目的 探討芪玉三龍湯（QYSLD）通過PTEN通路誘導(dǎo)M2/M1巨噬細(xì)胞極化的機(jī)制。方法 Lewis肺癌細(xì)胞株（LLC）皮下接種于C57BL/6小鼠右前肢腋下構(gòu)建非小細(xì)胞肺癌（NSCLC）動(dòng)物模型，將模型成功小鼠隨機(jī)分成模型組和QYSLD（80.48 g·kg^-1）組，腫瘤體積約150 mm³開始ig給藥，每天給藥1次，連續(xù)給藥15 d，模型組ig 0.9%氯化鈉溶液。轉(zhuǎn)錄組學(xué)分析小鼠的移植瘤樣本，進(jìn)行轉(zhuǎn)錄組測(cè)序及生物信息學(xué)分析；流式細(xì)胞術(shù)檢測(cè)腫瘤組織M1和M2型巨噬細(xì)胞、CD8⁺T、CD4⁺T細(xì)胞的百分比。體外培養(yǎng)小鼠腹腔原代巨噬細(xì)胞，40 ng·mL^-1的IL-4刺激構(gòu)建M2型模型，分為模型組、20%空白血清組、20% QYSLD含藥血清（以13.96 g·kg^-1的QYSLD每隔12 h ig給予大鼠1次，持續(xù)3 d，制備含藥血清）組、脂多糖（LPS，陽(yáng)性藥，20 ng·mL^-1）組，流式細(xì)胞儀檢測(cè)細(xì)胞CD206、活性氧（ROS）水平，實(shí)時(shí)熒光定量PCR法檢測(cè)精氨酸酶-1（Arg-1）、CC趨化因子配體24（CCL24）、趨化因子配體17（CXCL17）、白細(xì)胞介素（IL）-10、腫瘤壞死因子（TNF）-α、PTEN的mRNA相對(duì)表達(dá)水平； ELISA法檢測(cè)上清液中誘導(dǎo)型一氧化氮合酶（iNOS）、IL-10、IL-12和環(huán)氧合酶-2（COX-2）水平； M2型小鼠腹腔原代巨噬細(xì)胞設(shè)置為模型組、20%空白血清組、20% QYSLD含藥血清組，PTEN抑制劑組、QYSLD含藥血清＋PTEN抑制劑組，與CD4⁺T、CD8⁺T細(xì)胞共培養(yǎng)，ELISA法檢測(cè)各組細(xì)胞上清中干擾素-γ（IFN-γ）、IL-2的分泌水平；流式細(xì)胞術(shù)檢測(cè)CD279、CD69、CD366的表達(dá)情況。結(jié)果 體內(nèi)實(shí)驗(yàn)中，轉(zhuǎn)錄組測(cè)序結(jié)果表明，M1/M2型腫瘤相關(guān)巨噬細(xì)胞（TAMs）和CD4⁺/CD8⁺T細(xì)胞與QYSLD對(duì)NSCLC的治療相關(guān)； QYSLD降低了腫瘤組織的葡萄糖細(xì)胞內(nèi)穩(wěn)態(tài)（P＜0.05），CD4⁺T細(xì)胞、單核細(xì)胞、NK細(xì)胞對(duì)碳水化合物分解代謝的依賴性較高。相對(duì)于模型組，QYSLD組NSCLC組織內(nèi)M1/M2相對(duì)增加（0.24→0.37），CD4⁺/CD8⁺T細(xì)胞的比值相對(duì)減少（0.39→0.20）。體外實(shí)驗(yàn)中，相對(duì)于空白血清，含有QYSLD的血清可以有效減少巨噬細(xì)胞表面CD206以及M2型巨噬細(xì)胞相關(guān)Arg-1、CCL24、CXCL17、IL-10 mRNA的表達(dá)（P＜0.05、0.01、0.001），并增加M1型巨噬細(xì)胞相關(guān)TNF-α mRNA及PTEN mRNA的表達(dá)（P＜0.01、0.001）；含藥血清顯著降低IL-10蛋白分泌（P＜0.01），顯著增加M1型巨噬細(xì)胞相關(guān)蛋白IL-12、iNOS、COX-2的分泌（P＜0.01）。與空白血清組比較，在加入QYSLD后CD4⁺T細(xì)胞和CD8⁺T細(xì)胞均增加IFN-γ、IL-2（P＜0.01）和CD69的表達(dá)，二者被活化。與20%含藥血清組相比，加了PTEN抑制劑后，IL-2、IFN-γ（P＜0.01）、CD69、CD279表達(dá)下調(diào)。結(jié)論 QYSLD通過激活PTEN通路促進(jìn)TAMs向M1型極化以及T細(xì)胞活化，改善腫瘤免疫微環(huán)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

國(guó)家自然科學(xué)基金青年基金項(xiàng)目（82004314）;安徽省中醫(yī)藥傳承創(chuàng)新科研項(xiàng)目（2024ZYYXH160）;安徽醫(yī)科大學(xué)校科研基金立項(xiàng)資助項(xiàng)目（2023xkj128）