目的 研究组蛋白去乙酰化酶6(HDAC6)抑制剂tubastatin A对顺铂诱导的认知障碍的保护作用及其机制。方法 雄性C57BL/6小鼠分为正常对照组、模型组、模型+tubastatin A组和tubastatin A对照组。模型组以10 d为1个周期(第1~5天,每天1次ip给予顺铂2.3 mg·kg-1,第6~10天不注射),连续进行3个周期构建化疗相关的脑损伤模型。模型+tubastatin A组在每次顺铂注射前1 h ip 给予tubastatin A 25 mg·kg-1进行治疗。采用Morris水迷宫实验检测学习记忆功能;免疫荧光观察线粒体外膜转位酶20(TOMM20)在海马CA1区神经元胞体与辐射层的分布;生化法检测脑组织线粒体细胞色素c氧化酶(Cyto C)活性、ATP合成量和线粒体膜电位(MMP);Western印迹法检测海马组蛋白脱乙酰酶6(HDAC6)乙酰化底物α微管蛋白乙酰化水平;免疫荧光检测突触前标记物突触蛋白-1(synapsin-1)和突触后标记物突触后致密蛋白95(PSD95)的表达。结果 与对照组相比,模型组小鼠靶象限滞留时间明显减少(P<0.01),TOMM20聚集在海马CA1区神经元胞体,脑组织线粒体功能损伤,α微管蛋白乙酰化水平降低,synapsin-1和PSD95荧光强度均减少;与模型组相比,模型+tubastatin A组小鼠Morris水迷宫靶象限滞留时间显著增多(P<0.05),TOMM20均匀分布在海马CA1区神经元胞体与辐射层,脑组织线粒体Cyto C活性增加了35.2%(P<0.05),ATP合成量增加了58.6%(P<0.01),MMP增加了17.1%(P<0.05),α微管蛋白乙酰化水平升高(P<0.01),synapsin-1和PSD95荧光强度均增加(P<0.05)。结论 Tubastatin A对照组与正常对照组相比,神经行为学、线粒体功能和突触蛋白均没有显著性差异。Tubastatin A治疗通过增加海马组织α微管蛋白乙酰化水平,缓解海马线粒体转运障碍、线粒体失功能和突触损伤,从而改善顺铂诱导的小鼠学习记忆功能损伤。
Abstract
OBJECTIVE To investigate the neuroprotective effects of HDAC6 inhibitor tubastatin A on cisplatin-induced cognitive impairment in mice. METHODS Male C57BL/6 mice were randomly assigned into normal control group, model group, model+tubastatin A group, and tubastatin A group. The mice model of chemo brain was established by intraperitoneal injections with cisplatin for 3 cycles consisting of 5 daily injections followed by a 5-day rest with no injection. Tubastatin A (25 mg·kg-1) was administered intraperitoneally 1 h before cisplatin injection. The ability of learning and memory was assessed by Morris water maze. The mitochondrial axonal transport was analyzed with immunofluorescence, using TOMM20, a mitochondrial marker protein. The brain mitochondrial function, including cytochrome c oxidase activity, mitochondrial ATP production, and mitochondrial membrane potential (MMP) measurements, were measured by biochemical assay. The α-tubulin acetylation level was detected by Western blot. The levels of pre-synaptic marker synapsin-1 and post-synaptic marker PSD95 were examined by immunofluorescence. RESULTS Compared with the control group, the retention of target quadrant was significantly reduced in the cisplatin group. Cisplatin-treated mice exhibited a significantly impaired mitochondrial function as indicated by decreased cytochrome C oxidase activity, ATP production, and MMP, and mitochondrial transport deficits as evidenced by an obvious increase in the ratio of TOMM20 immunoreactivity in the soma to that in the stratum radiatum in the brain as compared with the controls. The levels of synapsin-1 and PSD95 in CA1 region of the hippocampus were remarkably decreased in cisplatin-treated mice. However, tubastatin A treatment significantly increased the retention of target quadrant, attenuated mitochondrial transport deficits and mitochondrial dysfunction, increased the α-tubulin acetylation level, and rescued the synaptic injury in brains of cisplatin group. CONCLUSION No significant difference is observed in the cognition, mitochondrial function and synaptic proteins between the normal control group and tubastatin A group. Tubastatin A can alleviate mitochondrial transport disorder, mitochondrial dysfunction, and cisplatin-elicited decrease in synaptic proteins by increasing the level of a-tubulin acetylation, thereby improving cisplatin-induced learning and memory impairment in mice.
关键词
顺铂 /
认知损伤 /
tubastatin A /
线粒体 /
突触功能
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Key words
cisplatin /
cognitive impairment /
tubastatin A /
mitochondria /
synaptic function
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中图分类号：
R965
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参考文献
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基金
国家自然科学基金项目资助(U1804174,81601225);河南省重点研发与推广专项(科技攻关)项目资助(192102310081);河南省高校科技创新人才支持计划资助(20HASTIT044);河南科技大学大学生研究训练计划项目资助(SRTP)(2019314)
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