α1-肾上腺素受体介导的Nampt调控途径促131I放射性涎腺炎腺体修复机制研究

Radioactive iodine 131 (131I) irradiation is an effective treatment for malignant tumors such as differentiated thyroid carcinomas. 131I targets thyroid tissue and is picked up by the salivary glands. Collateral damage results in the development of radiation sialadenitis, whose major pathological mechanism is apoptosis induced by β-irradiation. Our previous studies have shown that apoptosis is induced by a variety of stress conditions, such as ischemia/reperfusion injury, denervation, and X-irradiation, and is inhibited by the activation of α1-adrenergic receptor. However, so far little is known about the effects and mechanisms of the activation of α1-adrenergic receptor on salivary gland damage after 131I treatment. Therefore, the present study is aimed to investigate the molecular mechanisms of salivary gland damage by 131I, and determine the cytoprotection and regulating manner of the activation of α1-adrenergic receptor on nicotinamide phosphoribosyl transferase (Nampt) in submandibular gland after 131I irradiation. The half life of Nampt mRNA and the methylation pattern of the upstream regulatory sequence of Nampt promoter would first be determined; the RasGRP1/JNK/AP1, p38/ERK1/2/NF-κB, and AKT/SIRT1/FOXO1 signaling pathway would be demonstrated; the specific binding site of the transcriptional control effect of phenylephrine on Nampt promoter in 131I irradiated submandibular gland would next be investigated using the electrophoretic mobility shift assay and in vivo footprinting; the DNA regulatory sequence and pathway of Nampt promoter are planned to be demonstrated by the gene recombinant techniques of gene point deletion and mutation, and the transcriptional regulatory capacity of the promoter segments would be analyzed by luciferase activity; then, the protein-DNA interactions in the transcriptional factor and core promoter regions of Nampt would be investigated by yeast two-hybrid system and immune precipitation; finally, the regulation of microRNA was examined by mRNA microarrays and quantitative real-time PCR at the post-transcription level in 131I irradiated submandibular gland. The above study overall would be helpful to elucidate the regulation mechanism of the activation of α1-adrenergic receptor promoted glandular functional recovery after 131I irradiation, and may thus provide a novel gene therapy strategy to the prevention and treatment of 131I induced irradiated sialadenitis to the patients suffering malignant tumors.

在131I治疗甲状腺癌等恶性肿瘤的同时，涎腺受到内辐射损伤而发生放射性涎腺炎，其可能机制为β射线辐射导致腺泡细胞凋亡。我们前期研究表明，α1-肾上腺素受体（α1-AR）活化能介导应激状态下（如X射线辐射）颌下腺细胞保护作用，抑制腺泡细胞凋亡。但关于α1-AR在131I内放射损伤涎腺中的作用及机制尚未见报道。本项目拟研究131I辐射对涎腺损伤的分子机制，并探讨α1-AR活化介导的促细胞存活因子烟酰胺磷酸核糖转移酶（Nampt）的RasGRP1/JNK/AP1、p38/ERK/NF-κB和 AKT/SIRT1/FOXO1细胞信号转导通路、基因转录调控方式及microRNA在转录后水平的作用及靶基因的调节，以明确131I致涎腺损伤机制及α1-AR活化促腺体修复分子机制，阐明Nampt在涎腺非肿瘤疾病中的作用，为甲状腺癌等癌症病人因131I所致放射性涎腺炎的治疗提供创新性的治疗策略。

（1）项目的研究背景：分化型甲状腺癌是临床最常见恶性内分泌肿瘤，术后结合放射性碘（131I）的治疗，极大减少了肿瘤的复发和转移，显著提高十年生存率。然而，唾液腺是除甲状腺外的最高度富集131I的组织，不可避免地会受到131I的辐射损伤，严重影响病人生存质量。近年研究发现烟酰胺磷酸核糖转移酶(nicotinamide phosphoribosyltransferase, NAMPT)既是在诸多应激状态下的促细胞存活因子，又是辅酶I（NAD）生物合成的重要限速酶，而NAD是参与调控线粒体能量代谢的关键酶，是细胞生命活动能量的重要来源。我们课题组前期研究表明α1-肾上腺素受体激动剂苯肾上腺素（phenylephrine, PE）对X-线外辐射损伤的唾液腺具有细胞保护作用，其分子机制与NAMPT的表达上调密切相关。故本项目拟研究PE对131I致唾液腺内辐射损伤的作用及NAMPT途径相关分子机制。 .（2）主要研究内容：主要在动物（大鼠）唾液腺组织水平，发现了PE对131I致唾液腺内辐射损伤具有细胞保护作用,并证明PE维持了唾液腺细胞线粒体稳态，从而减少腺泡细胞凋亡并促进细胞存活。其机制涉及两个方面：一方面，PE活化α1-肾上腺素受体后PI3K/AKT通路而激活SIRT1/NAMPT信号途径，促进了NAD的合成和ATP的产生，从而促进腺泡细胞能量代谢；另一方面，PE活化α1-肾上腺素受体后，经PI3K/AKT激活PGC-1α，从而促进腺泡细胞线粒体合成代谢。.（3）重要结果：PE明显减轻131I所致颌下腺组织萎缩，维持了腺泡细胞内线粒体的数量和形态完整，防止了线粒体膜电势的下降，降低了线粒体凋亡相关蛋白cytochrome C和cleaved-caspase3的表达，减少了凋亡细胞数量，预防了腺体组织结构损伤。PE显著减少了131I所致的磷酸化PI3K/AKT蛋白表达的下降，并增加了SIRT1和NAMPT的蛋白表达，显著减轻了131I所致的NAD和ATP水平的下降，同时增加了腺泡细胞PGC-1α的表达。.（4）关键数据：在亚细胞水平发现PE显著增加线粒体数量和维持线粒体正常结构，预防131I所致的电离辐射损伤。.（5）科学意义：基于PE是临床常用药物，药代动力学和毒副作用明确，价格低廉且易于应用，将为临床防治131I所致放射性唾液腺炎提供新的策略。