N-甲基-4-异亮氨酸环孢素通过抑制线粒体通透性转换孔减轻凋亡诱导因子的核易位和细胞凋亡  

N-methyl-4-isoleucine Cyclosporine Alleviates the Nuclear Translocation of Apoptosis-inducing Factors and Apoptosis by Inhibiting Mitochondrial Permeability Conversion Pores

作　　者：曾玥 罗思晴 马鑫喆 潘泓乐 刘红梅[2] 张鸣号[2,3] ZENG Yue;LUO Siqing;MA Xinzhe;PAN Hongyue;LIU Hongmei;ZHANG Minghao(Ningxia Medical University,Yinchuan 750004,China;School of Basic Medical Sciences,Ningxia Medical University,Yinchuan 750004,China;Key Laboratory of Metabolic Cardiovascular Diseases,Yinchuan 750004,China)

机构地区：[1]宁夏医科大学,银川750004 [2]宁夏医科大学基础医学院,银川750004 [3]国家卫健委代谢性心血管疾病研究重点实验室,银川750004

出　　处：《宁夏医科大学学报》2022年第7期663-668,676,共7页Journal of Ningxia Medical University

基　　金：宁夏自然科学基金项目(2021AAC03122);宁夏回族自治区重点研发计划项目(2021BEG03093);宁夏回族自治区大学生创新创业训练计划项目(S202010752030)。

摘　　要：目的探讨鱼藤酮诱导小鼠海马神经元HT22细胞凋亡的机制,观察环孢素A(cyclosporin A,CsA)衍生物N-甲基-4-异亮氨酸环孢素(N-methyl-4-isoleucine-cyclosporin,NIM811)对鱼藤酮诱导的HT22细胞毒性的保护作用。方法使用线粒体复合物Ⅰ抑制剂鱼藤酮培养小鼠海马神经元HT22细胞,使用第3~7代的HT22细胞,分成control组、鱼藤酮组、鱼藤酮+NIM811组和NIM811组。NIM811在鱼藤酮刺激3 h前加入培养基。采用Alamar Blue法检测HT22细胞活性,二氢乙二胺(DHE)荧光探针测定细胞内活性氧(reactive oxygen species,ROS)的含量,四甲基罗丹明甲酯(tetramethylrhodamine methyl ester,TMRM)测定线粒体膜电位(mitochondrial membrane potential,MMP)。鱼藤酮孵育24 h后,提取细胞胞浆和胞核蛋白,Western blot检测细胞核中凋亡诱导因子(apoptosis-inducing factor,AIF)及胞浆中细胞色素C(cytochrome C,cyto C)、caspase-9、Bax和Bcl-2的蛋白表达。结果鱼藤酮使HT22细胞活力降低至56.26%(P<0.01),细胞明显缩小,核浓缩,细胞周围形成透明圈。ROS产生增加(P<0.05),MMP降低(P<0.01),AIF核易位,并引起线粒体cyto C释放和caspase-9激活,Bax蛋白表达增加(P<0.01),Bcl-2蛋白表达降低(P<0.01)。NIM811预处理增强了细胞活力,降低了ROS的产生,维持了线粒体膜电位,抑制了AIF核易位,减轻了线粒体cyto C的释放和caspase-9的激活,抑制Bax蛋白表达增加和Bcl-2蛋白表达降低。结论NIM811通过抑制ROS的产生,维持线粒体MMP,抑制线粒体通透性转换孔(mitochondrial permeability transition pore,MPTP)的形成,减少线粒体cyto C的释放,减轻AIF核易位,从而减轻鱼藤酮诱导的HT22细胞凋亡,对帕金森病具有保护作用。Objective To investigate the apoptosis mechanism of murine hippocampal HT22 cells induced by rotenone,and observe the protective effect of cyclosporine A(Cs A)derivative N-methyl-4-isoleucine cyclosporine(NIM811)against rotenone toxicity.Methods Mouse hippocampal neuron(HT22)cells were cultured with rotenone,a mitochondrial complex Ⅰ inhibitor.HT22 cells from generation 3 to 7 were divided into control group,rotenone group,rotenone + NIM811 group and NIM811 group.NIM811 was added to the culture medium 3 h before rotenone stimulation.Alamar Blue method was used to detect the cell activity,and dihydroethylenediamine(DHE)fluorescence probe was used to measure the intracellular reactive oxygen species(ROS).Mitochondrial membrane potential(MMP)was measured by tetramethylrhodamine methyl ester(TMRM).After incubation with rotenone for 24 hours,cytoplasmic and nuclear proteins were extracted.Western blot was used to detect the protein expressions of apoptosis-inducing factor(AIF)in the nucleus and cytochrome C(Cyto C),caspase-9,Bax and Bcl-2 in the cytoplasm.Results Rotenone reduces viability of HT22 cells to 56.26%(P<0.01).The cells were significantly shrunk,the nucleus was concentrated,and a transparent circle was formed around the cells.Rotenone induced the ROS production increased(P <0.05),MMP decreased(P <0.01),and AIF translocated to nuclear.Rotenone also caused mitochondrial cyto C release and caspase-9 activation.Bax protein expression increased(P<0.01),and Bcl-2 protein expression decreased(P<0.01).NIM811 pretreatment enhanced cell viability,decreased ROS production,maintained mitochondrial membrane potential,inhibited AIF nuclear translocation,alleviated mitochondrial cyto C release and caspase-9 activation,and inhibited increased Bax protein expression and decreased Bcl-2 protein expression.Conclusion NIM811 can inhibit rotenone-induced apoptosis of HT22 cells by inhibiting the production of ROS,maintaining mitochondrial MMP,inhibiting the formation of MPTP,reducing the release of mitochondrial cyto C,and al

关 键 词：鱼藤酮 小鼠海马神经元 细胞凋亡 N-甲基-4-异亮氨酸环孢素 氧化应激 线粒体损伤 

分 类 号：R965[医药卫生—药理学]