目的 探讨芪玉三龙汤（QYSLD）通过PTEN通路诱导M2/M1巨噬细胞极化的机制。方法 Lewis肺癌细胞株（LLC）皮下接种于C57BL/6小鼠右前肢腋下构建非小细胞肺癌（NSCLC）动物模型，将模型成功小鼠随机分成模型组和QYSLD（80.48 g·kg^-1）组，肿瘤体积约150 mm³开始ig给药，每天给药1次，连续给药15 d，模型组ig 0.9%氯化钠溶液。转录组学分析小鼠的移植瘤样本，进行转录组测序及生物信息学分析；流式细胞术检测肿瘤组织M1和M2型巨噬细胞、CD8⁺T、CD4⁺T细胞的百分比。体外培养小鼠腹腔原代巨噬细胞，40 ng·mL^-1的IL-4刺激构建M2型模型，分为模型组、20%空白血清组、20% QYSLD含药血清（以13.96 g·kg^-1的QYSLD每隔12 h ig给予大鼠1次，持续3 d，制备含药血清）组、脂多糖（LPS，阳性药，20 ng·mL^-1）组，流式细胞仪检测细胞CD206、活性氧（ROS）水平，实时荧光定量PCR法检测精氨酸酶-1（Arg-1）、CC趋化因子配体24（CCL24）、趋化因子配体17（CXCL17）、白细胞介素（IL）-10、肿瘤坏死因子（TNF）-α、PTEN的mRNA相对表达水平； ELISA法检测上清液中诱导型一氧化氮合酶（iNOS）、IL-10、IL-12和环氧合酶-2（COX-2）水平； M2型小鼠腹腔原代巨噬细胞设置为模型组、20%空白血清组、20% QYSLD含药血清组，PTEN抑制剂组、QYSLD含药血清＋PTEN抑制剂组，与CD4⁺T、CD8⁺T细胞共培养，ELISA法检测各组细胞上清中干扰素-γ（IFN-γ）、IL-2的分泌水平；流式细胞术检测CD279、CD69、CD366的表达情况。结果 体内实验中，转录组测序结果表明，M1/M2型肿瘤相关巨噬细胞（TAMs）和CD4⁺/CD8⁺T细胞与QYSLD对NSCLC的治疗相关； QYSLD降低了肿瘤组织的葡萄糖细胞内稳态（P＜0.05），CD4⁺T细胞、单核细胞、NK细胞对碳水化合物分解代谢的依赖性较高。相对于模型组，QYSLD组NSCLC组织内M1/M2相对增加（0.24→0.37），CD4⁺/CD8⁺T细胞的比值相对减少（0.39→0.20）。体外实验中，相对于空白血清，含有QYSLD的血清可以有效减少巨噬细胞表面CD206以及M2型巨噬细胞相关Arg-1、CCL24、CXCL17、IL-10 mRNA的表达（P＜0.05、0.01、0.001），并增加M1型巨噬细胞相关TNF-α mRNA及PTEN mRNA的表达（P＜0.01、0.001）；含药血清显著降低IL-10蛋白分泌（P＜0.01），显著增加M1型巨噬细胞相关蛋白IL-12、iNOS、COX-2的分泌（P＜0.01）。与空白血清组比较，在加入QYSLD后CD4⁺T细胞和CD8⁺T细胞均增加IFN-γ、IL-2（P＜0.01）和CD69的表达，二者被活化。与20%含药血清组相比，加了PTEN抑制剂后，IL-2、IFN-γ（P＜0.01）、CD69、CD279表达下调。结论 QYSLD通过激活PTEN通路促进TAMs向M1型极化以及T细胞活化，改善肿瘤免疫微环境。

Objective The purpose of this study was to explore the mechanism of Qiyu Sanlong Decoction (QYSLD) inducing M2 / M1 macrophage polarization through PTEN pathway. Methods Lewis lung cancer cell line (LLC) was subcutaneously inoculated into the right lower limb of C57BL/6 mice to establish a lung cancer animal model. The successfully modeled mice were randomly divided into the model group and the QYSLD (80.48 g·kg^-1) group. Intragastric administration was started when the tumor volume was approximately 150 mm³, once a day for 15 consecutive days. The model group was ig administered 0.9% sodium chloride solution. The tumor samples of the mice were subjected to transcriptome analysis, including transcriptome sequencing and bioinformatics analysis. The percentages of M1 and M2 type macrophages, CD8⁺ T cells, and CD4⁺ T cells in the tumor tissues were detected by flow cytometry. Mouse peritoneal primary macrophages were cultured in vitro, and the M2 type model was constructed by stimulating with 40 ng·mL^-1 IL-4. The groups included the model group, 20% blank serum group, 20% QYSLD-containing serum group (prepared by ig administering 13.96 g·kg^-1 QYSLD to rats once every 12 hours for 3 d), and the lipopolysaccharide (LPS, positive drug, 20 ng·mL^-1) group. The levels of CD206 and reactive oxygen species (ROS) in the cells were detected by flow cytometry. The relative mRNA expression levels of arginase-1 (Arg-1), CC chemokine ligand 24 (CCL24), chemokine ligand 17 (CXCL17), interleukin (IL)-10, tumor necrosis factor (TNF)-α, and PTEN were detected by real-time fluorescence quantitative PCR. The levels of inducible nitric oxide synthase (iNOS), IL^-10, IL^-12, and cyclooxygenase-2 (COX-2) in the supernatant were detected by ELISA. For the M2 type mouse peritoneal primary macrophages, the groups included the model group, 20% blank serum group, 20% QYSLD-containing serum group, PTEN inhibitor group, and QYSLD-containing serum + PTEN inhibitor group. They were co-cultured with CD4⁺ T and CD8⁺ T cells, and the secretion levels of interferon-γ (IFN-γ) and IL-2 in the supernatant of each group were detected by ELISA. The expression of CD279, CD69, and CD366 was detected by flow cytometry. Results In the in vivo experiments, the transcriptome sequencing results indicated that M1/M2 type tumor-associated macrophages (TAMs) and CD4⁺/CD8⁺ T cells were related to the treatment of non-small cell lung cancer (NSCLC) by QYSLD. QYSLD decreased the intracellular glucose homeostasis of tumor tissues (P < 0.05), and CD4+ T cells, monocytes, and NK cells had a higher dependence on carbohydrate catabolism. Compared with the model group, the ratio of M1/M2 in the NSCLC tissues of the QYSLD group increased (0.24→0.37), and the ratio of CD4⁺/CD8⁺ T cells decreased (0.39→0.20). In the in vitro experiments, compared with the blank serum, the serum containing QYSLD could effectively reduce the expression of CD206 on the surface of macrophages and the mRNA expression of Arg-1, CCL24, CXCL17, and IL-10 related to M2 type macrophages (P < 0.05, 0.01, 0.001), and increase the mRNA expression of TNF-α and PTEN related to M1 type macrophages (P < 0.01, 0.001). The drug-containing serum significantly reduced the secretion of IL^-10 protein (P < 0.01) and significantly increased the secretion of M1 type macrophage-related proteins IL^-12, iNOS, and COX-2 (P < 0.01). Compared with the blank serum group, after adding QYSLD, both CD4⁺ T cells and CD8⁺ T cells increased the expression of IFN-γ, IL-2 (P < 0.01), and CD69, indicating that they were activated. Compared with the 20% drug-containing serum group, after adding the PTEN inhibitor, the expressions of IL-2, IFN-γ (P < 0.01), CD69 and CD279 were down-regulated. Conclusion QYSLD promotes the polarization of tumor-associated macrophages to M1 type and the activation of T cells by activating the PTEN pathway, improves the tumor immune microenvironment.

R285.5

国家自然科学基金青年基金项目（82004314）;安徽省中医药传承创新科研项目（2024ZYYXH160）;安徽医科大学校科研基金立项资助项目（2023xkj128）