PARP1 对caspase-3介导的内耳边缘细胞凋亡的分子调控机制

Maintenance of the endocochlear potential is essential for normal hearing and depends primarily on the pumping mechanism of the marginal strial cell of cochlear laternal wall.Marginal cells(MCs) is rich of mitochondria and account for majority of adenosine triphosphate (ATP) and reactive oxygen species(ROS) generation that cause MCs to be the mainly attack target of such oxidative damage.Presbycusis, drug-induced deafness and noise-induced deafness drugs are associated with the oxidative stress injury of MCs.A large number of studies have confirmed that caspases-3 is an important effector molecule in the oxidative damage of inner ear.Our preliminary study reveal that high density fluorescence of PARP1 was found in the marginal stria tissue in rat cochlear laternal wall.We hypothesize that PARP1 and caspases-3 may be related to oxidative stress damage in the MCs.However, few reports focus on the relationship between PARP1 and inner ear injury under oxidative damage.It is controversial that what are the mechanisms PARP1 involved in the regulation of caspase-3 dependent mitochondrial apoptotic pathway of oxidative- stress damage in the rat marginal strial cell of inner ear.In order to find out its mechanism,we establishes an in vitro marginal strial cellular oxidative-stress model with glucose oxidase (GO).Gene expression was down-regulated in Ad-siPARP1 transfected MCs.The cell viability assays,reactive oxygen species,mitochondrial transmembrane potential(MMP),apoptosis,the translocation of Bax and cytC,cleaved caspase-3,PARP1 and PAR were tested with CCK-8 kit,real-timePCR,westernblot,flowcytometry and immune fluorescent tracer. Bases on the above finding,we want to clarify the the possible role of PARP1 in the classical caspase-3 dependent pathways in oxidase-induced oxidative-stress damage of marginal strial cell.It will provide a new therapeutic target for the treatment in the process of oxidative stress injury of inner ear and provide idears for the research and development of cell protective drugs.

听觉损伤与活性氧的过度蓄积密切相关，血管纹边缘细胞是活性氧作用的重要靶细胞，caspases-3是内耳氧化应激损伤中的重要效应分子。本课题组研究证实，氧化应激损伤可增加血管纹细胞中PARP1的表达，推断PARP1和caspases-3在血管纹细胞氧化应激损伤中存在相关性。目前 PARP1 与caspase-3参与内耳细胞凋亡的分子机制，尚待研究证实。我们以葡萄糖氧化酶制备边缘细胞氧化应激损伤模型，通过Ad-siPARP1下调 PARP1的表达，采用分子生物学技术，对不同组间细胞的活性、ROS、线粒体膜电位（MMP）、凋亡、Bax及细胞色素C的转位、caspase-3剪切片段、PARP1及PAR进行检测。通过以上研究阐明在氧化应激损伤过程中，PARP1对caspase-3途径介导的边缘细胞凋亡的分子调控机制，以期为内耳氧化应激损伤提供新的治疗靶点，为新的细胞保护药物的研发提供思路。

氧化应激理论认为活性氧过度堆积引发的DNA损伤是启动内耳衰老的重要事件，血管纹边缘细胞是活性氧作用的重要靶细胞，caspases-3是内耳氧化应激损伤的重要效应分子，聚腺苷二磷酸核糖聚合酶1（PARP1）是DNA损伤修复的重要酶类。本课题组前期研究显示，大鼠耳蜗外侧壁组织有PARP1的表达；60mU/ml的葡萄糖氧化酶持续作用4小时，血管纹边缘细胞中PARP1及其活性片段表达增加。为探讨PARP1和caspases-3在血管纹细胞氧化应激损伤中是否存在相关性及其可能的分子机制。我们以葡萄糖氧化酶制备边缘细胞氧化应激损伤模型，通过对PAR、NAD+/NADH、ROS检测，确定细胞是否达到有效的氧化应激损伤；应用caspase-3特异性抑制剂Z- VAD-fmk后PARP1剪切片段显著减少，证实了PARP1和caspases-3在血管纹细胞氧化应激损伤中确实存在相关性。以Ad-siPARP1下调 PARP1的表达，通过对Bax/BcL-2、线粒体膜电位（MMP）、Cyt-C线粒体转位、capase-3表达量及活性、PARP1表达量及剪切片段PAR的检测，我们发现，边缘细胞氧化损伤后，在线粒体中细胞色素C蛋白表达明显降低，而Bax蛋白表达明显升高（P<0.05），而在胞浆中正好相反（P<0.05）；通过电镜检测发现，氧化应激条件下边缘细胞中的细胞色素C从线粒体转位至胞浆，同时Bax从胞浆转位至线粒体。阐明PARP1通过细胞色素C和Bax的线粒体/胞浆转位与capase-3途径相互作用来介导葡萄糖氧化酶诱导的内耳边缘细胞凋亡（已发表SCI论文1篇）。我们进一步完成了PARP1对AIF通路介导的细胞死亡调控（已发表SCI论文1篇）的研究。目前PARP1抑制剂已获批在临床上应用于肿瘤治疗领域，希望本研究能够为内耳氧化应激损伤提供新的治疗靶点，为内耳新的细胞保护药物的选择提供新的思路。