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

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。

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.