BACKGROUND:The increased transcytosis in endothelial cells (EC) represents a significant secondary pathological event that results in BBB disruption following cerebral ischemia-reperfusion injury (CIRI). The Wnt/β-catenin pathway has been recognized for its vital role in maintaining BBB integrity, but its impact on transcytosis in brain ECs following CIRI remains unclear. Prior research has demonstrated that Fuzheng Jiedu Tongluo Granule (FZJDTL) was effective in treating post-stroke damage. Nonetheless, the precise mechanism underlying FZJDTL's protective effects on the BBB remains ambiguous and necessitates further exploration.
PURPOSE:This research sought to examine how FZJDTL protected the BBB in the aftermath of CIRI, and to clarify that the key mechanism pathway was via Wnt/ β-catenin/Mfsd2a/Cav-1 axis regulating endothelium transcytosis and maintaining BBB integrity.
METHODS:The chemical profile of FZJDTL was carried out through LC-MS. The potential protective effect mechanisms of FZJDTL on CIRI were first elucidated by an integrated approach combining transcriptomics analysis and network pharmacology. Molecular docking analysis was performed to investigate the interactions between the active ingredients of FZJDTL and the proteins Wnt3a, β-catenin, Mfsd2a, and Cav-1. Surface plasmon resonance (SPR) was further employed to validate the binding capacity. In vivo experiments were conducted using a MCAO/R rat model to assess the effects of FZJDTL on CIRI. TTC and HE staining were conducted to assess pathological injury. The function of BBB was evaluated employing Evans blue (EB) penetration, and the number of Cav-1 mediated transcytosis vesicles in murine cerebral capillary ECs was quantified by TEM. Immunofluorescent staining was employed to observe Cav-1 expression in brain. The mRNA and protein expression levels of Wnt3a, β-catenin, Mfsd2a, Cav-1, and claudin-5 were determined by RT-qPCR and Western Blotting analysis. Furthermore, a mouse brain microvascular endothelial cell (bEnd.3) model subjected to oxygen-glucose deprivation/ reoxygenation (OGD/R) was established in the in vitro experiment. The efficacy of FZJDTL for providing protection was assessed through the evaluation of cell viability using the CCK8 assay, measuring BBB integrity through transepithelial electrical resistance (TEER) value and sodium fluorescein (SF) leakage, and analyzing claudin-5 protein expression.
RESULTS:Eighty-four components were identified in FZJDTL. Comprehensive network pharmacology and transcriptomics analysis hinted that FZJDTL alleviated CIRI by inhibiting transcytosis vesicles via triggering the Wnt/β-catenin signaling pathway. Given that activation of the vesicle inhibitory protein Mfsd2a downregulates the expression of the vesicle transport protein Cav-1 via the Wnt/β-catenin signaling pathway, it was prompted the hypothesis that FZJDTL inhibited transcytosis through Wnt/β-catenin modulation, thereby alleviating CIRI. The molecular docking analysis indicated that lithospermic acid B, luteolin, adenosine, hydroxysafflor yellow A, paeonol, and formononetin may serve as potential key active ingredients interacting with the proteins Wnt3a, β-catenin, Mfsd2a, and Cav-1. The SPR results confirmed that Wnt3a bound to lithospermic acid B, paeonol, formonoetin, luteolin, adenosine, and hydroxysafflor yellow A. In the in vivo experiments, treatment with FZJDTL dose-dependently reduced neurological function score and cerebral infarct volume, it also ameliorated cerebral histopathological injury. TEM results revealed that FZJDTL inhibited the increase of transcytosis vesicles in MCAO/R rats. The therapeutic effect of FZJDTL on post-CIRI by mitigating BBB dysfunction as demonstrated by a reduction in macromolecule (EB and albumin) leakage in MCAO/R rats, as well as proved by increased TEER value and reduced macromolecule SF leakage after OGD/R in bEend.3 cells. FZJDTL alleviated MCAO/R- or OGD/R-induced BBB damage via upregulation of the Wnt/β-catenin/Mfsd2a signaling pathway, which subsequently suppressed downstream Cav-1 expression, reduced Cav-1-mediated transcytosis vesicles, and increased expression of claudin-5, a tight junction protein critical to the structural integrity of the BBB.
CONCLUSIONS:FZJDTL alleviated CIRI by modulating the Wnt/β-catenin signal pathway to promote Mfsd2a expression, inhibit the expression of Cav-1, further impeding vascular endothelial transcytosis, and ultimately protecting the function of the BBB.