补肺阳方通过miRNA-146a—NF-κB信号通路对COPD肺阳虚证大鼠免疫功能调控的研究

Immune imbalance which charactered by the decrease of the CD4+/CD8+ ratio of T lymphocytes is one of the important mechanism in the pathogenesis of COPD. The maturation and differentiation of T lymphocytes was regulated by NF- kappa B. The expression of NF- kappa B can be upregulated by the combination with miRNA-146a which can also promote the proliferation and differentiation of T cells. Above all speculates that miRNA-146a - NF- kappa B is an important signal pathway in pathogenesis of COPD immune imbalance. "Tonifying the lung Yang prescription" which confirmed can treats COPD lung Yang Deficiency Syndrome Treated and improve the immunity of the patients from our prescription of data mining and literature..Our project aims to establish rat models of COPD lung Yang deficiency syndrome by two modeling method and then intervene this disease by the application of "tonifying the lung Yang prescription". In our study, we will observe its effect on COPD lung Yang through the observation of the rat’s ordinary circumstances, the lung function and the trachea tissue structure change of lung. The immune function of rats with COPD lung Yang deficiency syndrome will be observed through the detection of CD4+/CD8+ and the expression of miRNA-146a and NF- kappa B. Based on the findings from our studies, we hope to identify the regulation role of "tonifying the lung Yang prescription" on immune function through miRNA-146a and NF- kappa B which can provide research ideas and new experimental evidence for the research of COPD lung Yang deficiency syndrome and the treatment of COPD by traditional Chinese medicine.

以T淋巴细胞的CD4+/CD8+比值降低为特点的免疫失衡是COPD发病的重要机制之一，NF-κB可调控T细胞的成熟与分化，而miRNA-146a能通过结合并上调NF-κB的表达促进T细胞的增殖和分化。因此miRNA-146a—NF-κB是COPD免疫失衡发病机制中一条重要的信号通路。“补肺阳方”是本课题组前期挖掘文献数据得出的治疗肺阳虚证的方剂，临床实验证实能有效提高患者免疫力。.本项目拟建立COPD肺阳虚证大鼠模型并用补肺阳方干预，通过观察大鼠肺功能及组织结构变化，研究补肺阳方对模型大鼠呼吸功能改善情况；通过检测CD4+/CD8+及miRNA-146a、NF-κB表达，研究补肺阳方对模型大鼠免疫功能调控作用。从而验证“补肺阳方可能通过miRNA-146a—NF-κB信号通路对COPD患者的免疫失衡状态发挥作用”的假说，为“肺阳虚证”及中药治疗COPD作用机制研究提供新的思路和实验依据。

本项目采用烟熏加气管滴注脂多糖的方法建立COPD大鼠模型，通过常温游泳加冰水游泳的方法二次造模制作病证结合COPD肺阳虚证大鼠模型，造模完成后进行补肺阳方高、中、低剂量组及对照药物灌胃治疗。治疗结束后，通过观察大鼠一般状况及肺功能的变化，研究补肺阳方对COPD肺阳虚证大鼠症状的改善情况；通过石蜡包埋肺、气管，切片进行形态学观察，扫描电子显微镜观察气管超微结构，研究补肺阳方对COPD肺阳虚证大鼠呼吸系统形态结构变化的作用；通过称重胸腺、脾脏并计算其指数，流式细胞仪检测脾脏CD4+、CD8+并计算其比值，研究补肺阳方对COPD肺阳虚证大鼠免疫功能的改善作用；通过免疫荧光检测脾淋巴细胞NF-κB、RT-PCR检测miRNA的表达情况，研究补肺阳方对COPD肺阳虚证大鼠免疫功能的调控作用。.结果显示：.1.各组大鼠的一般情况及肺、气管光镜和气管电镜的病理结果及肺功能指标均符合COPD的病理表现，并出现了阳虚不温及气虚的症候表现，说明 COPD肺阳虚证模型复制成功。.2.造模后大鼠的胸腺指数显著降低，脾脏指数呈升高趋势。脾脏CD4+/CD8+比值造模后各组显著降低，治疗后有所恢复，说明造模使大鼠免疫能力下降，而服用补肺阳方可以达到纠正COPD免疫失衡的作用。.3.模型大鼠脾脏淋巴细胞中NF-κB表达增强，经补肺阳方干预后NF-κB表达减弱，这与CD4+/CD8+比值的变化基本一致。.4.模型组大鼠脾脏miRNA-146a的表达降低，各治疗组表达较模型组进一步降低。.通过本项目研究，探讨了补肺阳方对 COPD 免疫功能的调控作用。初步阐释了COPD肺阳虚证大鼠miRNA-146a—NF-κB通路对免疫失衡的影响。初步阐明了补肺阳方通过miRNA-146a—NF-κB通路改善COPD肺阳虚证大鼠免疫失衡状态的作用机制，为中医肺阳虚证的研究及中医药治疗COPD的作用机制提供理论及实验依据。