Sema4D在病毒诱发哮喘NEI网络紊乱中的作用及咳喘宁的整体调节机理

Viral infection is the important inducement of infant asthma occurrence, the mechanism has not yet been elucidated. Recent studies suggest that the nerve endocrine immune (NEI) network dysfunction plays an important role in the pathogenesis of asthma has been, but its exact mechanism is still not clear, effective means has not been found to regulate the network in the prevention and treatment of asthma at present. Recent studies have confirmed that the brain signal protein 4D (Sema4D) is the link of NEI three systems, it may be involved in the pathogenesis of asthma, but the specific mechanism is not clear. The whole regulation is the characteristic and advantage of Chinese medicine. Our preliminary research showed that kechuanning can prevent and cure asthma induced by virus by influencing the PI3K signal path, and the signal path is closely related to the asthma neurogenic inflammation, so it could be inferred that kechuanning can display Chinese medicine regulating holistic function by regulating Sema4D and adjusting NEI network disorder. The technologies such as molecular biology、 gene targeting technology will be used to confirm this hypothesis. It plays a role in the asthmatic NEI network disorders will be confirmed from the aspect the research of the effect of Sema4D on the three systems for the first time. That kechuanning can improve asthma NEI network disorder by adjusting the Sema4D is the main mechanism. The scientific hypothesis will provide the new theory basis for the asthma NEI network mechanism, and will provide the practice basis for the whole regulation mechanism of traditional Chinese medicine in the prevention and treatment of asthma induced by virus.

病毒感染是婴幼儿哮喘发生的重要诱因，机制尚未阐明。新近认为神经内分泌免疫(NEI)网络功能紊乱在哮喘发病中发挥了重要作用，但其确切机制还不清楚，目前未找到调节这一网络防治哮喘的有效手段。近年研究证实，脑信号蛋白4D（Sema4D）是NEI三大系统间的纽带，可能参与了哮喘发病，但其具体机制尚不明确。整体调节是中医药的特点与优势，我们前期证实咳喘宁能通过影响PI3K通路防治病毒诱发哮喘，而此通路与哮喘神经源炎症密切相关，因而推测其可能通过调节Sema4D，改善哮喘NEI网络紊乱而发挥中医药的整体调节作用。为证实这一假说，我们将采用分子生物学、基因打靶等技术，首次从研究Sema4D对三大系统的影响，证实其在哮喘NEI网络紊乱中发挥了作用，咳喘宁通过调节Sema4D，改善哮喘NEI网络紊乱是其主要作用机理的科学假说，为哮喘NEI网络机制提供新的理论依据，为中医药防治哮喘的整体调节机理提供实践依据。

目的：通过干预 Sema4D 对病毒诱发哮喘中 NEI 网络失衡的影响，探讨咳喘宁口服液对哮喘机体整体调控作用的可能途径，为进一步阐明咳喘宁防治本病的机理提供理论与新的实验依据。方法：建立大鼠 RSV 复合 OVA 哮喘模型和Sema4D 基因敲除模型小鼠，观察 Sema4D 在哮喘肺与内脏感觉传入系统的表达变化、对哮喘大鼠 Th1/Th2、HPA 轴功能及离体气道上皮增殖与迁移的影响、对 NGF 与 PI3K- Akt 信号传导通路的调节作用以及咳喘宁口服液的调节机理。结果：①大鼠体内Sema4D 表达增强， CD72降低 ，Plexin-B1 表达增强，SIRPα及配体CD47表达明显增高， α-SMA、MMP-2 的表达显著上调，且气道上皮有部分脱落，上皮胶原沉积明显增多，诱导促炎因子TNF-α刺激嗜酸性粒细胞分泌IL-6，抑制Th1并促进Th2分化，Th2免疫反应过度，使Th1/Th2失衡，IL-4、IL-8分泌增多，机体无法产生足够的IFN-γ，导致炎症的发生，加重哮喘；抑制 Sema4D表达可减少炎症因子的产生，调节 Th1/Th2平衡；Sema4D可促进 NGF 的表达，刺激大鼠脑内c-fos蛋白的表达及SP的释放而进一步抑制机体HPA轴内分泌激素的释放，从而加重哮喘。②Sema4D 基因敲除模型小鼠Sema4D和Plexin-B1明显 缺失，CD72增强，SIRPα及配体CD47的表达减少，肺组织中 MMP-2、α-SMA 的表达下降，血清中IL-4、IL-6、IL-8、TNF-α水平降低，IFN-γ增多，NGF、c-fos蛋白和SP的表达降低，证实Sema4D 参与病毒诱发哮喘气道免疫炎症、HPA 轴功能及 NGF 表达。③咳喘宁口服液可干预病毒诱发哮喘模型大鼠 Sema4D、Plexin-B1 的表达，增加CD72含量， 抑制SIRPα及配体CD47的表达，下调肺组织中 MMP-2、α-SMA 的表达，减少胶原沉积，改善气道重塑，血清中IL-4、IL-6、IL-8、TNF-α水平降低，IFN-γ分泌增多，维持 Th1/Th2 平衡来调节机体免疫，抑制 NGF 的表达，改善Sema4D对哮喘大鼠HPA轴的抑制。结论：Sema4D 在哮喘 NEI 网络紊乱中发挥了作用，咳喘宁口服液通过调节Sema4D，改善哮喘NEI网络紊乱。