摘要
目的 探讨白及多糖(BSP)对巨噬细胞M1型极化过程的调节作用及其潜在机制.方法 诱导THP-1细胞分化为M0或M1型巨噬细胞,并给予不同质量浓度(0.001、0.010、0.100、1.000、10.000 μg·mL-1)的BSP处理,观察细胞贴壁程度及形态,通过CCK-8法检测BSP对细胞活力的影响.选择合适的剂量后,运用实时荧光定量PCR(qRT-PCR)技术分析BSP对M1型巨噬细胞标记基因[白细胞介素(IL)-1β、IL-6、肿瘤坏死因子-α(TNF-α)、诱导型一氧化氮合酶(iNOS)]和M2型巨噬细胞标记基因(IL-10、CD206)表达的影响;通过酶联免疫吸附实验检测BSP对上清液中IL-1β、IL-6和TNF-α释放的影响;通过Western blotting法检测BSP对核因子-κB(NF-κB)和信号转导子和转录激活子(STAT1)信号通路相关蛋白表达的影响.结果 BSP在0.001～10.000 μg·mL-1对M0和M1型巨噬细胞没有明显毒性.BSP显著抑制M1型巨噬细胞IL-1β、IL-6、TNF-α和 iNOS的mRNA表达水平(P＜0.05、0.01、0.001),并显著降低IL-1β、IL-6和 TNF-α 的释放(P＜0.01、0.001),显著抑制IL-1β、p65和STAT1的磷酸化蛋白的表达(P＜0.05).BSP对M0型巨噬细胞无显著影响.结论 BSP能够有效抑制M0型巨噬细胞向M1极化的转变,其机制主要通过抑制NF-κB和STAT1信号通路的活化实现.
Abstract
Objective To investigate the effect of Bletilla striata polysaccharide(BSP)on the polarization process of Ml macrophages and its potential mechanisms.Methods THP-1 cells were induced to differentiate into M0 or M1 macrophages and treated with different concentrations(0.001,0.010,0.100,1.000,and 10.000 µg·mL-1)of BSP.The CCK-8 assay was employed to assess cell adhesion and viability following BSP treatment.After selecting an appropriate dose,real-time fluorescence quantitative PCR(qRT-PCR)was used to analyze the expression of M1 macrophage marker genes[Interleukin(IL)-1β,IL-6,tumor necrosis factor-α(TNF-α),inducible nitric oxide synthase(iNOS)]and M2 macrophage marker genes(IL-10,CD206).The release of inflammatory cytokines IL-1β,IL-6,and TNF-α in the supernatant was measured using an enzyme-linked immunosorbent assay,and Western blotting analysis was performed to investigate the effect of BSP on Nuclear factor kappa B(NF-κB)and signal transducer and activator of transcription 1(STAT1)signaling pathways.Results BSP exhibited no significant toxicity to M0 and M1 macrophages within the tested concentration range.BSP significantly downregulated the mRNA expression of M1 macrophage markers(IL-1β,IL-6,TNF-α,and iNOS)and reduced the secretion of these inflammatory cytokines(P＜0.05,0.01,and 0.001).Furthermore,BSP inhibited the protein expression of IL-1β and phosphorylation of p65 and STAT1 proteins.BSP had no significant effect on M0 macrophages.Conclusion BSP effectively suppresses the transformation of M0 macrophages into the M1 phenotype,primarily through the inhibition of NF-κB and STAT1 signaling pathways.
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