NGF通过抑制Notch通路诱导哮喘小鼠肾上腺髓质嗜铬细胞转分化的机制

We have proposed in our previous studies that the insufficiency of epinephrine level under the condition of asthma was due to neuronal conversion of adrenal medulla chromaffin cells induced by elevated nerve growth factor (NGF) level, which is a significant neuroendocrine pathogenesis of asthma; however, the specific mechanism is still unknown. Mash1 is known as a downstream gene of Notch1-Hes1-|Mash1 pathway, it plays an important role in neuron differentiation. It have been found that Mash1 was significantly elevated in chromaffin cells of asthma rats and had a positive correlation with NGF. A previous study demonstrated that NGF could inhibit the activity of Hes1 by protein kinase C. And decreased glucocorticoids(GC) level among asthma could decline the activation function to Notch pathway. Therefore, we hypothesized that elevated NGF level as well as decreased GC level in asthma could reduce the activation of Notch1/Hes1 pathway, so as to increase the Mash1 level, which could induce neuron transdifferentiation of chromaffin cells and subsequently decrease circulating EPI, eventually cause bronchospasm. The project intends to establish asthma mouse model（wild type and Mash1 knock down）, and primary culture mouse chromaffin cells in order to study the role of Notch pathway in NGF, GC-mediated chromaffin cells neuron transdifferentiation, and application of NGF antibody, GC to them so as to further investigate the neuroendocrine pathogenesis of asthma and also provide new ideas to asthma treatment.

我们前期研究发现神经生长因子（NGF)升高促使肾上腺髓质嗜铬细胞向神经元转分化，导致肾上腺素分泌不足可能是哮喘发病重要神经内分泌机制，但具体机制不清。Mash1作为Notch1-Hes1-|Mash1通路下游靶基因, 是神经分化关键因子。在哮喘大鼠嗜铬细胞中Mash1异常增高，且与NGF正相关。研究表明，NGF可通过蛋白激酶C抑制Hes1活性。而哮喘引起糖皮质激素（GC）下降可降低其对Notch通路活化作用。因此，我们推测哮喘中NGF升高、GC下降可降低Notch1/Hes1通路活性，使Mash1升高促使嗜铬细胞表型转化，导致肾上腺素下降，引起支气管痉挛。本项目建立哮喘小鼠模型(野生型、Mash1基因沉默)，并培养原代小鼠嗜铬细胞，拟研究Notch通路在NGF、GC介导嗜铬细胞神经转分化中的作用，并用NGF抗体、GC处理，以从神经内分泌机制进一步探讨哮喘发病机制，为哮喘治疗提供新的思路。

我们前期在卵清蛋白（OVA）致敏的支气管哮喘动物模型中发现神经生长因子（NGF)升高促使肾上腺髓质嗜铬细胞（AMCC）从内分泌表型向神经表型转分化，导致肾上腺素（EPI）分泌不足，这可能是哮喘发病重要神经内分泌机制，但具体机制不清。Mash1作为Notch1通路下游靶基因, 是神经分化关键因子，我们发现其与AMCC神经转分化相关。有研究表明，嗜铬细胞的分化取决于内环境中NGF与糖皮质激素（GC）的水平，GC可维持嗜铬细胞分泌表型。为了从细胞层面证实以上发现，我们以原代培养的大鼠AMCC为研究对象，分别加入Mash1 siRNA和Mash1过表达质粒，再使用NGF、GC进行处理，观察AMCC是否出现形态和功能改变。结果发现，NGF可能通过抑制Notch1/Hes1通路活性，导致Mash1表达增高，促使AMCC向神经元表型转化，最终导致肾上腺素分泌下降。GC对该过程有抑制作用，可部分逆转AMCC表型转分化，使肾上腺素水平上升。本部分从神经内分泌机制进一步探讨哮喘发病机制，为哮喘治疗提供新的思路。此外新近研究发现，Notch1通路也参与了哮喘Th1/Th2免疫失衡过程。而GC作为治疗哮喘最有效的抗炎药物，其具体机制有待研究。为了探讨GC是否通过Notch1通路调节Th细胞免疫过程，我们以OVA致敏的哮喘小鼠模型为研究对象，观察GC对Notch1以及Th细胞的作用，并对比Notch1通路特异性抑制剂GSI作用。研究发现，GC可能通过抑制OVA致敏哮喘小鼠中过度活化的Notch1通路来逆转Th1/Th2免疫失衡，GSI也表现出了同样的作用，但这两者的作用没有协同性。本部分实验不仅是对GC经典抗炎机制的补充，也证实Notch1通路可能成为治疗哮喘的新靶点，GSI可能成为治疗哮喘的新药物，尤其是激素耐药的哮喘患者。