配伍红花籽油增强脑细胞及其线粒体对自由氧基介导的细胞损伤的抵抗机制研究
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摘要
目的:①制备由α-亚麻酸和亚油酸按不同比例混合而成的标准配伍红花籽油;②建立H_2O_2诱导的脑细胞(PC12细胞株)损伤(凋亡)模型及研究配伍红花籽油增强脑细胞抵抗自由氧基介导的细胞损伤的机制;③利用H_2O_2/Fe~(2+)诱导的线粒体损伤体系研究配伍红花籽油抑制脂质过氧化的能力。
方法:①制备配伍红花籽油并利用GC(气相色谱法)法测定α-亚麻酸和亚油酸含量;②建立H_2O_2诱导的PC12细胞凋亡模型,利用MTT法、流式细胞技术及荧光成像技术等方法对不同浓度的配伍红花籽油(0.5%DMSO溶液)药物干预进行分析,确定PC12细胞的活力和量效关系;③用改良的Clark技术提取大鼠脑线粒体,建立H_2O_2/Fe~(2+)氧化损伤体系,检测配伍红花籽油药物干预前后的线粒体褐脂质、膜的流动性、ATPase活性、膨胀度等指标变化。
结果:①利用本实验方法提取的混合十八碳二、三烯酸中α-亚麻酸(ALA)、亚油酸(LA)的含量分别达62.49%和22.91%,满足配伍红花籽油的要求;②H_2O_2诱导细胞损伤(凋亡)的最低浓度为200μM,配伍红花籽油对脑细胞(PC12细胞)抵抗自由氧基损伤产生作用的最低浓度为0.3mg/ml;③配伍红花籽油浓度在0.3-0.5mg/ml时,可以明显提高线粒体抑制脂质过氧化的能力,从而改善线粒体膜流动性、膨胀度和ATP酶(ATPase)活性。
结论:①提取与制备配伍红花籽油的方法具有可重复性和可行性;②H_2O_2诱导的细胞损伤模型可以用于配伍红花籽油的细胞药物干预试验,而且具有可重复性和稳定性等特点;③配伍红花籽油脑细胞自由氧基介导的细胞损伤抵抗能力可能是通过间接增强脑细胞内在的防护能力而实现的,而不是药物与自由氧基的直接接触所致;配伍红花籽油可能通过亚细胞器—线粒体途径起增强细胞抗氧化能力的作用。
Objective:①To prepare of compatibility safflower seed oil composed ofα-linolenic acid and linoleic acid in different proportion;②Establishment of a brain cell(PC12 cell line) damage model(apoptosis) induced by H_2O_2 stimulation,and studies on the mechanisms of compatibility safflower seed oil to enhance the resistance capacity of brain cells against free oxygen radical mediated cell damage.③To study the capacity of compatibility safflower seed oil against lipid overoxidation in a mitichondrian damage system induced by H_2O_2/Fe~(2+).
Methods:①To prepare compatibility safflower seed oil,and determine theα-linolenic acid and linoleic acid contents by GC method(gas phase chromatography);②To establish a free oxygen damage(apoptosis) model of PC12 brain cells,and to analyze the drug treatment of compatibility safflower seed oil in different dosage by methods of MTT,fluorescence imaging and flow cytometry to evaluate the relationship between cell activity and the dose-effect③To prepare mitochondria from rat brain by modified methods by Clark and establish an oxidative damage system with H_2O_2/Fe~(2+), and determine brownish lipid,membrane fluidity and ATPase activity before and after treatment of compatibility safflower seed oil.
Results:①The contents ofα-linolenic acid and linoleic acid were about 62.49% and 22.91%,respectively,to meet the requirements of compatibility safflower seed oil;②The lowest dosage to establish the H_2O_2-induced cell damage was 200μM;the lowest dosage of compatibility safflower seed oil to make a significant resistance of PC12 cells against free oxygen mediated damage was 0.3mg/ml.③The ability of rat mitochondria to inhibit lipid overoxidation was significantly enhanced when the dosage of compatibility safflower seed oil kept at 0.3-0.5mg/ml.
Conclusions:①The method to prepare compatibility safflower seed oil proved to be reproducible and feasible;②H_2O_2-induced cell damage model could be applied for the drug treatment with compatibility safflower seed oil,and was reproducible and stable.③The ability of compatibility safflower seed oil to resist free oxygen radical mediated cell damage maybe achieved by indirectly enhancing self-defense capacity of brain cells, instead of the direct contact between the drug and the free oxygen radicals. Compatibility safflower seed oil may work through the sub-cellular compartment-mitochondria by enhancing the anti-oxidation capacity of the cell.
方法:①制备配伍红花籽油并利用GC(气相色谱法)法测定α-亚麻酸和亚油酸含量;②建立H_2O_2诱导的PC12细胞凋亡模型,利用MTT法、流式细胞技术及荧光成像技术等方法对不同浓度的配伍红花籽油(0.5%DMSO溶液)药物干预进行分析,确定PC12细胞的活力和量效关系;③用改良的Clark技术提取大鼠脑线粒体,建立H_2O_2/Fe~(2+)氧化损伤体系,检测配伍红花籽油药物干预前后的线粒体褐脂质、膜的流动性、ATPase活性、膨胀度等指标变化。
结果:①利用本实验方法提取的混合十八碳二、三烯酸中α-亚麻酸(ALA)、亚油酸(LA)的含量分别达62.49%和22.91%,满足配伍红花籽油的要求;②H_2O_2诱导细胞损伤(凋亡)的最低浓度为200μM,配伍红花籽油对脑细胞(PC12细胞)抵抗自由氧基损伤产生作用的最低浓度为0.3mg/ml;③配伍红花籽油浓度在0.3-0.5mg/ml时,可以明显提高线粒体抑制脂质过氧化的能力,从而改善线粒体膜流动性、膨胀度和ATP酶(ATPase)活性。
结论:①提取与制备配伍红花籽油的方法具有可重复性和可行性;②H_2O_2诱导的细胞损伤模型可以用于配伍红花籽油的细胞药物干预试验,而且具有可重复性和稳定性等特点;③配伍红花籽油脑细胞自由氧基介导的细胞损伤抵抗能力可能是通过间接增强脑细胞内在的防护能力而实现的,而不是药物与自由氧基的直接接触所致;配伍红花籽油可能通过亚细胞器—线粒体途径起增强细胞抗氧化能力的作用。
Objective:①To prepare of compatibility safflower seed oil composed ofα-linolenic acid and linoleic acid in different proportion;②Establishment of a brain cell(PC12 cell line) damage model(apoptosis) induced by H_2O_2 stimulation,and studies on the mechanisms of compatibility safflower seed oil to enhance the resistance capacity of brain cells against free oxygen radical mediated cell damage.③To study the capacity of compatibility safflower seed oil against lipid overoxidation in a mitichondrian damage system induced by H_2O_2/Fe~(2+).
Methods:①To prepare compatibility safflower seed oil,and determine theα-linolenic acid and linoleic acid contents by GC method(gas phase chromatography);②To establish a free oxygen damage(apoptosis) model of PC12 brain cells,and to analyze the drug treatment of compatibility safflower seed oil in different dosage by methods of MTT,fluorescence imaging and flow cytometry to evaluate the relationship between cell activity and the dose-effect③To prepare mitochondria from rat brain by modified methods by Clark and establish an oxidative damage system with H_2O_2/Fe~(2+), and determine brownish lipid,membrane fluidity and ATPase activity before and after treatment of compatibility safflower seed oil.
Results:①The contents ofα-linolenic acid and linoleic acid were about 62.49% and 22.91%,respectively,to meet the requirements of compatibility safflower seed oil;②The lowest dosage to establish the H_2O_2-induced cell damage was 200μM;the lowest dosage of compatibility safflower seed oil to make a significant resistance of PC12 cells against free oxygen mediated damage was 0.3mg/ml.③The ability of rat mitochondria to inhibit lipid overoxidation was significantly enhanced when the dosage of compatibility safflower seed oil kept at 0.3-0.5mg/ml.
Conclusions:①The method to prepare compatibility safflower seed oil proved to be reproducible and feasible;②H_2O_2-induced cell damage model could be applied for the drug treatment with compatibility safflower seed oil,and was reproducible and stable.③The ability of compatibility safflower seed oil to resist free oxygen radical mediated cell damage maybe achieved by indirectly enhancing self-defense capacity of brain cells, instead of the direct contact between the drug and the free oxygen radicals. Compatibility safflower seed oil may work through the sub-cellular compartment-mitochondria by enhancing the anti-oxidation capacity of the cell.
引文
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