地黄饮子对脑缺血再灌注损伤大鼠保护作用及其机制
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摘要
目的:探讨地黄饮子对脑缺血再灌注损伤大鼠的保护作用及其初步机制。方法:建立大脑中动脉栓塞(MCAO)大鼠模型,随机分为6组,分别为假手术组,模型组,尼莫地平组(0.01 g·kg~(-1)),地黄饮子高、中、低剂量组(38.80,19.40,9.70 g·kg~(-1)),每组20只,连续给药28 d;术后第7,14,28天通过改良神经功能缺损评分(m NSS),第28天行2,3,5-氯化三苯基四氮唑(TTC)染色,苏木素-伊红(HE)染色观察地黄饮子的保护作用,应用免疫荧光技术观察侧脑室室管膜下区(SVZ)的5-溴脱氧尿嘧啶核苷(Brd U)阳性细胞数(第28天)以分析神经干细胞(NSCs)增殖情况,采用实时荧光定量聚合酶链式反应(Real-time PCR)观察Notch1,Jagged1,Hes1 mRNA的表达情况。结果:术后第7,14,28天地黄饮子高、中剂量组,尼莫地平组的m NSS明显低于同时间段的模型组(P<0.05,P<0.01);术后第28天地黄饮子高、中剂量组和尼莫地平组的脑梗死体积占脑组织体积的百分比均低于模型组(P<0.01); HE染色显示地黄饮子高、中剂量组,尼莫地平组大鼠脑组织坏死和软化灶不明显,并可见神经元细胞和神经胶质细胞增生;术后第28天,上述3组SVZ的Brd U阳性细胞数显著高于模型组(P<0.05,P<0.01),地黄饮子高剂量组Brd U阳性细胞数高于尼莫地平组(P<0.05);术后28 d地黄饮子高、中剂量组,尼莫地平组SVZ的Notch1,Jagged1,Hes1 mRNA表达水平明显高于模型组(P<0.05,P<0.01);尼莫地平组Hes1 mRNA水平高于地黄饮子高、中、低剂量组(P<0.05,P<0.01)。结论:地黄饮子能改善MCAO大鼠模型的神经功能缺损,减小脑梗死体积,减轻脑组织病理改变,从而对脑缺血再灌注损伤大鼠起到保护作用,可能与激活Notch信号通路,上调Notch1,Jagged1,Hes1 mRNA的表达,从而促进NSCs增殖有关。
Objective:To explore the protective effect and the preliminary mechanism of Dihuang Yinzi on cerebral ischemia-reperfusion injury in rats.Method:The middle cerebral artery occlusion(MCAO) model was established.Totally 90 SD rats were randomly divided into 6 groups:sham operation group,model group,nimodipine group(0.01 g·kg~(-1)) and high,medium,low-dose Dihuang Yinzi groups(38.80,19.40,9.70 g·kg~(-1)),with 20 rats in each group.The modified neurological severity score(m NSS) was assayed at the7 th,14 th,28 thdays after operation,and the volume of cerebral infarction,pathological changes of brain tissue,the BrdU positive cells and mRNA levels of Notch1,Jagged1 and Hes1 in subventricular zone(SVZ) were observed respectively by triphenyl tetrazolium chloride(TTC) stain,htorylin eastin(HE) stain,immunofluorescence technique and reverse transcriphase polymerase chain reaction(Real-time PCR) methods at the 28 thday after the operation.Result:The m NSS on the 7 th,14 th,28 thdays of high,medium-dose Dihuang Yinzi groups and nimodipine group were significantly lower than that of model group(P<0.05,P<0.01).On the 28 thday,the percentage of cerebral infarction volume in brain tissue volume of high,medium-dose Dihuang Yinzi groups and nimodipine group were smaller than that of model group(P<0.01).HE staining showed that the necrosis and softening lesions of brain tissue were not obvious in rats of high,medium-dose Dihuang Yinzi groups and nimodipine group,with neuronal cell and neuroglial cell proliferation.On the 28 thday,the BrdU positive cells in SVZ of the above 3 groups were significantly higher than model group(P<0.05,P<0.01),and the high-dose Dihuang Yinzi group was significantly higher than nimodipine group(P<0.05).On the 28 thday,the mRNA levels of Notch1,Jagged1 and Hes1 of high,medium-dose Dihuang Yinzi groups and nimodipine group were significantly higher than those of model group(P<0.05,P<0.01),the mRNA level of Hes1 of nimodipine group was higher than that of high,medium,low-dose Dihuang Yinzi groups(P<0.05,P<0.01).Conclusion:Dihuang Yinzi can improve the nerve function defect of MCAO rat model,and reduce the volume of cerebral infarction and the pathological changes of brain tissue,thus playing a protective role in cerebral ischemiareperfusion injury rats.Its mechanism may be related to the activation of the Notch signaling pathway,and the upregulation of expressions of Notch1,Jagged1 and Hes1 mRNA,thus promoting the proliferation of NSCs.
Objective:To explore the protective effect and the preliminary mechanism of Dihuang Yinzi on cerebral ischemia-reperfusion injury in rats.Method:The middle cerebral artery occlusion(MCAO) model was established.Totally 90 SD rats were randomly divided into 6 groups:sham operation group,model group,nimodipine group(0.01 g·kg~(-1)) and high,medium,low-dose Dihuang Yinzi groups(38.80,19.40,9.70 g·kg~(-1)),with 20 rats in each group.The modified neurological severity score(m NSS) was assayed at the7 th,14 th,28 thdays after operation,and the volume of cerebral infarction,pathological changes of brain tissue,the BrdU positive cells and mRNA levels of Notch1,Jagged1 and Hes1 in subventricular zone(SVZ) were observed respectively by triphenyl tetrazolium chloride(TTC) stain,htorylin eastin(HE) stain,immunofluorescence technique and reverse transcriphase polymerase chain reaction(Real-time PCR) methods at the 28 thday after the operation.Result:The m NSS on the 7 th,14 th,28 thdays of high,medium-dose Dihuang Yinzi groups and nimodipine group were significantly lower than that of model group(P<0.05,P<0.01).On the 28 thday,the percentage of cerebral infarction volume in brain tissue volume of high,medium-dose Dihuang Yinzi groups and nimodipine group were smaller than that of model group(P<0.01).HE staining showed that the necrosis and softening lesions of brain tissue were not obvious in rats of high,medium-dose Dihuang Yinzi groups and nimodipine group,with neuronal cell and neuroglial cell proliferation.On the 28 thday,the BrdU positive cells in SVZ of the above 3 groups were significantly higher than model group(P<0.05,P<0.01),and the high-dose Dihuang Yinzi group was significantly higher than nimodipine group(P<0.05).On the 28 thday,the mRNA levels of Notch1,Jagged1 and Hes1 of high,medium-dose Dihuang Yinzi groups and nimodipine group were significantly higher than those of model group(P<0.05,P<0.01),the mRNA level of Hes1 of nimodipine group was higher than that of high,medium,low-dose Dihuang Yinzi groups(P<0.05,P<0.01).Conclusion:Dihuang Yinzi can improve the nerve function defect of MCAO rat model,and reduce the volume of cerebral infarction and the pathological changes of brain tissue,thus playing a protective role in cerebral ischemiareperfusion injury rats.Its mechanism may be related to the activation of the Notch signaling pathway,and the upregulation of expressions of Notch1,Jagged1 and Hes1 mRNA,thus promoting the proliferation of NSCs.
引文
[1]孙晓杰,郭宝锋,王冰.重组人源Kunitz型蛋白酶抑制剂治疗大鼠脑皮质缺血/再灌注损伤[J].中国实验动物学报,2018,26(3):378-385.
[2]Dewar D,Yam P,McCulloch J.Drug development for stroke:importance of protecting cerebral white matter[J].Eur J Pharmacol,1999,375(1/3):41-50.
[3]Flanders K C,REN R F,Lippa C F.Transforming growth factor-betas in neurodegenerative disease[J].Prog Neurobiol,1998,54(1):71-85.
[4]张玉琴,李鸷,李煌,等.栝楼桂枝颗粒激活Nrf2信号通路减轻脑缺血再灌注损伤大鼠氧化应激损伤作用[J].中国实验方剂学杂志,2017,23(21):112-116.
[5]MING G L,SONG H.Adult neurogenesis in the mammalian central nervous system[J].Annu Rev Neurosci,2005,28:223-250.
[6]覃清霞,仲米存,吴宣,等.神经干细胞迁移研究平台的建立及补阳还五汤的干预影响[J].中国实验方剂学杂志,2017,23(6):131-136.
[7]LIU J,Solway K,Messing R O,et al.Increased neurogenesis in the dentate gyrus after transient global ischemia in gerbils[J].J Neurosci,1998,18(19):7768-7778.
[8]Lasky J L,WU H.Notch signaling,brain development,and human disease[J].Pediatr Res,2005,57(5 Pt 2):104-109.
[9]ZHANG X,HUANG G,LIU H,et al.Folic acid enhances Notch signaling,hippocampal neurogenesis,and cognitive function in a rat model of cerebral ischemia[J].Nutr Neurosci,2012,15(2):55-61.
[10]宫健伟,叶蕾,樊巧玲.地黄饮子对脑缺血再灌注模型大鼠Bax,Bcl-2,Caspase-3蛋白表达的影响[J].中国实验方剂学杂志,2013,19(5):248-251.
[11]李子军,刘春娜.地黄饮子对海马神经元缺氧损伤的保护机制[J].中成药,2012,34(8):1421-1424.
[12]闫妍,韩冉,高俊峰,等.地黄饮子改善AD大鼠脑组织线粒体生物合成与氧化损伤的机制[J].中国实验方剂学杂志,2018,24(21):105-110.
[13]白黎明,张晓双,武苗,等.地黄饮子对慢性脑低灌注大鼠学习记忆及海马ACh E,Ch AT的影响[J].中国实验方剂学杂志,2011,17(7):207-209.
[14]李胜志,李庆云,马睿杰,等.地黄饮子对缺血性脑损伤Nestin mRNA、SCF mRNA影响的实验研究[J].中医药信息,2008,25(1):77-79.
[15]Longa E Z,Weinstein P R,Carlson S,et al.Reversible middle cerebral artery occlusion without craniectomy in rats[J].Stroke,1989,20(1):84-91.
[17]冯珂,纪立金.健脾益智胶囊对MCAO大鼠海马β-catenin表达的影响[J].山东中医药大学学报,2015,39(2):160-162.
[18]程建华,刘双,韩钊,等.姜黄素通过调控Notch通路促进大鼠脑缺血后神经干细胞增殖和迁移[J].中国病理生理杂志,2013,29(5):878-882.
[19]CHEN J,LI Y,WANG L,et al.Therapeutic benefit of intravenous administration of bone marrow stromal cells after cerebral ischemia in rats[J].Stroke,2001,32(4):1005-1011.
[20]CHEN Y,Constantini S,Trembovler V,et al.An experimental model of closed head injury in mice:pathophysiology,histopathology,and cognitive deficits[J].J Neurotrauma,1996,13(10):557-568.
[21]Schallert T,Kozlowski D A,Humm J L,et al.Usedependent structural events in recovery of function[J].Adv Neurol,1997,73:229-238.
[22]LI Y,Chopp M,CHEN J,et al.Intrastriatal transplantation of bone marrow nonhematopoietic cells improves functional recovery after stroke in adult mice[J].J Cereb Blood Flow Metab,2000,20(9):1311-1319.
[23]Hatfield R H,Mendelow A D,Perry R H,et al.Triphenyltetrazolium chloride(TTC)as a marker for ischaemic changes in rat brain following permanent middle cerebral artery occlusion[J].Neuropathol Appl Neurobiol,1991,17(1):61-67.
[24]Swanson R A,Morton M T,Tsao-Wu G,et al.Asemiautomated method for measuring brain infarct volume[J].J Cereb Blood Flow Metab,1990,10(2):290-293.
[25]Livak K J,Schmittgen T D.Analysis of relative gene expression data using Real-time quantitative PCR and the 2(-Delta Delta C(T))Method[J].Methods,2001,25(4):402-408.
[26]王桂倩,谢雁鸣,刘峘,等.真实世界的参芎葡萄糖注射液治疗缺血性脑血管病临床联合用药特征分析[J].中国中药杂志,2017,42(14):2808-2813.
[27]汪红,钟晓明,陈梦静,等.丹酚酸B对急性脑缺血小鼠TLR4/NF-κB信号通路的影响[J].上海中医药大学学报,2016,30(2):57-61.
[28]支艳,杨明会.从补肾活血论治老年病[J].中华中医药杂志,2010,25(1):72-74.
[29]邓中甲.方剂学[M].北京:中国中医药出版社,2009:182.
[30]温彬宇,张志辰,高俊峰,等.地黄饮子抑制能量障碍诱导的APP/PS1小鼠内质网应激及神经元凋亡的作用机制[J].中国实验方剂学杂志,2018,24(21):111-117.
[31]陈攀,徐志伟,敖海清,等.基于海马神经干细胞的增殖与分化机制探讨肝肾“藏泄互用”关系的内涵[J].中华中医药杂志,2013,28(5):1202-1206.
[32]LAI E C.Notch signaling:control of cell communication and cell fate[J].Development,2004,131(5):965-973.
[33]Aster J C,Robertson E S,Hasserjian R P,et al.Oncogenic forms of NOTCH1 lacking either the primary binding site for RBP-Jkappa or nuclear localization sequences retain the ability to associate with RBP-Jkappa and activate transcription[J].J Biol Chem,1997,272(17):11336-11343.
[34]Oswald F,Winkler M,CAO Y,et al.RBP-Jkappa/SHARP recruits Ct IP/Ct BP corepressors to silence Notch target genes[J].Mol Cell Biol,2005,25(23):10379-10390.
[35]WANG X,MAO X,XIE L,et al.Involvement of Notch1signaling in neurogenesis in the subventricular zone of normal and ischemic rat brain in vivo[J].J Cereb Blood Flow Metab,2009,29(10):1644-1654.
[36]WANG L,Chopp M,ZHANG R L,et al.The Notch pathway mediates expansion of a progenitor pool and neuronal differentiation in adult neural progenitor cells after stroke[J].Neuroscience,2009,158(4):1356-1363.
[37]冯珂,纪立金.健脾益智胶囊对大脑中动脉栓塞大鼠海马神经干细胞增殖及Notch1、Jagged1表达的影响[J].中华中医药杂志,2013,28(4):921-924.
[2]Dewar D,Yam P,McCulloch J.Drug development for stroke:importance of protecting cerebral white matter[J].Eur J Pharmacol,1999,375(1/3):41-50.
[3]Flanders K C,REN R F,Lippa C F.Transforming growth factor-betas in neurodegenerative disease[J].Prog Neurobiol,1998,54(1):71-85.
[4]张玉琴,李鸷,李煌,等.栝楼桂枝颗粒激活Nrf2信号通路减轻脑缺血再灌注损伤大鼠氧化应激损伤作用[J].中国实验方剂学杂志,2017,23(21):112-116.
[5]MING G L,SONG H.Adult neurogenesis in the mammalian central nervous system[J].Annu Rev Neurosci,2005,28:223-250.
[6]覃清霞,仲米存,吴宣,等.神经干细胞迁移研究平台的建立及补阳还五汤的干预影响[J].中国实验方剂学杂志,2017,23(6):131-136.
[7]LIU J,Solway K,Messing R O,et al.Increased neurogenesis in the dentate gyrus after transient global ischemia in gerbils[J].J Neurosci,1998,18(19):7768-7778.
[8]Lasky J L,WU H.Notch signaling,brain development,and human disease[J].Pediatr Res,2005,57(5 Pt 2):104-109.
[9]ZHANG X,HUANG G,LIU H,et al.Folic acid enhances Notch signaling,hippocampal neurogenesis,and cognitive function in a rat model of cerebral ischemia[J].Nutr Neurosci,2012,15(2):55-61.
[10]宫健伟,叶蕾,樊巧玲.地黄饮子对脑缺血再灌注模型大鼠Bax,Bcl-2,Caspase-3蛋白表达的影响[J].中国实验方剂学杂志,2013,19(5):248-251.
[11]李子军,刘春娜.地黄饮子对海马神经元缺氧损伤的保护机制[J].中成药,2012,34(8):1421-1424.
[12]闫妍,韩冉,高俊峰,等.地黄饮子改善AD大鼠脑组织线粒体生物合成与氧化损伤的机制[J].中国实验方剂学杂志,2018,24(21):105-110.
[13]白黎明,张晓双,武苗,等.地黄饮子对慢性脑低灌注大鼠学习记忆及海马ACh E,Ch AT的影响[J].中国实验方剂学杂志,2011,17(7):207-209.
[14]李胜志,李庆云,马睿杰,等.地黄饮子对缺血性脑损伤Nestin mRNA、SCF mRNA影响的实验研究[J].中医药信息,2008,25(1):77-79.
[15]Longa E Z,Weinstein P R,Carlson S,et al.Reversible middle cerebral artery occlusion without craniectomy in rats[J].Stroke,1989,20(1):84-91.
[17]冯珂,纪立金.健脾益智胶囊对MCAO大鼠海马β-catenin表达的影响[J].山东中医药大学学报,2015,39(2):160-162.
[18]程建华,刘双,韩钊,等.姜黄素通过调控Notch通路促进大鼠脑缺血后神经干细胞增殖和迁移[J].中国病理生理杂志,2013,29(5):878-882.
[19]CHEN J,LI Y,WANG L,et al.Therapeutic benefit of intravenous administration of bone marrow stromal cells after cerebral ischemia in rats[J].Stroke,2001,32(4):1005-1011.
[20]CHEN Y,Constantini S,Trembovler V,et al.An experimental model of closed head injury in mice:pathophysiology,histopathology,and cognitive deficits[J].J Neurotrauma,1996,13(10):557-568.
[21]Schallert T,Kozlowski D A,Humm J L,et al.Usedependent structural events in recovery of function[J].Adv Neurol,1997,73:229-238.
[22]LI Y,Chopp M,CHEN J,et al.Intrastriatal transplantation of bone marrow nonhematopoietic cells improves functional recovery after stroke in adult mice[J].J Cereb Blood Flow Metab,2000,20(9):1311-1319.
[23]Hatfield R H,Mendelow A D,Perry R H,et al.Triphenyltetrazolium chloride(TTC)as a marker for ischaemic changes in rat brain following permanent middle cerebral artery occlusion[J].Neuropathol Appl Neurobiol,1991,17(1):61-67.
[24]Swanson R A,Morton M T,Tsao-Wu G,et al.Asemiautomated method for measuring brain infarct volume[J].J Cereb Blood Flow Metab,1990,10(2):290-293.
[25]Livak K J,Schmittgen T D.Analysis of relative gene expression data using Real-time quantitative PCR and the 2(-Delta Delta C(T))Method[J].Methods,2001,25(4):402-408.
[26]王桂倩,谢雁鸣,刘峘,等.真实世界的参芎葡萄糖注射液治疗缺血性脑血管病临床联合用药特征分析[J].中国中药杂志,2017,42(14):2808-2813.
[27]汪红,钟晓明,陈梦静,等.丹酚酸B对急性脑缺血小鼠TLR4/NF-κB信号通路的影响[J].上海中医药大学学报,2016,30(2):57-61.
[28]支艳,杨明会.从补肾活血论治老年病[J].中华中医药杂志,2010,25(1):72-74.
[29]邓中甲.方剂学[M].北京:中国中医药出版社,2009:182.
[30]温彬宇,张志辰,高俊峰,等.地黄饮子抑制能量障碍诱导的APP/PS1小鼠内质网应激及神经元凋亡的作用机制[J].中国实验方剂学杂志,2018,24(21):111-117.
[31]陈攀,徐志伟,敖海清,等.基于海马神经干细胞的增殖与分化机制探讨肝肾“藏泄互用”关系的内涵[J].中华中医药杂志,2013,28(5):1202-1206.
[32]LAI E C.Notch signaling:control of cell communication and cell fate[J].Development,2004,131(5):965-973.
[33]Aster J C,Robertson E S,Hasserjian R P,et al.Oncogenic forms of NOTCH1 lacking either the primary binding site for RBP-Jkappa or nuclear localization sequences retain the ability to associate with RBP-Jkappa and activate transcription[J].J Biol Chem,1997,272(17):11336-11343.
[34]Oswald F,Winkler M,CAO Y,et al.RBP-Jkappa/SHARP recruits Ct IP/Ct BP corepressors to silence Notch target genes[J].Mol Cell Biol,2005,25(23):10379-10390.
[35]WANG X,MAO X,XIE L,et al.Involvement of Notch1signaling in neurogenesis in the subventricular zone of normal and ischemic rat brain in vivo[J].J Cereb Blood Flow Metab,2009,29(10):1644-1654.
[36]WANG L,Chopp M,ZHANG R L,et al.The Notch pathway mediates expansion of a progenitor pool and neuronal differentiation in adult neural progenitor cells after stroke[J].Neuroscience,2009,158(4):1356-1363.
[37]冯珂,纪立金.健脾益智胶囊对大脑中动脉栓塞大鼠海马神经干细胞增殖及Notch1、Jagged1表达的影响[J].中华中医药杂志,2013,28(4):921-924.