虎杖苷对β淀粉样蛋白诱导体外皮质神经元线粒体氧化应激和功能障碍的保护作用  

Protective effect of polydatin on beta-amyloid-induced mitochondrial oxidative stress and dysfunction in cortical neurons invitro

作　　者：臧婷臻 郭阳 王凤云[2] 李延辉[2] 申艳霞 张振燕 Zang Tingzhen;Guo Yang;Wang Fengyun;Li Yanhui;Shen Yanxia;Zhang Zhenyan(Department of Neurolgy,Tsinghua University Hospital,Beijing 100084,China)

机构地区：[1]清华大学医院神经内科,北京100084 [2]聊城市人民医院神经内科

出　　处：《中华老年心脑血管病杂志》2024年第5期567-572,共6页Chinese Journal of Geriatric Heart,Brain and Vessel Diseases

基　　金：山东省医药卫生科技发展计划(2021WS108)。

摘　　要：目的探讨虎杖苷对β淀粉样蛋白25~35(Aβ_(25~35))诱导的原代培养大鼠皮质神经元损伤的保护作用及可能的作用机制。方法原代培养SD大鼠皮质神经元,分为对照组、虎杖苷组、Aβ_(25~35)组和联合组(Aβ_(25~35)+虎杖苷)。采用四甲基偶氮唑盐检测皮质神经元活力,2,7-二氯荧光素二乙酸酯探针或红色线粒体超氧化物荧光探针染色检测细胞内和线粒体活性氧水平,线粒体膜通透性转换孔(MPTP)试剂盒检测MPTP开放程度,蛋白免疫印记法检测细胞色素C及线粒体转录因子A(TFAM)表达。另外,测定细胞内电子传递链复合物(包括复合物Ⅰ、Ⅱ、Ⅲ、Ⅳ)活性和三磷酸腺苷(ATP)水平。采用高效液相色谱法测定线粒体中8-羟基脱氧鸟苷(8-OHdG)。结果与对照组比较,Aβ_(25~35)组皮质神经元细胞活力、线粒体荧光强度、线粒体呼吸链酶活性(复合物Ⅰ、Ⅱ、Ⅲ、Ⅳ)、细胞内ATP、线粒体内TFAM表达明显降低,差异有统计学意义(P<0.05,P<0.01);与Aβ_(25~35)组比较,虎杖苷组线粒体荧光强度、线粒体呼吸链酶活性(复合物Ⅰ、Ⅱ、Ⅲ、Ⅳ)、细胞内ATP、线粒体内TFAM表达明显增高,皮质神经元暴露3、6、12、24h细胞内和线粒体内活性氧明显降低(P<0.05,P<0.01),联合组细胞色素C细胞质/线粒体比值、线粒体内8-OHdG水平明显低于Aβ_(25~35)组[3.02±0.28 vs 5.73±0.45,P<0.05;(8.07±1.45)×10^(6)dG vs(16.07±2.29)×10^(6)dG,P<0.05]。结论虎杖苷可有效地保护皮质神经元免受Aβ_(25~35)诱导的损伤,至少部分作用是通过抑制线粒体氧化应激和改善线粒体功能实现。Objective To investigate the protective effect of polydatin(PLD)on primary cultured rat cortical neurons induced byβ-amyloid protein(Aβ_(25~35))and its possible mechanism.Methods Cortical neurons were isolated from SD rats and primarily cultured,and then divided into control group,PLD group,Aβ_(25~35)group and Aβ_(25~35)+PLD group.Cell viability was detected by MTT assay,intracellular and mitochondrial reactive oxygen species(ROS)levels were detected by dichlorofluorescein diacetic acid probe or MitoSOX Red staining,opening of mitochondrial membrane permeability transition pore(MPTP)was detected by MPTP kit,and contents of cytochrome C(Cyt C)and mitochondrial transcription factor A(TFAM)were detected by Western blotting.In addition,the activity of intracellular electron transport chain complexes(including complexesⅠ,Ⅱ,ⅢandⅣ)and the level of adenylate triphosphate(ATP)were determined.High performance liquid chromatography(HPLC)was conducted to determine the content of 8-hydroxydeoxyguanosine(8-OHdG)in the mitochondria.Results Compared with the control group,cell vitality,mitochondrial fluorescence intensity,activities of mitochondrial respiratory chain enzymes(complexⅠ,Ⅱ,ⅢandⅣ),intracellular ATP and mitochondrial TFAM expression were significantly decreased in the cortical neuron from the Aβ_(25~35)group(P<0.05,P<0.01).Compared with the Aβ_(25~35)group,mitochondrial fluorescence intensity,mitochondrial respiratory chain enzyme activity(complexⅠ,Ⅱ,Ⅲ,Ⅳ),intracellular ATP and mitochondrial TFAM expression were obviously increased in the PLD group,and intracellular and mitochondrial ROS levels were notably decreased after cortical neuron exposure for 3,6,12 and 24h(P<0.05,P<0.01).The ratio of cytoplasmic/mitochondrial Cyt C and mitochondrial level of 8-OHdG were statistically lower in the Aβ_(25~35)+PLD group than the Aβ_(25~35)group[3.02±0.28 vs 5.73±0.45,P<0.05;(8.07±1.45)×10^(6)dGvs(16.07±2.29)×10^(6)dG,P<0.05].Conclusion PLD can effectively protect cortical neurons against Aβ

关 键 词：虎杖苷 淀粉样β肽类 神经元 大脑皮质 线粒体 氧化性应激 大鼠 Sprague-Dawley 

分 类 号：R285.5[医药卫生—中药学]