安石榴苷通过靶向结合PTP1B调节小鼠巨噬细胞分型的机制研究

Excessive inflammatory response induced by macrophage activation contributes to diarrhea caused by Escherichia Coli. Macrophages with polarized phenotypes exhibit distinct functions, hinting that re-polarization of macrophages may be a potential method to change their roles in inflammatory response. Pomegranate is a proved traditional Chinese medicine that exhibits excellent efficacy on diarrhoeal disease. Our previous study demonstrated that punicalagin notably relieves the symptom of diarrhea and inhibits exceeding inflammatory response in macrophages; however, the underlying mechanisms remain unclear. Based on the prediction of Pharmmapper, a server designed to identify potential target candidates using pharmacophore mapping approach, together with inhibitory test, we speculated that PTP1B is a direct target of punicalagin and modulate macrophage polarization. To confirm our hypothesis, we first employ plasmon resonance technology to check the combination between punicalagin and PTP1B. Afterwards, we use LPS/IFNγ and IL-4 to induce M1 and M2 polarization, respectively, in mouse RAW264.7 macrophages. Using ELISA, immunofluorescence and flow cytometry, we try to determine the phenotype of macrophage when treated by punicalagin. Furthermore, we attempt to understand the underlying mechanisms in punicalagin-induced macrophage polarization via employing Western Blot, RT-PCR, RNAi and gene expression microarray. This study could bring new insights into the mechanisms of the anti-inflammatory property of punicalagin and PTP1B, which may provide new perspectives for novel therapeutic approaches using punicalagin when treating inflammatory diseases.

巨噬细胞介导的过度炎症是大肠杆菌性腹泻的重要病理基础。不同亚型巨噬细胞功能不同，改变分型是调节其功能的关键手段。中药石榴皮用于治疗腹泻已有明确疗效。前期研究发现，成分安石榴苷可以减轻小鼠腹泻并抑制巨噬细胞过度激活，但其分子机制仍不明确。近期通过反向药效团预测技术结合活性抑制试验，我们推测PTP1B是安石榴苷的作用靶点。本研究先通过Spr技术验证安石榴苷与PTP1B靶向结合，进而以RAW264.7小鼠巨噬细胞为研究对象，采用LPS/IFNγ和IL-4分别建立M1和M2型巨噬细胞模型，运用ELISA、免疫荧光和流式细胞术考察安石榴苷对巨噬细胞分型的调节，并通过Western Blot、RT-PCR、RNAi和表达谱芯片检测进一步探讨安石榴苷及PTP1B诱导巨噬细胞分型改变的分子机制。本研究旨在揭示安石榴苷的作用靶点，阐明安石榴苷/PTP1B改变巨噬细胞表型的分子机制，为其临床应用提供科学依据。

安石榴苷是中药石榴皮的主要活性成分，临床治疗感染性疾病和炎症性疾病效果确切，但相关机制不明确。炎症反应是一类以大量细胞因子和炎性介质释放、可以造成组织和器官不可逆损伤甚至机体死亡的病理过程。巨噬细胞作为天然免疫细胞，在识别和清除病原微生物及激发天然免疫反应的过程中发挥重要的作用。然而，过度激活的巨噬细胞会释放大量的细胞因子和炎性介质，导致急性炎症反应甚至败血症的发生。因此，抑制巨噬细胞的过度炎症反应是防止急性炎症反应和败血症等疾病发生的关键。巨噬细胞可以根据刺激源种类的不同激活并分化成为不同类型的巨噬细胞：经典激活型巨噬细胞（M1型）、损伤修复型巨噬细胞（亦称选择激活型或M2型）和调节型巨噬细胞。调节巨噬细胞由M1型向M2型转化是抑制其过度炎症反应的关键。研究结果发现，安石榴苷可以显著降低LPS诱导的M1型巨噬细胞表面CD 11c+ 受体的生成，并增加CD 206受体的数量。同时，安石榴苷可以显著降低巨噬细胞内iNOS蛋白的生成，诱导Arg-1蛋白的高表达，提高抗炎通路STAT-3/IL-10的激活。表明安石榴苷具有诱导巨噬细胞由M1型向M2型转化的调节作用。进一步研究证实安石榴苷可以直接靶向抑制PTP1B蛋白的活性，半数有效抑制浓度IC50为1.04 μM，Arg24是二者结合的关键氨基酸结合位点。通过ShRNA干扰等技术阐明PTP1B蛋白对巨噬细胞激活和分型的重要作用。借助基因组学等技术，我们发现HO-1蛋白作为PTP1B的下游分子，在M2型巨噬细胞中高表达。抑制HO-1表达后，安石榴苷诱导M2型巨噬细胞极化的作用也受到明显抑制，提示安石榴苷通过调节PTP1B/HO-1通路，影响巨噬细胞的激活和功能。