目的 探究二至丸调控肿瘤细胞-成骨细胞黏附干预乳腺癌骨内生长的机制。方法 采用MTT法、细胞划痕实验和结晶紫染色考察二至丸（19.53～5 000.00 μg·mL^-1）对4T1细胞活力、迁移及增殖的影响。将4T1-Luc细胞接种于小鼠胫骨构建乳腺癌骨内生长模型，通过生物发光成像、活体及离体Micro-CT、生存期和脏器指数评价二至丸（10、5 g·kg^-1）的体内药效及安全性，免疫组织化学检测荷瘤胫骨病灶组织中压电型机械敏感离子通道组件1（Piezo-1）和神经型钙黏蛋白（N-cdh）的表达。采用Fluo-4 AM探针检测4T1细胞内Ca²⁺水平，Western blotting检测Piezo-1、磷酸化钙调蛋白依赖性蛋白激酶Ⅱ（p-CaMKII）、磷酸化环磷酸腺苷反应元件结合蛋白（p-CREB）及N-cdh蛋白的表达。构建4T1细胞与成骨细胞直接接触共培养模型，通过细胞黏附、免疫荧光共定位和阻断实验评价N-cdh/钙黏蛋白11（Cdh-11）介导的细胞间黏附，并采用原子力显微镜（AFM）单细胞力谱技术测定两类细胞间的黏附力和黏附功。结果 与对照组比较，二至丸可降低4T1细胞活力，抑制其迁移和增殖（P＜0.01、0.001）。体内实验表明，与模型组比较，二至丸干预后小鼠胫骨局部生物发光信号减弱，骨内肿瘤负荷降低，活体及离体骨CT显示骨组织破坏趋势减轻，免疫组织化学结果进一步显示，二至丸可降低荷瘤胫骨病灶组织中Piezo-1和N-cdh的阳性表达（P＜0.01、0.001）；小鼠生存时间延长，且心、肾、脾、肝等脏器指数未见明显异常。细胞分子机制研究表明，二至丸下调4T1细胞中Piezo-1的表达，减少细胞内Ca²⁺水平，降低p-CaMKII和p-CREB表达，并降低N-cdh的表达（P＜0.05、0.01、0.001）。细胞黏附实验、共定位分析、阻断实验表明，二至丸可削弱N-cdh/Cdh-11介导的肿瘤细胞与成骨细胞黏附（P＜0.01、0.001）； AFM单细胞力谱技术进一步证实，二至丸降低两类细胞间黏附力和黏附功（P＜0.001）。结论 二至丸可抑制4T1乳腺癌细胞活力、迁移和增殖，降低乳腺癌胫骨内肿瘤负荷并减轻骨破坏，上述作用可能与Piezo-1/Ca²⁺/CaMKII/CREB/N-cdh相关信号变化及N-cdh/Cdh-11介导的肿瘤细胞-成骨细胞黏附减弱有关。

Objective To investigate how Erzhi Pills inhibits breast cancer growth in bone by regulating adhesion between tumor cells and osteoblasts. Methods MTT assay, wound-healing assay, and crystal violet staining were used to evaluate the effects of Erzhi Pills (19.53—5 000.00 μg·mL^-1) on the viability, migration, and proliferation of 4T1 cells. A mouse model of breast cancer growth in bone was established by injecting 4T1-Luc cells into the tibia. Bioluminescence imaging, in vivo and ex vivo micro-computed tomography (micro-CT), survival analysis, and organ index measurements were used to evaluate the efficacy and safety of Erzhi Pills (10, and 5 g·kg^-1) in vivo. Immunohistochemistry was used to examine the expression of piezo-type mechanosensitive ion channel component 1 (Piezo-1) and N-cadherin (N-cdh) in tibial tumor lesions. Intracellular Ca²⁺ levels were measured using the Fluo-4 AM probe. Western blotting was used to detect the protein levels of Piezo-1, phosphorylated calcium/calmodulin-dependent protein kinase II (p-CaMKII), phosphorylated cAMP response element-binding protein (p-CREB), and N-cdh. A direct-contact coculture model of 4T1 cells and osteoblasts was established. Cell adhesion assays, immunofluorescence colocalization, and blocking experiments were used to evaluate cell adhesion mediated by N-cdh/cadherin-11 (Cdh-11). Atomic force microscopy (AFM)-based single-cell force spectroscopy was used to measure the adhesion force and adhesion work between the two cell types. Results Erzhi Pills reduced 4T1 cell viability and inhibited cell migration and proliferation (P < 0.01, 0.001). In vivo, Erzhi Pills reduced the bioluminescence signal in the tibia and decreased the tumor burden in bone. In vivo and ex vivo micro-CT showed a trend toward reduced bone damage. Immunohistochemistry further showed that Erzhi Pills reduced Piezo-1 and N-cdh expression in tibial tumor lesions (P < 0.01, 0.001). Erzhi Pills prolonged mouse survival, while the heart, kidney, spleen, and liver indices showed no obvious abnormalities. Mechanistic experiments showed that Erzhi Pills reduced Piezo-1 expression and intracellular Ca²⁺ levels and decreased p-CaMKII, p-CREB, and N-cdh protein levels in 4T1 cells (P < 0.05, 0.01, 0.001). Cell adhesion, colocalization, and blocking experiments showed that Erzhi Pills weakened N-cdh/Cdh-11-mediated adhesion between tumor cells and osteoblasts (P < 0.01, 0.001). AFM-based single-cell force spectroscopy further confirmed that Erzhi Pills reduced the adhesion force and adhesion work between the two cell types (P < 0.001). Conclusion Erzhi Pills inhibits the viability, migration, and proliferation of 4T1 breast cancer cells, reduces breast cancer growth in the tibia, and alleviates bone damage. These effects may be associated with alterations in Piezo-1/Ca²⁺/CaMKII/CREB/N-cdh-related signaling and weakened N-cdh/Cdh-11-mediated adhesion between tumor cells and osteoblasts.

R965

山东省中医药科技项目（M20251207，M20251705）；山东省自然科学基金资助项目（ZR2025MS1285）