Background <p>Diabetic retinopathy (DR) is a leading cause of blindness. While anti-vascular endothelial growth factor (VEGF) therapy is effective, its utility is limited by variable patient response and the need for frequent injections. Therefore, identifying new therapeutic targets for DR is imperative. Emerging evidence indicates that astrocytes contribute to endothelial dysfunction in DR, suggesting that targeting astrocyte-endothelial cell crosstalk represents a promising therapeutic strategy.</p> Purpose <p>To evaluate the therapeutic potential of Salvianolic acid A (Sal A) for DR, elucidate the molecular mechanisms by which it modulates astrocyte-endothelial cell interactions, and develop a liposome-based nanodelivery system to enhance its efficacy.</p> Study Design <p>The protective effects and mechanisms of Sal A were systematically investigated using a streptozotocin (STZ)-induced diabetic mouse model, complemented by a suite of in vitro and molecular approaches including co-culture models, transcriptomic analysis, and target validation assays.</p> Methods <p>Retinal vascular structure and barrier function were assessed in vivo via immunofluorescence staining and Evans Blue leakage assays. Endothelial cell behaviors were examined in vitro using wound healing, Transwell migration, tube formation, and spheroid sprouting assays. Transcriptomic profiling was performed by RNA sequencing (RNA-seq). The direct target of Sal A was identified and validated using MS-based drug-affinity responsive target stability (DARTS) screening, cellular thermal shift assay (CETSA), and microscale thermophoresis (MST). Expression of key signaling molecules was measured by western blotting, enzyme-linked immunosorbent assay (ELISA), and quantitative real-time PCR (qRT-PCR). Liposome@Sal A was prepared and characterized for its physicochemical properties (dynamic light scattering, transmission electron microscopy), stability, and therapeutic efficacy in vitro and in vivo.</p> Results <p>Sal A treatment ameliorated retinal vascular abnormalities in diabetic mice, evidenced by increased VE-cadherin and NG2 expression, decreased α-smooth muscle actin (α-SMA) expression, and reduced acellular capillary formation, collectively restoring vascular integrity. Mechanistically, astrocyte-derived soluble Semaphorin 4D (sSema4D) promoted endothelial hyperactivation via the PlexinB1/RhoA/ROCK/pMLC2 signaling cascade. Sal A directly bound to the Arg92 residue of membrane-bound Sema4D on astrocytes, significantly inhibiting sSema4D shedding and its subsequent deleterious effects on endothelial cells. Furthermore, Liposome@Sal A enhanced retinal drug delivery and demonstrated superior therapeutic efficacy compared to free Sal A in diabetic mice.</p> Conclusion <p>Sal A preserves retinal vascular structure and function in DR by binding to astrocytic Sema4D at Arg92, thereby inhibiting sSema4D shedding and downstream PlexinB1/RhoA/ROCK/pMLC2 signaling, which modulates astrocyte-endothelial cell crosstalk. Liposomal encapsulation significantly potentiates the therapeutic efficacy of Sal A, positioning it as a promising drug candidate for DR treatment.</p>