清金消积丸调控M2-TAMs源性IL-6抑制A549细胞恶性行为

杨卯勤; 秦大凯; 任德祥; 夏小军

doi:10.13748/j.cnki.issn1007-7693.20251588

清金消积丸调控M2-TAMs源性IL-6抑制A549细胞恶性行为

Qingjin Xiaoji Pills Inhibit the Malignant Behavior of A549 Cells by Regulating M2-TAM-derived IL-6

摘要

摘要:
目的研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非小细胞肺癌(non-small cell lung cancer，NSCLC)及肿瘤相关巨噬细胞(tumor-associated macrophages，TAMs)的交集靶点，预测其核心靶点与通路。用佛波酯和IL-4将THP-1细胞诱导成M2型TAMs(M2-TAMs)，流式细胞术检测CD163的表达；CCK-8和ELISA筛选清金消积丸最佳浓度；ELISA、Western blotting、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试验检测细胞侵袭和迁移能力，Western blotting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，且对IL-6分泌抑制率最高；与si-IL6组相比，si-IL6+M2-CM组和si-IL6+rhIL-6组细胞凋亡率降低(P<0.01)，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 study the mechanism of Qingjin Xiaoji Pills in inhibiting the malignant behavior of lung cancer A549 cells.
METHODS Network pharmacology was employed to identify the overlapping targets among Qingjin Xiaoji Pills, non-small cell lung cancer(NSCLC), and tumor-associated macrophages(TAMs), and to predict the core targets and pathways involved. THP-1 cells were induced into M2-TAMs using phorbol 12-myristate 13-acetate and IL-4, and the expression of CD163 was assessed via flow cytometry. The optimal concentration of Qingjin Xiaoji Pills was screened by CCK-8 and ELISA assays. The effect of Qingjin Xiaoji Pills on the secretion of IL-6 by M2-TAMs was detected by ELISA, Western blotting 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 Qingjin Xiaoji 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. Western blotting was used to detect the expression of pathway-related proteins.
RESULTS Network pharmacological analysis predicted that IL-6 was a key target through which Qingjin Xiaoji Pills acts on NSCLC by regulating TAMs, and was significantly enriched in pathways such as PI3K/AKT. Experimental results showed that treatment with 10% Qingjin Xiaoji Pills-containing serum had no significant inhibitory effect on cell viability, while it exhibited the highest inhibition rate on IL-6 secretion. Compared with the si-IL6 group, the si-IL6+M2-CM and si-IL6+rhIL-6 groups showed decreased apoptosis(P<0.01) 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 Qingjin Xiaoji Pills are likely to inhibit the biological behaviors of lung cancer A549 cells by regulating the IL-6R/PI3K/AKT pathway mediated by M2-TAMs-secreted IL-6.

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