川芎嗪干预药物中毒性聋耳蜗mtRNA损伤作用与APE/Ref-1表达的相关性研究

In our previous study, we investigated the mechenism of tetramethylpyrazine in the repair of cochlear hair cells damage after drug-induced hearing loss. We found that gentamicin can promote the formation of free radicals, and lead to mitochondrial DNA mutations. The mammalian AP-endonuclease (APE1/Ref-1) is the rate-limiting enzyme in the DNA base excision repair (BER) pathway. It plays a central role in DNA damage, oxidative stress, and translational control of gene expression. APE/Ref-1 is also involved in apoptosis. In this study, We determined SOD, MDA, GSH and the levels of mtDNA damage, in order to explore the relationship between oxidative stress and mtDNA damage. Also, we examined the mechanisms underlying the effect of tetramethylpyrazine.we used immunocytochemistry, laser scanning confocal microscope and western blot to study the changes of cochlear subcellular localization in oxidative damages to mtDNA of APE1 after. tetramethylpyrazine used. We also investigated the relationship between APE1 and mtDNA damage and repair. We also used TUNEL stain, transmission electron microscopy and flow cytometry to examine the mechnism of cochlear apoptosis in oxidative damages to mtDNA after tetramethylpyrazine used. Also, we examined the mechanisms underlying the effect of tetramethylpyrazine. Our research will help to reveal the anti-oxidative damage effects of tetramethylpyrazine, and will provide evidence and a feasible strategy for gene therapy by targeting mtAPE1.

国家自然科学基金"川芎嗪对药物性聋耳蜗毛细胞损伤修复作用及其机制研究（30672739）"中发现：庆大霉素可促进机体氧自由基产生，导致mtDNA突变。APE/Ref-1是DNA碱基切除修复途径的限速酶，是连接DNA损伤、氧化应激及基因转录调控等重要的细胞生物学过程的桥梁分子，与细胞凋亡密切相关。我们①通过SOD、MDA、GSH、mtDNA氧化性损伤的检测,探讨氧化应激与mtDNA损伤关系及川芎嗪的干预作用机制；②采用免疫细胞化学、激光共聚焦和亚细胞组分Western blot方法，研究APE1在川芎嗪干预mtDNA氧化损伤性耳蜗亚细胞定位改变，探讨与mtDNA损伤修复调控的相关性；③应用TUNEL、透射电镜及流式细胞仪方法，观察川芎嗪干预mtDNA氧化损伤性耳蜗细胞凋亡的作用及机制，以期丰富川芎嗪抗氧化损伤作用的"化学成分－作用靶点"数据，为以mtAPE1为靶点的基因治疗提供理论及可行策略

国家自然科学基金"川芎嗪对药物性聋耳蜗毛细胞损伤修复作用及其机制研究（30672739）"中发现：庆大霉素可促进机体氧自由基产生，导致mtDNA突变。APE/Ref-1是DNA碱基切除修复途径的限速酶，是连接DNA损伤、氧化应激及基因转录调控等重要的细胞生物学过程的桥梁分子。APE/Ref-1能通过氧化还原机制调节多种转录因子生物DNA结合活性及其下游靶基因表达，从而参与氧化应激、细胞周期调控及凋亡等多种关键的细胞反应。APE/Ref-1在氧化应激中下调引起细胞凋亡的现象十分明确，但何种途径参与了APE/Ref-1表达的下调目前还不清楚。.我们①通过SOD、MDA、GSH、mtDNA氧化性损伤的检测,探讨氧化应激与mtDNA损伤关系及川芎嗪的干预作用机制；②采用免疫细胞化学、等方法，研究APE1在川芎嗪干预mtDNA氧化损伤性耳蜗亚细胞定位改变，探讨与mtDNA损伤修复调控的相关性；③应用TUNEL、透射电镜等方法，观察川芎嗪干预mtDNA氧化损伤性耳蜗细胞凋亡的作用及机制，以期丰富川芎嗪抗氧化损伤作用的"化学成分－作用靶点"数据，为以APE1为靶点的基因治疗提供理论及可行策略。.本项目在前期工作的基础上明确了庆大霉素通过促进过多氧自由基的产生使机体处于氧化应激状态，促进内耳部分细胞胞浆内APE/ Ref-1转位到胞核，诱导内耳的APE/ Ref-1mRNA和蛋白表达上调，进一步激活凋亡相关因子NF-kB、C-myc，进而激活耳蜗细胞中凋亡的关键酶和最终执行者caspase-3，促进耳蜗细胞产生凋亡现象，损伤听力；中药川芎嗪一方面减少自由基生成，另一方面上调和激活APE/Ref-1蛋白转位，活化转位后的APE/Ref-1蛋白可通过修复受损DNA，胞内氧化还原状态等多种途径拮抗ROS所致的内耳氧化损伤，调节凋亡相关因子NF-kB、C-myc等，进一步影响凋亡的关键酶和最终执行者caspase-3，从而干预GM的耳毒性，减少耳蜗组织细胞的凋亡。