RIP1 exacerbates BBB disruption by impairing autophagy-mediated A2 astrocyte polarization in hypertension-induced cerebral microhemorrhage in mice

Cerebral microhemorrhages (CMHs) are associated with cognitive decline and motor deficits. Inhibiting A1 astrocyte polarization can reduce brain injury and improve recovery in experimental intracerebral hemorrhage. RIP1 has been implicated in mediating neurological impairments following intracerebral hemorrhage, whereas whether it regulates astrocytic phenotypic switching to influence CMH progression remains to be investigated. Here, a mouse model of hypertension-induced CMHs was established by co-administration of Ang II and L-NAME. RIP1 was knocked down in vivo via tail vein injection of RIP1 shRNA. Following hypertension induction, daily neurological assessments showed progressively declining scores, indicating ongoing CMH development. RIP1 silencing delayed CMH onset, reduced cumulative incidence, and alleviated hypertension-induced deficits including gait abnormalities, impaired spatial learning and memory, blood-brain barrier (BBB) dysfunction, and A1 astrocyte polarization. For in vitro studies, primary mouse astrocytes were treated with hemoglobin to mimic the microhemorrhagic microenvironment, followed by RIP1 knockdown using RIP1 shRNA. RIP1 silencing attenuated hemoglobin-induced A1 polarization and facilitated a phenotypic shift from A1 to A2 in astrocytes. Furthermore, RIP1 knockdown counteracted the detrimental effects of A1-polarized astrocytes on endothelial function, as evidenced by enhanced proliferation, migration, and tube formation ability in endothelial cells. Mechanistic investigations revealed that RIP1 knockdown promoted the transition from A1 to A2 astrocytic phenotype by activating autophagy and suppressing the NF-κB-NLRP3 inflammasome pathway, thereby mitigating hypertension-induced BBB disruption following CMHs. In conclusion, RIP1 silencing alleviates BBB disruption following hypertension-induced CMHs by promoting autophagy-mediated A2 astrocyte polarization.