基于TGF-β1/Smad3信号通路探讨养阴清肺汤加味调控肺炎支原体感染肺上皮-间质转化的研究

Mycoplasma pneumoniae (MP) has wide pathogenicity. Severe mycoplasma pneumoniae pneumonia (SMPP) cases increase year by year. SMPP is highly related to MP characteristics for its antibiotic resistance, toxin release, mucus hypersecretion and hypercoagulability. Pulmonary epithelial-mesenchymal transition (EMT) is a new found pathogenesis of SMPP. On the basis of previous studies on dryness-heat injuring lung-fluid, the hypothesis that diagnosis and treatment for SMPP by dryness-toxin is drawn. And dryness-toxin rob lung collaterals is related to epithelial-mesenchymal transition in SMPP. Yangyin Qingfei Decoction is the traditional famous formula and has exact effect in clinics, pleasant embellish bitter drain, has function that can nourish yin to clear away the lung-heat and eliminate epidemic toxin, while adding honeysuckle and forsythia has benefit to patients with high fever. We applied the techniques of HPLC-MS to combine active ingredients quantification, serum pharmacology as well as serum pharmacochemistry in correlation analysis to explore material basics. Previous studies found that TGF-β, IL-17, TLR and other signal pathways were significantly enriched and related to EMT. Balb/c mice infected by high MP load is designed to observe dynamic change of EMT induced by TGF-β1/Smad3 signaling pathway with techniques of iTRAQ, ELISA, qPCR and so on. This study attempted to explore the content of dryness-toxin and lung collaterals through biological indicators, to compare effectiveness of modified Yangyin Qingfei Decoction, as well as to discuss the mechanism of integrated regulation through pulmonary EMT in SMPP.

肺炎支原体(MP)具有广泛致病性，重症MP肺炎（SMPP）逐年升高。SMPP与MP耐药、毒素释放、黏液多、高凝等有关，肺上皮-间质转化（EMT）是其发病新机制。在前期燥伤肺津基础上，从燥毒论治SMPP，提出“燥毒劫伤肺络”与SMPP肺EMT调控通路相关假说。经典名方养阴清肺汤临床疗效确切，甘润苦泄，养阴清肺，兼散疫毒；热甚者，加银花、连翘；采用HPLC-MS技术，从活性成分定量、血清药理学-药物化学关联分析，探索药效物质基础。前期对MP感染小鼠肺mRNA测序筛选，发现TGF-β、IL-17 、TLR等信号通路富集显著,恰与EMT相关。高载量MP感染BALB/c小鼠建立SMPP模型，采用iTRAQ、ELISA、qPCR等方法，观察TGF-β1/Smad3信号通路诱导EMT动态变化，阐明“燥毒”、“肺络”生物指标内涵，探讨养阴清肺汤加味效果差异性，揭示其改善SMPP肺EMT的调节机制。

肺炎支原体（MP）具有广泛致病性，重症MP肺炎（SMPP）有逐年增加的趋势。在前期研究基础上，本研究认为SMPP具有“燥毒劫伤肺络”的中医病机特点，提出“燥毒”、“肺络”与“肺EMT及调控通路元件”的生物学指标相关的假说，探讨养阴清肺汤加味（YQDP）通过TGF-β1/Smad3信号通路调控SMPP肺EMT的机制。研究首先采用了正交设计、Box-Behnken法、HPLC-MS等技术，从醇提工艺、复方指纹图谱、药代动力学等方面探索了YQDP的药效物质基础，进一步优化了YQDP的提取工艺，绘制了复方指纹图谱，结合大鼠体内药代动力学性质，绘制了药时曲线，初步探讨了YQDP药代动力学特征。其次，基于影像学、组织学及分子生物学检测技术，建立并评价了SMPP模型小鼠，并以此为基础开展了多项实验研究，系统地揭示了YQDP多通路、多指标干预SMPP的靶点机制。实验结果显示，YQDP可通过抑制性调控TGF-β1/Smad3信号通路来影响EMT的发生，还可通过TLR-2/MyD88/NF-κB以及ROS/NLRP3信号通路发挥抗炎作用，且YQDP的作用趋势与剂量呈现依赖性。在YQDP的“润燥”作用研究中，YQDP可促进AQP5、Occludin和ZO-1的表达，抑制MUC5ac的分泌来调节肺内的水液平衡，也可调控RORγt/Foxp3免疫平衡及肺表面活性物质SP-A、SP-D、DPPC动态的表达来影响Th17/Treg平衡，有效修复肺部屏障系统。本项研究还结合了4D-FastDIA定量蛋白质组学技术，探析YQDP干预SMPP小鼠肺组织蛋白的差异性表达，表明YQDP可有效治疗SMPP，其机制可能与调节先天免疫反应、炎症因子等途径相关。本课题从多层面、多角度揭示了YQDP基于“燥毒劫伤肺络”理论治疗SMPP的现代生物学内涵，为中药治疗SMPP提供了理论支持和实验依据。