Monocyte infiltration and macrophage polarization are widely considered as pivotal steps for the initiation and progression of atherosclerosis. Previous studies suggested that zanthoxylum piperitum had strong analgesic and anti-inflammatory effects. However, it remains unclear whether zanthoxylum piperitum inhibits inflammation via macrophage function. In the present study, we investigated the effects of xanthoplanine (the total alkaloid extract of zanthoxylum piperitum) on macrophage function. CCK-8 kit was performed to determine cell viability and the preferred concentration of xanthoplanine. We assayed the effects of xanthoplanine on markers of macrophage polarization and inflammation via quantitative PCR and enzyme-linked immunosorbent assay, and measured the production of reactive oxygen species (ROS) by flow cytometry. Immunoblots, co-immunoprecipitation, immunofluorescence and Luciferase activity were performed to investigate the molecular mechanism of STAT signaling pathway in response to xanthoplanine. We found that xanthoplanine (50 and 100 μM) significantly reduced M1 polarization and promoted M2 polarization. The contents of inflammatory cytokines measured by ELISA were markedly decreased in macrophages pretreated with xanthoplanine, compared with those induced by LPS and IFN-γ. In parallel, xanthoplanine alleviated the production of ROS in macrophages induced by LPS and IFN-γ. Moreover, xanthoplanine alleviated STAT5 phosphorylation and blocked STAT5 nuclear translocation without alterations in CrkL expression, subsequently interrupting the interaction between p-STAT5 and CrkL. Likewise, xanthoplanine prominently attenuated the transcription activity of STAT5 induced by LPS and IFN-γ but did not affect the transcription activity of STAT1 and STAT3. Xanthoplanine attenuated M1 phenotypic switch and macrophage inflammation via blocking the formation of CrkL-STAT5 complex.

单核细胞浸润和巨噬细胞极化被广泛认为是引发和发展动脉粥样硬化的关键步骤。先前的研究表明，花椒具有很强的镇痛和抗炎作用。然而，尚不清楚花椒是否通过巨噬细胞功能抑制炎症。在本研究中，我们研究了黄嘌呤（花椒总生物碱提取物）对巨噬细胞功能的影响。进行CCK-8试剂盒以确定细胞活力和黄嘌呤碱的优选浓度。我们通过定量PCR和酶联免疫吸附试验测定了黄嘌呤对巨噬细胞极化和炎症标志物的影响，并通过流式细胞术测量了活性氧（ROS）的产生。免疫印迹 进行了免疫共沉淀，免疫荧光和荧光素酶活性研究以研究STAT信号通路对黄嘌呤素的响应的分子机制。我们发现黄嘌呤（50和100μM）显着降低M1极化并促进M2极化。与用LPS和IFN-γ诱导的巨噬细胞相比，用黄嘌呤素预处理的巨噬细胞通过ELISA测定的炎性细胞因子含量显着降低。同时，黄嘌呤碱减轻了由LPS和IFN-γ诱导的巨噬细胞中ROS的产生。此外，黄嘌呤素可减轻STAT5磷酸化并阻断STAT5核易位，而不会改变CrkL的表达，从而中断p-STAT5和CrkL之间的相互作用。同样 黄嘌呤碱显着减弱了LPS和IFN-γ诱导的STAT5的转录活性，但不影响STAT1和STAT3的转录活性。Xanthoplanine通过阻止CrkL-STAT5复合物的形成来减轻M1表型转换和巨噬细胞炎症。