化瘀祛痰方对血管性痴呆的保护作用机制探讨
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摘要
目的:研究化瘀祛痰方对血管性痴呆(VD)沙鼠的保护作用,并初步探讨其作用机制是否与钙离子-钙调素依赖性蛋白激酶Ⅱ(Ca MKⅡ)/环磷腺苷效应元件结合蛋白(CREB)/脑源性神经营养因子(BDNF)信号通路相关。方法:将健康沙鼠40只,随机分为假手术组,模型组,化瘀祛痰方低、中、高(5. 35,10. 7,21. 4 g·kg~(-1))剂量组,每组8只,模型组及化瘀祛痰三组参考Levine法对全部沙鼠进行侧颈总动脉结扎再灌注法造VD模型;假手术组则不封闭颈部动脉,其他操作同手术组。术后沙鼠给予相应药物灌胃2次/日,21 d后进行水迷宫实验考察沙鼠的空间学习记忆能力。蛋白免疫印迹法(Western blot)及免疫组化分别测定沙鼠脑组织海马体的磷酸化(p)-Ca MKⅡ/Ca MKⅡ,p-CREB/CREB,BDNF蛋白的表达。结果:与假手术组比较,模型组沙鼠的上台潜伏期和平台穿梭次数明显降低,p-CaMKⅡ/Ca MKⅡ,p-CREB/CREB,BDNF蛋白表达均明显降低(P <0. 01);与模型组比较,化瘀祛痰方组沙鼠的上台潜伏期和平台穿梭次数明显升高,其中化瘀祛痰方中以高剂量组效果最为明显(P <0. 01),化瘀祛痰方可以有效的保护CA1区锥体细胞,明显上调p-CaMKⅡ/Ca MKⅡ,p-CREB/CREB,BDNF蛋白的表达(P <0. 01)。结论:化瘀祛痰方改善了血管性痴呆沙鼠的学习和记忆能力,作用机制可能与Ca MKⅡ/CREB/BDNF信号通路的激活相关。
Objective: To study the protective effect of Huayu Qutan decoction on vascular dementia( VD) gerbils and to explore whether its mechanism is related to Calcium ion-calmodulin-dependent protein kinaseⅡ( CaMK Ⅱ)/cyclic adenosine effect element binding protein( CREB)/brain-derived neurotrophic factor( BDNF) signaling pathway. Method: Forty healthy gerbils were randomly divided into sham operation group,model group,low,medium and high dose groups( 5. 35,10. 7,21. 4 g·kg~(-1)) of removing blood stasis and expelling phlegm. Eight gerbils in each group were divided into model group and removing blood stasis and expelling phlegm group. Gerbils were given corresponding drugs twice a day after operation. Water maze experiment was conducted 21 days later to investigate the spatial learning and memory ability of gerbils. The expression of p-CaMK Ⅱ/CaMK Ⅱ,p-CREB/CREB and BDNF in the hippocampus of gerbils were detected by Western blot and immunohistochemistry. Result: Compared with sham operation group,the incubation period and the number of platform trips of gerbil in the model group were significantly reduced,p-CaMK Ⅱ/CaMK Ⅱ,pCREB/CREB,and BDNF protein expression were significantly reduced( P < 0. 01). Compared with model group,the incubation period and the number of platform shuttling of gerbil in Huayu Qutan decoction group were significantly increased,among which the high-dose phlegm dispelling prescription had the most significant effect( P < 0. 01). The phlegm dispelling prescription could effectively protect pyramidal cells in CA1 region,significantly up-regulating the expression of p-CaMKⅡ/CaMKⅡ,p-CREB/CREB and BDNF protein( P < 0. 01).Conclusion: Huayu Qutan decoction improves the learning and memory abilities of gerbils with vascular dementia,and its mechanism may be related to the activation of CaMKⅡ/CREB/BDNF signaling pathway.
Objective: To study the protective effect of Huayu Qutan decoction on vascular dementia( VD) gerbils and to explore whether its mechanism is related to Calcium ion-calmodulin-dependent protein kinaseⅡ( CaMK Ⅱ)/cyclic adenosine effect element binding protein( CREB)/brain-derived neurotrophic factor( BDNF) signaling pathway. Method: Forty healthy gerbils were randomly divided into sham operation group,model group,low,medium and high dose groups( 5. 35,10. 7,21. 4 g·kg~(-1)) of removing blood stasis and expelling phlegm. Eight gerbils in each group were divided into model group and removing blood stasis and expelling phlegm group. Gerbils were given corresponding drugs twice a day after operation. Water maze experiment was conducted 21 days later to investigate the spatial learning and memory ability of gerbils. The expression of p-CaMK Ⅱ/CaMK Ⅱ,p-CREB/CREB and BDNF in the hippocampus of gerbils were detected by Western blot and immunohistochemistry. Result: Compared with sham operation group,the incubation period and the number of platform trips of gerbil in the model group were significantly reduced,p-CaMK Ⅱ/CaMK Ⅱ,pCREB/CREB,and BDNF protein expression were significantly reduced( P < 0. 01). Compared with model group,the incubation period and the number of platform shuttling of gerbil in Huayu Qutan decoction group were significantly increased,among which the high-dose phlegm dispelling prescription had the most significant effect( P < 0. 01). The phlegm dispelling prescription could effectively protect pyramidal cells in CA1 region,significantly up-regulating the expression of p-CaMKⅡ/CaMKⅡ,p-CREB/CREB and BDNF protein( P < 0. 01).Conclusion: Huayu Qutan decoction improves the learning and memory abilities of gerbils with vascular dementia,and its mechanism may be related to the activation of CaMKⅡ/CREB/BDNF signaling pathway.
引文
[1]陈梅,王立新.中药联合干细胞疗法治疗缺血性脑卒中的研究进展[J].中国实验方剂学杂志,2017,23(19):218-226.
[2]曲艳吉,卓琳,王华丽,等. 1980-2011年中国社区55岁及以上人群中血管性痴呆流行病学的Meta分析[J].中国卒中杂志,2013,8(7):533-543.
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[11]张久亮,柳翼,史载祥,等.缺血性脑卒中的中医病因病机再认识[J].中医杂志,2008,49(6):569-570.
[12] ZHANG Y,QIAO L,XU W, et al. Paeoniflorin attenuates cerebral ischemia-induced injury by regulating Ca2+/camkii/creb signaling pathway[J]. Molecules,2017,22(3):359.
[13] QU L,GAO Y,SUN H,et al. Role of pten-akt-creb signaling pathway in nervous system impairment of rats with chronic arsenite exposure[J]. Biol Trace Elem Res,2016,170(2):366.
[14] Schmid R S,Graff R D,Schaller M D,et al. Ncam stimulates the ras-mapk pathway and creb phosphorylation in neuronal cells[J]. Develop Neurobiol,2015,38(4):542-558.
[15] ZHU Y P,XI S H,LI M Y,et al. Fluoride and arsenic exposure affects spatial memory and activates the Erk/creb signaling pathway in offspring rats[J].Neurotoxicology,2017,59(3):56-64.
[16]荆玉谱.蜕皮激素引发的钙离子/钙调蛋白依赖的蛋白激酶Ⅱ信号转导途径以及蛋白激酶A信号转导途径对基因组转录的调控[D].济南:山东大学,2015.
[17] Vaynman S,YING Z,Gomez‐pinilla F. Hippocampal Bdnf mediates the efficacy of exercise on synaptic plasticity and cognition[J]. Eur J Neurosci,2015,20(10):2580-2590.
[18] DUAN S,WANG T,ZHANG J,et al. Huatuo Zaizao pill promotes functional recovery and neurogenesis after cerebral ischemia-reperfusion in rats[J]. BMC Complement Altern Med,2017,17(1):19.
[19]贾连群,杨关林,任路,等.化瘀祛痰方药对H2O2诱导人脐带静脉内皮细胞氧化应激保护作用的研究[J].中国中医药信息杂志,2011,18(9):44-45.
[20]赵钢,杨关林,李志明.化瘀祛痰颗粒剂对血管内皮细胞表达MCP-1的影响研究[J].实用中医内科杂志,2011,25(5):3-6.
[2]曲艳吉,卓琳,王华丽,等. 1980-2011年中国社区55岁及以上人群中血管性痴呆流行病学的Meta分析[J].中国卒中杂志,2013,8(7):533-543.
[3] XU W L,QIU C X,Gatz M,et al. Mid-and late-life diabetes in relation to the risk of dementia:a populationbased twin study[J]. Diabetes,2009,58(1):71-77.
[4] Yamagata Y,Kobayashi S,Umeda T,et al. Kinasedead knock-in mouse reveals an essential role of kinase activity of Ca2+/calmodulin-dependent protein kinase Iiαin dendritic spine enlargement, long-term potentiation,and learning[J]. J Neurosci,2009,29(23):7607-7618.
[5] Morioka M,Fukunaga K,Yasugawa S,et al. Regional and Temporal Alterations in Ca2+/calmodulin-dependent protein kinase Ii and calcineurin in the hippocampus of rat brain after transient forebrain ischemia[J]. J Neurochem,1992,58(5):1798-1809.
[6] Marie H,Morishita W,YU X,et al. Generation of silent synapses by acute in vivo expression of camkiv and creb[J]. Neuron,2005,45(5):741-752.
[7] Shieh P B,HU S C,Bobb K,et al. Identification of a signaling pathway involved in calcium regulation of BDNF expression[J]. Neuron,1998,20(4):727-740.
[8]贾连群,杨关林,任路,等.化瘀祛痰方药及其拆方含药血清对脂多糖诱导的人脐静脉内皮细胞炎症因子表达的影响[J].中国实验方剂学杂志,2011,17(7):144-148.
[9]贾连群,杨关林,任路,等.化瘀祛痰方药及其拆方对内皮细胞TLR4/NF-κB炎症信号通路干预的研究[J].中国老年学杂志,2012,32(23):5159-5161.
[10]王齐.二苯乙烯苷对改善全脑缺血再灌注致沙鼠海马损伤的研究[D].合肥:安徽医科大学,2013.
[11]张久亮,柳翼,史载祥,等.缺血性脑卒中的中医病因病机再认识[J].中医杂志,2008,49(6):569-570.
[12] ZHANG Y,QIAO L,XU W, et al. Paeoniflorin attenuates cerebral ischemia-induced injury by regulating Ca2+/camkii/creb signaling pathway[J]. Molecules,2017,22(3):359.
[13] QU L,GAO Y,SUN H,et al. Role of pten-akt-creb signaling pathway in nervous system impairment of rats with chronic arsenite exposure[J]. Biol Trace Elem Res,2016,170(2):366.
[14] Schmid R S,Graff R D,Schaller M D,et al. Ncam stimulates the ras-mapk pathway and creb phosphorylation in neuronal cells[J]. Develop Neurobiol,2015,38(4):542-558.
[15] ZHU Y P,XI S H,LI M Y,et al. Fluoride and arsenic exposure affects spatial memory and activates the Erk/creb signaling pathway in offspring rats[J].Neurotoxicology,2017,59(3):56-64.
[16]荆玉谱.蜕皮激素引发的钙离子/钙调蛋白依赖的蛋白激酶Ⅱ信号转导途径以及蛋白激酶A信号转导途径对基因组转录的调控[D].济南:山东大学,2015.
[17] Vaynman S,YING Z,Gomez‐pinilla F. Hippocampal Bdnf mediates the efficacy of exercise on synaptic plasticity and cognition[J]. Eur J Neurosci,2015,20(10):2580-2590.
[18] DUAN S,WANG T,ZHANG J,et al. Huatuo Zaizao pill promotes functional recovery and neurogenesis after cerebral ischemia-reperfusion in rats[J]. BMC Complement Altern Med,2017,17(1):19.
[19]贾连群,杨关林,任路,等.化瘀祛痰方药对H2O2诱导人脐带静脉内皮细胞氧化应激保护作用的研究[J].中国中医药信息杂志,2011,18(9):44-45.
[20]赵钢,杨关林,李志明.化瘀祛痰颗粒剂对血管内皮细胞表达MCP-1的影响研究[J].实用中医内科杂志,2011,25(5):3-6.
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