补肺益肾方通过树突细胞外泌体调节COPD大鼠Th17/Treg平衡的机制研究

Bufei Yishen Formula (BYF) is an indication for the COPD of "lung and kidney deficiency". We found that BYF can correct the IL-17-producing CD4+T helper/regulatory T cells(Th17/Treg) imbalance in COPD , inhibit the maturation of dendritic cells (DCs), and increase the DC-exosomes (DC-exo) significantly. Based on the increased expression of miRNA-206 in COPD, and exosomes are markers in the sputum of patients with COPD and are the main carriers of miRNA. The scientific hypothesis is proposed: the main mechanism of BYF regulates Th17/Treg axis COPD is regulates the level of miRNA in DC-Exo. The following work is planned: First,The effect of BYF on DCs, Th17 and Treg cells is detected by flow cytometry and ELISA; Second, DC-Exo is separated by ultra-high speed centrifugation and kit method, and DC-Exo is identified by fluoroscopy and nanometer The microarray gene chip was used to screen the differential gene, and the target gene was quantified by PCR. Last, The miRNA-206, miRNA-155 regulates the maturation of dendritic cells .To investigate the differentiation and mechanism of miRNA-regulated DC-Exo on Th17 and Treg cells in COPD rats, and to explain the mechanism of COPD treatment from BYF-DC-Exo-Th17/Treg.

补肺益肾方（BYF）主治“肺肾气虚”型COPD，研究发现BYF能纠正COPD大鼠Th17/Treg失衡状态，抑制树突细胞（DCs）成熟，给予该方血清的DCs外泌体（exo）显著增加。基于外泌体是COPD患者痰液中标志物且是miRNA的主要载体，在液体活检中应用广泛的现状，提出科学假说：BYF通过调节DC-Exo内miRNA水平，调节Th17/Treg轴是其治疗COPD的主要机制。拟开展以下工作①采用流式细胞术和ELISA法检测BYF对DCs、Th17和Treg细胞的影响；②采用超高速离心和试剂盒方法分离DC-Exo，采用透视电镜和纳米检测仪鉴定DC-Exo，采用微阵列基因芯片筛选DC-Exo差异基因，用PCR方法定量目标基因；③研究miRNA-206，miRNA-155调节树突细胞成熟度和对T细胞分化情况，从BYF-DC-Exo-Th17/Treg角度阐释该方治疗COPD的机制。

本研究基于代谢组学—血清药物化学相结合的方法研究补肺益肾方治疗COPD稳定期大鼠的的药效物质基础，分析补肺益肾方的药效物质基础为人参皂苷Re、Rb1、Rg1、淫羊藿苷、黄芪甲苷、芍药苷、贝母甲素、橙皮苷等8个成分。以其中的人参皂苷Rg1为主要药效成分，研究其对树突细胞成熟度的影响及其外泌体中差异miRNA的筛选，发现人参皂苷Rg1可通过调节树突细胞外泌体中的miRNA，促进树突细胞成熟，通过基因芯片技术筛选和转录组学研究发现，人参皂苷Rg1可能通过调节 imDC-exo中miRNA-695水平，下调PI3K-AKT通路，抑制COPD的发生。通过电穿孔的方法制备富含miRNA--695的外泌体，通过马尔文粒径仪以及透射电子显微镜对制备的富含miRNA的外泌体进行表征， Westernblot检测包载miRNA-695的外泌体较对照组对PI3K-AKT有较显著的影响作用，证实了人参皂苷Rg1刺激组可通过调节imDC-exo中miRNA-695水平，从而下调PI3K-AKT通路，抑制COPD的发生。