目的：探究三七总皂苷（PNS）通过JAK2/STAT3通路调控巨噬细胞极化对小鼠黑色素瘤B16-F10细胞的活力的影响。 方法：常规培养B16-F10细胞和巨噬细胞RAW264.7，MTT法检测不同浓度PNS对RAW264.7或B16-F10细胞存活率的影响。实 验分为空白组（仅B16-F10细胞）、对照组（B16-F10细胞与RAW264.7细胞共培养）、不同浓度（50、100、200 μg/mL）PNS组（B16- F10 细胞与 RAW 264.7 细胞共培养）及 200 μg/mL PNS + colivelin（[ B16-F10 细胞与 RAW264.7 细胞共培养，0.5 μmol/L colivelin JAK2/STAT3 通路激活剂）]组，MTT 法、流式细胞术检测各组共培养细胞的存活率和凋亡率，显微镜观察巨噬细胞形态变化； ELISA实验检测上清液中相关细胞因子TNF-α、IL-6、IL-1β的水平，qPCR法检测巨噬细胞极化相关基因诱导型一氧化氮合酶 （iNOS）、IL-12、CD206、精氨酸酶-1（Arg-1）mRNA表达，WB法检测细胞中JAK2、STAT3蛋白磷酸化水平。结果：不同浓度PNS 对单独培养的RAW264.7、B16-F10细胞的存活率均无明显影响（均P > 0.05）。与对照组相比，PNS呈浓度依赖性地促进共培养细 胞凋亡、IL-6、TNF-α、IL-1β 蛋白和 IL-12、iNOS mRNA 表达水平均显著增加（均 P < 0.05），降低共培养细胞的存活率、JAK2 与 STAT3蛋白磷酸化水平（均P < 0.05），PNS对共培养细胞的作用部分被colivenlin抑制。结论：PNS通过抑制JAK2/STAT3通路促 进M1巨噬细胞极化进而抑制小鼠黑色素瘤B16-F10细胞的活力。

To investigate the effect of total panax notoginseng saponin (PNS) on the survival of mouse melanoma B16-F10 cells by regulating macrophage polarization through JAK2/STAT3 pathway. Methods: B16-F10 cells and macrophage RAW264.7 were cultured regularly. The effects of different concentrations of PNS on the survival rate of RAW264.7 or B16-F10 cells were detected by MTT assay. The experiment was divided into the following groups: blank group (B16-F10 cells only), control group (B16-F10 cells cocultured with RAW264.7 cells), PNS groups of various concentrations (B16-F10 cells co-cultured with RAW 264.7 cells, treated with 50, 100, 200 μg/mL PNS), and PNS + colivelin [B16-F10 cells co-cultured with RAW264.7 cells, 200 μg/mL PNS, 0.5 μmol/L colivelin (JAK2/STAT3 pathway activator)] group. MTT assay and flow cytometry were applied to detect the survival rate and apoptosis rate of co-cultured cells in each group, and the morphological changes of macrophages were observed under a microscope. ELISA was applied to detect the levels of cytokines TNF- α, IL-6 and IL-1β in the supernatant. qPCR was applied to detect the mRNA expression of macrophage polarization-related genes, inducible nitric oxide synthase (iNOS), IL-12, CD206, and arginase-1 (Arg-1). Western blotting was applied to detect the phosphorylation levels of JAK2 and STAT3 proteins pathway in cells. Results: PNS of different concentrations did not significantly affect the viability of RAW264.7 cells or B16-F10 cells cultured alone (all P > 0.05). Compared with the control group, PNS significantly promoted cell apoptosis, protein levels of IL-6, TNF-α, IL-1β, and mRNA levels of IL-12 and iNOS in a concentration-dependent manner (all P < 0.05); additionally, PNS reduced the survival rate of co-cultured cells and the phosphorylation levels of JAK2 and STAT3 proteins (all P < 0.05). These effects of PNS on co-cultured cells were partially inhibited by colivelin. Conclusion: PNS inhibits the viability of mouse melanoma B16-F10 cells by promoting the polarization of M1 macrophages via inhibiting JAK2/STAT3 pathway.

江苏省卫生健康委医学科研指导性项目（No. 272）