墨旱莲双靶调控治疗肺纤维化的物质基础和作用机制研究

The incidence of pulmonary fibrosis (PF) is increasing in recent years，and the treatment of PF has been always a difficult problem. Inhibition promoting fibrosis cell factors on the synergy of multiple targets in different signaling pathways is a new trend of PF treatment. On the view of traditional Chinese Medicine, PF is considered as Feilou or Feibi. According to the TCM theory, nourishing kidney Yin is helpful to treat PF from root causes. Our previous work indicated that the extract of Herba Ecliptae, which nourishes kidney Yin, can significantly relieve symptoms of fibrosis in mice. As main components, wedelactone (WEL) of coumarin type and ecliptasaponin A (ESA) of triterpenoid type showed significant effects against PF. Our primary analysis showed that tyrosine kinase FGFR2 is likely to be the main target of WEL, and toll-like receptor TLR4 may be related with active metabolite of ESA. On the view of these findings, the project intends to find out the mechanism of double targets of FGFR2 and TLR4 involved in WEL and/or ESA metabolite regulation in the process of PF on the base of structure-activity relationship of these two type ingredients, especially the mechanisms of WEL ameliorating PF and ESA regulating TLR4 pathway, as well as their mutual relationship on regulation of IL-17A and Th1/Th2 balance. All this illustrates the mechanism of Herba Ecliptae against PF through symptoms and root cause in modern scientific methods, and provides a cellular mechanism for immunologic balance in controlling PF.

肺纤维化的发病率逐年上升，其治疗一直是医学领域的难题。在不同信号转导通路的协同抑制作用是治疗肺纤维化药物的新研究趋势。中医将肺纤维化归为肺痿或肺痹，滋补肾阴中药有利于免疫调节角度对肺纤维化疾病进行治疗。本课题组发现益肾阴中药墨旱莲可显著对抗实验性肺纤维化，香豆素类蟛蜞菊内酯（WEL）和三萜旱莲苷A（ESA）都可显著改善博来霉素诱导的小鼠纤维化；WEL显著降低模型肺组织FGF-2表达，基于Docking技术推测FGFR2可能为WEL的靶标蛋白。ESA在大鼠肠道代谢为刺囊酸，刺囊酸抑制LPS/TLR4结合而参与机体免疫调节。本项目拟在澄清墨旱莲改善肺纤维化的物质基础上，以两类成分WEL和ESA为代表，研究肺纤维化进程中FGFR2与TLR4靶蛋白所介导的下游信号通路间联系，及其在肺微环境IL-17A表达和Th1/Th2平衡的调控作用，用现代科学诠释墨旱莲作于多靶点标本兼治肺纤维化的分子机制。

肺纤维化是一种代表性肺间质疾病。墨旱莲Herba Ecliptae为菊科鳢肠Eclipta prostrata L.的干燥地上部分。采用一次性气管滴注博来霉素诱导小鼠实验性肺纤维化模型。墨旱莲提取物（2.5g/kg或1.25g/kg）可明显改善模型小鼠体重下降、生存率，降低肺系数以及肺组织中MDA，NO，HYP的含量，降低HYP，α-SMA），以及与降低TGF-β1，COX-2，MMP-2，FGF-2等蛋白表达，抑制肺组织胶原的生成和沉积从而干预肺纤维化进程。墨旱莲中的蟛蜞菊内酯（WEL，40，10 mg/kg）和三萜酸类物质旱莲苷A(ESA，80，20 mg/kg)均可有效改善博来霉素诱导的小鼠肺泡炎症及纤维化进程。两种联合用药并没有出现明显药效加合现象。体内代谢分析表明：ESA在大鼠体内口服生物利用度仅为0.2%，在肠道菌群作用下代谢为具有改善急性肺损伤作用的苷元刺囊酸；刺囊酸绝对生物利用度为8.7%，明显高于ESA。刺囊酸的表观分布容积（13.5 L/kg）远大于ESA（1.38 L/kg），表明刺囊酸更易进入组织。WEL在大鼠体内主要的代谢途径是C-5位羟基葡萄糖醛酸化，口服生物利用度较低为2.03%，在肺组织中分布较多。WEL和ESA灌胃联合给药后显著降低WEL生物利用度，但对ESA基本没有影响。WEL抗肺纤维化作用机制研究表明，WEL作为植物雌激素可与ERβ结合调节下游炎症信号通路，同时介导AMPK磷酸化调节TGFβ1/Smad经典纤维化信号通路。中医理论中大肠与肺相表里，长期肠道炎症可引起肺损伤。 WEL明显改善DSS诱导的小鼠溃疡性结肠炎，激活AMPK抑制NLRP3炎症小体激活相关炎症通路，同时提高肠系膜淋巴结中Treg细胞含量，并显著抑制IL-17A蛋白表达，免疫调节实验性结肠炎中Treg/Th17。ESA及其代谢产物刺囊酸基于TLR4受体信号通路发挥抗炎作用进而改善博来霉素诱导的小鼠肺纤维化进程。综上，采用现代科学方法诠释中药墨旱莲双靶干预肺纤维化的物质基础和作用机制，即香豆素类成分WEL直接阻止肺纤维化——治标实，而三萜皂苷类如旱ESA调节机体免疫能力——治本虚。为寻找有效抗肺纤维化进药物提供新的研究思路。