黄芪多糖调节程序性死亡分子 1/程序性死亡配体 1信号通路对非霍奇金淋巴瘤发展和免疫逃逸的影响

【目的】 观察黄芪多糖对非霍奇金淋巴瘤（NHL）的治疗作用及机制。【方法】 培养WSU-DLCL2、BJAB、SP53等3种NHL细胞，给予不同浓度（0、5、10、50、100、200、400 μmol/L）黄芪多糖处理，应用细胞计数试剂盒8（CCK-8）法检测细胞活力，筛选黄芪多糖最佳浓度、对黄芪多糖敏感的细胞。将WSU-DLCL2细胞分为对照组、黄芪多糖组、BMS-1[程序性死亡分子1（PD-1）/程序性死亡配体1（PD-L1）通路抑制剂]组、黄芪多糖+BMS-1组，检测各组细胞菌落形成、凋亡、迁移和侵袭情况，Western Blot 法检测 PD-1/PD-L1 信号通路蛋白表达。WSU-DLCL2 细胞和 CD8+ T 细胞共培养后，检测 CD8+ T 细胞百分比、凋亡率和上清液中干扰素（IFN）-γ、肿瘤坏死因子（TNF）-α水平。【结果】 后续实验选择200 μmol/L 黄芪多糖处理WSUDLCL2细胞。与对照组比较，黄芪多糖组、BMS-1组和黄芪多糖+BMS-1组WSU-DLCL2细胞菌落形成数、迁移细胞数、侵袭细胞数，CD8+ T 细胞凋亡率，及 WSU-DLCL2细胞 PD-1、PD-L1蛋白表达水平显著下降（P＜0.05），WSU-DLCL2细胞凋亡率、CD8+ T细胞比例及共培养体系上清液中IFN-γ、TNF-α水平显著升高（P＜0.05）；与黄芪多糖组、BMS-1组比较，黄芪多糖+BMS-1 组 WSU-DLCL2 细胞菌落形成数、迁移细胞数、侵袭细胞数，CD8+ T 细胞凋亡率，及 WSU-DLCL2 细胞PD-1、PD-L1 蛋白表达进一步下降（P＜0.05），WSU-DLCL2 细胞凋亡率、CD8+ T 细胞比例及共培养体系上清液中 IFN-γ、TNF-α水平进一步升高（P＜0.05）。【结论】 黄芪多糖可抑制NHL细胞增殖、迁移和侵袭，并促进其凋亡，减少免疫逃逸，其机制可能与抑制PD-1/PD-L1信号通路有关。

Effects of Astragalus Polysaccharide on the Development and Immune Escapeof Non-Hodgkin’s Lymphoma by Regulating Programmed DeathMolecule 1/Programmed Death Ligand 1 Signaling Pathway

Objective To observe the therapeutic effect and mechanism of astragalus polysaccharide on nonHodgkin’s lymphoma（NHL）. Methods Three kinds of NHL cells， WSU-DLCL2， BJAB and SP53， werecultured and treated with different concentrations（0， 5， 10， 50， 100， 200， 400 μmol/L） of astragaluspolysaccharide. The cell viability was detected by cell counting kit-8（CCK-8）method to screen the optimalconcentration of astragalus polysaccharide and the cells sensitive to astragalus polysaccharide. WSU-DLCL2 cellswere divided into control group，astragalus polysaccharide group，BMS-1 [programmed death molecule 1（PD-1）/programmed death ligand 1（PD-L1）pathway inhibitor] group， astragalus polysaccharide+BMS-1 group. Thecolony formation，apoptosis，migration and invasion of each group were detected，and the protein expression ofPD-1/PD-L1 signaling pathway was detected by Western Blot. After co-culture of WSU-DLCL2 cells and CD8+ Tcells，the percentage and apoptosis of CD8+ T cells，and the levels of interferon（IFN）-γ and tumor necrosis factor（TNF）-α in the supernatant were detected. Results The follow-up experiments selected 200 μmol/L astragalosideto treat WSU-DLCL2 cells. Compared with the control group，the number of WSU-DLCL2 cell colony formation，the number of migrating WSU-DLCL2 cells，the number of invading WSU-DLCL2 cells，the apoptosis rate ofCD8+ T cells， and the protein expression levels of PD-1 and PD-L1 in WSU-DLCL2 cells were significantlydecreased in the Astragali polysaccharide group，the BMS-1 group and the Astragali polysaccharide+BMS-1 group（P＜0.05），and the apoptosis rate of WSU-DLCL2 cells，the proportion of CD8+ T cells，and the levels of IFN-γ and TNF- α in the supernatant of co-culture system were significantly increased（P＜0.05）； compared withastragali polysaccharide group and BMS-1 group，the number of WSU-DLCL2 cell colony formation，migratingWSU-DLCL2 cells，invading WSU-DLCL2 cells，CD8+ T-cell apoptosis rate，and protein expressions of PD-1，PD-L1 in WSU-DLCL2 cells were further decreased in astragali polysaccharide+BMS-1 group（P＜0.05），andthe rate of WSU-DLCL2 cell apoptosis，the proportion of CD8+ T-cells，and the levels of IFN-γ，TNF-α in thesupernatant of co-culture system were significantly further elevated （P＜0.05）. Conclusion Astragaluspolysaccharide can inhibit NHL cell proliferation， migration and invasion， promote its apoptosis and reduceimmune escape，and its mechanism may be related to the inhibition of PD-1/PD-L1 signaling pathway.