ROS抑制DUSP6活性在ERK1/2诱导的放射性脑损伤中的作用机制研究

Radiation-induced brain injury (RIBI) is a serious complication which is commonly associated with significant functional morbidity and decreased quality of life in patients treated with brain radiotherapy. The mechanisms involved in radiation-induced cellular injury and death remain incompletely understood. Our preliminary studies have found that nerve cells underwent two forms of death, autophagy and apoptosis, after irradiation. In this project, we will establish radiation-induced nerve injury models both in vitro and in vivo, try to clarify that reactive oxygen species (ROS) generated after irradiation inhibit the activity of ERK-specific phosphatases (dual specificity phosphatase 6,DUSP6), which affecting the kinetics of extracellular signal-regulated kinase 1/2 (ERK1/2) activity in a given cellular compartment, provoking a sustained activation of ERK1/2, leading to the progressive accumulation of death-promoting factors up to a level that induces cell death. Sustained cytoplasmic ERK1/2 activity might promote autophagy, whereas sustained nuclear sequestration of ERK1/2 activity might trigger apoptosis. And both autophagy and apoptosis contribute to radiation-induced cellular injury and death. Meanwhile, we will observe the degree of autophagy and apoptosis during cell death at different time points after irradiation, together with the level of cytoplasmic and nuclear activated ERK1/2, to further understand the influence of ERK1/2 on autophagy and apoptosis, and the relationship between autophagy and apoptosis. Furthermore, we will explore interventions such as scavenging ROS, enhancing capacity of autophagy or interfering the activity of ERK1/2, to provide potential therapeutic targets in the treatment of RIBI.

放射性脑损伤是头颈部肿瘤放疗后严重影响病人生活质量的并发症，但是放射性神经细胞损伤的机制未明。我们在既往发现放疗后神经元可以出现自噬性死亡和凋亡两种死亡形式基础上，通过建立体外和体内放射性神经损伤的模型，探讨放疗后由于细胞的自噬作用不能完全清除ROS的毒性，放疗后脑内产生的ROS通过影响DUSP6的活性，引起ERK1/2活性调节的反馈机制出现障碍，导致ERK1/2活化程度的改变以及其从胞浆向胞核内的核转入水平的变化，从而在胞浆和胞核内分别启动神经细胞的自噬和凋亡两种不同的死亡形式。同时动态观察细胞自噬和凋亡水平的变化，明确放疗后胞浆内和胞核内活化的ERK1/2水平对细胞自噬和凋亡的影响以及两者之间的相互关系。并进一步探讨可能的干预靶点，为今后进一步试图通过清除ROS，增强自噬或干预ERK1/2的活性而保护神经细胞免受放疗所导致的死亡提供新的治疗靶点，为放射性脑损伤的防治提供新的理论依据。

双特异性磷酸酶6 （DUSP6）是双特异性蛋白磷酸酶亚家族的成员，其缺失可引起ERK1/2活性调节的反馈机制出现障碍，进而调控神经细胞的自噬和凋亡等。在本课题里，我们建立了DUSP6基因敲除小鼠模型（DUSP6-/-）及细胞DUSP6过表达载体（pcDNA-DUSP6），并在动物实验上探讨DUSP6对放疗后小鼠ERK1/2蛋白表达、细胞亚定位和认知行为能力的影响等，且在细胞实验上研究DUSP6对谷氨酸诱导的海马神经元细胞的细胞死亡、细胞凋亡、细胞自噬及ERK1/2磷酸化水平的影响。.我们首次证明了DUSP6会加剧放疗后小鼠的认知障碍，其机制尚在进一步研究中。我们首次证明DUSP6对谷氨酸诱导的小鼠海马神经元细胞系（HT22）和原代培养的海马神经元（pc-HNeu）具有神经保护作用。通过细胞增殖测定、流式细胞术、蛋白免疫印迹、透射电子显微镜等方法，我们发现DUSP6过表达会减少谷氨酸诱导的细胞死亡、细胞凋亡、细胞自噬和ERK1/2蛋白磷酸化。.所有这些结果表明，DUSP6在谷氨酸诱导的海马神经元中具有重要的神经保护作用，同时在放射性认知损伤中亦起到重要的影响，为确立DUSP6作为防治神经细胞损伤的新靶标提供科学依据。