益气温阳方调控NK细胞线粒体分裂/融合平衡干预变应性鼻炎的机制研究

Abnormal NK cell activation in the Th2-type response is of great significance, which is an important pathological factor for allergic rhinitis (AR) and other diseases. However, up to now, the mechanism of abnormal NK cell activation in AR has not been elucidated. Researches have shown that the dynamic imbalance of mitochondria is an important intrinsic factor in the abnormal changes of cells associated with allergic diseases, and it is closely related to the activation of NK cells. Our previous research showed that Yiqi Wenyang Recipe could effectively regulate NK cell to treat AR. Preliminary experiment prompted that the regulation of immune response by Yiqi Wenyang Recipe may be related to the improvement of mitochondrial function. Therefore, we propose the hypothesis that hat the dynamic imbalance of mitochondria is the pathological mechanism of abnormal NK cell activation in AR, and Yiqi Wenyang Recipe may play an important role in improving AR by restoring mitochondrial homeostasis and inhibiting the abnormal changes of NK cells. In order to verify this hypothesis, we will use AR mouse model, primary NK cells, NK-92 cells and adopt multiple techniques such as molecular biology, cell biology, siRNA, lentiviral vector transfection and other techniques to clarify the important role of dynamic imbalance of mitochondria in the activation of NK cells and the pathogenesis of AR, and reveal the mechanism of Yiqi Wenyang Recipe in regulating NK cells from aspects of molecular, cell, tissue and other levels, which will provide new ideas for the prevention and treatment research of Traditional Chinese Medicine in AR.

NK细胞异常活化在Th2型反应中具有重要意义，为变应性鼻炎(AR)等疾病重要病理因素。但至今，AR中NK细胞异常活化的机制尚不明确。研究表明，线粒体动态失衡是变应性疾病相关细胞异常变化的重要内在因素，与NK细胞活化密切关联。我们前期研究表明，益气温阳方能有效调控NK细胞干预AR。预实验结果则提示，益气温阳方调控免疫应答与改善线粒体功能相关。为此，课题组提出假说，线粒体动态失衡是AR中NK细胞异常活化的病理机制；益气温阳方则可能通过恢复线粒体动态平衡，抑制NK细胞异常变化，以起到改善AR的作用。为了验证该假说，课题组将通过AR小鼠模型、原代NK细胞、NK-92细胞，采用分子生物学、细胞生物学、siRNA、慢病毒载体转染等技术，从分子、细胞、动物整体水平等多个层面阐明线粒体动态失衡在NK细胞活化、AR发病中的重要作用，揭示益气温阳方调控NK细胞的机制，为中医药AR防治研究提供新的思路。

目的 揭示线粒体动态失衡是NK细胞异常活化及变应性鼻炎（AR）进展的重要机制，而益气温阳方可能通过恢复线粒体分裂/融合平衡，减少下游ROS等大量生成，抑制NK细胞分化NK2型分化，以有效干预AR的作用机理，为中医药AR防治研究提供新的思路。.方法 以线粒体分裂抑制剂（Mdivi-1）干预OVA建立的小鼠模型，采用ELISA法、病理组织学染色、免疫荧光、荧光探针、透射电镜结合激光共聚焦等技术，观察小鼠过敏症状及变态反应性炎症，鼻黏膜、脾脏及局部NK细胞JAK2等信号分子表达，鼻黏膜及脾脏ROS水平，鼻黏膜、脾脏及局部NK细胞线粒体形态；建立NK细胞体外NK2活化模型，以Mdivi-1干预，观察重建线粒体动态平衡对NK细胞活化表型、JAK2、STAT6信号活化及ROS内生水平的影响。分别以益气温阳方及君药成分黄芪甲苷干预小鼠AR模型，结合HE染色、ELISA、免疫荧光染色、荧光探针、电镜、激光共聚焦等技术，观察益气温阳方及君药成分黄芪甲苷对AR小鼠行为学变化，鼻黏膜、脾脏及局部NK细胞JAK2、STAT6信号分子活化，鼻黏膜及脾脏内生ROS水平，鼻黏膜、脾脏及局部NK细胞Drp1活化水平及线粒体形态的影响。.结果 Mdivi-1的体内、外研究结果表明，抑制线粒体过度分裂，可有效下调内生ROS水平，抑制JAK2、STAT6等信号分子活化，逆转NK细胞的NK2型活化，亦改善AR相关的变应性炎症，缓解AR相关症状。益气温阳方及其君药有效成分黄芪甲苷可有效改善AR小鼠鼻腔过敏症状及变应性炎症反应，调节鼻黏膜、脾脏及局部NK细胞IL-4、FN-γ表达；抑制AR小鼠鼻黏膜及脾脏JAK2、STAT6蛋白的磷酸化及内生ROS水平的升高，逆转其鼻黏膜、脾脏及局部NK细胞Drp1活化、线粒体过度分裂现象。.结论 线粒体动态失衡为NK细胞异常活化内在机制之一，其或是调控NK细胞NK2型活化介导的变应性炎症反应的重要靶点。益气温阳方及其君药有效成分黄芪甲苷可通过抑制Drp1介导的线粒体过度分裂，重建线粒体动态平衡，调节下游ROS内生及相关信号分子活化，以抑制AR小鼠NK细胞NK2型活化，发挥改善AR的效应。本研究为益气温阳方开展高质量大型临床研究及进行深度临床推广奠定了重要理论基础。