Compound Danshen Dripping Pills retards the progression of cerebral cavernous malformations via strengthening vascular integrity and ameliorating inflammatory response.

BACKGROUND: Cerebral cavernous malformations (CCMs) are characterized by abnormal clusters of dilated, thin-walled capillaries in the brain that are prone to bleeding, which give rise to a range of neurological symptoms including seizures and stroke. Current therapeutic strategies are restricted to surgical resection, highlighting the requirement for effective and efficient treatments.

OBJECTIVE: In this study, we investigated the therapeutic potential of Compound Danshen Dripping Pills (CDDP) as a traditional Chinese medicine (TCM) against the progression of CCMs.

METHODS: Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was employed to characterize the chemical composition of CDDP and identify its brain-penetrating ingredients. Lesion burden was assessed by macroscopic observation, micro-computed tomography (microCT), and histological analysis. Vascular integrity and function were evaluated by immunofluorescence staining. Blood flow was assessed by laser speckle contrast imaging and permeability was examined using Evans blue dye and FITC-dextran. Multi-omics approaches, including RNA sequencing (RNA-seq), proteomics, and metabolomics, were conducted to decipher molecular mechanisms. Western blot and quantitative Real-time PCR (qPCR) were performed to detect key signaling pathways. Brain-penetrating components were identified by UPLC-MS/MS, followed by molecular docking and molecular dynamics simulations for target proteins (MEKK3 and NF-κB). Surface plasmon resonance (SPR) assay was performed to validate the direct binding affinity of the identified key components to their respective target proteins. The functional impacts of target binding were assessed in HEK293T cells overexpressing MEKK3 (HEK293T/MEKK3-OE) by examining the phosphorylation levels of downstream mediators. KRIT1-knockdown human cerebral microvascular endothelial cells (HCMEC/D3) stimulated by lipopolysaccharide (LPS) were treated with identified components (ginsenoside F3 and tanshinone I), and assessed for trans-endothelial electrical resistance (TEER) and expression of critical inflammatory cytokines.

RESULTS: UPLC-MS/MS identified 36 ingredients in CDDP, including phenolic acids, alkaloids, and ginsenosides. CDDP treatment dose-dependently reduced CCM lesion burden in Krit1iECKO mice, with 0.2 g/kg demonstrating optimal efficacy comparable to propranolol. Immunofluorescence revealed that CDDP significantly enhanced vascular integrity by upregulating Claudin-5 and VE-cadherin expression, increasing pericyte coverage, and normalizing basement membrane support. Functional assays demonstrated that CDDP restored cerebral blood flow and reduced vascular permeability. Integrated transcriptomics, proteomics, and metabolomics analysis revealed that CDDP significantly downregulated the MEKK3-MEK5-ERK5-KLF2/4-p-MLC2 signaling axis and suppressed inflammatory networks involving NF-κB, ICAM1, VCAM1, IL-6, IL-1β, and neutrophil extracellular traps (CitH3). Diprovocim-induced exacerbation of CCM lesions was effectively reversed by CDDP, confirming the involvement of MAPK and NF-κB pathways. Brain tissue analysis identified 11 brain-penetrating components, including salvianolic acids and ginsenosides. Molecular docking and molecular dynamics simulations revealed that ginsenoside F3 exhibited optimal binding affinity with NF-κB, while tanshinone I strongly bound to MEKK3. SPR assay further confirmed the direct binding, with ginsenoside F3 binding to NF-κB and tanshinone I binding to MEKK3. Functional validation in HEK293T/MEKK3-OE cells demonstrated that tanshinone I markedly suppressed MEKK3-driven phosphorylation of MEK5 and ERK5, while ginsenoside F3 significantly attenuated NF-κB phosphorylation. Administration of ginsenoside F3, tanshinone I, or their combination in Krit1iECKO mice led to reductions in cerebellar hemorrhagic lesions and vascular leakage, with the combination group exhibiting the most prominent therapeutic effect. In vitro validation in KRIT1-knockdown HCMEC/D3 cells demonstrated that ginsenoside F3, tanshinone I, and their combination significantly restored TEER values and reduced IL-1β and IL-6 expression, with the combination showing synergistic effects.

CONCLUSION: CDDP exerts therapeutic effects against CCM progression by strengthening vascular integrity, restoring endothelial barrier function, and suppressing inflammation through inhibition of the MEKK3-MEK5-ERK5-KLF2/4-p-MLC2 and NF-κB signaling pathways. Brain-penetrating components, particularly ginsenoside F3 and tanshinone I, directly targeted key proteins (NF-κB and MEKK3) and synergistically protected endothelial function. These findings provide preclinical evidence supporting CDDP as a promising multi-target therapeutic strategy for CCMs.