海风藤水煎剂对纤维状Aβ_(1-42)介导的b End.3细胞损伤的影响
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摘要
目的:探究海风藤水煎剂对纤维状Aβ_(1-42)介导的b End.3细胞损伤的影响及可能机制。方法:将b End.3细胞分为阴性对照组,模型组(Aβ_(1-42)20μmol/L),海风藤水煎剂低中高剂量组(Aβ_(1-42)20μmol/L+海风藤0.5,1,5 mg/m L);(1)利用噻唑蓝(MTT)法检测海风藤水煎剂对Aβ_(1-42)介导的b End.3细胞活力的影响;(2)利用Hoechst33258染色法鉴定b End.3细胞的凋亡情况;(3)Western Blot法检测死亡受体凋亡信号通路相关蛋白Caspase-8,Cleaved Caspase-8,Caspase-3及Cleaved Caspase-3的表达水平。结果:MTT与Hoechst33258染色结果显示,与模型组相比,海风藤低、中、高剂量组能显著提高b End.3细胞活性,抑制Aβ_(1-42)介导的b End.3细胞凋亡(P<0.05),且保护作用与海风藤水煎剂浓度成正相关;Western blot显示,与模型组相比,海风藤预处理后,可明显减Cleaved Caspase-8/Caspase-8、Cleaved Caspase-3/Caspase-3的比值(P<0.05)。结论:海风藤能够提高b End.3细胞可能通过抑制Aβ_(1-42)诱导的死亡受体信号通路发挥其神经保护作用。
Objective:To uncover the effect of haifengteng on the damaged mouse brain microvascular endothelial cells(b End.3)induced by Aβ_(1-42)and explore the mechanism of its protection.Methods:b End.3 cells were divided into five groups:control,model(Aβ_(1-42)20μmol·L~(-1)),haifengteng low,middle,high dose groups(Aβ_(1-42)20μmol/L with haifengteng 0.5,1,5 mg·m L~(-1)).MTT assay to detect the effect of haifengteng or with Aβ_(1-42)on cells activity,respectively;Hoechst33258 staining was used to evaluate the effect of haifengteng on apoptosis in b End.3 cells induced by Aβ_(1-42).Western blot method was to detect the related proteins levels of apoptosis:Caspase-8,Cleaved Caspase-8,Caspase-3,Cleaved Caspase-3.Results:The result of MTT showed haifengteng had non-toxic side effect to b End.3 cells(P>0.05).MTT and Hoechst33258 staining results all showed that haifengteng low,middle and high dose groups could significantly improve the activity of b End.3 cells and inhibit the apoptosis induced by Aβ_(1-42)verse model(P<0.05).The result of Western blot showed that the ratio of Cleaved Caspase-8/Caspase-8 and Cleaved Caspase-3/Caspase-3 obviously reduce verse model after pretreated with haifengteng(P<0.05).Conclusion:Haifengteng may exert its nerve protective effect via inhibiting the death receptor apoptosis pathway induced by Aβ_(1-42).
Objective:To uncover the effect of haifengteng on the damaged mouse brain microvascular endothelial cells(b End.3)induced by Aβ_(1-42)and explore the mechanism of its protection.Methods:b End.3 cells were divided into five groups:control,model(Aβ_(1-42)20μmol·L~(-1)),haifengteng low,middle,high dose groups(Aβ_(1-42)20μmol/L with haifengteng 0.5,1,5 mg·m L~(-1)).MTT assay to detect the effect of haifengteng or with Aβ_(1-42)on cells activity,respectively;Hoechst33258 staining was used to evaluate the effect of haifengteng on apoptosis in b End.3 cells induced by Aβ_(1-42).Western blot method was to detect the related proteins levels of apoptosis:Caspase-8,Cleaved Caspase-8,Caspase-3,Cleaved Caspase-3.Results:The result of MTT showed haifengteng had non-toxic side effect to b End.3 cells(P>0.05).MTT and Hoechst33258 staining results all showed that haifengteng low,middle and high dose groups could significantly improve the activity of b End.3 cells and inhibit the apoptosis induced by Aβ_(1-42)verse model(P<0.05).The result of Western blot showed that the ratio of Cleaved Caspase-8/Caspase-8 and Cleaved Caspase-3/Caspase-3 obviously reduce verse model after pretreated with haifengteng(P<0.05).Conclusion:Haifengteng may exert its nerve protective effect via inhibiting the death receptor apoptosis pathway induced by Aβ_(1-42).
引文
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[2]Axel M,Samuel RB,Melanie DS.Blood-Brain Barrier Breakdown in the Aging Human Hippocampus[J].Neuron January,2015,85(2):296-302.
[3]Zhen Z,Amy RN,Christer B,et al.Establishment and Dysfunction of the Blood-Brain Barrier[J].J Cell,2015,163(5):1064-1078.
[4]Clifford PM,Zarrabi S,Siu G,et al.Abeta peptides can enter the brain through a defective blood-brain barrier and bind selectively to neurons[J].Brain Res,2007,1142:223-236.
[5]何芳,尹飞,彭镜,等.永生化小鼠bEnd.3细胞株具有脑微血管内皮细胞的屏障特性[J].中国当代儿科杂志,2010(6):474-478.
[6]史国娟,贺燕勤,于顾然.补肾益精方含药血清对D-半乳糖致原代培养神经元损伤的影响[J].江苏中医,2015,47(2):80-82.
[7]王丹丹,史国娟,于顾然.补肾益精方含药血清对β-淀粉样蛋白干预血脑屏障体外模型渗透性的影响[J].江苏中医,2015,47(10):72-74.
[8]Kuo YC,Yang NS,Chou CJ,et al.Regulation of Cell Prolife ration,Gene Expression,Production of Cytokines,and Cell Cycle Progression in Primary Human TLymphicytes by Pipelactam SIsolated from Piperkadsura[J].Mol Pharmacol,2000,58(5):1057-1066.
[9]Stohr JR,Xiao PG,Bauer R.Constituents of Chinese Piperspeciesand their inhibitory activity on prostaglandin and leukotrienebio synthesis in vitro[J].Journal of Ethno pharmacology,2001,75(2-3):133-139.
[10]王林,姚俊,耿智敏.海风藤酮治疗大鼠实验性急性胰腺炎[J].第四军医大学学报,2006,27(13):1166-1168.
[11]Tsai JY,Chou CJ,Chen CF,et al.Anti-oxidant activity of pipelactam S:prevention of copper-induced LDL peroxidation an damelioration of free radical-induced oxidative stress of endotherlial cells[J].Planta Med,2003,69(1):3.
[12]韩恩吉,胡洪涛,何秀全,等.海风藤抑制淀粉样前体蛋白基因表达的研究[J].中国中药杂志,1998,23(11):691.
[13]罗焕敏,肖飞,李晓光.广东海风藤提取物对痴呆鼠脑内β淀粉样前体蛋白基因表达的影响[J].中国老年学杂志,2003,23(6):360-361.
[14]Han EJ,Hu HT,He XQ,et al.Selective inhibition of haifengtengin gene expression of beta-amyloid precursor protein[J].Chinese Journal of Clinical Rehabiliatation,2004,8(1):2592-2593.
[15]朱经艳,张春凤,杨中.海风藤对叠氮钠诱导的PC12细胞损伤的保护作用[J].中医药学报,2011,3(39):50-52.
[16]韩恩吉,许军,Rajiv Joseph.海风藤抑制淀粉样蛋白诱导神经细胞胞浆钙离子升高的研究[J].山东医科大学学报,1998,3(36):239-241.
[17]Jekabsone A,Mander PK,Tickler A,et al.Fibrillar beta-amyloid peptide Aβ1-40 activates microglial proliferation via stimulating TNF-αrelease and H2O2-derived from NADPH oxidase:a cell culture study[J].Journal of Neuroiflammation,2006,3:24.
[18]陆韵薇,于顾然,符布清.生地黄水煎剂对H2O2诱导的PC12细胞凋亡的保护作用研究[J].江苏中医,2016,48(10):107-109.
[19]Buchhave P,Minthon L,Zetterberg H,et al.Cerebrospinal fluid levels ofβ-amyloid 1-42,but not of tau,are fully changed already 5to 10 years before the onset of Alzheimer dementia[J].Arch Gen Psychiatry,2012,69(1):98-106.
[20]Choi SH,Kim YH,Hebisch M,et al.A three-dimensional human neural cell culture model of Alzheimer's disease[J].Nature,2014,515(7526):274-278.
[21]Feng J,Meng C,Xing D.Abeta induces PUMA activation:a new mechanism for Abeta-mediated neuronal apoptosis[J].Neurobiology of aging,2015,36(2):789-800.
[22]Folin M,Baiguera S,Fioravanzo L,et al.Caspase-8 activation and oxidative stress are involved in the cytotoxic effect of beta-amyloid on rat brain microvascular endothelial cells[J].Int J Mol Med.2006,17(3):431-435.
[23]Zhao L,Qian ZM,Zhang C,et al.Amyloid beta-peptide 31-35-induced neuronal apoptosis is mediated by Caspase-dependent pathways via c AMP-dependent protein kinase A activation[J].Aging cell,2008,7(1):47-57.