Ciprofol protects against hypoxia/reoxygenation-induced injury in cardiac microvascular endothelial cells via the DYRK1A/PI3K/AKT signaling pathway.

OBJECTIVE: Myocardial ischemia/reperfusion (I/R) injury is a major complication following revascularization therapy for ischemic heart disease. Ciprofol (CIP) has been proven to protect cardiomyocytes from I/R-induced apoptosis, yet its role and underlying molecular mechanisms in vascular endothelial cells under hypoxia/reoxygenation (H/R) conditions remain incompletely elucidated. This study aimed to investigate the protective effects of CIP on I/R injury and its associated molecular mechanisms. METHODS: C57BL/6 J mice were subjected to myocardial I/R injury. An in vitro H/R model was established in HUVECs to mimic myocardial I/R injury. The histopathological analysis was conducted by H&E staining. After H/R stimulation and/or CIP treatment, multiple cellular biological functions and molecular markers were assessed: cell viability and proliferation were detected via CCK-8 assay and EdU staining; colony formation ability was evaluated by cell cloning assay; cell migration capacity was measured through wound-healing assay; apoptosis was analyzed using TUNEL staining and Annexin V-FITC/PI flow cytometry; DNA damage was assessed by detecting the expression of ATM, ATR, γ-H2AX, and PARP1; cell cycle progression was determined by the expression of Cyclin B and cdc2; and the intrinsic mitochondrial apoptotic pathway was examined via the expression of Bcl-2, Bax, and Caspase-3. RNA-seq and untargeted metabolomics were conducted to identify the potential therapeutic mechanisms of CIP. DYRK1A, a key candidate gene, was validated by RNA-seq, metabolomics, and qRT-PCR. Overexpression and knockdown experiments were performed to confirm the role of the DYRK1A/PI3K/AKT signaling axis in CIP-mediated protection against H/R injury. RESULTS: The levels of cardiomyocyte apoptosis and myocardial enzymes were decreased in CIP-treated mice compared with the H/R group. In H/R-injured HUVECs, CIP dose-dependently restored cell viability, enhanced EdU incorporation, and accelerated cell invasion ability. Simultaneously, CIP reduced apoptosis and suppressed activation of caspase-3. Meanwhile, CIP alleviated DNA damage responses, shown decreased levels of γ-H2AX, ATM, and ATR, and restored PARP1 expression. Mechanistically, CIP significantly modulated tyrosine metabolism and downregulated DYRK1A expression, leading to the activation of the PI3K/AKT pathway. Notably, overexpression of DYRK1A abolished the protective effects of CIP, whereas DYRK1A knockdown reversed H/R-induced cell injury and further potentiated the protective function of CIP, confirming that the PI3K/AKT signaling pathway is a critical mediator of this process. CONCLUSION: CIP promotes proliferation, migration and invasion, while inhibiting apoptosis, in H/R-injured HUVECs by regulating the DYRK1A/PI3K/AKT signaling axis. These findings highlight CIP as a promising endothelial-protective agent for the treatment of I/R-related vascular disorders.