洛伐他汀对抗β-淀粉样肽引起的体外培养神经细胞毒蕈碱型乙酰胆碱能受体表达降低及氧化应激水平增高的作用

发布时间：2019-06-03 06:43

【摘要】：目的:阿尔茨海默氏病(Alzheimer's disease,AD)的病因及发病机制仍未完全清楚,极大的限制了临床治疗药物的开发,由于β-淀粉样肽(β-Amyloid peptide,Aβ)的神经毒性作用是AD发病过程的关键环节,因此,本课题采用传统上降低胆固醇的史他汀类药物一洛伐他汀,研究其对抗Aβ引起的神经毒性作用,特别是对抗Aβ神经毒性作用过程中毒蕈碱型乙酰胆碱能受体(Muscarinic acetylcholine receptors,mAChRs)表达和氧化应激水平等的改变,探讨他汀类药物在AD治疗中的非胆固醇依赖性神经保护作用。方法:选择体外培养的原代大鼠海马神经细胞和人骨髓神经母细胞瘤细胞(SH-SY5Y),采用免疫荧光双染法检测NeuN和GFAP进行原代大鼠海马神经细胞纯度鉴定。用洛伐他汀、Aβ寡聚体(β-Amyloid peptide oligomers,AβOs)分别或合并处理细胞24 h～48 h,采用CCK-8法检测细胞的存活率;蛋白印迹法(Western blotting)和实时荧光定量PCR(Real-time PCR)检测细胞中 M1 mAChR 和 M3 mAChR 的蛋白及 mRNA表达水平;生物化学方法测定细胞内胆固醇、丙二醛(Malondialdehyde,MDA)含量、超氧化物歧化酶(Superoxide Dismutase,SOD)和谷胱甘肽过氧化物酶(Glutathione peroxidase,GSH-Px)活性及氧自由基(OH-、H2O2、O2·-)等的变化。结果:(1)原代培养的大鼠海马神经细胞纯度达到85%以上;0.O1μmol/L或0.1μmol/L洛伐他汀处理细胞,不会引起细胞活性和胆固醇水平明显改变(P0.05)。(2)洛伐他汀对 mAChRs 的作用:0.01μmol/L 或 0.1μmol/L 洛伐他汀处理神经细胞24 h,M1 mAChR和M3 mAChR的蛋白及mRNA表达水平明显增加(P0.05)。(3)洛伐他汀抗AβOs对mAChRs的作用:0.5μmol/L AβOs处理神经细胞48 h,M1 mAChR和M3 mAChR的蛋白及mRNA表达水平明显下降(P0.05),但预先用0.11μmol/L洛伐他汀处理细胞24h,可减轻AβOs导致的M1 mAChR和M3 mAChR的蛋白及mRNA表达水平改变(P0.05)。(4)洛伐他汀对杭AβOs的细胞毒性作用:0.5μmol/L AβOs处理两种神经细胞48 h,MDA含量增加、SOD和GSH-Px活性降低、氧自由基(OH-、H2O2、O2·-)水平明显增加(P0.05);给予AβOs前预先用0.1μmol/L洛伐他汀处理神经细胞24 h,可减弱AβOs引起的MDA含量、SOD及GSH-Px活性、氧自由基水平等改变(P0.05)。结论:(1)0.01μmol/L及0.1μmol/L洛伐他汀处理细胞,不会引起细胞活性和胆固醇水平明显改变。(2)洛伐他汀可能通过上调M1 mAChR和M3 mAChR的表达对抗Aβ毒-性作用。(3)洛伐他汀可缓解AβOs引起的MDA含量增加、SOD及GSH-Px活性降低、氧自由基(OH-、H2O2、O2·-)水平升高等毒性作用。综上,洛伐他汀可能以非胆固醇依赖的方式,缓解Aβ引起的mAChRs表达降低及氧化应激水平升高,这为他汀类药物治疗AD提供了实验依据。
[Abstract]:Objective: the etiology and pathogenesis of Alzheimer's disease (Alzheimer's disease,AD) are still not fully understood, which greatly limits the development of clinical therapeutic drugs, due to 尾-Amyloid peptide,. The neurotoxicity of A 尾) is the key link in the pathogenesis of AD. Therefore, this paper studied the neurotoxicity induced by A 尾 by using Lovastatin, a traditional cholesterol lowering drug, to study the neurotoxicity induced by A 尾. In particular, the expression of muscarinic acetylcholine receptor (Muscarinic acetylcholine receptors,mAChRs) and the change of oxidative stress water during antagonizing A 尾 neurotoxicity were studied to explore the cholesterol-independent neuroprotective effect of statins in the treatment of AD. Methods: primary rat hippocampal neurons and human bone marrow neuroblastoma cells (SH-SY5Y) were cultured in vitro. NeuN and GFAP were detected by immunofluorescence double staining to identify the purity of primary rat hippocampal neurons. Lovastatin and A 尾 oligomer (尾-Amyloid peptide oligomers, A 尾 Os) were used to treat the cells for 24 h and 48 h, respectively. The survival rate of the cells was detected by CCK-8 assay. The expression of M1 mAChR and M3 mAChR protein and mRNA in cells were detected by (Western blotting) and real-time fluorescence quantitative PCR (Real-time PCR). Determination of intracellular cholesterol, malondialdehyde (Malondialdehyde,MDA) content, activities of Superoxide Dismutase,SOD and Glutathione peroxidase,GSH-Px and oxygen free radical (OH-,H2O2,) by biochemical method The change of O2 -), etc. Results: (1) the purity of primary cultured rat hippocampal neurons was over 85%; 0.O1 渭 mol / L or 0.1 渭 mol / L lovarastatin, There was no significant change in cell activity and cholesterol level (P 0.05). (2). The effect of lovarastatin on mAChRs was 0.01 渭 mol / L or 0.1 渭 mol / L for 24 h. The expression levels of M1 mAChR and M3 mAChR protein and mRNA were significantly increased (P 0.05). (3). The effect of lovarastatin on mAChRs was treated with 0.5 渭 mol / L A 尾 Os for 48 h. The expression levels of M1 mAChR and M3 mAChR protein and mRNA were significantly decreased (P 0.05), but the cells were treated with 0.11 渭 mol / L lovarastatin for 24 h. It could reduce the expression of M1 mAChR and M3 mAChR protein and mRNA induced by A 尾 Os (P 0.05). (4). The cytotoxicity of lovarastatin on hang A 尾 Os was increased after treatment with 0.5 渭 mol / L A 尾 Os for 48 h. The activities of SOD and GSH-Px decreased and the level of oxygen free radical (OH-,H2O2, O2 -) increased significantly (P 0.05). Pretreatment with 0.1 渭 mol / L lovarastatin for 24 h before administration of A 尾 Os could weaken the changes of MDA content, SOD and GSH-Px activity and oxygen free radical level induced by A 尾 Os (P 0.05). Conclusion: (1) the cells were treated with 0.01 渭 mol / L and 0.1 渭 mol / L lovarastatin. It did not cause significant changes in cell activity and cholesterol level. (2) Lovastatin may antagonize A 尾 toxicity by up-regulating the expression of M1 mAChR and M3 mAChR. (3) Lovastatin can alleviate the increase of MDA content induced by A 尾 Os. The activities of SOD and GSH-Px decreased and the level of oxygen free radical (OH-,H2O2, O 2 -) increased. In summary, lovarastatin may alleviate the decrease of mAChRs expression and the increase of oxidative stress level induced by A 尾 in a cholesterol-independent manner, which provides experimental basis for statins in the treatment of AD.
【学位授予单位】：贵州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96

【参考文献】