NGF调控嗜铬细胞Ca2+通道致RSV感染小鼠肾上腺素异常释放机制

The secretion of epinephrine include synthesis and release stages. We found that the occurrence of asthma after the Respiratory syncytial virus (RSV) infection is closely related to the the lack of epinephrine secretion, which May due to the elevated nerve growth factor (NGF) induced transformation of adrenal chromaffin cells into neurons and lead to the decrease of synthesis of epinephrine. The release of epinephrine depends on the activation of chromium membrane Ca2+ channel to produce enough intracellular calcium concentrations ([Ca2+]i). the nicotinic acetylcholine receptor (nAChR) subtypes located in the chromaffin cell membrane activated Ca2+ channel and regulate the [Ca2+]i. NGF can inducce the expression of α7nAChR increased and activated Ca2+ channel which may elevated [Ca2+]i and caused the release of epinephrine. We hypothesized that due to the synthesis dysfunction of epinephrine after RSV infection, through a negative feedback mechanism, NGF induce the expression of α7nAChR elevated so that Ca2+ channel is excessiviy activated and [Ca2+]i increased which led to the threshold reduction of epinephrine release. Therefore,to test this hypothesis we plans to carry out the following works: 1、to determine inadequate secretion of epinephtine after RSV infection is closely related to the abnormal release of epinephrine of chromaffin cells, which is relevant with excessive activation of Ca2+ channels； 2、to explore that NGF may induce α7nAChR over-expression in chromaffin cells and regulate Ca2+ channels which may lead to the decline in epinephrine release threshold and participate in the pathogenesis of asthma.

肾上腺素的分泌过程包括合成和释放两个阶段。我们发现：RSV感染后哮喘的发生与机体肾上腺素分泌不足密切相关，可能因RSV感染引起NGF升高，诱导嗜铬细胞向神经元转化致肾上腺素合成减少。肾上腺素的释放需活化嗜铬细胞Ca2+通道产生足够浓度的细胞内钙离子浓度( [Ca2+]i)，[Ca2+]i由烟碱样乙酰胆碱受体（nAChR）活化Ca2+通道调控。NGF可诱导α7nAChR表达上调并活化Ca2+通道致[Ca2+]i升高。据此推测：RSV感染后肾上腺素合成减少，可能通过负反馈机制使NGF诱导嗜铬细胞α7nAChR升高，过度活化Ca2+通道使[Ca2+]i升高致肾上腺素释放阈值降低。为验证此假说我们拟开展以下研究：1、明确RSV感染后肾上腺素分泌不足与嗜铬细胞对肾上腺素的异常释放状态密切相关2、探讨NGF可能通过诱导嗜铬细胞α7nAChR过表达调控Ca2+通道致肾上腺素释放阈值降低而参与哮喘发病。

流行病学资料显示反复感染RSV的婴幼儿后期出现哮喘的风险明显增高，其发病机制尚未明确。本研究立足于我们的前期研究提出：肾上腺素的分泌不足除与NGF诱导嗜铬细胞发生神经元转变导致肾上腺素合成下降外，肾上腺素的释放过程同样存在异常，因此，我们分别从体内和体外实验观察NGF调控嗜铬细胞表型和内分泌功能变化、α7nAChR亚型表达变化、Ca2+通道电流以及[Ca2+]i变化。体外实验显示：与单独转染α7nAChR相比，在转录和蛋白表达水平，NGF处理组的α7nAChR的表达明显升高，细胞内Ca2+信号明显增强，DEX处理组的α7nAChR的表达明显下降，Ca2+信号信号明显减弱。ELISA显示：与转染组相比，NGF处理组肾上腺素浓度明显下降，与转染＋NGF组相比，DEX处理组肾上腺素浓度明显升高。体内试验显示：与对照组相比，在转录和蛋白表达水平，模型组、转染组和anti-NGF组的α7nAChR 表达明显升高；与模型组相比，转染组、DEX组、同时转染+NGF 处理组和转染+ DEX 处理组的α7nAChR 的表达明显下降，其中转染+ DEX 处理组有更加显著的抑制α7nAChR 表达的作用。电镜示对照组的细胞嗜铬颗粒分布较均匀；Model 组部分细胞膜出现皱缩并可见杵状和绒毛状突起、颗粒内容较浅淡，shα7nAChR 组嗜铬颗粒数目变少，突触囊泡数量减少；anti-NGF和DEX 组嗜铬颗粒数目变少，突触囊泡减少，转染+ anti-NGF和转染+ DEX 组部分线粒体出现了轻微皱缩现象，免疫荧光显示：与对照组相比，模型组的SYN和NF的表达明显升高；与模型组相比，转染组、anti-NGF和DEX组的SYN和NF的表达明显下降，同时转染和NGF 处理与转染和DEX处理组SYN和NF表达下降更加明显。ELISA显示：与对照组相比，各组大鼠肾上腺素浓度均降低，与模型组相比，转染组、转染+ anti-NGF组和转染+DEX组肾上腺素浓度均降低，与转染组相比，anti-NGF组、地塞米松组以及转染+ anti-NGF组和转染+DEX组肾上腺素浓度均降低，与地塞米松组相比，转染+ anti-NGF组和转染+DEX组肾上腺素浓度均降低。据此，我们推论：NGF可能通过诱导嗜铬细胞α7nAChR亚型表达升高，调控Ca2+通道过度活化致肾上腺素释放阈值下降而参与哮喘的发病。