羟基红花黄色素A抑制慢阻肺发病过程中TNF受体1介导的炎症信号转导作用的研究

In recent years the incidence of chronic obstructive pumonary disease(COPD) increased and it has been becoming a serious public health problem.Chronic inflammation is the main pathological mechanism of COPD. There is no very good drug to attenuate this injury up to now. Tumor Necrosis Factor α(TNFα) is an important cytokine in inflammation of COPD. After TNFα induction TNF receptor1（TNFR1） can interact with adaptor proteins to trigger inflammation signal transduction and studies of this have been a hotspot in recent years. Hydroxysafflor yellow A(HSYA) is the main effective ingredient of Carthamus tinctorius L..We found that HSYA was effective to attenute chronic inflammatory injury in COPD rat and can attenuate pulmonary fibrosis and TGFβ1 upregulation in mice. HSYA was effective to inhibit cellular inflammatory factor expression. In this project we plan to observe the effect of HSYA to inhibit lung inflammatory signal transduction,attenuate abnormal cytokine expression,pulmonary inflammatory injury and tissue remodeling in COPD rat. We want to observe the time effect of HSYA to inhibit chronic inflammation in COPD mouse. We also want to observe the TNFα receptor antagonistic effect of HSYA. We intend to study the inhibition effect of HSYA on TNFα induced interactions of TNFR1 with some adaptor proteins to trigger NFκB and MAPK activation to induce inflammatory signal transduction,abnormal cytokine expression and related cellular injuries. Besides we plan to observe the changes of the effect of HSYA after TNF antagonist addition.In this study we want to prove TNFR1 being the target of chronic inflammatory attenuating effect of HSYA and to establish a foundation for the development of new drug to treat COPD.

慢阻肺(COPD)患病率上升已成严重的公共卫生问题，该病主要机理为肺部慢性炎症,现无理想的治疗药物。COPD炎症主要因子TNFα致其受体TNFR1与接头蛋白作用激活炎症信号转导是近年研究热点。羟基红花黄色素A（HSYA）为红花有效成分，预试发现它可抑制COPD大鼠炎症损伤及小鼠肺纤维化与TGFβ1表达上调、抑制TNFα引发细胞炎症因子表达升高。本课题观察HSYA抑制COPD大鼠肺部炎症信号转导、细胞因子表达异常、炎症损伤及组织重构的药效，探索HSYA缓解COPD小鼠慢性炎症的时效关系，观察HSYA拮抗TNFα与受体结合，研究HSYA抑制TNFα引发的TNFR1与接头蛋白作用激活NFκB及MAPK的炎症信号转导、缓解细胞因子表达异常及细胞损伤的作用，并以TNF拮抗剂拮抗TNFR1信号转导考察HSYA的药效变化，阐明TNFR1为HSYA缓解COPD慢性炎症的靶点,为开发抗COPD新药提供依据。

本课题观察了HSYA腹腔注射缓解大鼠慢阻肺炎症损伤和组织重构的作用，发现HSYA可缓解慢阻肺（COPD）大鼠肺组织肺组织的炎症细胞浸润与肺水肿，缓解COPD大鼠肺组织多种炎症因子mRNA 及NFκBp65的表达升高与p38MAPK的磷酸化，HSYA还可缓解COPD大鼠肺组织TGF-β1、α-SMA和collagen1 mRNA表达升高和血浆TGF-β1水平的升高，缓解COPD大鼠肺组织羟脯氨酸水平的升高和胶原沉积加剧和小气道的增厚，可治疗COPD大鼠肺部的慢性炎症损伤与组织重构。COPD造模后不同时间的实验结果表明该药可缓解COPD早期的炎症损伤、COPD中期的慢性炎症为主与组织重构启动的损伤以及晚期组织重构为主的损伤。我们观察了HSYA缓解小鼠肺纤维化的药效，发现HSYA可缓解博来霉素诱发肺纤维化小鼠动脉氧饱和度的下降，缓解肺组织的实变、羟脯氨酸水平升高与胶原沉积，还可抑制肺纤维化小鼠肺组织TGF-β1、CTGF、α-SMA和 Collagen I mRNA及α-SMA蛋白水平的升高水平。细胞实验结果表明，HSYA可抑制TGF-β1 诱导多种成纤维细胞的活化，该成分对TGF-β1诱发的成纤维细胞增殖、迁移有抑制作用，HSYA还可抑制TGF-β1诱发的成纤维细胞p38 MAPK、ERKMAPK与Smad2/3的磷酸化、Smad2的核转移及TGF-β1诱发的α-SMA 、 COL1A1和FN mRNA及α-SMA、COL1A1和FN蛋白的表达升高，HSYA还可抑制TGF-β1诱发的成纤维细胞Smad 3 与Col I启动子中转录因子结合片段SBE的结合，加入特异性TGFBRI受体激酶抑制剂或沉默TGFBRII受体的表达可取消HSYA抑制TGF-β1诱发的成纤维细胞活化的药效，且HSYA可特异性地抑制FITC-TGF-β1与成纤维细胞受体的结合，说明HSYA 抑制TGF-β1活化成纤维细胞的靶点是TGFBRII受体。通过本课题的实验证明HSYA腹腔注射可缓解大鼠COPD及小鼠肺纤维化，HSYA为红花抗COPD的有效成分，其抑制COPD组织重构的重要靶点为TGFBRII受体，其主要作用机理为抑制该受体介导的细胞信号转导，我们的工作为以HSYA为主要成分的红花黄色素制剂开发为治疗COPD或肺纤维化的药物奠定了理论基础。我们完成了计划内和计划外的工作，按期超额完成了任务。