清心开窍方对Aβ_(25-35)诱导原代海马神经元损伤IGF-1R、IRS-1及Aβ mRNA表达的影响
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摘要
目的:研究清心开窍方对Aβ_(25-35)诱导原代海马神经元细胞损伤胰岛素样生长因子1受体(insulinlike growth factor 1 receptor,IGF-1R)、胰岛素受体底物蛋白1(insulin receptor substrate-1,IRS-1)及β-淀粉样蛋白(β-amyloid,Aβ)基因表达的影响,探讨清心开窍方对神经元细胞的保护作用及其机制。方法:制备SD乳鼠原代海马神经元细胞,分为6组:正常对照组、模型组、尼莫地平组(1×107mol·L~(-1))、清心开窍方低、中、高剂量组(6.25、12.5、25 mg/m L)。支持培养至第7天,各治疗组分别给予相应药物预处理24 h后,模型组及各治疗组加入老化处理的Aβ_(25-35)(终浓度为10μmol·L~(-1))共同作用24 h,建立AD细胞模型。正常对照组给予相等体积的培养液继续孵育24 h。采用CCK-8法检测各组的细胞活力,RT-PCR法检测各组IGF-1R、IRS-1和AβmRNA的表达水平。结果:模型组与正常对照组比较细胞活性明显降低(P<0.01),而清心开窍方能有效改善细胞活性;RT-PCR法结果显示模型组IGF-1R mRNA明显降低(P<0.01),IRS-1和AβmRNA表达均明显增加(P<0.01),而不同浓度的清心开窍方处理后均能提高IGF-1R mRNA的表达(P<0.01或P<0.05),减少IRS-1和AβmRNA表达(P<0.01或P<0.05)。结论:清心开窍方能减轻β-淀粉样蛋白对神经元细胞的毒性作用,提高细胞存活率,保护海马神经元细胞。
Objective: To study the effect of Qingxin Kaiqiao Recipe on the expressions of IGF1 R,IRS1 and Aβ mRNA in Aβ_(25-35) injuried primary rats hippocampus neurons and explore the protective effect of Qingxin Kaiqiao Recipe on neurons and its mechanism of anti-AD formation. Methods: Get hippocampus neurons of newborn Sprague-Dawley( SD) rats for vitro culture,and they were divided into 6 groups: normal model,Nimodipine group,Qingxin Kaiqiao Decoction low,middle and high dose groups( 6. 25 mg · mL~(-1),12. 5 mg·mL~(-1),25 mg·mL~(-1)). Different doses of Qingxin Kaiqiao Decoction groups and Nimodipine group were pre-incubated for 24 h. Then all of groups expect normal group were cultured with Aβ_(25-35)( final concentration of 10 μmol·L-1) to establish the neuron cell injury,and the normal group was given an equal volume of culture medium and incubated for 24 h. After 24 h,the cell viability was detected by CCK-8 method,and the expressions of IGF-1 R,IRS-1 and Aβ mRNA were detected by RT-PCR. Results:Compared with the normal control group,the neurons activity was significantly lower in the model group( P < 0. 01),and Qingxin Kaiqiao Decoction can significantly improve it. The results of RT-PCR showed that the expression of IGF-1 R mRNA was significantly decreased and IRS-1 and Aβ mRNA were increased in model group compared with those of the normal control group( P < 0. 01),while the different doses of Qingxin Kaiqiao Decoction groups could increase IGF-1 R mRNA,and reduce the expressions of IRS-1 and Aβ mRNA( P < 0. 01 or P < 0. 05). Conclusion: Qingxin Qingxin Decoction can alleviate the toxicity of β-amyloid on neuronal cells,increase cell survival rate and protect hippocampal neurons.
Objective: To study the effect of Qingxin Kaiqiao Recipe on the expressions of IGF1 R,IRS1 and Aβ mRNA in Aβ_(25-35) injuried primary rats hippocampus neurons and explore the protective effect of Qingxin Kaiqiao Recipe on neurons and its mechanism of anti-AD formation. Methods: Get hippocampus neurons of newborn Sprague-Dawley( SD) rats for vitro culture,and they were divided into 6 groups: normal model,Nimodipine group,Qingxin Kaiqiao Decoction low,middle and high dose groups( 6. 25 mg · mL~(-1),12. 5 mg·mL~(-1),25 mg·mL~(-1)). Different doses of Qingxin Kaiqiao Decoction groups and Nimodipine group were pre-incubated for 24 h. Then all of groups expect normal group were cultured with Aβ_(25-35)( final concentration of 10 μmol·L-1) to establish the neuron cell injury,and the normal group was given an equal volume of culture medium and incubated for 24 h. After 24 h,the cell viability was detected by CCK-8 method,and the expressions of IGF-1 R,IRS-1 and Aβ mRNA were detected by RT-PCR. Results:Compared with the normal control group,the neurons activity was significantly lower in the model group( P < 0. 01),and Qingxin Kaiqiao Decoction can significantly improve it. The results of RT-PCR showed that the expression of IGF-1 R mRNA was significantly decreased and IRS-1 and Aβ mRNA were increased in model group compared with those of the normal control group( P < 0. 01),while the different doses of Qingxin Kaiqiao Decoction groups could increase IGF-1 R mRNA,and reduce the expressions of IRS-1 and Aβ mRNA( P < 0. 01 or P < 0. 05). Conclusion: Qingxin Qingxin Decoction can alleviate the toxicity of β-amyloid on neuronal cells,increase cell survival rate and protect hippocampal neurons.
引文
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[19]Jeong S.Molecular and Cellular Basis of Neurodegeneration in Alzheimer’s Disease[M].Molecules&Cells,2017.
[20]古文娟,刘頔,张孟仁,等.人参皂苷Rb1对高糖培养海马神经元胰岛素信号转导途径的影响[J].中国中药杂志,2014,39(6):1064-1068.
[21]Li H,Song Y,Jian W,et al.c AMP/PKA signaling pathway contributes to neuronal apoptosis via regulating IDE expression in a mixed model of type 2 diabetes and Alzheimer’s disease[M].Journal of Cellular Biochemistry,2017.
[2]Alford S,Patel D,Perakakis N,et al.Obesity as a risk factor for Alzheimer’s disease:weighing the evidence[J].Obesity Reviews,2017(Suppl 5):519-522.
[3]Laws S M,Gaskin S,Woodfield A,et al.Insulin resistance is associated with reductions in specific cognitive domains and increases in CSF tau in cognitively normal adults[J].Scientific Reports,2017,7(1):9766.
[4]Talbot K,Wang H Y,Kazi H,et al.Demonstrated brain insulin resistance in Alzheimer’s disease patients is associated with IGF-1 resistance,IRS-1 dysregulation,and cognitive decline[J].Journal of Clinical Investigation,2012,122(4):1316-1338.
[5]王宇锋.颜乾麟运用清法治疗阿尔茨海默病的经验[J].江苏中医药,2004,25(3):10-11.
[6]洪文.张景岳关于痴呆病的学术思想[J].河北中医,2000(7):551-552.
[7]颜乾麟,邢斌.阿尔茨海默病中医治疗目的和方法的拓展[J].中医杂志,2003,44(10):725-726.
[8]胡玉萍,王平,孔明望,等.从痰热论治老年痴呆[J].中医杂志,2013,54(12):1071-1072.
[9]王逸如,张益慧,陈志裕,等.清心开窍方有效成分对AD大鼠学习记忆能力及其海马区Bax、Bcl-2、Caspase-3和βAPP表达的影响[J].中国中西医结合杂志,2017(2):189-197.
[10]Hu H Y,Cui Z H,Li H Q,et al.Fumanjian,a Classic Chinese Herbal Formula,Can Ameliorate the Impairment of Spatial Learning and Memory through Apoptotic Signaling Pathway in the Hippocampus of Rats with Aβ1-40-Induced Alzheimer’s Disease[J].Evidence-based complementary and alternative medicine:e CAM,2014,2014:942917.
[11]Hu H,Wang Y,Zhang Y,et al.Effect of Qingxinkaiqiao compound on cortical mRNA expression of the apoptosis-related genes Bcl-2,BAX,caspase-3,and Aβin an Alzheimer’s disease rat model[J].中医杂志(英文版),2016,36(5):654-662.
[12]叶光华,胡海燕,陈翔,等.清心开窍方含药脑脊液对Aβ25-35所致PC12细胞损伤的保护作用[J].中国中药杂志,2012,37(21):3271-3274.
[13]李仪奎.中药药理实验方法学[M].上海:上海科学技术出版社,1991.
[14]Boura-Halfon S,Zick Y.Phosphorylation of IRS proteins,insulin action,and insulin resistance[J].American Journal of Physiology Endocrinology&Metabolism,2009,296(4):E581.
[15]Carro E,Trejo J C,Bohl D,et al.Blockade of the insulinlike growth factor I receptor in the choroid plexus originates Alzheimer’s-like neuropathology in rodents:New cues into the human disease?-Neurobiology of Aging[J].Neurobiology of Aging,2006,27(11):1618.
[16]Steen E,Terry B M,Rivera E J,et al.Impaired insulin and insulin-like growth factor expression and signaling mechanisms in Alzheimer’s disease--is this type 3 diabetes?[J].Journal of Alzheimers Disease Jad,2005,7(1):63.
[17]Liu Xin,Zhang Yina.Study of the correlation about IGF-I receptor andβ-amyloid in Alzheimer’s disease[J].Natl Med J China,2012,92(15):1062-1065.
[18]Linda Adlerz,Sofia Holback,Gerd Multhaup,et al.IGF-linduced processing of the amyloid precursor protein family is mediated by different signaling pathways[J].The journal of biologicalchemistry,2007,282(14):10203-10209.
[19]Jeong S.Molecular and Cellular Basis of Neurodegeneration in Alzheimer’s Disease[M].Molecules&Cells,2017.
[20]古文娟,刘頔,张孟仁,等.人参皂苷Rb1对高糖培养海马神经元胰岛素信号转导途径的影响[J].中国中药杂志,2014,39(6):1064-1068.
[21]Li H,Song Y,Jian W,et al.c AMP/PKA signaling pathway contributes to neuronal apoptosis via regulating IDE expression in a mixed model of type 2 diabetes and Alzheimer’s disease[M].Journal of Cellular Biochemistry,2017.
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