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.

Tongguanteng Injection  /  doxorubicin  /  triple-negative breast cancer  /  macrophage polarization  /  immunomodulation  /  JAK-STAT signaling pathway