The Role Of GCN2 In The Inflammatory Response And Mechanism Research After Intracerebral Hemorrhage In Mice

Part ? Role for GCN2 after intracerebral hemorrhage in miceObjective:In the intracerebral hemorrhage(ICH)model,we study the dynamic changes and cell localization of general control non-derepressible-2(GCN2)and its downstream eukaryotic initiation factor 2 alpha(eIF2?)and Activating Transcription Factor 4(ATF4).We also apply multiple doses of GCN2 in mice after surgery.Based on the results,we will choose the best dose to test long-term behavior.Methods:1.8-week-old adult male CD1 mice were selected and randomly divided into 6 groups(n=6),namely:sham operation group,3h group,6h group,12h group,24h group and Group 72h.Western blot was used to determine the expression of GCN2 protein and its downstream p-eIF2? protein in brain tissue,and the cell location of GCN2 protein and eIF2?protein was observed by immunofluorescence detection.2.To test the short term neurological function of the sham group,ICH group and different doses of exogenous GCN2(low dose group 0.15?g/piece,medium dose group 0.5?g/piece and high dose group 1.5?g/piece)though brain water content and Garcia test;3.8 To evaluate the long-term neurological function of ICH mice by water maze and rotarod experiments with the best dose of GCN2.Results:1.GCN2 protein started to increase at 3h after ICH,reached a peak at 24h,increased by 86%compared with sham group,and decreased at 72h.p-eIF2? increased at 3h after ICH,reached a peak at 6h,and the expression level was doubled compared to sham group.2.GCN2 and eIF2? proteins in microglia,astrocytes and neuronal cells are clearly expressed.3.The middle-dose exogenous GCN2(0.5?g/piece)is superior to other groups in the determination of cerebral edema and short-term behavior,and was chosen as the best dose for treatment.4.The results of long-term behaviors showed behavior of the GCN2 treatment group were better than the ICH group.Conclusion:1.GCN2 proteins increase with time after the ICH and decreased after reaching the peak.p-eIF2? reaches the peak in short term and maintains high expression,and eIF2? does not change significantly;2.GCN2 and eIF2? were obviously expressed in microglia,and they expressed in neuronal cells and astrocytes as well.3.GCN2 has a protective effect on the short-term behavior and long-term behavior of mice after ICH.Part ? Mechanism study of GCN2 inhibiting inflammation after intracerebral hemorrhageObjective:To study the mechanism of GCN2 inhibition of inflammation though inhibiting eIF2? and ATF4 using specific siRNA.Methods:8-week-old male CD1 mice were randomly divided into 6 groups(n=6):sham group,ICH group,ICH+GCN2 group,ICH+Scramble siRNA+GCN2 group,ICH+eIF2? siRNA+GCN2 group,In the ICH+ ATF4 siRNA+GCN2 group.The Scramble siRNA,eIF2? siRNA and ATF4 siRNA(100 pmol dissolved in 2 ?l of solvent)were injected into the brain of mice in the sham group and the ICH group 48 hours before the operation.The modified Garcia neurological score,forelimb position test,and corner turn test were performed at 24 hours after ICH surgery.Western blot was used to test all protein level.Results:1.The levels of inflammatory markers NLRP3 and TNF-? after ICH increased by 113%compared with the sham group,and the level of IL-1? increased by 52%compared with the sham group;2.After treatment with exogenous GCN2,the level of NLRP3 decreased by about 31%compared with the ICH group,TNF-? level decreased by about 28%compared with ICH group,and IL-1? level decreased by about 57%compared with ICH group;3.After blocking eIF2?,p-eIF2? expression decreased by about 41%compared with scramble siRNA group,lower than In the sham group,the expression of eIF2?decreased by about 27%compared with the scramble siRNA group,and the expression of ATF4 decreased by about 55%compared with the scramble siRNA group,but GCN2 expression was not affected;4.After blocking ATF4,the expression of ATF4 decreased by about 56%compared with the scramble siRNA group,but the expressions of p-eIF2? and GCN2 were not affected;5.Compared with the control group,after the suppression of p-eIF2? and ATF4,the level of NLRP3 in the same group increased by 67%and 57%,IL-1?,respectively increased by about 1.2 times and 1 times,TNF-? increased by about 27%and 33%compared with the control group,the expression level was close to the ICH group;6.After blocking p-eIF2? or ATF4,the Garcia score,corner experiment and forelimb test of the mice in the GCN2 treatment group decreased significantly compared with the control group..Conclusion:1.Brain dysfunction after cerebral hemorrhage is related to inflammation;2.GCN2 plays an anti-inflammatory and improves short-term neurological function after ICH through the p-eIF2?/ATF4 pathway.