黏菌素诱导神经细胞线粒体自噬与凋亡的交叉对话及p53双重调控机制研究

We have previously proved that colistin could induce neurotoxicity via oxidatie stress and mitochondrial apoptotic pathway. In the present project, we will extend our studies to investigate the cross-talk of both apoptosis and mitophagy, and the molecular coincident regulation mechansim. Using the rat chromaffin glioma cells (PC12), by assay methods of confocal fluorescence microscope, western blotting, RT-PCR, flow cytometry and gene silencing, the change trend with time or dose of autophagy and apoptosis will be determined first; and then the interaction of apoptosis and autophagy, and the cross-talk between various regulatory proteins were determined by applying chemical inhibitors or inducers of apoptosis or autophagy, and Bcl-2 expression or Beclin-1 shRNA interfere; Early we have proved colistin could cause mitochondrial p53-dependent apoptosis, in this project, by using the p53 shRNAi and chemical p53 inhibitors, we will explore the dual regulation of apoptosis and autophagy for p53 and p53/AMPK/mTOR pathway transduction mechanism; Finally by the animal in vivo tests, we will verify the neuroprotective effect for autophagy and p53 inhibition against colistin-induced neurotoxicity in mice. The above research will provide a new thinking to further explore the action mechanism of colistin-induced neurotoxicity and find anagotoxic target drugs or preventive measures.

本项目在前期已证明黏菌素可通过氧化应激和线粒体凋亡途径引起神经毒性的基础上，进一步探究线粒体自噬和凋亡之间的交叉对话及分子调控机制。采用大鼠嗜铬神经瘤细胞，通过共聚焦荧光显微镜、western blotting、RT-PCR、流式细胞仪、基因沉默等技术，首先确定自噬和凋亡随时间和剂量的变化规律；再通过凋亡和自噬抑制剂、激活剂及Bcl-2表达、Beclin-1 shRNAi等条件下凋亡和自噬测定及蛋白表达，进一步确定二者相互作用及各种调控蛋白之间的交叉对话联系；前期实验已证明黏菌素能引起神经元线粒体p53依赖性凋亡，本项目采用p53 shRNAi及化学抑制剂，探讨p53对凋亡和自噬的双重调节作用及其p53/AMPK/mTOR通路转导机制；最后采用动物体内试验来验证自噬及p53抑制对小鼠黏菌素所致神经毒性的保护作用，为深入探讨黏菌素神经毒性的作用机制及寻找对抗性靶标药物或预防措施提供新思路。

“超级细菌”的出现预示着黏菌素广阔的再开发和应用前景，但神经毒性仍然是限制其应用的瓶颈。由于神经毒性的确切机制尚不清楚，致使临床因使用黏菌素而发生神经毒性时没有相对有效的防治措施。本项目以PC12细胞为研究对象，探讨了黏菌素诱导神经细胞线粒体自噬与凋亡的交叉对话及p53双重调控机制。获得如下研究成果：（1）黏菌素（125 μg/mL）诱导的自噬先于凋亡出现，且其出现可能与线粒体受损有关。（2）细胞自噬抑制凋亡发生，即细胞自噬可降低黏菌素神经毒性而起到保护细胞的作用，p53在黏菌素诱导的细胞自噬和细胞凋亡中发挥着双向调节作用，自噬和凋亡间的趋势变换与p53在细胞内的定位有关。（3）p53基因的沉默使得黏菌素诱导的PC12细胞自噬现象在12 h时下调、24 h上调，并且上调的自噬促进黏菌素诱导的细胞凋亡；p53基因的过表达抑制黏菌素诱导的自噬并加速了细胞凋亡的发生。（4）通过对JNK信号通路在黏菌素诱导自噬机制的初探，发现JNK参与黏菌素诱导的自噬，JNK-Bcl2-Bax信号通路调节黏菌素诱导的细胞自噬与凋亡，加速了黏菌素诱导的自噬，并提前了凋亡的发生，同时有ROS-JNK-p53活化环路参与在其中。（5）硫酸黏菌素诱导小鼠坐骨神经产生的自噬对其神经具有保护作用，其作用机制可能与氧化应激有关。本项目将为寻找和开发对抗黏菌素神经毒性的靶标药物或预防措施提供新途径。