Ginsenoside Rh3 induces barrier-associated injury in an in vitro blood-brain barrier model with involvement of MMP2/9.

The blood-brain barrier (BBB) is essential for maintaining central nervous system homeostasis, and its disruption is associated with numerous neurological disorders. Ginsenoside Rh3 (GRh3), a bioactive saponin with recognized anticancer properties, has previously been shown to exert cytotoxic effects on neural cells, raising concerns regarding its neurovascular safety. In this study, we evaluated the effects of GRh3 in an in vitro co-culture transwell BBB model comprising mouse-derived bEnd.3 endothelial cells, pericytes, and C8-D1A astrocytic cells. Exposure to GRh3 (0.4-1.6 μM) for 24 h reduced transendothelial electrical resistance (TEER), increased extracellular lactate dehydrogenase (LDH) release, decreased bEnd.3 cell viability, and reduced the expression of the tight junction (TJ) proteins ZO-1, Claudin-5, and Occludin. These alterations were accompanied by increased MMP2 and MMP9 levels and decreased TIMP1 expression. Molecular docking suggested potential interactions between GRh3 and MMP2/9. Moreover, siRNA-mediated knockdown of MMP2 or MMP9 partially restored TEER, reduced GRh3-associated LDH release, and attenuated the loss of TJ proteins. Collectively, these findings indicate that GRh3 impairs barrier-associated properties, at least in part through an MMP2/9-mediated mechanism, raising concerns regarding its neurovascular safety. However, because the barrier-associated changes occurred concurrently with detectable cytotoxicity, a direct barrier-specific effect cannot be distinguished from secondary consequences of cellular injury or cell loss. Further studies using minimally cytotoxic exposure conditions and in vivo models are required to establish the neurovascular relevance of these findings.