Changes of ferroptosis in hippocampal neurons of rats with multiple cerebral concussion / 中华行为医学与脑科学杂志

ABSTRACT

Objective:To observe the ferroptosis in hippocampal neurons of rats with multiple cerebral concussion (MCC) .Methods:Ninety clean grade male Wistar rats with a body mass of (250±10) g were randomly divided into control group (12 rats) and model group (78 rats) according to the random number table method. The rat of MCC model was prepared by hitting the frontotemporal lobe of rats with free fall method once a day for 3 consecutive days.Then the MCC model rats were randomly divided into 12 h, 24 h, 48 h, 72 h, 7 d and 14 d groups with 12 rats in each group. The balance beam experiment was used to detect the motor coordination function of the rats. The levels of interleukin-β(IL-1β), interleukin-6(IL-6), serum glutathione (GSH), neuron-specific enolase (NSE) in serum of rats were detected by ELISA.The content of iron ion in hippocampus was detected by colorimetry. The mRNA and protein levels of glutathione peroxidase 4(GPX4), ferritin heavy (FTH) and ferritin light(FTL) in the hippocampus were detected by RT-PCR and Western blot. Prussian blue staining was used to observe the iron deposition in brain tissue.The ultrastructural changes of hippocampal neurons and mitochondria were observed by transmission electron microscope. SPSS 24.0 statistical software was used for one-way ANOVA among groups, and LSD test was used for multiple pairwise comparison.Results:In the balance beam experiment, the passing time and motor coordination score of rats in each group were significantly different ( F=30.08, 60.34, both P<0.05). The passing time and motor coordination score of rats in the 48 h group ((87.00±4.74) s, (4.75±0.43)) were significantly higher than those in the control group ((35.13±6.99) s, (0.75±0.23)) (both P<0.05). There was significant difference in the total iron ion content, Fe 2+ content and Fe 3+ content in hippocampus of rats in each group ( F=25.20, 94.42, 40.25, all P<0.05), and the content of Fe 2+ in hippocampus of 48 h group was significantly higher than that of the control group ((10.17±0.05) ng/μL, (8.65±0.01) ng/μL)( P<0.05). In the results of RT-PCR and Western blot, the mRNA and protein levels of GPX4, FTH and FTL in hippocampus of each group were significantly different ( F=37.94, 82.09, 49.01, 71.63, 28.94, 15.78, all P<0.05). The mRNA level and protein level of GPX4 ((1.09±0.01), (0.23±0.01) )and FTL ((1.60±0.03), (0.64±0.02)) in 24 h group were significantly higher than those of the control group (GPX4: (1.00±0.02), (0.17±0.01)), FTL: ((1.00±0.04), (0.32±0.01))(all P<0.05). The mRNA level and protein level of FTH ((0.24±0.03), (0.07±0.01)) in 24 h group were significantly lower than those of the control group((1.00±0.01), (0.67±0.03))(both P<0.05). The results of electron microscope showed that the hippocampal neuronal cells of the model rats were reduced, the nucleolus was broken and the nuclear membrane was shrunk in varying degrees, the mitochondria were swollen and deformed and there were vacuoles, and the cristae in the mitochondria decreased or disappeared. Conclusion:The levels of inflammatory reaction and oxidative stress in the multiple cerebral concussion model rats increase, and the hippocampal neurons show the characteristics of ferroptosis, especially at 24 h and 48 h.