Receptor interacting protein kinase 1 and receptor interacting protein kinases 3 mediate glutamate induced cell death in HT-22 hippocampal neuronal cells

Abstract

Objective To explore whether receptor interacting protein (RIP)1/RIP3 pathways participate in glutamate induced cell death in HT-22 neuronal cells and investigate the potential neuroprotection of necrostatin-1 in glutamate induced cell death in HT-22. Methods (1) In vitro cultured mouse hippocampal neuronal HT-22 cells were divided into control group, zVAD group, necrostatin-1 (Nec-1) group, glutamate group, glutamate+zVAD group, glutamate+zVAD+Nec-1 group and glutamate+Nec-1 group; they were treated with zVAD, Nec-1 and glutamate at the final concentrations of 20 μmol/L, 30 μmol/L and 3 mmol/L for 24 h. Cell viability was detected using a luminescence-based commercial kit Cell Titer-Glo (CTG). Necrotic cell death was measured by propidium iodide (PI) and HE stainings. (2) HT-22 cells were divided into control group I, glutamate group I and glutamate+Nec-1 group I; MitoSox Red was used to detect mitochondrial reactive oxygen species (ROS) level. (3) HT-22 cells were divided into control group II, glutamate group II and glutamate+tertiary butyl-hydroxyanisole (BHA) group; the final concentration of BHA was 100 μmol/L; necrotic cell death was measured by PI and HE stainings after 24 h of treatment. (4) HT-22 cells were divided into RIP3 siRNA and control group III, and then, they were transfected with RIP3 siRNA or negative siRNA, respectively; the RIP3 protein expression was determined by Western blotting after 72 h of treatment. (5) HT-22 cells were divided into negative siRNA+Control, RIP3 siRNA, negative siRNA+glutamate and RIP3 siRNA+glutamate groups; the cells were transfected with RIP3 siRNA or Negative siNRA, respectively; 48 h later, the glutamate groups were treated with 3 mmol/L glutamate; PI positive cells and cell viability were measured by PI and HE stainings and CTG at 24 h after glutamate treatment. Results (1) As compared with control group, percentage of PI positive cells was greatly increased and cell viability was decreased in glutamate group and glutamate+zVAD group, with statistically significant differences (P<0.05); as compared with those in the glutamate group, percentage of percentage of PI positive cells was was significantly decreased and cell viability was statistically increased in glutamate+Nec-1 group (P<0.05). (2) ROS level in HT-22 cells of the glutamate group was significantly increased than that in the control group I (P<0.05); however, ROS level in HT-22 cells of glutamate+Nec-1 group I was significantly decreased than that in glutamate group I (P<0.05). (3) Percentage of PI positive cells in the glutamate group II was significantly higher than that in the control group II (P<0.05), and that in the glutamate+BHA group was statistically lower than that in the glutamate group II (P<0.05). (4) The RIP3 protein expression in the RIP3 siRNA group was obviously down-regulated as compared with that in the control group III. (5) As compared with those in the negative siRNA group, percentage of PI positive cells was statistically increased and cell viabilities were statistically decreased in glutamate group (P<0.05); however, percentage of PI positive cells was significantly decreased and cell viability was significantly increased in RIP3 siNRA+glutamate group as compared with those in the glutamate group (P<0.05). Conclusion RIP1/RIP3 pathway and ROS might mediate glutamate induced cell death in HT-22 cells. Key words: Glutamate; Necroptosis; Necrostatin-1; Receptor interacting protein 1/3; HT-22 cell