NGF及其PI3-K/Akt信号通路在黏菌素致周围神经毒性中的作用及机制研究

The rat pathological model of colistin-caused peripheral neurotoxicity established in our earlier research will be used as the study object in the present project. First, we will explore the correlation between the endogenous nerve growth factor (NGF) and the colistin-induced peripheral neurotoxicity by determining the expression of NGF and its receptors combining with the neurotoxicity characteristics. Then, the nephroprotective effect of NGF was examined by measuring the behaviour function, electrophysiological parameters, physiology-biochemical indexs, and morphological quantitative analysis of electron microscope in peripheral nerves; the cascading effects of NGF on the colistin-caused pathological course (e.g. reducing cell autophagy, enhancing oxidative stress, increasing apoptosis, decreasing endogenous protective substance and abnormal expression of injure-related proteins) were determined by means of RT-PCR, western-blot, immuno-histochemistry to clarify their interaction relationship. Furthermore, expression of the downstream molecules in the PI3-K/Akt signaling pathway was analyzed using Schwann, dorsal root ganglia and neuronsmotor neuron cells to confirm the effect of PI3-K/Akt signaling pathway on the colistin-caused neurotoxicity and the accurate effect target of NGF. The ultimate aim of our study is to clarify the nephroprotective effect of NGF against the colistin-induced peripheral neurotoxicity and its signal transduction mechanism at levels of body, organ, cell and molecule, which will provide a new idea of co-administration of NGF to increase the therapeutic index of colistin and the subsequent screen of serial efficient gene-targeted drugs.

以前期已建立的黏菌素周围神经毒性大鼠病理模型为研究对象，通过测定神经生长因子（NGF）及其受体表达变化，结合周围神经毒性特点，探讨内源性NGF与黏菌素周围神经毒性作用的相关性；通过周围神经行为学功能、神经电生理参数、神经生理生化指标的测定及周围神经形态计量分析，探讨外源性NGF对黏菌素周围神经毒性的干预作用；采用RT-PCR、Western-blot、免疫组化等方法进行神经细胞自噬、氧化应激、凋亡和损伤相关蛋白表达分析，探讨NGF对黏菌素周围神经毒性病理进程的影响及相互作用关系；利用周围神经元培养体系，对PI3-K/Akt信号通路的下游靶蛋白进行表达分析，阐明黏菌素对PI3-K/A通路的影响机制，明确NGF作用靶点。在整体、器官、细胞、分子水平从NGF及PI3-K/Akt信号通路角度探讨黏菌素致周围神经毒性作用及神经保护机制，为NGF临床联合用药，提高黏菌素治疗指数及靶标药物筛选提供新思路

“超级细菌”的出现预示着黏菌素广阔的再开发和应用前景，但其神经毒性问题已成为限制应用的瓶颈。为解决这一问题，本项目尝试将神经生长因子NGF引入到黏菌素临床治疗方案中，另外针对NGF信号转导通路中的特异性靶点，进一步筛选对神经毒性高效干预的药物（如黄芩苷、维生素C、红景天苷），使黏菌素具备更广阔的应用前景。目前已确定黏菌素可引起小鼠周围神经毒性，确证每日静脉注射15mg/kg硫酸黏菌素，连续给药7d，可成功建立小鼠黏菌素神经毒性模型。黏菌素可导致小鼠神经组织内NGF及其受体蛋白TrkA及p75NTR的表达升高，揭示黏菌素神经毒性与神经细胞NGF配体-受体结合相关；通过神经行为学、电生理学、神经生理生化指标、病理组织学检查，确定外源性NGF及黄芩苷对黏菌素诱发的神经毒性具有一定的保护作用，并使神经组织中TrkA、p75NTR的mRNA表达水平降低及坐骨神经中凋亡相关因子P53、BAX和碱性髓鞘蛋白（MBP）表达明显降低；黏菌素可致PC12细胞形态结构改变，并引起神经元细胞内氧化应激反应，通过死亡受体、线粒体、内质网三条途径诱导PC12细胞凋亡，导致中枢神经系统损伤，提示细胞凋亡可能在黏菌素所致神经毒性中起重要作用，且线粒体氧化应激是黏菌素致小鼠神经毒性的主要途径；维生素C可维持黏菌素损伤PC12细胞的正常形态，提高细胞活力，降低黏菌素对PC12细胞膜的损伤和氧化应激，使由线粒体途径介导的细胞凋亡得到抑制。表明维生素C能有效拮抗黏菌素诱发的神经毒性，为黏菌素与维生素C在临床上的联合应用提供理论依据；黏菌素也能够引起PC12细胞自噬，对细胞凋亡可能是一种促进机制；NGF和黄芩苷能提高PC12细胞活性，不仅能够抑制黏菌素介导的PC12细胞凋亡，也可抑制PC12细胞自噬。体内实验也表明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，作用机制可能与氧化应激有关；在MTT法构建黏菌素体外损伤雪旺细胞的模型的基础上，证明了黏菌素能引起雪旺细胞凋亡，诱发神经毒性，造成细胞内氧化应激损伤，使PI3K/Akt信号通路失活，这可能也是黏菌素引发神经毒性的机制之一。红景天苷能有效拮抗黏菌素引发的雪旺细胞损伤，其神经保护作用可能与其降低氧化应激损伤，激活PI3K/Akt信号通路密切相关。以上研究不仅阐明NGF对黏菌素引起神经毒性的干预作用及机制，也为黏菌素的临床合理应用提供新思路。