Effect of moxibustion combined with chemotherapy on immune checkpoints in tumor tissue of breast cancer-bearing mice

To observe the effects of moxibustion combined with chemotherapy on immune checkpoints including programmed cell death protein 1 (PD-1), T cell immunoglobulin domain and mucin domain (TIM-3) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) in the tumor tissue of breast cancer-bearing mice, so as to explore the effect and mechanisms of moxibustion combined with chemotherapy on breast cancer.

Forty BALB/c female mice were randomly divided into model, chemotherapy, moxibustion and combination groups, with 10 mice in each group. 4T1 tumor cells were inoculated into the fat pad under the left fourth nipple of the mice to establish the breast cancer-bearing mice model. The chemotherapy group was injected intraperitoneally with doxorubicin (DOX) solution at 2.5 mg/kg once every 3 days; the moxibustion group received bilateral “Zusanli” (ST36) moxibustion with 2 moxa cones per point, treated every 2 days; the combination group received both DOX injection and moxibustion intervention. The above interventions lasted for 21 days. The body weight and tumor volume of the mice were recorded daily. HE staining was used to observe tumor tissue pathological morphology. Immunohistochemistry and Western blot were used to detect the positive expression and protein expression levels of PD-1, TIM-3, and CTLA-4 in the tumor tissue.

Compared with the model group after intervention, the body weight was increased (P<0.01, P<0.05), the tumor volume was decreased (P<0.01), the pathological morphology of tumor tissue showed varying degrees of tumor cells degeneration, and the positive expression and protein expression levels of PD-1, TIM-3, and CTLA-4 in tumor tissue were decreased (P<0.01, P<0.05) of mice in the chemotherapy, moxibustion and combination groups.Compared with the chemotherapy group, the body weight of mice in the moxibustion group was higher (P<0.01); the tumor volume of mice in the combination group was smaller (P<0.01); the positive expression and protein expression levels of PD-1, TIM-3, and CTLA-4 were further reduced (P<0.01, P<0.05) of mice in the moxibustion and combination groups. Compared with the moxibustion group, the body weight was decreased (P<0.01), the tumor volume was smaller (P<0.01), the degree of tumor cell degeneration was higher, and the positive expression and protein expression levels of PD-1, TIM-3, and CTLA-4 were decreased (P<0.01) of mice in the combination group.

Moxibustion combined with chemotherapy shows more significant effects in inhibiting tumor growth in breast cancer-bearing mice compared to moxibustion or chemotherapy alone. It reduces the expression of PD-1, TIM-3, and CTLA-4 in tumor tissues, suggesting that lowering immune checkpoint expression levels may be one of the mechanisms by which moxibustion combined with chemotherapy drugs treats tumors.