基于LncRNABCAR4调控EGFR/AKT通路探讨滋阴潜阳化痰散结法治疗Graves病的机制

The excessive growth of thyroid epithelial cells induced by TRAb is a fundamental pathological link within the onset of GD.Restraint of the excessive growth is crucial to GD’s treatment.According to the latest research, LncRNA-BCAR4-mediated EGFR/AKT pathway has a close relationship with thyroid cells’ growth.Our team has been using the Chinese medicine named Jia Kang Ning for many years,which is on behalf of nourishing Yin,latent Yang,resolving phlegm and dispersing knots.Previous animal experiments showed that Jiakangning capsule inhibited the expression of AKT and bcl2.We infer that Jia Kang Ning could cut down the EGFR pathway by inhibiting the expression of LncRNA BCAR4.GD is treated by inhibiting the expression of AKT and regulating the dynamic equilibrium of thyroid cells.In this study, immunization Ad-TSHR289 is used to construct a mouse GD model and M22 is used to stimulate Nthy-ori-3-1 cells to establish a GD cell model.In combination with vivo and vitro trials,western blot,qRT-PCR,siRNA and other technologies are used with the LncRNA BCAR4-EGFR/AKT pathway as a breakthrough point to further explore the mechanism of the treatment of GD with Chinese medicine Jia Kang Ning,so as to provide new ideas and targets for the treatment of GD.

TRAb诱导的甲状腺上皮细胞过度增殖是GD发病的基本病理环节。抑制甲状腺细胞过度增殖是GD治疗的关键。最新研究提示，LncRNABCAR4介导的EGFR/AKT通路和甲状腺细胞增殖密切相关。本课题组多年来采用中药甲亢宁代表的滋阴潜阳化痰散结法治疗GD且前期动物实验表明甲亢宁对AKT、bcl2的表达均有抑制作用。我们推测甲亢宁可能通过抑制LncRNABCAR4的表达，从而下调EGFR通路，抑制AKT的表达而抑制TSAb刺激的甲状腺细胞增殖、促其凋亡来治疗GD。本研究拟通过免疫注射Ad-TSHR289法构建小鼠GD模型，M22刺激Nthy-ori-3-1细胞建立GD细胞模型，体内外实验相结合，应用蛋白印迹、qRT-PCR、siRNA等技术，以LncRNABCAR4-EGFR/AKT通路为切入点，进一步探讨中药甲亢宁治疗GD的机制，为治疗GD提供新的思路和靶标。

TRAb诱导的甲状腺上皮细胞过度增殖是Graves病（以下简称GD）发病的基本病理环节。抑制甲状腺细胞过度增殖是GD治疗的关键。最新研究提示，LncRNABCAR4介导的EGFR/AKT通路和甲状腺细胞增殖密切相关。本课题组多年来采用中药甲亢宁代表的滋阴潜阳化痰散结法治疗GD且前期动物实验表明中药甲亢宁对AKT、bcl2的表达均有抑制作用。我们推测甲亢宁可能通过抑制LncRNABCAR4的表达，从而下调EGFR通路，抑制AKT的表达而抑制TSAb刺激的甲状腺细胞增殖、促其凋亡来治疗GD，认为该假说符合逻辑，科学合理。通过研究，我们得出结论：①中药甲亢宁在一定程度上改善甲亢小鼠模型的病理结构，下调Ki67的表达水平；②中药甲亢宁能够下调甲亢小鼠甲状腺组织EGFRmRNA、AKTmRNA的表达，上调baxmRNA的表达，下调EGFR、p-EGFR、AKT、p-AKT、bcl-2、CyclinD1蛋白的表达；③中药甲亢宁能够调控cAMP的水平，能够抑制Nthy-ori-3-1甲亢细胞模型增殖，促进细胞凋亡；④中药甲亢宁能够下调Nthy-ori-3-1细胞模型中LncRNABCAR4mRNA、EGFRmRNA、AKTmRNA、bcl-2mRNA的表达，下调EGFR、p-EGFR、AKT、p-AKT、bcl-2、CyclinD1蛋白的表达；⑤LncRNABCAR4-siRNA可以协同中药甲亢宁所致的抑制Nthy-ori-3-1甲亢细胞模型增殖、促其细胞凋亡的作用；⑥中药甲亢宁治疗Graves病的机制可能与调控LncRNABCAR4的表达，从而抑制EGFR/AKT信号通路，抑制甲状腺细胞增殖、促进其凋亡有关。本研究通过体内外实验相结合，以LncRNABCAR4-EGFR/AKT通路为切入探讨中药甲亢宁治疗GD的机制，从而为滋阴潜阳化痰散结法治疗GD提供科学依据，为临床上治疗GD提供新的思路和靶标。