Based On Iron Death To Study The Pathological Mechanism Of Secondary Brain Injury After Cerebral Hemorrhage And The Intervention Effect Of Naotai Fang

ObjecttiveTo explore the mechanism and dynamic change of ferroptosis in nerve cells mediated by iron deposition in the acute stage of intracerebral hemorrhage(ICH),and to observe the intervention effect and mechanism of Naotai Fang(NTF)on ferroptosis in nerve cells after ICH.Methods(i)Cortical neurons of newborn SD rats were cultured,and hemoglobin(HGB)induced neuron simulation was used to establish a neuronal iron overload and oxidative stress injury cell model after ICH.The neurons were divided into normal group,model group,blank serum control group,Deferoxamine(DFX)group,N-acetylcysteine(NAC)group and NTF containing serum group.Cell survival rate was measured by CCK8,intracellular iron content was measured by Calcein-AM staining,glutathione(GSH),glutathione peroxidase 4(GPX-4)and lipid reactive oxygen species(lipid ROS)content in supernatants were measured by ELISA.The expression of neuronal transferrin receptor(Tf R),iron regulatory protein 2(IRP-2)and ferroportin 1(Fpn-1)were detected by immunofluorescence and high connotation cell imaging analysis(HCA).(ii)Rat models of acute ICH were established by autologous blood injection.They were divided into normal group,sham-operated group,IC H 6h group,ICH 24 h group,IC H 3d group and IC H 7d group.Samples were taken from each group at corresponding time points.Another batch of the same rat models were established.They were divided into sham operated group,ICH model group,deferoxamine(DFX)group,N TF conventional dose(NTFC)group and NTF high dose(N TFH)group.Each group was made after modeling and 7d administration.H E staining was used to observe the pathological changes of brain tissue,Zea longa 5-grade scoring method was used to assess the neurological deficit,Pr ussian blue staining was used to detect the iron deposition,biochemical kit was used to detect the lipid ROS content in the focus brain tissue and Western blot was used to detect the iron metabolism proteins of Tf R,IRP-2,Fpn-1 and the ferroptosis marker of COX-2.Results(i)After HGB stimulate,the survival rate of neurons decreased,the intracellular iron content,the cell supernatant lipid ROS level and the expression of Tf R and Fpn-1 increased,the expression of IRP-2,GSH and GP X-4 decreased significantly,the difference was statistically significant(P < 0.05,P < 0.01).After the intervention of corresponding substances in each group,the cell survival rate,GSH and GPX-4 content in DFX group,NAC group and NTF group increased,while the intracellular iron content and the lipid ROS level decreased(P < 0.05,P < 0.01).In DFX group and NTF group,the expression of Tf R and IRP-2 decreased,and the expression of Fpn-1 increased(P < 0.05,P < 0.01).GSH content was positively correlated with GPX-4 content(r =0.992,P < 0.05),GSH content was negatively correlated with lipid ROS content(r =-0.996,P < 0.05),lipid ROS content was positively correlated with iron content(r =0.859,P < 0.05),lipid ROS content was negatively correlated with cell survival rate(r =-0.991,P < 0.05).(ii)After molding,the pathological damage of brain tissue in ICH model group increased gradually at each time point,and the score of neurological deficit increased gradually,the difference was statistically significant(P < 0.05,P < 0.01).The iron content,lipid ROS content and COX-2 expression were significantly increased,while GSH content and GPX-4 expression were significantly decreased at 24 hours,3 days and 7 days after modeling(P < 0.05,P < 0.01).The expression of Tf R was significantly increased at 6 hours after ICH modeling,decreased at 3 days and increased again at 7 days;the expression of IRP-2 was significantly decreased at 24 hours after ICH modeling,further decreased at 3 da ys and increased again at 7 days;the expression of Fpn-1 was significantly increased at 24 hours after ICH modeling,remained at a high level at 3 days and decreased significantly at 7 days(P < 0.05,P < 0.01).(iii)After the intervention of DFX,NAC,NTFC and NTFH,the pathological damage of brain tissue was obviously reduced,the neurological deficit score was significantly reduced,the GSH content and GPX-4 expression in brain tissue were significantly increased,the iron deposition,lipid ROS content and COX-2 expression were significantly reduced,the difference was statistically significant(P < 0.05,P < 0.01).The iron deposition of NTFC group was higher than that of DFX group,but the GSH content,GPX-4 expression,lipid ROS content and COX-2 expression were not significantly different from those of DFX group(P < 0.05,P < 0.01).The iron deposition of N TFC group was higher than that of DFX group,while the GSH content,GPX-4 expression were higher than those of DFX group,and the lipid ROS content and COX-2 expression were lower than those of DFX group.The effects of DFX on Tf R were better than NTFC and NTFH,and NTFH on Fpn-1 were better than DFX and NTFC;NTFC on GSH,GPX-4,lipid ROS and COX-2 were weaker than NAC(P < 0.05,P < 0.01),and N TFH had no significant difference with NAC.There was no significant difference in iron deposition between N TFC group and NTFH group,but GSH content in NTFH group were higher than that of NTFC group,lipid ROS content and COX-2 expression were lower than NTFC group,and the improvement of brain pathological damage and neurological deficit score was better than NTFC group(P < 0.05,P < 0.01).Conclusions(i)The degradation of HGB after ICH results in iron overload of nerve cells,and ferroptosis caused by iron overload is the pathological mechanism of secondary brain injury in the acute stage of ICH.(ii)In the acute stage of ICH,the amount of iron deposition in the focal brain tissue of rats increased grad ually.The iron deposition exhausted GSH and GPx-4,which led to the accumulation of lipid ROS and the ferroptosis in the nerve cells,leading to the secondary brain injury.(iii)N TF can play a neuroprotective role by regulating the expression of iron metabolizing protein and anti-oxidation signal molecule of nerve cells in the acute stage of IC H,reducing the iron deposition of brain tissue,reducing the peroxidation injury of nerve cells,and inhibiting the ferroptosis of nerve cells.(iiii)Iron deposition is not the only determinant of ferroptosis in nerve cells.High dose NTF may also play a neuroprotective role by inhibiting ferroptosis in nerve cells through other targets.