41)、(1.00±0.63)、(1.17±0.41),两组比较差异有统计学意义(P<0.05)。Bielschowsky轴突银染色显示,MMF组脊髓轴突损伤评分(1.00±0.63),与EAE组(1.83±0.75)相比,差异有统计学意义(P<0.05)。电镜结果示,MMF治疗组髓鞘与轴突的超微结构改变较EAE模型组明显减轻。(3)EAE模型组大鼠脾脏淋巴细胞刺激指数为(2.48±0.29),MMF治疗组刺激指数降低为(1.68±0.24),两组比较差异有统计学意义(p<0.05)。EAE模型组大鼠中枢神经系统中ICAM-1、VCAM-1阳性血管数及MMP-9阳性细胞率分别为(43.61±5.05)、(41.28±4.33)、(46.93±6.50)%,MMF组各指标表达情况分别为(10.89±1.76)、(11.39±2.08)、(20.58±3.34)%,两组间各指标比较差异均有统计学意义(p<0.05)。
【结论】(1)本研究中复制的急性EAE模型成功、稳定、可靠。(2)霉酚酸酯对急性EAE具有明显的治疗作用,可有效改善EAE的临床表现,减轻中枢神经系统炎症细胞浸润,降低神经髓鞘的脱失和神经轴突的损伤。(3)霉酚酸酯治疗EAE的作用机制是多方面的,不仅与抑制免疫反应有关,还可能通过降低EAE中枢神经系统中ICAM-1、VCAM-1及MMP-9的表达发挥其治疗作用。
Objective:To explore the efficacy and therapeutic mechanism of mycophenolate mofetil(MMF) in treating rats with experimental autoimmune encephalomyelitis(EAE),which is an animal model of multiple sclerosis , by observing clinical features, pathological changes of central nervous system and detecting the ability of spleen lymphocyte proliferation,the expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1 and matrix metalloproteinase-9 in central nervous system.
Methods:(1) The guinea pig spinal cord homogenate in complete freund's adjuvant and bordetella pertussis vaccine were both used to induce the acute EAE in Wistar rats. (2) The experimental rats were divided into normal control group, EAE model group and MMF treated group randomly.There are 12 rats in each group. Respectively, rats were given the two agents by oral gavage for a week from 10th day after immunization. The dosage of MMF was 30mg·kg~(-1)·d~(-1).The rats in EAE model group were treated in stead of equal normal saline (N.S). (3) After immunization, changes of body weight and clinical scores of the experimental animals were recorded everyday. 16th day after immunization, a part of rats in each group were killed . The pathological changes of central nervous system was observed by HE staining , Luxol Fast Blue-HE staining, Bielschowsky staining and electron microscope. The ability of spleen lymphocyte proliferation was deteceted by CCK-8 method.The expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1 and matrix metalloproteinase-9 in central nervous system were examined by immunohistochemical method. The other rats in each group were killed until 30th day after immunization to observe the whole course of disease.
Results:(1)The rats immunized in our study showed typical clinical manifestation and pathological changes of acute EAE. (2)In EAE model group,maximum clinical score was(2.83±1.27), maximum loss of body weight was(34.58±13.47)g, disease duration was (16.33±1.15)d. In MMF treated group, maximum clinical score was(1.75±1.12), maximum loss of body weight was(24.67±8.42)g,disease duration was (11.67±1.53)d.The differences of maximum clinical score, maximum loss of body weight and disease duration between MMF treated group and EAE model group were statistically significant(P<0.05).Observed in HE staining ,the inflammatory infiltration of Brain ,Cerebellum and Spinal cord in EAE group were (2.50±0.55),(3.50±0.84),(3.83±0.41)respectively,which were (1.5±0.55), (2.00±0.63),(2.17±0.98)in MMF group.The statistical difference was found between two groups(P<0.05).Showed by Luxol Fast Blue-HE staining, the demyelination of Brain ,Cerebellum and Spinal cord in EAE group were (1.33±0.52), (2.00±0.63), (2.17±0.75) respectively , which were (0.83±0.41),(1.00±0.63),(1.17±0.41) in MMF group. The statistical difference was detected(P<0.05).In Bielschowsky staining, axonal damage of spinal cord were significantly improved in MMF treated group,which was (1.00±0.63) compared with EAE group ,which was (1.83±0.75)(P<0.05). Compared with EAE model group, ultrastructural changes of myelin and axon in MMF treated group were relieved. (3) Stimulation index(SI) of Spleen lymphocyte proliferation in EAE group was(2.48±0.29), which was (1.68±0.24)in MMF treated group .A statistical difference in SI was found between MMF treated group and EAE model group(P<0.05).There was statistical difference in positive blood vessel numbers of ICAM-1 and VCAM-1 between EAE model group ,which were (43.61±5.05) and (41.28±4.33) respectively and MMF treated group, which were(10.89±1.76) and (11.39±2.08) respectively(P<0.05).MMP-9 positive ratio was (46.93±6.50)% in EAE model group and (20.58±3.34)% in MMF treated group. A statistical difference was detected between two groups(P<0.05).
Conclusion:(1)The method establishing acute EAE model was successful, stable and reliable in our study. (2) Mycophenolate mofetil is an effective drug in treating EAE as it can relieve the clinical signs and ameliorate pathological lesion in central nervous system of acute EAE rats. (3) The therapeutic mechanism of mycophenolate mofetil on EAE is related to suppressing immune response and may be also associated with its contribution to inhibit the expression of ICAM-1, VCAM-1 and MMP-9 in central nervous system.
【方法】(1)采用豚鼠全脊髓匀浆和完全弗氏佐剂(complete freund's adjuvant,CFA)制成的混合乳剂作为抗原,辅以同时皮内注射百日咳疫苗,复制Wistar大鼠急性EAE。(2)将实验动物随机分为3组,正常对照组、EAE模型组和MMF治疗组,每组12只。自免疫后第10天起,MMF治疗组以霉酚酸酯30mg·kg~(-1)·d~(-1)的剂量连续灌胃治疗1周,EAE模型组用等量生理盐水代替。(3)免疫后每日观察实验动物的体重变化并进行临床症状评分;免疫后第16天,3组各取部分大鼠处死。HE染色、Luxol Fast Blue-HE髓鞘染色、Bielschowsky轴突银染色及电镜检查观察中枢神经系统的病理改变;CCK-8法检测脾脏淋巴细胞增殖能力;免疫组织化学方法检测中枢神经系统中ICAM-1、VCAM-1和MMP-9蛋白的表达。各组其余大鼠观察至免疫后第30天处死,每天仍进行体重称量及临床症状评分,以了解整个病程变化。
【结果】(1)建模大鼠出现的临床表现、病理改变均符合急性EAE的病变特点。(2)EAE模型组大鼠临床症状高峰评分为(2.83±1.27),体重下降最高值为(34.58±13.47)g,病程持续(16.33±1.15)d。MMF治疗组大鼠临床症状减轻,症状高峰评分为(1.75±1.12),体重下降最高值(24.67±8.42)g,病程持续(11.67±1.53)d,与EAE组相比差异均有统计学意义(P<0.05)。HE染色显示,EAE组大鼠脑、小脑、脊髓的炎症细胞浸润评分分别为(2.50±0.55)、(3.50±0.84)、(3.83±0.41),经MMF治疗后相应部位评分分别为(1.5±0.55)、(2.00±0.63)、(2.17±0.98),两组比较差异有统计学意义(P<0.05)。Luxol Fast Blue-HE髓鞘染色显示,EAE组大鼠脑、小脑、脊髓的髓鞘脱失评分分别为(1.33±0.52)、(2.00±0.63)、(2.17±0.75),MMF治疗组相应部位评分分别为(0.83±0.41)、(1.00±0.63)、(1.17±0.41),两组比较差异有统计学意义(P<0.05)。Bielschowsky轴突银染色显示,MMF组脊髓轴突损伤评分(1.00±0.63),与EAE组(1.83±0.75)相比,差异有统计学意义(P<0.05)。电镜结果示,MMF治疗组髓鞘与轴突的超微结构改变较EAE模型组明显减轻。(3)EAE模型组大鼠脾脏淋巴细胞刺激指数为(2.48±0.29),MMF治疗组刺激指数降低为(1.68±0.24),两组比较差异有统计学意义(p<0.05)。EAE模型组大鼠中枢神经系统中ICAM-1、VCAM-1阳性血管数及MMP-9阳性细胞率分别为(43.61±5.05)、(41.28±4.33)、(46.93±6.50)%,MMF组各指标表达情况分别为(10.89±1.76)、(11.39±2.08)、(20.58±3.34)%,两组间各指标比较差异均有统计学意义(p<0.05)。
【结论】(1)本研究中复制的急性EAE模型成功、稳定、可靠。(2)霉酚酸酯对急性EAE具有明显的治疗作用,可有效改善EAE的临床表现,减轻中枢神经系统炎症细胞浸润,降低神经髓鞘的脱失和神经轴突的损伤。(3)霉酚酸酯治疗EAE的作用机制是多方面的,不仅与抑制免疫反应有关,还可能通过降低EAE中枢神经系统中ICAM-1、VCAM-1及MMP-9的表达发挥其治疗作用。
Objective:To explore the efficacy and therapeutic mechanism of mycophenolate mofetil(MMF) in treating rats with experimental autoimmune encephalomyelitis(EAE),which is an animal model of multiple sclerosis , by observing clinical features, pathological changes of central nervous system and detecting the ability of spleen lymphocyte proliferation,the expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1 and matrix metalloproteinase-9 in central nervous system.
Methods:(1) The guinea pig spinal cord homogenate in complete freund's adjuvant and bordetella pertussis vaccine were both used to induce the acute EAE in Wistar rats. (2) The experimental rats were divided into normal control group, EAE model group and MMF treated group randomly.There are 12 rats in each group. Respectively, rats were given the two agents by oral gavage for a week from 10th day after immunization. The dosage of MMF was 30mg·kg~(-1)·d~(-1).The rats in EAE model group were treated in stead of equal normal saline (N.S). (3) After immunization, changes of body weight and clinical scores of the experimental animals were recorded everyday. 16th day after immunization, a part of rats in each group were killed . The pathological changes of central nervous system was observed by HE staining , Luxol Fast Blue-HE staining, Bielschowsky staining and electron microscope. The ability of spleen lymphocyte proliferation was deteceted by CCK-8 method.The expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1 and matrix metalloproteinase-9 in central nervous system were examined by immunohistochemical method. The other rats in each group were killed until 30th day after immunization to observe the whole course of disease.
Results:(1)The rats immunized in our study showed typical clinical manifestation and pathological changes of acute EAE. (2)In EAE model group,maximum clinical score was(2.83±1.27), maximum loss of body weight was(34.58±13.47)g, disease duration was (16.33±1.15)d. In MMF treated group, maximum clinical score was(1.75±1.12), maximum loss of body weight was(24.67±8.42)g,disease duration was (11.67±1.53)d.The differences of maximum clinical score, maximum loss of body weight and disease duration between MMF treated group and EAE model group were statistically significant(P<0.05).Observed in HE staining ,the inflammatory infiltration of Brain ,Cerebellum and Spinal cord in EAE group were (2.50±0.55),(3.50±0.84),(3.83±0.41)respectively,which were (1.5±0.55), (2.00±0.63),(2.17±0.98)in MMF group.The statistical difference was found between two groups(P<0.05).Showed by Luxol Fast Blue-HE staining, the demyelination of Brain ,Cerebellum and Spinal cord in EAE group were (1.33±0.52), (2.00±0.63), (2.17±0.75) respectively , which were (0.83±0.41),(1.00±0.63),(1.17±0.41) in MMF group. The statistical difference was detected(P<0.05).In Bielschowsky staining, axonal damage of spinal cord were significantly improved in MMF treated group,which was (1.00±0.63) compared with EAE group ,which was (1.83±0.75)(P<0.05). Compared with EAE model group, ultrastructural changes of myelin and axon in MMF treated group were relieved. (3) Stimulation index(SI) of Spleen lymphocyte proliferation in EAE group was(2.48±0.29), which was (1.68±0.24)in MMF treated group .A statistical difference in SI was found between MMF treated group and EAE model group(P<0.05).There was statistical difference in positive blood vessel numbers of ICAM-1 and VCAM-1 between EAE model group ,which were (43.61±5.05) and (41.28±4.33) respectively and MMF treated group, which were(10.89±1.76) and (11.39±2.08) respectively(P<0.05).MMP-9 positive ratio was (46.93±6.50)% in EAE model group and (20.58±3.34)% in MMF treated group. A statistical difference was detected between two groups(P<0.05).
Conclusion:(1)The method establishing acute EAE model was successful, stable and reliable in our study. (2) Mycophenolate mofetil is an effective drug in treating EAE as it can relieve the clinical signs and ameliorate pathological lesion in central nervous system of acute EAE rats. (3) The therapeutic mechanism of mycophenolate mofetil on EAE is related to suppressing immune response and may be also associated with its contribution to inhibit the expression of ICAM-1, VCAM-1 and MMP-9 in central nervous system.
引文
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[1] Ikuo Tsunoda, Tomoko Tanaka, Emily Jane Terry, et al.Contrasting Roles for Axonal Degeneration in an Autoimmune versus Viral Model of Multiple Sclerosis When Can Axonal Injury Be Beneficial?[J].Am J Pathol, 2007, 170(1): 214–226.
[2] Fox EJ. Immunopathology of multiple sclerosis[J].Neurology,2004,63(12 Suppl 6):S3-S7.
[3] 戚晓昆, 蒲传强, 朱克等. 实验性变态反应性脑脊髓炎模型病理及组化研究[J]. 中华神经科杂志,1996,2:97-99.
[4] Bjartlnar C,Kinkel RP,Kidd G,et a1. Axonal loss in normal-appearing white matter in a patient with acute MS[J]. Neurology, 2001, 57(7): 1248-1252.
[5] Trapp BD, BoL, Mork S, et a1. Pathogenesis of tissue injury in MS lesions[J]. J Neuroimmunol. l999;98(1): 49-56.
[6] Kornek B, Storch MK, Weissert R, et al. Multiple sclerosis and chronic autoimmune encephalomyelitis: a comparative quantitative study of axonal injury in active, inactive, and remyelinated lesions[J]. Am J Pathol, 2000 , 157(1):267-276.
[7] Bjartmar C, Trapp BD. Axonal degeneration and progressive neurologic disability in multiple sclerosis[J]. Neurotox Res,2003,5(1-2):157-l64.
[8] De Stefano N, Narayanan S, Francis GS,et a1. Evidence of axonal damage in the early stages of multiple sclerosis and its relevance to disability[J].Arch Neurol,2001,58(1):65-70.
[9] Bitsch A, Schuchardt J, Bunkowski S, et a1. Acute axonal injury in multiple sclerosis[J]. Brain, 2000, 123 ( Pt 6):1174-1183.
[10] Bjartmar C, Kidd G, Mork S, et a1. Neurological disability correlates with spinal cord axonal loss and reduced N-acetyl aspartate in chronic multiple sclerosis pa tients. Ann Neurol, 2000, 48(6): 893-901.
[11] Wujek JR, BjartmarC, RicherE, et al. Axon loss in the spinal cord determines permanent neurological disability in an animal model of multiple sclerosis[J].JNeuropathol Exp Neurol,2002,61(1):23-32.
[12] Confavreux C, Vukusic S, Moreau T, et al. Relapses and progression of disability in multiple sclerosis[J].N Engl J Med, 2000, 343(20):1430-1438.
[13] Brosnan CF, Selmaj K ,Schroeder CE,et al. Recombinant human lymphokines induce changes in visual evoked potentials in the rabbit[J].Ann NY Acad Sci,1988, 540:571-572.
[14] Bjartmar C, Wujek JR, Trapp BD. Axonal loss in the pathology of MS consequences for understanding the progressive phase of the disease[J].J Neurol Sci, 2003 , 206(2):165-171.
[15] Mancardi G,Hart B, Roccatagliata L, et al.Demyelination and axonal damage in a non-human primate model of multiple sclerosis[J].J Neurol Sci,2001;184(1): 41-49.
[16] Filippi M, Bozzali M, Rovaris M, et a1. Evidence for widespread axonal damage at the earliest clinical stage of multiple sclerosis[J].Brain, 2003 ,126(Pt2): 433-437.
[17] Rogove AD, Siao C, Keyt B, et a1. Activation of microglia reveals a non-proteolytic cytokine function for tissue plasminogen activator in the central nervous system[J]. J Cell Sci,1999, 112(Pt 22): 4007-4016.
[18] Nagai N, Urano T, Endo A, et al.Neuronal degeneration and a decrease in laminin-like immunoreactivlty is associated with elevated tissue-type plasminogen activator in the rat hippocam pus after kainic acid injection[J].Neurosci Res,1999,33(2):147-154.
[19] Nicot A, Ratnakar PV, Ron Y,et al. Regulation of gene expression in experimental autoimmune encephalomyelitis indicates early neuronal dysfunction[J].Brain, 2003,126(2):398-4l2.
[20] Newman TA, Woolley ST, Hughes PM, et a1.T-cell- and macrophage-mediated axon damage in the absence of a CNS-specific immune response: involvement of metalloproteinases[J].Brain, 2001,124(Pt 11):2203-2214.
[21] Sarchielli P,Greco L,Floridi A, et al. Excitatory amino acids and multiple sclerosis: evidence from cerebrospinal fluid[J].Arch Neurol, 2003, 60(8) :1082–1088.