【摘要】 为探讨芍药苷(paeoniflorin, PF)对人支气管上皮细胞(BEAS-2B)的影响及其机制，利用佛波酯诱导THP-1细胞分化为巨噬细胞，CCK-8法检测PF对THP-1巨噬细胞的毒性，1μg/mL的LPS培养THP-1巨噬细胞以诱导炎症，分别用1、10和30μmol/L的PF处理24、48和72 h, CCK-8法检测细胞活力，筛选PF的最佳作用浓度和时间。在Transwell上室中接种BEAS-2B细胞，下室接种THP-1巨噬细胞，将THP-1巨噬细胞分成4组：对照组、LPS组、PF组、LPS+PF组。CCK-8法检测BEAS-2B细胞的存活率，FACS检测BEAS-2B细胞的凋亡率，ELISA检测炎性因子IFN-γ、IL-4、IL-17C、IL-10的水平，Western blotting检测CD63、CD9、凋亡转接基因2互作蛋白X(apoptosis-linked gene 2-interacting protein X, Alix)表达水平，FACS检测THP-1巨噬细胞中M1、M2型细胞标志物CD80和CD206的表达。结果显示，PF的最佳作用浓度和时间分别为10μmol/L和48 h。与对照组比较，LPS组细胞存活率，IL-4、IL-10水平，M2型巨噬细胞比例显著降低(P<0.01),细胞凋亡率，IFN-γ、IL-17C水平，CD63、CD9、Alix蛋白表达水平，M1型巨噬细胞比例显著升高(P<0.01);与LPS组比较，LPS+PF组细胞存活率，IL-4、IL-10水平，M2型巨噬细胞比例显著升高(P<0.01),细胞凋亡率，IFN-γ、IL-17C水平，CD63、CD9、Alix蛋白表达水平，M1型巨噬细胞比例显著降低(P<0.01)。该研究提示，PF能够提高BEAS-2B细胞的存活率，减少细胞凋亡，抑制外泌体的分泌和炎症反应，其机制可能与PF诱导巨噬细胞的M2型极化有关。更多还原

【Abstract】 To investigate the effect and mechanism of paeoniflorin(PF) on human bronchial epithelial cells(BEAS-2B), THP-1 cells were induced to differentiate into macrophages by phorbol 12-myristate 13-acetate, and the toxicity of PF to THP-1 macrophages was detected by CCK-8 assay. THP-1 macrophages were cultured with 1 μg/mL LPS to induce inflammation, and treated with 1, 10, and 30 μmol/L PF for 24, 48, and 72 h, respectively. Cell viability was detected by CCK-8 assay, and the optimal concentration and treating time of PF were determined. BEAS-2B cells were inoculated in the upper chamber of Transwell and THP-1 macrophages were inoculated in the lower chamber of Transwell. THP-1 macrophages were divided into 4 groups: control group, LPS group, PF group, and LPS+PF group. The survival rate of BEAS-2B cells was detected by CCK-8 assay, the apoptosis rate of BEAS-2B cells was detected by FACS, the levels of inflammatory cytokines IFN-γ, IL-4, IL-17C, and IL-10 were detected by ELISA, and the expression levels of CD63, CD9, and apoptosis-linked gene 2-interaction protein X(Alix) were detected by Western blotting. The expressions of M1 and M2 markers CD80 and CD206 in THP-1 macro-phages were detected by FACS. The results showed that the optimal concentration and treating time of PF were 10 μmol/L and 48 h. Compared to those in the control group, the cell survival rate, IL-4 and IL-10 levels and the proportion of M2-type macrophages in the LPS group were significantly decreased(P<0.01). On the other hand, the apoptosis rate, IFN-γ and IL-17C levels, CD63, CD9, Alix protein expression levels and the proportion of M1-type macrophages were significantly increased(P<0.01). Compared to those in the LPS group, the cell survival rate, IL-4 and IL-10 levels and the proportion of M2-type macrophages in the LPS+PF group were significantly increased(P<0.01), while the apoptosis rate, IFN-γ and IL-17C levels, CD63, CD9, Alix protein expression levels and the proportion of M1-type macrophages were significantly decreased(P<0.01). These results suggest that PF can improve the survival rate of BEAS-2B cells, reduce apoptosis, and inhibit exosome secretion and inflammatory response. The underlying mechanism may be related to PF induced M2-type polarization of macrophages.更多还原