高碘抑制自噬协同DEHP致甲状腺毒性作用的机制研究

Di-2-ethylhexyl phthalate(DEHP) are used extensively for flexibility and durability of plastics, which are widely distributed environmental contaminants and interference with human health. DEHP exposures are associated with altered thyroid function. Due to the national iodine supplement, the problem of excessive iodine intake on thyroid increase seriously. So we are facing dual threat of DEHP exposure and excessive iodine intake. In this study, we sought to examine the synergistic effect of DEHP and excessive iodine intake on thyroid toxicity. First, we found that DEHP is able to significantly enhance autophagic markers of thyroid in rats exposed to DEHP alone, while autophagic markers and T3, T4 level further decrease in DEHP and excessive iodine intake exposed rats. But the mechanism of how excessive iodine intake sensitizes DEHP-induced thyroid toxicity is still not known. Next, we provided evidence that excessive iodine intake induced Bcl2 mRNA level . Bcl2 bind to Beclin 1, and disturb class III PI3kinase complex formation, which is responsible for autophagosome formation. Therefore iodine induced Bcl-2, restrain the protective effect of autophagy, and can be an explanation for the aggravating hypothyroidism. In this study, we utilize FRTL-5 cell and Bcl-2 gene knockout mouse, through Molecular biology method such as by RNAi, to examine the effect of DEHP on autophagy and Bcl-2 gene, and to evaluate whether such effect is relevant to the sensitization effect of DEHP on hypothyroidism induced by excessive iodine intake.

DEHP是常用增塑剂，会释放入环境并危害人体健康，且对甲状腺毒性作用明显；全民补碘推行后，过量碘摄入对甲状腺的副作用日渐突出，两者均为备受关注的公共卫生问题。本课题组发现DEHP暴露大鼠T4下降；甲状腺滤泡变小；胶质浓度下降，起保护作用的自噬被明显诱导；增加碘摄入后自噬被抑制，T3、T4进一步下降。碘如何协同DEHP引起甲减是亟待明确的问题。我们还发现高碘上调Bcl-2mRNA表达，Bcl-2是干扰III型PI3K复合物形成进而抑制自噬的关键基因。本研究拟以此为切入点，通过自噬诱导剂、抑制剂和ATG7siRNA调控自噬，应用Bcl-2siRNA和Bcl-2基因敲除小鼠，构建不同程度Bcl-2基因表达的体内外实验平台，深入探讨自噬在DEHP甲状腺毒性中的保护作用，证实高碘诱导Bcl-2表达抑制自噬，协同DEHP致甲状腺毒性的作用机制，为制定DEHP的安全限量标准及合理的碘摄入量提供理论依据。

本研究围绕DEHP甲状腺毒性、氧化应激及自噬在其中的作用机制展开研究。在按照研究工作计划进行过程中发现DEHP对大鼠具有甲状腺毒性作用，随着DEHP染毒剂量增加血清T4下降，甲状腺滤泡变小、胶质浓度下降；证实DEPH通过抑制体内抗氧化酶的活力，引起机体抗氧化能力与脂质过氧化的失衡是其产生甲状腺毒性的重要机制。且DEHP对甲状腺组织的氧化损伤是不可逆的毒性作用。另外，DEHP可以上调大鼠甲状腺组织的自噬活性。应用人正常甲状腺细胞株Nthy-ori 3-1进行研究发现，DEHP处理后的细胞LC3-Ⅱ蛋白累积，且加入氯喹后LC3-Ⅱ蛋白水平进一步增加。提示DEHP可能在自噬的早期起诱导作用，是DEHP甲状腺毒性作用中的自我保护机制。而DEHP和高碘双重暴露的大鼠，血清T4进一步下降并出现T3的下降。DEHP通过氧化应激引起甲减的同时自噬被明显诱导，而增加碘摄入后自噬反被抑制，进一步提示自噬在这一过程中发挥重要的作用。发现高碘显著上调Bcl-2的mRNA表达，提示高碘通过诱导Bcl-2表达抑制自噬。本课题组同时深入研究DEHP代谢产物MEHP对细胞自噬的影响，发现MEHP可能在自噬的晚期起抑制作用，且MEHP通过抑制自噬小体的降解抑制Nthy-Ori3-1细胞自噬。本研究对自噬及氧化损伤及自噬在高碘协同DEHP甲状腺毒性作用中的机制进行了深入的研究并取得了一定的进展，为今后的研究确立了方向。