芍药苷通过介导THP-1源巨噬细胞极化抑制支气管上皮细胞炎症反应

Paeoniflorin inhibits the inflammatory response of bronchial epithelial cells by mediating the polarization of THP-1-derived macrophages

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。

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 1μg/mL LPS to induce inflammation,and treated with 1,10,and 30μmol/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 10μmol/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-γand 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-γand 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.