基于IL-17信号通路探讨药用真菌对非小细胞肺癌肿瘤微环境的影响

陈俊颖; 张奇; 崔明琦; 王晓倩; 王升厚; 逄洪波; 王泽

doi:10.13386/j.issn1002-0306.2025090299

基于IL-17信号通路探讨药用真菌对非小细胞肺癌肿瘤微环境的影响

Effects of Medicinal Fungi on the Tumor Microenvironment in Non-Small Cell Lung Cancer through the IL-17 Signaling Pathway

摘要

摘要: 目的：从肿瘤微环境的角度，探讨灵芝、暴马丁香桑黄和蛹虫草的抗肿瘤新机制。方法：采用水浴浸提结合真空冷冻干燥技术制备灵芝（RL）、暴马丁香桑黄（BJ）和蛹虫草（XG）的水提物（FAE），通过MTT法和Annexin V-FITC/PI双染法检测FAE及各药用真菌条件培养基（CM）对非小细胞肺癌细胞A549增殖活性的影响。以4 mg/mL FAE处理细胞后，转录组测序并结合KEGG通路富集分析IL-17信号通路对各FAE的响应特征；采用免疫细胞化学染色、抗体封闭实验及ELISA实验进一步检测各FAE对黏蛋白MUC5AC和MUC5B，趋化因子IL6、CCL2、CCL20、CXCL8和CXCL10表达量的影响。结果：FAE和CM均可抑制A549细胞增殖并诱导其凋亡。转录组测序结果显示，RL组与BJ组以上调基因为主，XG组以下调基因为主，且均在肿瘤微环境形成相关信号通路存在显著富集；各组IL-17信号通路（ko04657）下游关键效应分子表达水平存在显著性改变（P<0.05）。三种FAE均可显著抑制肺癌细胞的MUC5B的表达（P<0.01），但BJ处理对MUC5AC的表达无显著影响（P>0.05）。MUC5AC抗体封闭显著削弱RL和XG对细胞增殖的抑制作用（P<0.05），MUC5B抗体封闭后，RL组和BJ组对细胞增殖的抑制作用显著下降（P<0.05）。此外，三种FAE对细胞因子IL6、CCL2、CCL20和CXCL8的分泌水平具有显著抑制的作用（P<0.01）。结论：灵芝、暴马丁香桑黄与蛹虫草均可通过重塑肿瘤微环境，协同发挥抗肿瘤作用，这为其开发为辅助肿瘤治疗的功能性食品提供了新理论支撑。

Abstract: Objective: From the perspective of the tumor microenvironment, this study explored the anti-tumor mechanisms of Ganoderma lucidum, Phellinus linteus, and Cordyceps militaris. Methods: Aqueous extracts (FAEs) of Ganoderma lucidum (RL), Phellinus linteus (BJ), and Cordyceps militaris (XG) were prepared via water bath extraction and vacuum freeze-drying. The effects of the FAEs and their conditioned media (CM) on the proliferative activity of A549 non-small cell lung cancer cells were determined using the MTT assay and Annexin V-FITC/PI double staining. Transcriptome sequencing was performed on cells treated with 4 mg/mL FAE, and KEGG pathway enrichment analysis was used to identify the response characteristics of the IL-17 signaling pathway to each extract. Additionally, immunocytochemical staining, antibody blocking assays, and ELISA were used to detect the effects of the FAEs on the expression levels of the mucins MUC5AC and MUC5B and the chemokines IL-6, CCL2, CCL20, CXCL8, and CXCL10. Results: The three FAEs and CM inhibited the proliferation of the A549 cells and induced their apoptosis. Transcriptome analysis results revealed that upregulated genes were predominant in the RL and BJ groups, whereas downregulated genes were predominant in the XG group. All groups showed significant enrichment in signaling pathways related to tumor microenvironment formation. Additionally, significant alterations were observed in the expression levels of key effector molecules downstream of the IL-17 signaling pathway (ko04657) in all groups. All three FAEs significantly inhibited MUC5B expression (P<0.01), however, BJ treatment did not significantly affect MUC5AC expression (P>0.05) MUC5AC antibody blocking weakened the inhibitory effects of RL and XG on cell proliferation (P<0.05), whereas MUC5B antibody blocking significantly reduced the anti-proliferative effects of RL and BJ on the cells (P<0.05). Furthermore, all three FAEs significantly inhibited the secretion of IL-6, CCL2, CCL20, and CXCL8 (P<0.01). Conclusion: In conclusion, Ganoderma lucidum, Phellinus linteus, and Cordyceps militaris can all remodel the tumor microenvironment to synergistically exert anti-tumor effects. This provides a new theoretical basis for their development as functional foods for adjuvant cancer therapy.