碘过量对甲状腺氧化应激-抗氧化防御影响的线粒体机制

Epidemiological studies have shown that iodide excess plays a key role in hypothyroidism, hyperthyroidism, euthyroid goiter or thyroid autoimmunity. Iodide is a fundamental constituent of thyroid hormones. Mitochondria are the primary site of action of thyroid hormones and the major intracellular source of reactive oxygen species (ROS), the mitochondria contain specific receptors for the thyroid hormones. Although the involvement of mitochondria and an apoptotic process have been hypothesized, the effect of iodide excess on the mitochondrial mechanism of oxidative stress and antioxidative defense remain poorly defined..Recently, we have identified a direct correlation between mitochondrial superoxide production and iodide excess induced oxidative stress, which suggest that mitochondrial superoxide production may be a reliable metabolic sensor in thyroid, the imbalance between oxidative stress and antioxidative defenses may lead to disclose the underlying mitochondrial mechanisms in the development of thyroid cytotoxicity..Targeting mitochondrial oxidative stress, we plan to do as follows:.(1) In vitro systems. To assess the role played by iodide-induced mitochondrial oxidative stress in the thyroid cells, we investigated the concentration-response and time-course effects; Besides, we investigate the effect of pharmacologic strategies on iodide excess induced mitochondrial oxidative stress-antioxidative defenses, mitochondrial permeability transition pore (mPTP) and apoptosis, by using the inhibitor of the enzymatic activity of thyroperoxidase (TPO), the competitive inhibitor of iodide transport (perchlorate), superoxide dismutase (SOD) mimics or SOD inhibitor, the inhibitor of complex I (rotenone) or the antagonist of complex III (antimycin) of the electron transport chain (ETC)..(2) In vivo systems. We aim to investigate the effect of short-term iodide excess on the mitochondrial mechamism of oxidative stress-antioxidative defenses in thyroid. In addition,as mammary glands also have the ability to concentrate iodide, we plan to investigate the the effect of iodide excess on the mitochondrial mechamism of oxidative stress-antioxidative defenses in the breast of lactating rats..This study will bring new insights on the mitochondrial mechanism of iodide excess induced oxidative stress-antioxidative defenses in thyroid and breast.

碘过量在甲减、甲亢、甲状腺自身免疫性疾病的发生、发展以及甲肿流行中起重要作用。碘是甲状腺激素合成的必需元素，线粒体含有甲状腺激素的特异受体。我们发现：线粒体超氧化物生成是碘过量诱导的甲状腺线粒体氧化应激的敏感指标，线粒体氧化应激与抗氧化防御的失衡可能揭示甲状腺细胞毒性产生的机制。在新发现的基础上,我们拟(1)在细胞水平,研究碘对甲状腺线粒体氧化应激的浓度-效应和时间-效应；通过阻断甲状腺过氧化物酶、竞争性抑制碘转运、抑制或增强超氧化物歧化酶、抑制呼吸链复合体I或III等手段，明确碘过量诱导线粒体氧化应激-抗氧化防御产生、以及线粒体通透性转换孔（mPTP）开放与细胞凋亡的线粒体机制。(2)在动物水平，研究短期碘过量对甲状腺线粒体氧化应激-抗氧化防御的机制;研究碘过量对泌乳母鼠乳腺的线粒体氧化应激-抗氧化防御机制;为防治碘致性甲状腺疾病及非甲状腺碘转运组织（乳腺）的疾病提供新思路和理论依据。

在体外研究，利用线粒体荧光探针MitoSOX, 我们发现早期高浓度碘处理大鼠甲状腺FRTL细胞，2 h后线粒体超氧化物产生显著增加、脂质过氧化、细胞色素C 大量释放、细胞膜破坏，24 h 后出现明显的DNA 碎片和凋亡小体的形成；而且，抗甲状腺药丙基硫氧嘧啶（PTU，300 μmol/L）、TSH（10 mU/mL）、NIS 竞争抑制剂过氯酸盐（KClO4，30 μmol/L）干预后，线粒体超氧化物产生明显减少；反之，SOD 抑制剂二乙基二硫代氨基甲酸银（DETC，2mmol/L）可促进碘过量诱导的甲状腺线粒体超氧化物产生。线粒体呼吸链抑制剂也参与了高碘诱导的ROS产生。线粒体复合体Ⅰ抑制剂鱼藤酮（Rotenone）常被用作植物杀虫剂，线粒体复合体Ⅲ抑制剂Antimycin A 常用于鱼类养殖的杀虫剂，可通过食用植物或鱼类被人体摄入。我们发现碘化钾处理FRTL细胞后2 h，线粒体超氧化物产生显著增加，且SOD 抑制剂(DETC 2mM)、鱼藤酮（0.5 μmol/L）、Antimycin A（10 μmol/L）均可促进线粒体超氧化物产生，降低细胞活力，而SOD 1000 unit/mL对FRTL细胞具有保护作用。.在活体动物研究，我们将Wistar大鼠随机分为适碘组、10 倍和100倍高碘组处理7、14和28天，发现各组间甲状腺激素水平无明显差别，随着碘摄入量的增加，尿碘和组织碘浓度显著增高，碘过量诱导甲状腺的氧化应激线粒体超氧化物产生增加的同时，通过Nrf2-Keap 1通路诱导抗氧化防御的增强以维持碘过量早期甲状腺功能的稳定；哺乳期（哺乳7天、14天和21天）适碘、10倍和100倍高碘喂养的动物模型研究发现随着碘摄入量的增加，母鼠尿碘浓度和甲状腺和乳腺的组织碘浓度也显著增加，FT3水平下降，大鼠甲状腺和乳腺组织线粒体超氧化物生成增加的同时，T3依赖型甲状腺激素受体α1、β1、钠碘转运体也显著下降。100倍高碘的甲状腺组织免疫组化可见出现淋巴细胞浸润。另外，我们利用金属硫蛋白基因敲除小鼠短期高碘饮食模型研究发现：缺乏金属硫蛋白保护的甲状腺在碘过量处理后会产生更强的氧化应激和甲状腺细胞的损害。