Conditioned medium from Sanhuang Decoction-pretreated MDA-MB-231 cells attenuates endothelial inflammation and barrier dysfunction

Background Tumor-cell-derived inflammatory signals can alter endothelial activation and barrier integrity in triple-negative breast cancer. Whether pretreating tumor cells with Sanhuang Decoction (SHD) changes the subsequent conditioned-medium (CM)-associated endothelial phenotype remains unclear. Methods MDA-MB-231 cells were exposed to SHD, washed twice with phosphate-buffered saline, and cultured in fresh drug-free medium before CM collection. Tumor-cell migration, CXCL8 mRNA, and viable donor-cell-normalized CXCL8 secretion were assessed, with selected CCK-8 and CXCL8 validation in MDA-MB-468, BT-549, and Hs578T cells. Human umbilical vein endothelial cells (HUVECs) were exposed to Control-CM, 231-CM, or CM from SHD-pretreated MDA-MB-231 cells and evaluated by RNA sequencing, RT-qPCR, western blotting, immunofluorescence, FITC-dextran permeability, CellTracker-based transendothelial migration, and representative transmission electron microscopy. A direct-SHD HUVEC western blot control and the PI3K agonist 740 Y-P were used to examine direct-exposure effects and pathway participation. Public-data, LC-HRMS/MS profiling, HPLC-DAD emodin marker analysis, network, and enrichment analyses provided study context. Results SHD reduced MDA-MB-231 migration, decreased CXCL8 mRNA across the TNBC validation panel, and reduced viable donor-cell-normalized CXCL8 secretion in all four TNBC cell lines, most consistently at 6 mg/mL. In HUVECs, SHD6-CM was associated with opposite-direction changes in a subset of 231-CM-associated transcriptomic features involving cytoskeletal, vascular-wall interaction, PI3K/AKT, and junction-related programs. It reduced VCAM-1, ICAM-1, and PI3K/AKT phosphorylation while increasing ZO-1, VE-cadherin, and Occludin-related readouts. Direct SHD exposure did not reproduce this coordinated HUVEC protein pattern. SHD6-CM was also associated with lower FITC-dextran permeability and fewer CellTracker Green-positive MDA-MB-231 cells crossing the endothelial monolayer. Addition of 740 Y-P attenuated these molecular and functional changes, consistent with partial PI3K/AKT participation. Conclusion CM from SHD-pretreated MDA-MB-231 cells was associated with reduced endothelial inflammatory activation and improved barrier-related readouts in this in vitro model. CXCL8 remains an associated candidate mediator within a multicomponent secretory response, rather than an established sole mediator, and pathway-specific experiments are needed to define the contribution of PI3K/AKT.