基于下丘脑ERα介导的IGF-1/NKB crosstalk 探究DEHP诱导雌性性早熟的分子机制

Precocious puberty in girls is related to DEHP, but its mechanism is still unclear. Researchers have found that the hypothalamic IGF-1/PI3K/Akt/mTOR and NKB/NK3R signaling pathways play important roles in regulating the reproduction and development during puberty. Our previous studies have confirmed that DEHP could cause early puberty timing and increase the levels of ERα, IGF-1, NKB and GnRH in hypothalamus of female rats, and we have also found that inhibition of ERα can block the activation of IGF-1 and NKB by DEHP, suggesting that the regulation of ERα in the hypothalamus on downstream IGF-1 and NKB might be critical to DEHP-induced female sexual precocity. In this study, we intend to use molecular biology techniques to observe the effects of DEHP on biomarkers of precocious puberty in female rats and establish a model of female precocious puberty induced by DEHP, and to investigate the expression of genes and proteins related to IGF-1 and NKB pathway mediated by ERα in rat hypothalamus after treatment with inhibitors. In addition, the hypothalamic neurons of female suckling mice were used as in vitro models to verify the molecular mechanism of MEHP interfering with the release of GnRH from hypothalamic neurons by activating ERα that regulates IGF-1 and NKB pathways, and to explore whether the two pathways participate in the regulation through crosstalk mechanism. This study will provide a new target for the prevention and control of precocious puberty in girls.

女童性早熟的发生与DEHP暴露有关，但致病机制尚不明确。目前认为下丘脑IGF-1与NKB通路在调控机体青春期的生殖发育中均发挥重要作用。前期研究证实，DEHP导致青春期雌性大鼠性发育启动时间提前，并上调下丘脑ERα、IGF-1、NKB与GnRH的表达；抑制ERα可阻断DEHP对IGF-1及NKB的激活，提示下丘脑中ERα对下游IGF-1及NKB的调控可能是DEHP诱导雌性性早熟的关键。基于此，本研究拟通过体内实验观察DEHP对雌性大鼠性启动相关标志物的影响并建立其诱导的雌性性早熟模型，探讨抑制剂处理后大鼠下丘脑ERα介导的IGF-1与NKB通路相关基因和蛋白的表达情况。另外，以雌性乳鼠下丘脑神经元细胞为体外模型，验证MEHP激活ERα调控下游IGF-1与NKB通路干扰下丘脑神经元释放GnRH的分子机制，并探索两通路是否通过crosstalk机制参与调控，为防治女童性早熟提供新思路和新靶点。

邻苯二甲酸二-(2-乙基己基)酯（DEHP）暴露与女童性早熟的发生密切相关，但致病机制尚不清楚。本研究旨在阐明DEHP对性早熟发病机制的影响，并探讨两者之间的关系。选择青春期雌性大鼠60只（15日龄），随机分为5组，即即溶剂对照组（玉米油）、阳性对照组（50 μg/kg/d 雌二醇）和低剂量组（0.2 mg/kg/d DEHP，1/15000LD50）、中剂量组（1 mg/kg/d DEHP，1/3000LD50）、高剂量组（5 mg/kg/d DEHP，1/600LD50），每组12只，连续染毒4周。观察青春期雌性大鼠阴道开口时间，测定下丘脑和血清中IGF-1、GnRH以及血清性激素水平，检测下丘脑组织中IGF-1信号通路基因和蛋白表达情况。选取36只实验动物建立DEHP（5 mg/kg/d）暴露诱导雌性性早熟的动物模型，利用脑立体定位注射技术向大鼠下丘脑注射IGF-1R及mTOR特异性抑制剂，观察抑制剂处理后雌性大鼠性早熟相关指标的变化规律，检测IGF-1/NKB通路及其下游相关基因和蛋白的表达情况。.结果显示，DEHP导致雌性大鼠阴道开口时间提前，下丘脑神经元中的尼氏小体数量增加，下丘脑细胞凋亡减少，血清和下丘脑中的IGF-1和GnRH水平升高，下丘脑中NKB水平升高，DEHP暴露组和E2组大鼠下丘脑ERα、IGF-1、IGF-1R、NKB、NK3R和GnRH mRNA及蛋白表达水平明显高于对照组。利用脑立体定位技术进行抑制剂活体干预实验，结果显示抑制下丘脑ERα可阻断DEHP对IGF-1及NKB的激活。 IGF-1R特异性抑制剂处理后，下丘脑NKB蛋白表达水平与对照组相比明显升高，而IGF-1R和GnRH蛋白表达水平显著下降。.综上，DEHP可能通过激活ERα干扰下丘脑IGF-1与NKB信号通路，激活下丘脑GnRH神经元，促进GnRH释放，从而导致女性性发育的开始。我们的研究结果为DEHP诱导的青春期雌性大鼠生殖和发育毒性提供了一种新的分子机制，并将为DEHP诱导的性早熟的预测和逆转提供一个新的靶点。