南蛇藤素通过调控M2型巨噬细胞极化提高A549细胞对顺铂的敏感性

Celastrol regulates M2-type tumor-associated macrophage polarization to enhance cisplatin sensitivity of A549 cells

目的:探讨南蛇藤素(CEL)通过调控M2型巨噬细胞极化提高肺癌细胞A549对顺铂敏感性的作用及可能机制。方法:体外培养巨噬细胞RAW264.7和肺癌细胞A549,CCK-8检测CEL对细胞活性的影响。IL-4/IL-13诱导RAW264.7细胞极化后给予CEL,qRT-PCR和免疫荧光实验检测M2型巨噬细胞相关标志物表达。Western blot检测RAW264.7细胞p-PI3K、PI3K、p-AKT和AKT表达。将A549细胞分为对照组、CEL组、M2组(A549细胞与M2型巨噬细胞共培养)、M2+CEL组(A549细胞与CEL处理的M2型巨噬细胞共培养)和M2+LY-294002组(A549细胞与LY-294002处理的M2型巨噬细胞共培养),给予顺铂处理后,CCK-8检测细胞活性,Western blot检测Bcl-2和Bax蛋白表达。结果:80 nmol/L CEL抑制RAW264.7细胞活性但对A549细胞活性无影响。40 nmol/L CEL处理RAW264.7细胞后,IL-4/IL-13诱导的M2型巨噬细胞标志物MRC1、Arg1和CD163表达明显降低,p-PI3K和p-AKT蛋白表达下降。A549细胞经20μmol/L顺铂处理后,与对照组相比,M2组细胞活性明显升高,Bcl-2表达下降但Bax表达升高。与M2组相比,M2+CEL组和M2+LY-294002组细胞活性显著降低,Bcl-2表达升高而Bax表达降低。结论:CEL可能通过PI3K/AKT信号通路抑制M2型巨噬细胞极化从而提高A549细胞对顺铂的敏感性。

Objective:To explore effect and possible mechanism of celastrol(CEL)on cisplatin sensitivity of lung cancer cells A549 by regulating polarization of M2 type macrophages.Methods:Macrophages RAW264.7 and lung cells A549 were cultured in vitro,and effect of CEL on cell viability was detected by CCK-8. RAW264.7 cells were induced polarization by IL-4/IL-13 and treated with CEL. qRT-PCR and immunofluorescence were used to detect expressions of M2 type macrophages related markers. Western blot was used to detect expressions of p-PI3K,PI3K,p-AKT and AKT in RAW264.7 cells. A549 cells were divided into control group,CEL group,M2 group(A549 cells and M2-type macrophages co-culture),M2+CEL group(A549 cells and CEL-treated M2-type macrophages co-culture)and M2+LY-294002 group(A549 cells and LY-294002-treated macrophages co-culture). After treatment with cisplatin,cell viability was detected by CCK-8,and expressions of Bcl-2 and Bax proteins were detected by Western blot.Results:80 nmol/L CEL inhibited viability of RAW264.7 cells but had no effect on viability of A549 cells. In RAW264.7 cells,IL-4/IL-13-induced M2 type macrophages markers MRC1,Arg1 and CD163 expressions and expressions of p-PI3K and p-AKT proteins were significantly decreased after 40 nmol/L CEL treatment. After treated with 20 μmol/L cisplatin,compared with control group of A549 cells,cell viability of M2 group was significantly increased,expression of Bcl-2 was decreased but expression of Bax was increased.Compared with M2 group,activities of A549 cells in M2+CEL group and M2+LY-294002 group were significantly decreased,and expression of Bcl-2 was increased while expression of Bax was decreased.Conclusion:CEL may inhibit M2-type macrophages polarization through PI3K/AKT signaling pathway,thereby increasing sensitivity of A549 cells to cisplatin.