清金消积丸调控M2-TAMs来源的IL-6介导的IL-6R/PI3K/AKT通路抑制肺癌A549细胞恶性行为

QingjinXiaoji Pills inhibits the malignant behavior of lung cancer A549 cells by regulating the IL-6R/PI3K/AKT pathway mediated by IL-6 secreted by M2-TAMs.

关键词:  清金消积丸  非小细胞肺癌  肿瘤相关巨噬细胞  白细胞介素-6  肿瘤微环境

Key words:  QingjinXiaoji Pills  non-small cell lung cancer  tumor-associated macrophages  interleukin-6  tumor microenvironment

摘要:

目的：探究清金消积丸抑制肺癌A549细胞恶性行为的作用机制。方法：采用网络药理学方法筛选清金消积丸作用靶点与NSCLC及TAMs的交集靶点，预测其核心靶点与通路。用PMA和IL-4将THP-1细胞诱导成M2-TAMs，流式细胞术检测CD163的表达；CCK8和ELISA筛选清金消积丸最佳浓度；ELISA、WB、RT- PCR检测清金消积丸对M2-TAMs分泌IL-6的影响；IL6-siRNA转染A549细胞，RT-PCR验证转染效率；用清金消积丸干预后的M2-TAMs条件培养基来培养经过IL6-siRNA处理的A549细胞，分组为：si-IL6组、si-IL6+M2-CM组、si-IL6+QJXJW-M2-CM组、si-IL6+TCZ-M2-CM组、si-IL6+rhIL-6组；CCK-8检测各组IL6-siRNA的A549细胞增殖活性，流式细胞术检测细胞凋亡，Transwell实验检测细胞侵袭和迁移能力，WB检测通路蛋白的表达。结果：网络药理学预测提示IL-6是清金消积丸通过TAMs作用于NSCLC的关键靶点，并显著富集于PI3K-Akt等通路。实验显示，10%清金消积丸含药血清对细胞存活率无明显抑制且能显著抑制IL-6分泌（P<0.01）；与si-IL6组相比，si-IL6+M2-CM组和si-IL6+rhIL-6组细胞凋亡率降低，增殖率、侵袭与迁移细胞数增加（P<0.01）；与si-IL6+M2-CM组比较，si-IL6+QJXJW-M2-CM组细胞凋亡率升高（P<0.01），增殖率、侵袭与迁移细胞数降低（P<0.01），且IL-6R/PI3K/AKT通路的蛋白表达均显著降低（P<0.01）。结论：清金消积丸可能通过调控M2-TAMs来源的IL-6介导的IL-6R/PI3K/AKT通路抑制肺癌A549细胞生物学行为。

Abstract:

Objective:To explore the mechanism of QingjinXiaoji Pills in inhibiting the malignant behavior of lung cancer A549 cells. Methods:Network pharmacology was employed to identify the overlapping targets among QingjinXiaoji Pills, NSCLC, and TAMs, and to predict the core targets and pathways involved.THP-1 cells were induced into M2-TAMs using PMA and IL-4, and the expression of CD163 was assessed via flow cytometry. The optimal concentration of QingjinXiaoji Pills was screened by CCK8 and ELISA assays. The effect of QingjinXiaoji Pills on the secretion of IL-6 by M2-TAMs was detected by ELISA, WB and RT-PCR. IL6-siRNA was transfected into A549 cells, and the transfection efficiency was verified by RT-PCR. A549 cells of IL6-siRNA were cultured with M2-TAMs-CM after being intervened with QingjinXiaoji Pills, and divided into si-IL6 group, si-IL6+M2-CM group, si-IL6+QJXJW-M2-CM group, si-IL6+TCZ-M2-CM group, si-IL6+rhIL-6 group.Cell proliferation was measured by CCK-8, apoptosis by flow cytometry, and invasion and migration abilities by Transwell assay. WB was used to detect the expression of pathway-related proteins. Results:Network pharmacological analysis predicted that IL-6 is a key target through which QingjinXiaoji Pills acts on NSCLC by regulating TAMs, and was significantly enriched in pathways such as PI3K-Akt. Experimental results showed that 10% serum containing QingjinXiaoji Pills showed no significant effect on cell viability but significantly inhibited IL-6 secretion (P<0.01). Compared with the si-IL6 group, the si-IL6+M2-CM and si-IL6+rhIL-6 groups showed decreased apoptosis and increased proliferation, invasion, and migration (P<0.01). Compared with the si-IL6+M2-CM group, the si-IL6+QJXJW-M2-CM group showed increased apoptosis (P<0.01), decreased proliferation, invasion, and migration (P<0.01), and significantly reduced protein expression of the IL-6R/PI3K/AKT pathway (P<0.01).Conclusion: QingjinXiaoji Pills likely suppress the behavior of lung cancer A549 cells by modulating the IL-6R/PI3K/AKT pathway mediated by M2-TAMs-secreted IL-6.