Dihydroartemisinin promotes neurological rehabilitation after ischemic stroke by targeting TAK1-CREB1 signaling pathway to inhibit ferroptosis

Dihydroartemisinin (DHA) dose-dependently ameliorates ischemic brain injury and promotes neurological recovery in mice after ischemic stroke. • DHA inhibits ferroptosis in ischemic brain tissue via regulating iron homeostasis, lipid peroxidation and core ferroptosis-related proteins. • Astrocytic TAK1-CREB1 signaling pathway is identified as the key molecular target mediating the neuroprotective effects of DHA. • This study provides a promising drug repurposing candidate and novel therapeutic target for the clinical treatment of ischemic stroke. Background Ischemic stroke accounts for 87% of all stroke cases, and remains a leading cause of mortality and disability worldwide. Current reperfusion therapies are limited by narrow therapeutic windows, and cerebral ischemia-reperfusion injury (CIRI) remains a major clinical challenge. Ferroptosis, an iron-dependent form of regulated cell death characterized by lethal lipid peroxidation, has been identified as a key pathological driver of CIRI. Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, has shown neuroprotective effects in ischemic stroke, but its underlying mechanism, especially the regulatory effect on ferroptosis via the TAK1-CREB1 signaling axis, remains unclear. Objective To investigate the neuroprotective effect of DHA in ischemic stroke and its molecular mechanism related to the TAK1-CREB1 signaling pathway and ferroptosis regulation. Methods Wild-type (WT) C57BL/6 J mice and astrocyte-specific CREB1 conditional knockout (CREB1 CKO) mice were used to establish a photothrombotic stroke (PTs) model. Neurological function was evaluated by modified neurological severity score (mNSS), Bederson score, Garcia scale and hanging test. Cerebral infarct volume was measured by TTC staining. Cerebral blood flow (CBF) was detected by laser speckle contrast imaging. Histopathological changes were observed by HE and Nissl staining. Mitochondrial ultrastructure was examined by transmission electron microscopy (TEM). The levels of malondialdehyde (MDA), glutathione (GSH), NADPH/NADP + ratio, ferrous iron (Fe 2+ ) and total iron in brain tissue were detected by biochemical kits. The expression of ferroptosis-related proteins and CREB1 was detected by Western blot and immunofluorescence staining. Results DHA treatment dose-dependently improved neurological function, reduced infarct volume, restored CBF, and ameliorated histopathological damage in PTs mice. TEM revealed that DHA reversed ferroptosis-typical mitochondrial changes in ischemic brain tissue. Biochemically, DHA decreased MDA, Fe²⁺, and total iron, while increasing GSH and NADPH/NADP⁺ ratio. Mechanistically, DHA dose-dependently upregulated GPX4, xCT, and FTH1, and downregulated ACSL4. Notably, astrocyte-specific CREB1 knockout nearly completely abolished DHA's neuroprotective and anti-ferroptosis effects. Conclusion DHA promotes neurological rehabilitation after ischemic stroke by targeting the astrocytic TAK1-CREB1 signaling pathway to inhibit ferroptosis. This study provides a novel theoretical basis and promising candidate drug for the clinical treatment of ischemic stroke.