Receptor-interacting protein 3-phosphorylated Ca2+/calmodulin-dependent protein kinase II and mixed lineage kinase domain-like protein mediate intracerebral hemorrhage-induced neuronal necroptosis

Necroptosis-mediated cell death is an important mechanism in intracerebral hemorrhage (ICH)-induced secondary brain injury (SBI). Our previous study has demonstrated that receptor-interacting protein 1 (RIP1) mediated necroptosis in SBI after ICH. However, further mechanisms, such as the roles of receptor-interacting protein 3 (RIP3), mixed lineage kinase domain-like protein (MLKL), and Ca²⁺/calmodulin-dependent protein kinase II (CaMK II), remain unclear. We hypothesized that RIP3, MLKL, and CaMK II might participate in necroptosis after ICH, including their phosphorylation. The ICH model was induced by autologous blood injection. First, we found the activation of necroptosis after ICH in brain tissues surrounding the hematoma (propidium iodide staining). Meanwhile, the phosphorylation and expression of RIP3, MLKL, and CaMK II were differently up-regulated (western blotting and immunofluorescent staining). The specific inhibitors could suppress RIP3, MLKL, and CaMK II (GSK'872 for RIP3, necrosulfonamide for MLKL, and KN-93 for CaMK II). We found the necroptosis surrounding the hematoma and the concrete interactions in RIP3-MLKL/RIP3-CaMK II also both decreased after the specific intervention (co-immunoprecipitation). Then we conducted the short-/long-term neurobehavioral tests, and the rats with specific inhibition mostly had better performance. We also found less blood–brain barrier (BBB) injury, and less neuron loss (Nissl staining) in intervention groups, which supported the neurobehavioral tests. Besides, oxidative stress and inflammation were also alleviated with intervention, which had significant less reactive oxygen species (ROS), tumor necrosis factor (TNF)-α, lactate dehydrogenase (LDH), Iba1, and GFAP surrounding the hematoma. These results confirmed that RIP3-phosphorylated MLKL and CaMK II participate in ICH-induced necroptosis and could provide potential targets for the treatment of ICH patients.