目的 探究通关藤注射液（Tongguanteng Injection，TGT）调节巨噬细胞极化进而协同多柔比星（doxorubicin，DOX）抑制三阴性乳腺癌（triple-negative breast cancer，TNBC）生长的作用机制。方法 构建4T1乳腺癌荷瘤小鼠模型，随机分为模型组、TGT组、DOX组及TGT联合DOX治疗组，观察肿瘤体积及质量，评估体内抗肿瘤疗效；采用苏木素-伊红（hematoxylin-eosin，HE）、TUNEL及免疫组化染色检测肿瘤组织病理、细胞凋亡及剪切型半胱氨酸天冬氨酸蛋白酶-3（cleaved cystein-asparate protease-3，cleaved Caspase-3）、B细胞淋巴瘤-2（B-cell lymphoma-2，Bcl-2）、Bcl-2相关X蛋白（Bcl-2 associated X protein，Bax）表达；通过免疫器官指数、血常规分析评估小鼠免疫功能；ELISA、qRT-PCR及免疫荧光染色检测肿瘤组织炎症因子水平和巨噬细胞极化标志物CD86、诱导型一氧化氮合酶（inducible nitric oxide synthase，iNOS）、CD206、精氨酸酶-1（arginase-1，Arg-1）的表达；流式细胞术及免疫组化分析肿瘤组织CD4⁺ T、CD8⁺ T细胞浸润比例；qRT-PCR检测T细胞耗竭相关因子及细胞毒性因子的mRNA表达；转录组测序及京都基因与基因组百科全书（Kyoto encyclopedia of genes and genomes，KEGG）富集分析挖掘核心信号通路并进行验证。结果 与模型组比较，TGT组、DOX组及TGT联合DOX治疗组小鼠的肿瘤体积和质量显著降低（P＜0.01、0.001），肿瘤组织出现不同程度坏死，肿瘤细胞凋亡率显著升高（P＜0.001），其中TGT联合DOX治疗组上述变化更为显著（P＜0.05、0.01、0.001）。与模型组相比，TGT组的促炎因子肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）、白细胞介素-1β（interleukin-1β，IL-1β）表达水平显著升高（P＜0.01、0.001），抗炎因子IL-4、IL-10表达水平显著降低（P＜0.05、0.01）；M1型巨噬细胞（CD86⁺/F4/80⁺）比例增加（P＜0.001），标志物CD86、iNOS mRNA表达水平显著升高（P＜0.05、0.01）；M2型巨噬细胞（CD206⁺/F4/80⁺）比例减少（P＜0.001），标志物CD206、Arg-1 mRNA表达水平显著降低（P＜0.01、0.001）；CD8⁺ T细胞比例显著升高（P＜0.05、0.001），CD4⁺ T细胞比例显著降低（P＜0.01）；T细胞耗竭指标程序性死亡受体-1（programmed cell death protein 1，PD-1）、T细胞免疫球蛋白黏蛋白分子-3（T cell immunoglobulin and mucin domain-containing protein-3，Tim-3）、淋巴细胞活化基因-3（lymphocyte activation gene-3，Lag-3）mRNA表达水平显著下调（P＜0.05、0.01），T细胞毒性因子穿孔素（perforin）、γ干扰素（interferon γ，IFN-γ）、颗粒酶B（granzyme B）mRNA表达水平显著上调（P＜0.05、0.001）；TGT联合DOX治疗组上述变化更加显著（P＜0.05、0.01、0.001）。转录组及KEGG富集分析显示，与模型组比较，联合治疗组差异基因显著富集于Janus激酶（Janus kinase，JAK）-信号转导和转录激活蛋白（signal transducer and activator of transcription，STAT）信号通路；免疫组化染色结果表明TGT联合DOX显著下调肿瘤细胞中p-JAK2和p-STAT3蛋白的表达（P＜0.001）。结论 TGT联合DOX显著抑制TNBC的生长，促进肿瘤相关巨噬细胞由M2型向M1型极化，促进CD8⁺ T细胞浸润并减轻其耗竭，其作用机制与抑制JAK-STAT信号通路的磷酸化有关。

Objective To investigate the mechanism by which Tongguanteng Injection (通关藤注射液, TGT) modulates macrophage polarization to synergize with doxorubicin (DOX) in inhibiting triple-negative breast cancer (TNBC) growth. Methods A 4T1 TNBC- bearing mouse model was constructed, randomly divided into model group, TGT group, DOX group and TGT combined with DOX treatment group. Tumor volume and weight were observed to evaluate in vivo antitumor efficacy. Hematoxylin-eosin (HE), TUNEL and immunohistochemical staining were employed to examine histopathological morphological changes in tumor tissues, tumor cell apoptosis, and protein expressions of cleaved cystein-asparate protease-3 (Caspase-3), B-cell lymphoma-2 (Bcl-2), and Bcl-2 associated X protein (Bax). Immune organ indices and routine blood analysis were conducted to assess immune function in mice. ELISA, qRT-PCR and immunofluorescence staining were performed to measure inflammatory cytokine levels and expressions of macrophage polarization markers CD86, inducible nitric oxide synthase (iNOS), CD206, arginase-1 (Arg-1) in tumor tissues. Flow cytometry and immunohistochemical analysis were used to determine the infiltration ratios of CD4⁺ T and CD8⁺ T cells, while qRT-PCR was employed to detect mRNA expression levels of T cell exhaustion-related factors and cytotoxic factors. Transcriptome sequencing and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis were conducted to identify core signaling pathways and perform validation. Results Compared with model group, the tumor volume and weight were significantly reduced in TGT group, DOX group and TGT combined with DOX treatment group (P < 0.01, 0.001), varying degrees of necrosis were observed in tumor tissues, the apoptosis rate of tumor cells was significantly increased (P < 0.001), these changes were more pronounced in TGT + DOX combination therapy group (P < 0.05, 0.01, 0.001). Compared with model group, the expression levels of pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were significantly increased in TGT group (P < 0.01, 0.001), while the expression levels of anti-inflammatory cytokines IL-4 and IL-10 were significantly decreased (P < 0.05, 0.01). The proportion of M1-type macrophages (CD86⁺/F4/80⁺) was increased (P < 0.001), with significantly elevated mRNA expression levels of markers CD86 and iNOS (P < 0.05, 0.01). The proportion of M2-type macrophages (CD206⁺/F4/80⁺) was decreased (P < 0.001), with significantly reduced mRNA expression levels of markers CD206 and Arg-1 (P < 0.01, 0.001). The proportion of CD8⁺ T cells was significantly increased (P < 0.05, 0.001), while the proportion of CD4⁺ T cells was significantly decreased (P < 0.01). The mRNA expression levels of T cell exhaustion markers programmed cell death protein-1 (PD-1), T cell immunoglobulin and mucin domain-containing protein-3 (Tim-3) and lymphocyte activation gene-3 (Lag-3) were significantly downregulated (P < 0.05, 0.01), whereas the mRNA expression levels of T cell cytotoxic factors perforin, interferon-γ (IFN-γ) and granzyme B were significantly upregulated (P < 0.05, 0.001). These changes were more pronounced in TGT combined with DOX treatment group (P < 0.05, 0.01, 0.001). Transcriptome and KEGG enrichment analyses revealed that, compared with model group, differentially expressed genes in the combination therapy group were significantly enriched in Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway. Immunohistochemical staining results indicated that TGT combined with DOX significantly downregulated the expressions of p-JAK2 and p-STAT3 proteins in tumor cells (P < 0.001). Conclusion TGT combined with DOX significantly inhibits the growth of TNBC, promotes the polarization of tumor associated macrophages from M2 to M1, promotes CD8⁺ T cell infiltration and reduces their exhaustion. Its mechanism is related to the inhibition of phosphorylation of JAK-STAT signaling pathway.

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国家自然科学基金资助项目（82204838）；河南省医学科技攻关计划省部共建项目（SBGJ202302100）；河南省高校科技创新团队（23IRTSTHN026）；河南省中医药科学研究专项课题（2024ZY3012，2022ZYZD01）；河南省医学科技攻关计划项目（LHGJ20250411）；河南省高校科技创新人才支持计划（25HASTIT061）；河南省第三批中医药学科拔尖人才项目（豫卫中医药科教函[2025]14号）；河南省自然科学基金面上科学基金项目（262300421585）