Neuroprotective effects of Rosavin via HIF-1α signaling in a rat model of ischemic stroke.

BACKGROUND: Ischemic stroke is a common cerebrovascular disease and a major global cause of permanent disability and death. Angiogenesis plays a critical role in neurological recovery during cerebral ischemia. Rosavin is the main active ingredient of Rhodiola rosea for ischemic stroke treatment.

PURPOSE: The study investigates the neuroprotective effects of Rosavin on ischemic stroke rats through activating HIF-1α signaling.

METHODS: In vivo studies, MCAO/IR model was employed and treatment with Rosavin, and the concentration of Rosavin in brain tissue was determined using LC-MS/MS at different time points. After 7 days of treatment, mNSS were used to assess the degree of neurological deficits in rats for evaluating the neuroprotective effects of Rosavin. MRI was used to measure cerebral infarct volume, and the Morris water maze was employed to evaluate spatial learning and memory abilities. IL-1β, TNF-α, and IL-6 mRNA was analyzed by RT-qPCR, NeuN, Bax and Bcl-2 was detected by Western blotting. In vitro studies, the effects of Rosavin on angiogenesis were assessed using OGD/R-induced HUVEC model. Moreover, Network pharmacology was used to predict the potential mechanisms of Rosavin. To investigate Rosavin protects against MCAO/IR-induced neurological damage via HIF-1α signaling, rats were treated with the HIF-1α inhibitor YC-1. After 7 days, Western blotting and RT-qPCR were used to detect the expression levels of NeuN, HIF-1α, VEGFA, and EPO in the ischemic cortical brain tissue. Immunofluorescence staining was performed to evaluate the expression of HIF-1α, VEGFA, CD31, and NeuN.

RESULTS: Rosavin (ip.) was detectable in the rat brain, with peak concentration achieved at 20 min post-administration. Rosavin significantly reduced cerebral infarct volume and mNSS scores in MCAO/IR rats. Additionally, Rosavin decreased the escape latency and increased the number of platform crossings in the Morris water maze test. In ischemic brain tissue, Rosavin reduced the number of apoptotic cells and Bax expression while increasing Bcl-2, NeuN, and CD31. Network pharmacology analysis identified 174 potential therapeutic targets of Rosavin for ischemic stroke, with enrichment analysis suggesting that it activated HIF-1α signaling. Rosavin upregulated HIF-1α protein and nuclear localization, and VEGF and EPO in the ischemic cortical brain tissue. In vitro studies showed that Rosavin inhibited OGD/R-induced tube formation in HUVECs and increased CD31, VEGFA, and EPO. These effects of Rosavin were abolished by HIF-1α inhibitor YC-1. Rosavin's interaction with the VHL protein instead of PHD2. Expression of HIF-1α and CD31 increased by Rosavin, which were failed after siVHL.

CONCLUSION: Rosavin can penetrate the blood-brain barrier, improve neurological deficits, reduce cerebral infarct volume, and enhance spatial learning and memory abilities in MCAO/IR rats, thereby exerting neuroprotective effects. The underlying mechanism involves stabilizing HIF-1α protein through inhibiting VHL, increasing its nuclear translocation, which upregulates the expression of its target genes VEGF and EPO.