补阳还五汤对自身免疫性脑脊髓炎模型小鼠神经保护作用的机制探讨
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摘要
目的:探讨补阳还五汤(BYHWT)在实验性自身免疫性脑脊髓炎(EAE)发展的不同阶段的神经保护作用和机制。方法:采用髓鞘少突胶质细胞糖蛋白35-55多肽(MOG_(35-55))诱导36只C57BL/6雌性小鼠建立实验性自身免疫性脑脊髓炎(EAE)模型,随机分为EAE 9,17,28 d组以及BYHWT 9,17,28 d组。BYHWT各组于免疫后第3天开始灌胃给药(50 g·kg~(-1)·d~(-1)),EAE组以相同方式等体积给予生理盐水,每天1次,连续至免疫后9,17,28 d。采用国际通用的五级临床症状评分观察BYHWT对EAE小鼠的干预作用;采集小鼠脊髓标本,进行苏木素-伊红(HE)染色和固蓝(LFB)染色观察BYHWT的神经保护作用;采用蛋白免疫印迹法(Western blot)检测脊髓神经营养因子(BDNF)和生长相关蛋白-43(GAP-43)等的表达。结果:补阳还五汤治疗后可明显抑制脊髓炎细胞浸润,减轻髓鞘脱失;与EAE组比较,BYHWT各给药组Nogo-A在脊髓表达显著下调(P <0. 01),与EAE 17,28 d组比较,BYHWT 17 d组和28 d组BDNF在脊髓中表达明显上调(P <0. 05,P <0. 01);与EAE 9,17 d组比较,BYHWT 9,17 d组GAP-43在脊髓中表达显著上调(P <0. 01)。结论:补阳还五汤可以通过上调NTFs类物质的表达,下调神经抑制因子的表达,改善局部神经生长微环境而发挥神经保护的作用。
Objective: To explore the neuroprotective effect and mechanism of Buyang Huanwu Tang( BYHWT) on experimental autoimmune encephalomyelitis( EAE) at different stages. Method: The 36 female C57 BL/6 mice were immunized subcutaneously with myelin oligodendrocyte glycoprotein peptides( MOG_(35-55)),then randomly divided into 9,17,28 d EAE control group. Each BYHWT group was orally given drugs on the 3 rd day after immunization( 50 g·kg~(-1)·d~(-1)),and EAE control group was given the same volume of normal saline in the same way once a day for 9,17 and 28 d after immunization. The effect of BYHWT on EAE mice was observed with internationally accepted clinical score. Brain and spinal cord specimens were collected at 9,17 and 28 d after immunization. The neuroprotective effect of BYHWT was observed by hematoxylin-eosin( HE) staining and solid blue staining( LFB). The expressions of BDNF and GAP-43 in spinal cord and brain were detected by Western blot. Result: After treatment,BYHWT can significantly inhibit myelitis cell infiltration and alleviate myelin loss.Compared with EAE group,the expression of Nogo-A in the spinal cord of each BYHWT group was significantly down-regulated( P < 0. 01),and the expression of BDNF in the spinal cord was significantly up-regulated( P <0. 05,P < 0. 01) in the BYHWT group 17 and 28 d group compared with EAE group 17 and 28 d group.Compared with EAE 9,17 d group,GAP-43 expression was significantly up-regulated in the spinal cord of BYHWT 9,17 d group( P < 0. 01). Conclusion: BYHWT can improve the local nerve growth microenvironment and promote the expression of NTFs,reduce the expressions of neuroinhibitory factors,and play a role in neuroprotection.
Objective: To explore the neuroprotective effect and mechanism of Buyang Huanwu Tang( BYHWT) on experimental autoimmune encephalomyelitis( EAE) at different stages. Method: The 36 female C57 BL/6 mice were immunized subcutaneously with myelin oligodendrocyte glycoprotein peptides( MOG_(35-55)),then randomly divided into 9,17,28 d EAE control group. Each BYHWT group was orally given drugs on the 3 rd day after immunization( 50 g·kg~(-1)·d~(-1)),and EAE control group was given the same volume of normal saline in the same way once a day for 9,17 and 28 d after immunization. The effect of BYHWT on EAE mice was observed with internationally accepted clinical score. Brain and spinal cord specimens were collected at 9,17 and 28 d after immunization. The neuroprotective effect of BYHWT was observed by hematoxylin-eosin( HE) staining and solid blue staining( LFB). The expressions of BDNF and GAP-43 in spinal cord and brain were detected by Western blot. Result: After treatment,BYHWT can significantly inhibit myelitis cell infiltration and alleviate myelin loss.Compared with EAE group,the expression of Nogo-A in the spinal cord of each BYHWT group was significantly down-regulated( P < 0. 01),and the expression of BDNF in the spinal cord was significantly up-regulated( P <0. 05,P < 0. 01) in the BYHWT group 17 and 28 d group compared with EAE group 17 and 28 d group.Compared with EAE 9,17 d group,GAP-43 expression was significantly up-regulated in the spinal cord of BYHWT 9,17 d group( P < 0. 01). Conclusion: BYHWT can improve the local nerve growth microenvironment and promote the expression of NTFs,reduce the expressions of neuroinhibitory factors,and play a role in neuroprotection.
引文
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[2]于婧文,李艳花,张辉,等.新型Rho激酶抑制剂FSDC11化合物抑制EAE的作用研究[J].中国神经免疫学和神经病学杂志,2015,22(3):156-161.
[3]张辉,张海飞,李艳花,等. ROCK抑制剂在中枢神经再生和修复中的作用[J].中国免疫学杂志,2013,29(11):1227-1229,1233.
[4]王义周,寇爽,刘妍,等.中医药促进中枢神经再生的研究进展[J].中国实验方剂学杂志,2010,16(12):197-201.
[5]曲铁兵,俞天虹,刘志婷,等.补阳还五汤及其拆方对大鼠脑缺血后神经发生的影响[J].中国中西医结合杂志,2014,34(3):342-347.
[6]刘建春,张红珍,郭文娟,等.补阳还五汤防治多发性硬化作用机理概述[J].中医杂志,2016,57(13):1155-1159.
[7]王晓庆,田倩倩,李艳花,等.补阳还五汤对EAE小鼠免疫调节的作用[J].中国免疫学杂志,2017,33(1):52-57.
[8]田倩倩,李艳花,尉杰忠,等.补阳还五汤对实验性自身免疫性脑脊髓炎单核巨噬细胞的免疫调控作用[J].中国病理生理杂志,2017,33(2):200-207.
[9]田倩倩.补阳还五汤对实验性自身免疫性脑脊髓炎单核巨噬细胞的免疫调控研究[D].太原:山西中医药大学,2017.
[10]郑华珠,王利胜,吴银爱,等. UPLC-MS/MS联合细胞膜固相色谱法分析补阳还五汤作用于神经元样PC12细胞的效应成分[J].中国实验方剂学杂志,2018,24(4):14-20.
[11] LOU Z Y,CHEN C,HE Q,et al. Targeting CB 2receptor as a neuroinflammatory modulator in experimental autoimmune encephalomyelitis[J]. Mol Immunol,2011,49(3):453-461.
[12]张若楠,柴智,樊慧杰,等.五子衍宗丸对实验性自身免疫性脑脊髓炎小鼠防治作用及其机制研究[J].中华中医药杂志,2018,33(4):1316-1319.
[13] Urban J L,Kumar V,Kono D H,et al. Restricted use of T cell receptor V genes in murine autoimmune encephalomyelitis raises possibilities for antibody therapy[J]. Cell,1988,54(4):577-592.
[14]樊永平,王少卿.论多发性硬化血瘀与活血化瘀治法[J].中华中医药杂志,2018,33(9):3819-3821.
[15]王义周,寇爽,刘妍,等.中医药促进中枢神经再生的研究进展[J].中国实验方剂学杂志,2010,16(12):197-201.
[16]郭文娟,张红珍,李艳花,等.补阳还五汤促进中枢神经再生作用研究进展[J].中国中医基础医学杂志,2016,22(3):434-436.
[17] Herzog C,Otto T. Regeneration of olfactory receptor neurons following chemical lesion:time course and enhancement with growth factor administration[J]. Brain Res,1999,849(1/2):155-161.
[18] O'kusky J R,Ye P,D'ercole A J. Insulin-like growth factor-1 promotes neurogenesis and synaptogenesis in the hippocampal dentate gyrus during postnatal development[J]. J Neurosci,2000,20(22):8435-8442.
[19] Prinjha R,Moore S E,Vinson M,et al. Neurobiology:inhibitor of neurite outgrowth in humans[J]. Nature,2000,403(6768):383-384.
[20]刘会贤,刘敬霞,俞维,等.补阳还五汤和星蒌承气汤对脑缺血大鼠神经元突触重塑及胶质源性神经营养因子和神经生长因子表达的影响[J].中国老年学杂志,2015,35(3):702-705.
[21] Lichtman J W,Taghert P H. Trophic factor theory matures[J]. Nature,1987,326(6111):336.
[22]籍新潮,徐如祥.脑源性神经营养因子在中枢神经系统损伤中的多种神经保护作用及其机制的研究进展[J].中华神经创伤外科电子杂志,2016,2(3):168-172.
[23] Kwon B K,LIU J,Messerer C,et al. Survival and regeneration of rubrospinal neurons 1 year after spinal cordinjury[J]. Proc Natl Acad Sci USA,2002,99(5):3246-3251.
[24] de Groot D M,Coenen A J M,Verhofstad A,et al. In vivo induction of glial cell proliferation and axonal outgrowth and myelination by brain-derived neurotrophic factor[J]. Mol Endocrinol,2006,20(11):2987-2998.
[25] XIAO J,Hughes R A,Lim J Y,et al. A small peptide mimetic of brain-derived neurotrophic factor promotes peripheral myelination[J]. J Neurochem,2013,125(3):386-398.
[26] Frey D,Laux T,XU L,et al. Shared and unique roles of CAP23 and GAP-43 inaction regulation, neurite outgrowth,and anatomical plasticity[J]. Cell Biol,2000,149(7):1443-1454.
[27]刘健,杨小玉,杨茂光,等.中枢神经损伤后GAP-43蛋白对神经再生及轴突导向作用及其机制的研究进展[J].吉林大学学报,2013,39(1):180-183.
[28] Gupta S K,Mishra R,Kusum S,et al. GAP-43 is essential for the neurotrophic effects of BDNF and positive AMPA receptor modulator S18986[J]. Cell Death Differ,2009,16(4):624-637.
[29] Walmsley A R,Mir A K. Targeting the Nogo-A signalling pathway to promote recovery following acute cns injury[J]. Curr Pharm Des,2007,13(24):2470-2484.
[30]赵喆,邓其跃. Nogo-A在中枢神经系统损伤后再生及发育中的作用[J].局解手术学杂志,2012,21(6):659-661.