为了研究新型四草方（4HF）与多柔比星（doxorubicin）联合治疗三阴性乳腺癌（TNBC）的体内免疫调节和抗肿瘤机制。我们培养了小鼠源性三阴性乳腺癌细胞系4T1，在小鼠乳腺中接种了这些细胞。将66只小鼠随机分为6组（每组11只）：天然组、对照组、LD 4HF组（低剂量4HF）、HD 4HF组（高剂量4HF）、LD 4HF + D组（低剂量4HF和多柔比星）、D组（多柔比星）。除了天然组，每只小鼠皮下接种了5 × 105个4T1细胞悬浮于100 µL生理盐水中的乳腺脂肪垫中。从肿瘤细胞接种的那天起，肿瘤生长了6天。LD组和HD组每天口服658 mg/kg和2,630 mg/kg的4HF。LD 4HF+D组每天口服658 mg/kg的4HF，并每周腹腔注射多柔比星（5 mg/kg）。D组每周腹腔注射多柔比星（5 mg/kg）。天然处理组每天口服0.2 mL双蒸馏水和每周腹腔注射0.1 mL生理盐水。对照组每天口服0.2 mL双蒸馏水。治疗持续30天。治疗结束后，收集小鼠器官，通过免疫表型分析、基因和多重分析、组织学染色和肠道菌群分析来研究免疫活性。 4HF和多柔比星联合治疗小鼠明显减少肿瘤和脾脏负担（P<0.05），改变了低氧和整体免疫淋巴细胞结构，并调节肠道菌群以促进抗肿瘤免疫活性。此外，瘤体中的免疫抑制基因、细胞因子和趋化因子，如C-C基序趋化因子2和白细胞介素-10被明显下调（P<0.05）。4HF-多柔比星联合治疗表现出协同作用，最有效地激活抗肿瘤免疫反应（P<0.05）。 上述结果为评估4HF在乳腺癌中的免疫调节机制提供了证据，并支持其作为辅助治疗药物或免疫补充剂的临床意义。 © 2023年。中华中西医结合杂志社和施普林格出版社德国的一部分。

To investigate the in vivo immunomodulatory and anti-tumor mechanisms of the combined treatment of novel Four-Herb formula (4HF) and doxorubicin in triple-negative breast cancer (TNBC).Murine-derived triple-negative mammary carcinoma cell line, 4T1 cells, was cultured and inoculated into mouse mammary glands. Sixty-six mice were randomly assigned into 6 groups (n=11 in ench): naïve, control, LD 4HF (low dose 4HF), HD 4HF (high dose 4HF), LD 4HF + D (low dose and doxorubicin), and D (doxorubicin). Apart from the naïve group, each mouse received subcutaneous inoculation with 5 × 105 4T1 cells resuspended in 100 µL of normal saline in the mammary fat pads. Starting from the day of tumor cell inoculation, tumors were grown for 6 days. The LD and HD groups received daily oral gavage of 658 and 2,630 mg/kg 4HF, respectively. The LD 4HF+D group received daily oral gavage of 658 mg/kg 4HF and weekly intraperitoneal injection of doxorubicin (5 mg/kg). The D group received weekly intraperitoneal injections of doxorubicin (5 mg/kg). The treatment naïve mice received daily oral gavage of 0.2 mL double distilled water and 0.1 mL normal saline via intraperitoneal injection once a week. The control group received daily oral gavage of 0.2 mL double-distilled water. The treatment period was 30 days. At the end of treatment, mice organs were harvested to analyze immunological activities via immunophenotyping, gene and multiplex analysis, histological staining, and gut microbiota analysis.Mice treated with the combination of 4HF and doxorubicin resulted in significantly reduced tumor and spleen burdens (P<0.05), altered the hypoxia and overall immune lymphocyte landscape, and manipulated gut microbiota to favor the anti-tumor immunological activities. Moreover, immunosuppressive genes, cytokines, and chemokines such as C-C motif chemokine 2 and interleukin-10 of tumors were significantly downregulated (P<0.05). 4HF-doxorubicin combination treatment demonstrated synergetic activities and was most effective in activating the anti-tumor immune response (P<0.05).The above results provide evidence for evaluating the immune regulating mechanisms of 4HF in breast cancer and support its clinical significance in its potential as an adjunctive therapeutic agent or immune supplement.© 2023. The Chinese Journal of Integrated Traditional and Western Medicine Press and Springer-Verlag GmbH Germany, part of Springer Nature.