Effect Of Ceftriaxone On Ferroptosis After Early Brain Injury In Rats With Subarachnoid Hemorrhage

Objectives 1 To investigate ferroptosis and neurological impairment during early brain injury（EBI）in rats.2 To investigate the effect of ceftriaxone（CTX）on ferroptosis in rats with early brain injury and its possible mechanism.Methods Experiment 1:Forty-eight healthy male SD rats were stochastically assigned into four groups:Sham group,SAH 6h group,SAH 24h group and SAH 72h group,with12 rats in each group.The SAH model was made by intravascular puncture.The blood vessel was not punctured in Sham group.Experiment 2:Forty-eight healthy male SD rats were stochastically assigned into four groups:Sham group,SAH group,SAH+CTX group and SAH+CTX+Nrf2 inhibitor group（SAH+CTX+ML385 group）,with 9 rats in each group.CTX was injected into SAH+CTX group 5 days in advance(200mg·kg^-1,once a day,the injection lasted for five days).Rats in SAH+CTX+ML385 group were injected intraperitoneally with ML385 7 days in advance(30mg·kg^-1)once a day for 7 consecutive days.Take 24 hours after SAH as the detection point.Then,the neurological function score was measured.Calculate the water content of rat brain tissue.The morphology of nerve cells in brain tissue was observed by HE staining and the number of nerve cell necrosis was calculated.The iron deposition in brain tissue was observed.The contents of iron,MDA,GSH and the vitality of GPX4 were measured.The expression of Nrf2 and GPX4 protein in brain tissue was detected by Western Blot.Results Experiment 1:Compared with Sham group,the neurological function score of SAH 6h,SAH 24h,SAH 72h group decreased significantly（P<0.05）.The brain water content in SAH 24h group and SAH 72h group was significantly rised,compared with Sham group（P<0.05）.The brain water content between SAH 6h group and Sham group was similar（P>0.05）.The number of neuronal necrosis increased significantly in SAH 24h and SAH 72h groups,compared with sham operation group（P<0.05）.The number of neuronal necrosis between SAH 6h group and Sham group was similar（P>0.05）.Prussian blue staining showed that there was no obvious iron deposition in Sham group.There was only a small amount of iron deposition after 6 hours with subarachnoid hemorrhage.The amount of iron deposition in brain tissue increased significantly after 24 hours and 72hours of subarachnoid hemorrhage,compared with Sham group（P<0.05）.Compared with Sham group,the contents of iron and lipid peroxide MDA in brain tissue of SAH 24h group and SAH 72h group were significantly increased,while GSH content and GPX4activity were significantly decreased.Experiment 2:The neurological function score of SAH group was significantly dreased,compared with Sham group（P<0.05）.Compared with SAH group,the neurological function score of SAH+CTX group was significantly higher（P<0.05）.Compared with SAH+CTX group,the neurological function score of SAH+CTX+ML385 group decreased significantly（P<0.05）.The brain water content in SAH group and SAH72h group increased significantly,compared with Sham group（P<0.05）.The water content of brain tissue in SAH+CTX group was significantly lower than that in SAH group（P<0.05）.The water content of brain tissue in SAH+CTX+ML385group was significantly higher than that in SAH+CTX group（P<0.05）.The number of nerve cell necrosis in SAH group was significantly increased,compared with Sham group（P<0.05）.Compared with SAH group,the number of nerve cell necrosis in SAH+CTX group was significantly decreased（P<0.05）.The number of nerve cell necrosis in SAH+CTX+ML385 group was significantly higher than that in SAH+CTX group.Prussian blue staining showed that there was no iron deposition in sham operation group.Compared with Sham group,the amount of iron deposition in brain tissue of SAH group was significantly increased（P<0.05）.Compared with SAH group,the deposition of brain tissue in SAH+CTX group decreased significantly（P<0.05）.Compared with SAH+CTX group,the amount of iron deposition in SAH+CTX+ML385 group was significantly higher than that in SAH+CTX+ML385 group.Compared with Sham group,the contents of iron and lipid peroxide MDA in brain tissue of SAH group increased significantly,while GSH content and GPX4 activity decreased significantly（P<0.05）.Compared with SAH group,the contents of iron and lipid peroxide MDA in brain tissue of SAH+CTX group decreased significantly,while GSH content and GPX4 significantly increased（P<0.05）.Compared with SAH+CTX group,the contents of iron and lipid peroxide MDA in brain tissue of SAH+CTX+ML385 group increased significantly,while GSH content and GPX4decreased significantly（P<0.05）.The Nrf2 and GPX4 protein expression in brain tissue of SAH group decreased significantly,compared with Sham group（P<0.05）.Compared with SAH group,the expression levels of Nrf2 and GPX4 protein in brain tissue of SAH+CTX group significantly increased（P<0.05）.Compared with SAH+CTX group,the expression of Nrf2 and GPX4 protein in brain tissue of SAH+CTX+ML385 group decreased significantly（P<0.05）.Conclusions Ceftriaxone can alleviate ferroptosis and protect neurological function in early brain injury after SAH,and its mechanism may be related to the up-regulation of Nrf2/GPX4 pathway.Figure16;Table8;Reference 176...