ADR, an optimized combination of ASIV, DLA, and R1, ameliorates blood–brain barrier disruption following rtPA thrombolysis at 4.5 h after stroke onset in mice via multitarget regulation

INTRODUCTION: Administration of recombinant tissue plasminogen activator (rtPA) beyond 4.5 h after ischemic stroke exacerbates blood-brain barrier (BBB) disruption, leading to vasogenic cerebral edema and hemorrhage. However, current therapies remain ineffective. OBJECTIVE: This study aimed to develop and validate an optimized multicomponent combination, termed ADR, consisting of Astragaloside IV (ASIV), 3,4-dihydroxyphenyl lactic acid (DLA), and Notoginsenoside R1 (R1), to prevent BBB damage following rtPA thrombolysis at 4.5 h after stroke onset in mice and to explore the underlying mechanisms. METHODS: ADR was optimized using a uniform design-entropy weight-regression model. Its efficacy was assessed in mice receiving rtPA at 4.5 h after stroke onset. The pharmacokinetic (PK) and pharmacodynamic (PD) properties of the components were evaluated. Multi-omics analysis, molecular docking, surface plasmon resonance (SPR), and cellular thermal shift assays (CETSA) were performed to identify key targets, followed by functional validation through gene silencing or overexpression in vitro and in vivo. RESULTS: The optimized ADR improved cerebral blood flow, reduced infarct size and neuronal apoptosis, ameliorated neurological deficits, and enhanced survival rates. It effectively inhibited microvascular leakage, hemorrhage, and leukocyte adhesion. PK and PD studies, along with in vivo pharmacological evaluation of the individual components, demonstrated that the combination produced synergistic effects. Integrated analyses identified five key molecules. Molecular docking, SPR, and CETSA confirmed that LMO7 was exclusively modulated by ADR, whereas CAPG was regulated by both ADR and all three components. Additionally, MOBP, HMGB2, and TAGLN2 were targeted by ASIV, DLA and R1, respectively. These findings were further confirmed by silencing LMO7 or overexpressing CAPG, MOBP, HMGB2, or TAGLN2 in vitro and in vivo. CONCLUSION: Our study demonstrated that ADR prevented BBB disruption following rtPA thrombolysis in mice with ischemic stroke through multitarget regulation and provided valuable insights into the integration of Traditional Chinese Medicine and modern pharmacology.