TRAb对脑血管内皮细胞功能影响与Graves病小鼠延髓蛋白质组学及转录组学的研究

Acute thyrotoxic myopathy (ATM) is a rare but serious complication of hyperthyroidism. It includes different degrees of encephaledema or cerebral hemorrhage with obscure pathogenesis, presenting as abrupt onset of bulbar palsy. The conditions could deteriorate rapidly with attendant morbidity. Currently with treatment as ATD and glucocorticoid, most patients can get a complete recovery. However, the specific mechanism of ATM is still not understood. In the previous data of our group, we found that patients with ATM were prone to have a relatively high levels of thyrotrophin receptor antibody (TRAb) and patients of hyperthyrotism had higher levels of blood endothelium-derived relaxing factor. These findings are support of our hypothesis that the excess of TRAb may influence the function of vascular endothelial cell (VEC) through influencing TSHR-related pathway. Therefore, in this study we are focusing on the influence of TRAb on the penetration function, inflammatory response, and oxidative stress of VEC in levels of molecule, cell and animal model. At the same time, we will combine application of proteomics and transcriptomics to explore distinction of medulla and cerebrospinal fluid between TRAb (+) and TRAb (-) rats in mRNA and protein levels to search for potential targeted factors. This research is important and valuable for further exploring of the pathogenesis of acute thyrotoxic bulbar palsy in ATM, and also shed light on the functional mechanism of glucocorticoid’ intervening role.

急性甲亢肌病（ATM）是甲亢较少见的严重并发症，存在不同程度的脑水肿或脑出血，表现为急性延髓麻痹，病情变化迅速、危及生命，目前的治疗策略为应用抗甲状腺药物和糖皮质激素，可迅速缓解症状。ATM具体发病机制尚不明确。本课题组的前期研究发现，发生ATM患者存在高水平的血浆促甲状腺激素受体抗体（TRAb），且甲亢患者血浆中血管舒张因子均增加，推测可能与过高的TRAb结合促甲状腺激素受体（TSHR）后对下游通路的影响有关。本课题拟在分子、细胞及动物层次，从脑血管内皮细胞的舒缩功能、炎症反应、氧化应激等多个角度出发，研究TRAb对脑血管内皮细胞功能的影响，同时应用蛋白质组学及转录组学整合研究策略，筛选并验证由TRAb导致的Graves病小鼠延髓及脑脊液的差异分子，为进一步探讨ATM及其延髓麻痹的机制、以及糖皮质激素的干预研究提供新的理论依据和实验基础。

急性甲亢肌病（ATM）是甲亢较少见的严重并发症，具体发病机制尚不明确。促甲状腺激素受体抗体(TRAb)作为一种Graves病中特异性存在的甲状腺自身免疫抗体，可与促甲状腺激素受体（TSHR）结合后激活受体后相关信号通路，从而在Graves病的发病过程中起到重要作用。本研究通过小鼠脑血管内皮（bEnd.3）细胞实验、Graves病小鼠模型及临床ATM标本转录组学研究，探索TRAb在ATM发病过程中的作用及其机制。本研究结果显示：小鼠延髓组织局部及脑血管内皮细胞上表达TSHR，刺激性TRAb（M22）可在mRNA水平和蛋白水平上诱导bEnd.3细胞TSHR的表达增加，并增加细胞内cAMP水平，证实bEnd.3细胞表达功能性的TSHR。进一步研究发现，M22经TSHR参与了NF-κB信号通路激活，促使IKBα的磷酸化及p65入核，增加下游炎症因子IL-1β、TNF-α以及内皮功能相关蛋白MMP9、AQP4、 Occludin的表达改变，导致bEnd.3细胞屏障模型TEER值下降及Na-F透过增加，提示内皮屏障功能损伤、通透性增加。而甲强龙干预可通过抑制NFκB通路，有效地逆转M22对bEnd.3细胞屏障的损伤。临床ATM患者外周血转录组学显示，与甲亢个体相比，差异mRNA主要富集于细胞因子、免疫补体途径等通路上，并包括体外实验证实的NF-κB信号通路在内；此外，炎症因子还可能通过影响细胞活性氧簇合成以及相关免疫细胞的趋化，参与ATM的发病过程。至此，本研究通过体内外实验、临床标本转录组学多个层次证实了TRAb通过作用于脑血管内皮NF-κB通路进而损伤屏障功能，及糖皮质激素可有效治疗，未来可进一步探索其在ATM患者的临床应用价值。