基于LPS-巨噬细胞炎症模型和蛋白芯片技术探讨白及抗炎组分作用机理

Inflammation is a basic pathological process in clinical, research and development of high efficiency, low toxicity, components and mechanism basically clear of traditional Chinese medicine for anti-inflammatory is important in the future to prevent and control the adverse effects on the body caused by inflammation. Our prior research on the animal model of silicosis showed that Effective Fraction of Bletilla sfriata (EFBS) may play a role in anti-pulmonary fibrosis through inhibition of IL-1β, TNF-α inflammatory factors; and further LPS-induced macrophage inflammatory model research confirmed that EFBS can significantly inhibit the expression of inflammatory cytokines IL-1β, IL-6 and TNF-α etc., showing a strong anti-inflammatory activity, suggesting that EFBS may inhibit NF-κB, MAPKs signaling pathways to endue its anti-pulmonary fibrosis effect. Therefore, this study intends to use protein microarrays to screen the targets of anti-inflammatory effects of EFBS, and further verified by fluorescence quantitative PCR and Western blot techniques etc., so as to uncover the underlying mechanism of the anti-inflammatory effects of EFBS. In addition, the target of the main active monomer components of EFBS will be located according to the candidate targets screening by protein microarrays, and the possible synergistic interaction between the main monomer components will be discussed as well. This research can provide evidence for the multi-target, multi-effect and synergistic effect of traditional Chinese medicine, and lay solid foundation for the scientific development and utilization of EFBS.

炎症是常见且重要的基本病理过程，研发高效、低毒、成分基本明确、机理基本清楚的创新抗炎中药，对预防和控制炎症对机体造成的不利影响具有重要意义。项目组前期对矽肺动物模型研究发现白及药效组分（EFBS）可通过抑制IL-1β、TNF-α等炎症因子发挥抗肺纤维化作用；并进一步通过LPS诱导的巨噬细胞炎症模型证实，EFBS可显著抑制炎症因子IL-1β、IL-6、TNF-α等基因的表达，显示出极强的抗炎活性，提示EFBS可能通过抑制NF-κB、MAPKs炎症信号通路发挥作用。为此，本研究拟采用蛋白芯片技术筛选EFBS的抗炎作用靶点，并采用qRT-PCR及Western blot等技术进行验证，以期阐明EFBS抗炎作用机理；同时以蛋白芯片筛选确定的EFBS作用靶点为候选靶点，对EFBS中主要药效单体成分作用靶点进行定位，并对各主要单体成分间可能互作特点进行探讨，为中药多靶点、多效应、协同作用特征提供依据。

中药白及具有很好的抗炎和治疗肺纤维化作用，在本项目的支持下，以LPS诱导的RAW264.7细胞炎症为模型，评价了白及药效部位EFBS体外抗炎活性；采用LPS诱导的急性肺损伤模型，分析了药效部位对肺损伤的保护作用；采用磷酸化抗体芯片分析了EFBS以及单体贝母兰宁抗炎的可能作用通路。通过研究，我们发现，白及药效部位EFBS对ERK1/2、p38磷酸化水平无显著影响，40 μg/mL处理浓度时对JNK磷酸化水平有显著抑制作用；相对而言，EFBS对NF-κB和其上游调控蛋白IKBα磷酸化水平以及下游调控基因iNOS和COX2蛋白表达表现出显著的剂量依赖性抑制作用。因此，EFBS可能主要通过抑制NF-κB活化从而抑制IL-1β、IL-6、TNF-α、iNOS和COX2等下游蛋白表达。此外，成分敲除实验结果表明，EFBS的四个成分在抑制p65入核和IL-1β等炎症因子表达作用中具有重要贡献，从药效学角度阐明了四个成分作为EFBS指标成分的合理性。动物实验结果表明EFBS对LPS诱导的急性肺损伤具有较好的保护作用，也进一步证实EFBS确实可以通过NF-κB通路发挥抗炎活性。蛋白磷酸化芯片结果表明，分别有23和32个蛋白磷酸化水平经EFBS和贝母兰宁预处理而显著下调，其中大部分蛋白富集于MAPK,PI3K/AKT通路。Western blot结果证实贝母兰宁可剂量依赖性抑制PTEN、AKT、IκBα、p65和p27Kip1磷酸化，验证了芯片结果的可靠性。共聚焦显微镜结果表明，5μg/mL贝母兰宁可显著抑制p65入核，进一步证实其可以通过抑制NF-κB活化而发挥抗炎活性。另外，Western blot和流式细胞术确证，贝母兰宁可以通过PTEN依赖性抑制LPS诱导的p27Kip1磷酸化降解，从而实现对G1期的细胞周期阻滞作用；但贝母兰宁并不能完全通过PTEN/AKT通路抑制NF-κB活化而抑制IL-1β等炎症因子表达，表明贝母兰宁通过其他更为关键的通路靶点发挥对NF-κB的调控作用，这些结果体现了中药多靶点、多效应的作用特点。因此，本研究为今后白及科学开发利用奠定了良好基础。