The <scp>CCL11</scp> ‐ <scp>CCR3</scp> Axis Regulates the Aggravation of Neuroinflammation in Astrocytic Necroptosis After Cerebral Ischemia

In ischemic stroke (IS), the programmed death of neurons is a key factor contributing to poor prognosis and neurological dysfunction. Recently, it has been discovered that necroptosis, as a form of programmed cell death, promotes neuroinflammatory damage after IS; however, its mechanism is still unclear. Recent studies have shown that CCL11, as an eosinophil chemokine, may induce neuronal cytotoxicity by triggering the production of reactive oxygen species (ROS) in microglia. Meanwhile, CCR3, serving as the major functional membrane receptor target of CCL11, plays a crucial role in transducing CCL11 signals. This study establishes a mouse model of IS using permanent middle cerebral artery ligation (pMCAL) and utilizes methods such as Western blot (WB), quantitative polymerase chain reaction (qPCR), and immunofluorescence (IF) to detect changes in necroptosis markers and chemokines. The aim was to analyze the dynamic progression of necroptosis after stroke and its potential regulatory mechanisms. In in vivo experiments, the recovery levels of necroptosis and neuroinflammation were observed by administering a C-C motif chemokine receptor 3 (CCR3) inhibitor via cerebral stereotaxic injection. In vitro experiments utilized an oxygen-glucose deprivation (OGD) model to simulate the in vivo ischemic state of astrocytes. Exogenous CCL11 was administered, and the expression of CCR3 on astrocytes was inhibited to observe changes in necroptosis and apoptosis markers, including receptor-interacting protein kinase 1 (RIPK1), receptor-interacting protein kinase 1 (RIPK3), and mixed lineage kinase domain-like protein (MLKL), as well as inflammatory factors interleukin-1 beta (IL-1β), NOD-like receptor thermal protein domain-associated protein 3 (NLRP3), and apoptosis-associated speck-like protein containing a CARD(ASC). The results indicated that CCL11 stimulation significantly activated necroptosis and apoptosis in astrocytes, while blocking the CCR3 receptor on astrocytes resulted in a significant inhibition of this process. By collecting supernatants from various groups of astrocytes and stimulating neurons, it was further observed that necroptosis, induced by CCL11-CCR3 stimulation, significantly disrupts synaptic connections in neurons. We conclude that the CCL11-CCR3 axis plays a key role in the necroptosis of astrocytes after IS.