Hemin induces necroptosis of neurons through interleukin-1 receptor after intracerebral hemorrhage

Abstract

Objective To investigate the effect of hemin on neuronal necroptosis and its mechanism in mice after intracerebral hemorrhage. Methods (1) Experiment one: 24 C57BL/6 mice were divided into sham-operated group, saline control group, 5 mmol/L hemin group, and 10 mmol/L hemin group (n=6); mice in the latter two groups were injected 20 μL 5 mmol/L or 10 mmol/L heme solution into the corpus striatum to prepare cerebral hemorrhage models, and the normal saline group was injected 20 μL normal saline; Western blotting was used to detect the protein expressions of heme oxygenase-1 (HO-1), receptor-interacting protein (RIP)1, RIP3, and mixed lineage kinase domain like protein (MLKL). Three C57BL/6 mice were injected 20 μL 10 mmol/L heme solution into the corpus striatum to prepare cerebral hemorrhage models; 24 h after that, double-labelling immunofluorescence was used to detect the expressions of Caspases-3 and neuron-specific nuclear protein (NeuN) in the hippocampus tissues. (2) Experiment two: the primary cultured hippocampal neurons were stimulated with 0, 10, 20, 40 and 80 μmol/L heme for 6 h and 40 μmol/L heme for 0, 1, 2, 4 and 6 h, respectively; lactic dehydrogenase (LDH) method was used to determine the neuron damage. After the primary cultured hippocampal neurons being stimulated with 40 μmol/L heme for 0, 1, 2, 4 and 6 h, respectively, the protein expressions of interleukin-1 receptor (IL-1R)1, RIP1, RIP3, MLKL and caspase-3 in the neurons were detected by Western blotting, and the level of inflammatory factor interleukin-1β (IL-1β) was detected by ELISA. After primary cultured hippocampal neurons being stimulated with 0, 40 μmol/L heme for 6 h, the expressions of NeuN and Caspase-3 in the neurons were detected by double-labelling immunofluorescence. (3) Experiment three: the primary cultured hippocampal neurons were pre-incubated with 0, 0.5, 1.0, and 1.5 μg/mL IL-1R antagonist for one h, and then, stimulated with 40 μmol/L heme for 6 h; the toxic injury of neurons was detected by LDH method, and the protein expressions of IL-1R1, RIP1, RIP3 and MLKL in neurons were detected by Western blotting. Results (1) As compared with those in the sham-operated group and saline control group, protein expressions of HO-1, RIP1, RIP3 and MLKL in the 5 mmol/L heme group and 10 mmol/L heme group were significantly increased (P<0.05); at 24 h after 10 mmol/L heme injection, Caspase-3 expression was found in frozen sections of hippocampal tissues of mice, but no co-localization of Caspase-3 and NeuN was found. (2) As compared with those in the 0 μmol/L heme group, LDH release rates in the heme groups of different concentrations were significantly increased and concentration-dependent (P<0.05); as compared with the heme group of 0 h stimulation, the heme groups of different times of stimulation had significantly increased release rates of LDH in time dependent manner (P<0.05). As compared with the heme group of 0 h stimulation, the heme groups of different times of stimulation had significantly increased IL-1R1, RIP1, RIP3, and MLKL protein expressions (P<0.05); Caspase-3 showed no obvious changes. As compared with the heme group of 0 h stimulation, the heme groups of different times of stimulation had significantly increased IL-1β levels in time dependent manner (P<0.05). No obvious Caspase-3 expression was noted in the heme group of 6 h stimulation. (3) As compared with the 40 μmol/L heme group, pre-incubation groups of 0.5, 1.0, and 1.5 μg/mL IL-1R antagonist had significantly reduced LDH release rates and protein expressions of IL-1R1, RIP1, RIP3 and MLKL (P<0.05). Conclusions Hemin could induce neuron cell death directly via a necroptosis pathway rather than Caspase3-mediated apoptosis. The cytotoxicity of hemin could be alleviated by blocking IL-1R. Key words: Intracerebral hemorrhage; Hemin; Neuron; Necroptosis; Interleukin-1 receptor