To explore whether receptor interacting protein kinase 1 (RIP1)/RIP3 pathways participate in hemin induced cell death in HT-22 hippocampal neuronal cells and investigate the potential neuroprotection of necrostatin-1 in hemin induced cell death in HT-22.

First, different concentrations of hemin (0, 25, 50, 100 μmol/L) were added to HT-22 cells. Propidium iodide (PI) positive cells and cell viability were measured at 24 h after hemin treatment. Then, necrostatin-1, zVAD and reactive oxygen species (ROS) scavenger butylated hydroxyanisole (BHA) were applied to hemin-treated HT-22 cells. PI⁺ cells and cell viability were measured at 24 h after hemin treatment. MitoSox Red was used to indicate ROS level. Last, the effect of RIP3 in hemin induced HT-22 cell death was explored through RIP3 knockdown using siRNA. PI⁺ cells, cell viability and ROS lever were measured at 24 h after hemin treatment.

Hemin could induce a dose dependent cell death in HT22 neural cells. RIP1 specific inhibitor necrostatin-1 significantly inhibited cell death induced by hemin in HT-22 cells, greatly reducing PI⁺ cells, dramatically improving cell viability and decreasing ROS accumulation. BHA could significantly inhibit PI⁺ cells induced by hemin in HT-22 cells. Furthermore, silencing of RIP3 using siRNA attenuated hemin induced cell death in HT-22 cells, greatly reducing PI⁺ cells, dramatically improving cell viability and decreasing ROS accumulation.

These data revealed that RIP1/ RIP3 pathway and ROS might mediate hemin induced cell death in HT-22 hippocampus neural cells, and necrostatin-1 played a neuroprotection role in hemin induced cell death in HT-22.