Electroacupuncture stimulation at CV4 sites prevents breast cancer-induced osteoporosis by activating the Wnt/β-catenin signaling pathway

Osteoporosis is a common condition among breast cancer patients, characterized by decreased bone density due to chemotherapy and hormone therapy. However, the application of electroacupuncture (EA) stimulation at Guanyuan (CV4) sites has been found to be beneficial in enhancing bone mineral density. In this study, we aimed to investigate the efficacy of EA stimulation at CV4 sites in breast cancer-induced osteoporosis and elucidate the potential underlying mechanisms. A breast cancer model in mice was established, EA stimulation at CV4 sites was performed, and hematoxylin-eosin (HE) stain was conducted to assess the bone mineral density and trabecular bone volume of femur tissues. Additionally, Tartrate-resistant acid phosphatase (TRAP) staining was performed to identify and visualize osteoclasts. Furthermore, Western blots were used to evaluate the expression levels of various proteins, including c-src, Cathepsin K (CtSK), matrix metalloproteinase 9 (MMP9), Collagen 1A1 (COL1A1), Osteopontin (OPN), Runt-related transcription factor 2 (RUNX2), Wnt3a and β-catenin, and Enzyme Linked Immunosorbent Assay (ELISA) to measure the concentration of Alkaline phosphatase (ALP), NO synthase 2 (NOS2), glutathione reductase (GSR), superoxide dismutase (SOD), reactive oxygen species (ROS) and lactate dehydrogenase (LDH) in the serum. The results revealed that EA stimulation in the breast cancer model resulted in higher bone mineral density and trabecular bone volume, along with a reduction in the number of osteoclasts. EA stimulation at CV4 site significantly decreased the protein levels of c-src, CtSK and MMP9, while also reducing ROS and LDH production. Conversely, it promoted the expression of COL1A1, OPN, RUNX2, Wnt3a and β-catenin. Additionally, it elevated the concentration of ALP, NOS2, GSR and SOD in the serum. Overall, EA stimulation at the CV4 site may inhibit bone loss and promote bone differentiation by activating the Wnt/β-catenin pathway, indicating its potential use for breast cancer-induced osteoporosis treatment.

Breast cancer; CV4; Electroacupuncture stimulation; Osteoporosis; Wnt/β-catenin

[1] Yu L, Dong L, Wang Y, Liu L, Long H, Li H, et al. Reversible regulation of SATB1 ubiquitination by USP47 and SMURF2 mediates colon cancer cell proliferation and tumor progression. Cancer Letters. 2019; 448: 40–51.

[2] Malik JA, Ahmed S, Jan B, Bender O, Al Hagbani T, Alqarni A, et al. Drugs repurposed: an advanced step towards the treatment of breast cancer and associated challenges. Biomedicine & Pharmacotherapy. 2022; 145: 112375.

[3] Arellano DL, Juárez P, Verdugo-Meza A, Almeida-Luna PS, Corral-Avila JA, Drescher F, et al. Bone microenvironment-suppressed T cells increase osteoclast formation and osteolytic bone metastases in mice. Journal of Bone and Mineral Research. 2022; 37: 1446–1463.

[4] Amorim D, Brito I, Caseiro A, Figueiredo JP, Pinto A, Macedo I, et al. Electroacupuncture and acupuncture in the treatment of anxiety—a double blinded randomized parallel clinical trial. Complementary Therapies in Clinical Practice. 2022; 46: 101541.

[5] He J, Yang L, Qing Y, He C. Effects of electroacupuncture on bone mineral density, oestradiol level and osteoprotegerin ligand expression in ovariectomised rabbits. Acupuncture in Medicine. 2014; 32: 37–42.

[6] Feng Y, Lin H, Zhang Y, Li L, Wu X, Wang T, et al. Electroacupuncture promotes insulin-like growth factors system in ovariectomized osteoporosis rats. The American Journal of Chinese Medicine. 2008; 36: 889–897.

[7] Wang H, Wang D, Li Z, Liu S, Dong J, Zhang J, et al. Electroacupuncture stimulation at BL20, BL23 and SP6 prevents hind limb unloading—induced osteoporosis in rats. Acupuncture in Medicine. 2021; 39: 656–662.

[8] Fan H, Ji F, Lin Y, Zhang M, Qin W, Zhou Q, et al. Electroacupuncture stimulation at CV4 prevents ovariectomy-induced osteoporosis in rats via Wnt-β-catenin signaling. Molecular Medicine Reports. 2016; 13: 2485–2491.

[9] Xie G, Liu W, Lian Z, Xie D, Yuan G, Ye J, et al. Spleen tyrosine kinase (SYK) inhibitor PRT062607 protects against ovariectomy-induced bone loss and breast cancer-induced bone destruction. Biochemical Pharmacology. 2021; 188: 114579.

[10] Qian C, Bian H, Jiang T. Traditional Chinese medicine for bone metastasis of breast cancer: a systematic review and meta-analysis. European Journal of Gynaecological Oncology. 2022; 43: 13–20.

[11] Zhang CQ, Shi N, Ouyang G. Effect of electroacupuncture on intestinal calcium absorption and expression of transmembrane calcium transport-related receptors in ovariectomized rats. Acupuncture Research. 2020; 45: 702–707. (In Chinese)

[12] Liu T, Jiang L, Xiang Z, Li J, Zhang Y, Xiang T, et al. Tereticornate a suppresses RANKL-induced osteoclastogenesis via the downregulation of c-Src and TRAF6 and the inhibition of RANK signaling pathways. Biomedicine & Pharmacotherapy. 2022; 151: 113140.

[13] Kozawa E, Nishida Y, Cheng XW, Urakawa H, Arai E, Futamura N, et al. Osteoarthritic change is delayed in a Ctsk-knockout mouse model of osteoarthritis. Arthritis & Rheumatism. 2012; 64: 454–464.

[14] Gavrylyak IV, Greben NK, Bilous VL, Korsa VV, Zhaboiedov DG, Ağca CA, et al. The levels of hypoxiaand angiogenesis-related regulators and matrix metalloproteinase 9 activity in tear fluid of patients with non-penetrating ocular traumas. Medical Perspectives. 2022; 27: 168–176.

[15] Tanjaya J, Ha P, Zhang Y, Wang C, Shah Y, Berthiaume E, et al. Genetic and pharmacologic suppression of PPARγ enhances NELL-1-stimulated bone regeneration. Biomaterials. 2022; 287: 121609.

[16] Pan H, Jin R, Li M, Liu Z, Xie Q, Wang P. The Effectiveness of acupuncture for osteoporosis: a systematic review and meta-analysis. The American Journal of Chinese Medicine. 2018; 46: 489–513.

[17] Jin X, Jin W, Li G, Zheng J. Zeaxanthin attenuates OVA-induced allergic asthma in mice by regulating the p38 MAPK/β-catenin signaling pathway. Allergologia et Immunopathologia. 2022; 50: 75–83. (In Spanish)

[18] Zhang YY, Li XH, Wu MX. Effect of electroacupuncture at Wnt/β-catenin signaling pathway on inhibiting cartilage degeneration in rats with knee osteoarthritis. Chinese Acupuncture & Moxibustion. 2019; 39: 1081–1086. (In Chinese)