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.
ABSTRACT
Objective The aim of this study was to investigate the anti-tumor effect on sequential injection of heterogeneic lymphocyte(HL)and autogeneic lymphocyte(AL). Methods The HL was prepared by using CC3HF1 mice as feeders. CB6F1 mice were used as recipients,and Hepa1-6 cells were inoculated into the recepients′groin subcutis. A cryoprecipitate was extracted from mouse plasma by freeze-thaw method to prepare fibrin Glue(FG);FG was combined with HL or AL to be FG-HL or FG-AL. The experimental treatment consisted of two stages. At first stage(15 d),FG-HL were injected on the surface of the tumor-bearing tissue of the recipients as the experimental group,and FG-phosphate buffer saline(FG-PBS)were injected on the surface of the tumor-bearing tissue of the rest recipients as the control group. The immunological factors such as tumor cell killing rate of the spleen lym-phocytes and numbers of lymphocytes,CD8 +T and NK in the two groups were detected,respectively. At later stage(10 d),a part of mice were randomly selected from the experimental and control groups,and the lymphocytes( AL) were used to form FG-AL,which were injected on the surface of tumor-bearing tissues in the rest of mice. Tumors in mice of the two groups were compared for tumor volume and tumor inhibition rate. Results The tumor cell killing rate of AL in the experimental group(26. 70 ± 7. 22) was signifi-cantly higher than that in the control group(5. 70 ± 2. 68)(P<0. 01). Numbers of mouse spleen lymphocytes,CD8 +T cells and NK cells were significantly higher than the corresponding values of the control group(P<0. 05). After the two-stage treatment,the aver-age tumor volume of the experimental group[(1.20 ±0.33)cm3]was significantly smaller than that of control group[(2.05 ±0.37) cm3](P<0. 01). The tumor inhibition rate in the experiment group was 41. 5% when compared to the control group. Conclusion Local injections of FG-HL followed by FG-AL can significantly inhibit the growth of transplanted tumor in mice;it is expected to become an anti-tumor biological therapy.