Anti-metastasis evaluation
The anti-metastatic effect of icariin was evaluated in a lung metastatic 4T1 model. A total of 1×106 eT1-Luciferase cells were injected into Balb/c mice via the tail vein to establish the pulmonary metastasis model. A total of 18 mice were equally and randomly divided into three groups (control, 5 mg/kg, and 10 mg/kg). The mice received icariin treatment 2 days after inoculation via intraperitoneal injection once daily for two weeks. At the determined time (5, 10, or 15 days after treatment), the mice were anesthetized, intraperitoneally administered with 100 μL of D-luciferin (30 mg/mL in PBS), and the bioluminescence from metastatic lung tumors was analyzed using an IVIS Lumina in vivo Imaging System (PerkinElmer, USA). At the termination of the experiment, the lungs were isolated from the euthanized mice, and the metastatic nodules were counted and fixed with paraformaldehyde for further evaluation using hematoxylin and eosin (H&E) staining.