| 白薇超,刘 亚,庞亚梅,李 宏,宁 谦.雷公藤内酯甲通过抑制巨噬细胞M2极化发挥抗肺癌作用[J].现代生物医学进展英文版,2024,(16):3013-3019. |
| 雷公藤内酯甲通过抑制巨噬细胞M2极化发挥抗肺癌作用 |
| Wilforlide A Plays an Anti-lung Cancer Role by Inhibiting Macrophage M2 Polarization |
| Received:March 11, 2024 Revised:March 31, 2024 |
| DOI：10.13241/j.cnki.pmb.2024.16.003 |
| 中文关键词: 雷公藤内酯甲 雷公藤 非小细胞肺癌 巨噬细胞M2极化 免疫微环境 |
| 英文关键词: Wilforlide A (WA) Tripterygium wilfordii Hook F Non-small cell lung cancer Macrophage M2 polarization Immune microenvironment |
| 基金项目:陕西省重点研发计划项目（2022SF-141） |
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| 中文摘要: |
| 摘要 目的：探讨雷公藤内酯甲（Wilforlide A，WA）对肺癌细胞增殖和侵袭及巨噬细胞M2极化的影响。方法：（实验一）：将肺癌细胞LLC按照WA处理浓度分为0、0.01、0.1、1和10 μg/L组。WA孵育48 h后，采用MTT法、Annexin V-FITC/PI染色法和Transwell法检测细胞增殖、凋亡和侵袭。采用Western blot检测Bcl-2、Bax、MMP-2和MMP-9蛋白表达水平。（实验二）：将单核巨噬细胞RAW264.7按照WA处理浓度分为0、0.01、0.1、1和10 μg/L组。采用MTT法检测细胞增殖。然后再将RAW264.7细胞为Control组、IL-4组、WA组和IL-4+WA组。使用10 ng/mL的IL-4和1 μg/L的WA培养细胞24 h。采用ELISA法检测细胞培养上清液中CD206、Arg-1和IL-10水平。（实验三）：细胞分组如下：Blank组、RAW264.7组、IL-4 RAW264.7组、WA组和IL-4 RAW264.7+WA组。按照分组方案，将LLC细胞分别与RAW264.7细胞、IL-4诱导RAW264.7细胞以及10 μg/L的WA共培养24 h，然后检测细胞增殖、凋亡、侵袭及细胞培养上清液中的M2型巨噬细胞标志物水平。流式细胞仪检测CD86和CD206阳性百分比。结果：（实验一）：与0 μg/L组比较，0.1、1和10 μg/L组LLC细胞的相对细胞活力降低（P<0.05），细胞凋亡率和Bax蛋白相对表达水平均升高（P<0.05），侵袭细胞数量以及Bcl-2、MMP-2和MMP-9蛋白相对表达水平均降低（P<0.05）。（实验二）：与0 μg/L组比较，10 μg/L组RAW264.7细胞的相对细胞活力降低（P<0.05）。与Control组比较，IL-4组RAW264.7细胞的CD206、Arg-1和IL-10水平升高（P<0.05），WA组RAW264.7细胞的CD206、Arg-1和IL-10水平降低（P<0.05）。与IL-4组比较，IL-4+WA组RAW264.7细胞的CD206、Arg-1和IL-10水平降低（P<0.05）。（实验三）：与Blank组比较，RAW264.7组和IL-4 RAW264.7组的相对细胞活力和侵袭细胞数量均升高（P<0.05），细胞凋亡率降低（P<0.05）。与RAW264.7组比较，IL-4 RAW264.7组的相对细胞活力和侵袭细胞数量升高（P<0.05），细胞凋亡率降低（P<0.05），CD206、Arg-1和IL-10水平升高（P<0.05），CD86百分比降低，CD206百分比升高（P<0.05）。与IL-4 RAW264.7比较，IL-4 RAW264.7+WA组的相对细胞活力和侵袭细胞数量降低（P<0.05），细胞凋亡率升高（P<0.05），CD206、Arg-1和IL-10水平降低（P<0.05），CD86百分比升高，CD206百分比降低（P<0.05）。结论：本研究表明雷公藤内酯甲具有抗NSCLC作用，其机制与抑制巨噬细胞的M2型极化有关。 |
| 英文摘要: |
| ABSTRACT Objective: To explore the effect of Wilforlide A (WA) on lung cancer cell proliferation and invasion and macrophage M2 polarization. Methods: (Experiment 1): LLC cells were divided into 0, 0.01, 0.1, 1 and 10 μg/L groups according to WA concentration. After incubation with WA for 48 h, the cell proliferation, apoptosis and invasion were detected by MTT method, AnnexinV-FITC/PI staining method and Transwell method, respectively, the protein expression levels of Bcl-2, Bax, MMP-2 and MMP-9 were detected by Western blot. (Experiment 2): RAW264.7 cells were divided into 0, 0.01, 0.1, 1 and 10 μg/L groups according to WA concentration. The cell proliferation was detected by MTT assay. RAW264.7 cells were divided into Control group, IL-4 group, WA group and IL-4+WA group. The cells were cultured with 10 ng/mL of IL-4 and 1 μg/L of WA for 24 h. The levels of CD206, Arg-1 and IL-10 in cell culture supernatant were detected by ELISA. (Experiment 3): The cells were grouped as follows: Blank group, RAW264.7 group, IL-4RAW264.7 group, WA group and IL-4RAW264.7+WA group. LLC cells were co-cultured with RAW264.7 cells, IL-4-induced RAW264.7 cells and 10 μg/L WA for 24 h according to the grouping scheme. Cell proliferation, apoptosis, invasion, and M2 macrophage marker levels in cell culture supernatants were then detected by the above methods. The percentage of CD86 and CD206 positive were detected by flow cytometry. Results: (Experiment 1): Compared with the 0 μg/L group, the relative cell viability of the 0.1, 1 and 10 μg/L groups decreased (P<0.05), the cell apoptosis rate and the relative protein expression level of Bax increased (P<0.05), and the number of invasive cells and the relative protein expression levels of Bcl-2, MMP-2 and MMP-9 proteins decreased (P<0.05). (Experiment 2): Compared with the 0 μg/L group, the relative cell viability of RAW264.7 cells in the 10 μg/L group decreased(P<0.05). Compared with the Control group, the levels of CD206, Arg-1 and IL-10 in the IL-4 group increased (P<0.05), while the levels of CD206, Arg-1 and IL-10 in the WA group decreased (P<0.05). Compared with the IL-4 group, the levels of CD206, Arg-1 and IL-10 in the IL-4+WA group decreased (P<0.05). (Experiment 3): Compared with the Blank group, the relative cell viability and number of invasive cells in the RAW264.7 group and the IL-4RAW264.7 group increased (P<0.05), and the cell apoptosis rate decreased (P<0.05). Compared with the RAW264.7 group, the relative cell viability and number of invasive cells in the IL-4RAW264.7 group increased (P<0.05), the cell apoptosis rate decreased (P<0.05), and the levels of CD206, Arg-1 and IL-10 increased(P<0.05) , CD86 percentage decreased and CD206 percentage increased (P<0.05). Compared with IL-4RAW264.7 group, the relative cell viability and number of invasive cells in the IL-4RAW264.7+WA group decreased (P<0.05), the cell apoptosis rate increased (P<0.05), and the levels of CD206, Arg-1 and IL-10 decreased (P<0.05), CD86 percentage increased and CD206 percentage decreased(P<0.05). Conclusion: This study shows that WA has anti-NSCLC effects, and its mechanism is related to inhibiting the M2 polarization of macrophages. |
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