白杨素对脑缺血-再灌注损伤的神经保护机制研究
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摘要
目的:探索白杨素(Chrysin)对脑缺血-再灌注损伤的神经保护作用及其机制。方法:体外实验通过噻唑兰比色法(MTT比色法)检测白杨素对谷氨酸神经损伤细胞的保护作用;免疫荧光染色检测白杨素诱导后的大鼠骨髓间充质干细胞(Bone marrow mesenchymal stem cells,MSC)相关神经营养因子的表达水平;体内药效学评价采用脑中动脉阻断法构建大鼠局灶性脑缺血再灌注模型(MCAO),对白杨素体内治疗脑缺血再灌注损伤作用进行评价。体内评价指标包括:神经功能评分、脑梗死体积、含水量,HE染色观察病理变化,免疫荧光染色检测大鼠脑组织脑源性神经营养因子(BDNF)、神经生长因子(NGF)的表达水平。结果:白杨素对正常PC12细胞在实验浓度条件下未表现出细胞毒性,且在谷氨酸(10 mM)损伤模型中白杨素(药物浓度为10μM)对损伤细胞具有保护作用;白杨素诱导MSC后不同时间细胞表面神经标志性蛋白:神经生长因子(NGF)、半乳糖神经酰胺(GalC)、巢蛋白(Nestin)、神经元特异烯醇化酶(NSE)、波形蛋白(Vimentin)均有不同程度的表达。体内实验中,与模型组相比,白杨素给药组改善了大鼠的神经功能缺损症状评分,减少脑梗死体积,改善了缺血半暗带损伤情况,上调了大鼠脑组织的神经营养因子NGF、BDNF蛋白的表达。结论:白杨素可通过上调神经营养因子NGF、BDNF表达量而对脑缺血-再灌注损伤具有一定的神经保护作用。
Objective: To explore the neuroprotective effect and mechanism of Chrysin on cerebral ischemia-reperfusion injury. Methods: The protective effect of Chrysin on glutamate-induced nerve injury cells was detected by thiazolyl colorimetry(MTT colorimetry) in vitro. The expression levels of neurotrophic factors related to rat bone marrow mesenchymal stem cells(MSCs) induced by Chrysin were detected by immunofluorescence staining. For in vivo pharmacodynamic evaluation, the rat model of focal cerebral ischemia-reperfusion(MCAO) was established by middle cerebral artery occlusion method, and the effect of in vitro treatment of cerebral ischemia-reperfusion injury was evaluated. In vivo evaluation indicators included neurological function score, cerebral infarction volume, and water content. HE staining was used to observe pathological changes. Immunofluorescence staining was used for detection of brain-derived neurotrophic factor(BDNF) and nerve growth factor(NGF) expression in rat brain tissues. Results: Chrysin(10 μM) did not express non-cytotoxicity to normal PC12 cells at the experimental concentration, but also had protective effects on injured cells which were injured by the glutamate(10 mM). The cell surface neuronal markers NGF, GalC,Nestin, NSE and Vimentin were expressed at different levels after induction of MSC. In vivo, compared with the model group, the Chrysin administration group improved the neurological deficit symptom score in rats, reduced the volume of cerebral infarction; improved the ischemic penumbra damage, and up-regulated the expression of NGF and BDNF factors in rat brains. Conclusion: Chrysin has neuroprotective effects on cerebral ischemia-reperfusion injury by up-regulating the expression of NGF and BDNF factors.
Objective: To explore the neuroprotective effect and mechanism of Chrysin on cerebral ischemia-reperfusion injury. Methods: The protective effect of Chrysin on glutamate-induced nerve injury cells was detected by thiazolyl colorimetry(MTT colorimetry) in vitro. The expression levels of neurotrophic factors related to rat bone marrow mesenchymal stem cells(MSCs) induced by Chrysin were detected by immunofluorescence staining. For in vivo pharmacodynamic evaluation, the rat model of focal cerebral ischemia-reperfusion(MCAO) was established by middle cerebral artery occlusion method, and the effect of in vitro treatment of cerebral ischemia-reperfusion injury was evaluated. In vivo evaluation indicators included neurological function score, cerebral infarction volume, and water content. HE staining was used to observe pathological changes. Immunofluorescence staining was used for detection of brain-derived neurotrophic factor(BDNF) and nerve growth factor(NGF) expression in rat brain tissues. Results: Chrysin(10 μM) did not express non-cytotoxicity to normal PC12 cells at the experimental concentration, but also had protective effects on injured cells which were injured by the glutamate(10 mM). The cell surface neuronal markers NGF, GalC,Nestin, NSE and Vimentin were expressed at different levels after induction of MSC. In vivo, compared with the model group, the Chrysin administration group improved the neurological deficit symptom score in rats, reduced the volume of cerebral infarction; improved the ischemic penumbra damage, and up-regulated the expression of NGF and BDNF factors in rat brains. Conclusion: Chrysin has neuroprotective effects on cerebral ischemia-reperfusion injury by up-regulating the expression of NGF and BDNF factors.
引文
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2 Richard M,Carine A,Olivier T,et al.Stroke and the immune system:from pathophysiology to new therapeutic strategies.Lancet Neurol,2011,10(5):471
3中华医学会神经病学分会.中国急性缺血性脑卒中诊治指南2014.中华神经科杂志,2015,48(4):246-257.
4张雯,宋俊科,杜立达,等.急性脑缺血治疗药物研究进展.神经药理学报,2014,4(4):50-58.
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7 Bonilla IE,Tanabe K,Sm S.Small proline-rich repeat 1A is expressed by axotomized neurons and promotes axonal outgrowth.J Neurosci,2002,22(4):1303-1315.
8 Lin B,Pirrung MC,Deng L,et al.Neuroprotection by small molecule activators of the nerve growth factor receptor.J Pharmacol Exp Ther,2007,322(1):59-69.
9 Capsoni S,Covaceuszach S,Ugolini G,et al.Delivery of NGF to the brain:intranasal versus ocular administration in anti-NGF transgenic mice.J Alzheimers Dis,2009,16(2):371-388.
10姜金生,韦英杰,贾晓斌,等.白杨素及其衍生物的药理作用和构效关系研究进展.中草药,2011,42(11):2345-2350.
11马慧萍,吴金华,蒙萍,等.白杨素对大鼠急慢性脑缺血损伤后氧化应激和Nrf2/HO-1途径的影响.解放军医药杂志,2015,27(12):15-20.
12赵煦萌.白杨素对实验性脑梗死大鼠脑保护作用及其机制的研究.石家庄:河北医科大学硕士研究生学位论文,2013.
13郑永唐,贲昆龙.测定细胞存活和增殖的MTT方法的建立.免疫学杂志,1992(4):266-269.
14林军,李艳芳,李冲,等.线栓法大鼠MCAO模型制作的要点及经验总结.医学综述,2018,24(17):3398-3402+3408.
15刘俊伟,任冶龙,刘旭玲,等.人参皂苷Rb1对大鼠局灶性脑缺血再灌注损伤后脑梗死体积及脑组织和血清IL-1β的影响.中国中西医结合杂志,2013,33(12):1696-1700.
16周飞,李福凤,程介士,等.电针对急性脑缺血大鼠局部脑血流和脑梗死体积的影响.上海针灸杂志,2003,22(5):3-6.
17叶龙彬,奚涛,陈峰,等.丹参酮ⅡA对大鼠局灶性脑缺血再灌注损伤的保护作用.中国药科大学学报,2004(3):73-76.
18 Grossman AW,Broderick JP.Advances and challenges in treatment and prevention of ischemic stroke.Ann Neurol,2013,74(3):363-372.
19赖文芳,红景天苷对脑缺血再灌注损伤大鼠的保护作用及机制研究.福建:福建中医药大学博士研究生学位论文,2016:1-4
20莲花,麻春杰,呼日乐巴根,等.额尔敦-乌日勒对MCAO/R大鼠脑前额叶皮质BDNF及NGF表达的影响.世界科学技术-中医药现代化,2016,18(7):1212-1218.
21 Hsu CY,An G,Liu JS,et al.Expression of immediate early gene and growth factor mRNAs in a focal cerebral ischemia model in the rat.Stroke,1993,24(12):178-181.
2 Richard M,Carine A,Olivier T,et al.Stroke and the immune system:from pathophysiology to new therapeutic strategies.Lancet Neurol,2011,10(5):471
3中华医学会神经病学分会.中国急性缺血性脑卒中诊治指南2014.中华神经科杂志,2015,48(4):246-257.
4张雯,宋俊科,杜立达,等.急性脑缺血治疗药物研究进展.神经药理学报,2014,4(4):50-58.
5 Emanueli C,Salis MB,Pinna A,et al.Nerve growth factor promotes angiogenis and arteriogenesis in ischemic hind limbs.Circulation,2002,106(17):225.
6 Yang JP,Liu HJ,Yang H,et al.Therapeutic time window for the neuroprotective effects of NGF when administered after focal cerebral ischemia.Neurol Sci,2011,32(3):433-441.
7 Bonilla IE,Tanabe K,Sm S.Small proline-rich repeat 1A is expressed by axotomized neurons and promotes axonal outgrowth.J Neurosci,2002,22(4):1303-1315.
8 Lin B,Pirrung MC,Deng L,et al.Neuroprotection by small molecule activators of the nerve growth factor receptor.J Pharmacol Exp Ther,2007,322(1):59-69.
9 Capsoni S,Covaceuszach S,Ugolini G,et al.Delivery of NGF to the brain:intranasal versus ocular administration in anti-NGF transgenic mice.J Alzheimers Dis,2009,16(2):371-388.
10姜金生,韦英杰,贾晓斌,等.白杨素及其衍生物的药理作用和构效关系研究进展.中草药,2011,42(11):2345-2350.
11马慧萍,吴金华,蒙萍,等.白杨素对大鼠急慢性脑缺血损伤后氧化应激和Nrf2/HO-1途径的影响.解放军医药杂志,2015,27(12):15-20.
12赵煦萌.白杨素对实验性脑梗死大鼠脑保护作用及其机制的研究.石家庄:河北医科大学硕士研究生学位论文,2013.
13郑永唐,贲昆龙.测定细胞存活和增殖的MTT方法的建立.免疫学杂志,1992(4):266-269.
14林军,李艳芳,李冲,等.线栓法大鼠MCAO模型制作的要点及经验总结.医学综述,2018,24(17):3398-3402+3408.
15刘俊伟,任冶龙,刘旭玲,等.人参皂苷Rb1对大鼠局灶性脑缺血再灌注损伤后脑梗死体积及脑组织和血清IL-1β的影响.中国中西医结合杂志,2013,33(12):1696-1700.
16周飞,李福凤,程介士,等.电针对急性脑缺血大鼠局部脑血流和脑梗死体积的影响.上海针灸杂志,2003,22(5):3-6.
17叶龙彬,奚涛,陈峰,等.丹参酮ⅡA对大鼠局灶性脑缺血再灌注损伤的保护作用.中国药科大学学报,2004(3):73-76.
18 Grossman AW,Broderick JP.Advances and challenges in treatment and prevention of ischemic stroke.Ann Neurol,2013,74(3):363-372.
19赖文芳,红景天苷对脑缺血再灌注损伤大鼠的保护作用及机制研究.福建:福建中医药大学博士研究生学位论文,2016:1-4
20莲花,麻春杰,呼日乐巴根,等.额尔敦-乌日勒对MCAO/R大鼠脑前额叶皮质BDNF及NGF表达的影响.世界科学技术-中医药现代化,2016,18(7):1212-1218.
21 Hsu CY,An G,Liu JS,et al.Expression of immediate early gene and growth factor mRNAs in a focal cerebral ischemia model in the rat.Stroke,1993,24(12):178-181.