Mechanism Of BRD4 Regulation Of Ferritinophagy-dependent Ferroptosis In Neuronal Cells After Experimental Subarachnoid Hemorrhage

Objective: Subarachnoid hemorrhage(SAH)is a common hemorrhagic cerebrovascular disease in neurosurgery,with low incidence,but high disability rate and high mortality.Early brain injury(EBI)after SAH is associated with poor prognosis after SAH.Neuronal death due to EBI is a key factor in poor prognosis for patients.Ferroptosis is a novel regulatory form of cell death,and studies have found that ferroptosis is involved in the EBI process.BRD4 is the most important protein in the bromine domain and superterminal domain(BET)family,and its main function is to regulate inflammation and oxidative stress.The literature confirms that in mouse ischemic stroke models,BRD4 expression is increased by intervention,and its expression can protect cerebral ischemic brain injury by inhibiting neuroinflammation and pyroptosis.However,BRD4 has not been reported in EBI after subarachnoid hemorrhage,and the role of BRD4 in ferroptosis has not been explored in detail.In this study,we aim to explore the role and underlying mechanism by which BRD4 regulates ferroptosis in neurons after experimental SAH.Methods:1.Experiments in mice: Experimental mouse SAH model was constructed using a modified single-clamp puncture method.Western Blot(WB)was used to explore endogenous BRD4 protein expression levels in brain tissue during post-SAH EBI.Immunofluorescence(IF)is used to observe its localization in nerve cells.The modified Garcia scale and Beam balance tests were used to assess neurological function in mice.2.Experiments in cell: mouse hippocampal neuronal cells(HT22)were stimulated to mimic in vitro SAH damage using oxyhemoglobin(OxyHb).2.1 Control group(control)and experimental group(OxyHb group),using WB,CCK8,cell fluorescence,MDA detection kit and other methods to detect ferroptosis related indicators.2.2 Construction of intracellular BRD4 knockdown strains in HT22 cells using commercial lentiviruses.OxyHb stimulated cells which were devided into control groups(HT22-NC,HT22-KD)and experimental groups(OxyHb-HT22-NC,OxyHb-HT22-KD).The above method was used to detect the changes of ferroptosis related indicators after RBD4 silencing.Results:1.BRD4 in the mouse brain tissue cortex was mainly colocalized with neurons,and immunofluorescence results showed that the expression of BRD4 in neurons in the postcortex of SAH was decreased.2.OxyHb induced ferroptosis in HT22: Stimulated with OxyHb decreased cell viability,accumulated intracellular iron content and increased ROS indexes of reactive oxygen species.The lipid peroxidation index MDA/BODIPY 581/591 increased significantly.Transmission electron microscopy showed that mitochondria were densed and smaller,and the outer mitochondrial membrane was significantly thickened.Western blot showed that the key antioxidants factors,GPX4 and Co Q10 B,were significantly decreased.2.Fer-1 inhibits OxyHb-induced ferroptosis in HT22: Fer-1 6 u M is the lowest concentration that exerts maximum inhibitory effect.Fer-1 6u M can rescue cell viability.Reduce the number of cell deaths and increase cell growth density.Significantly reduce the OxyHb-induced elevated lipid peroxide index of MDA and BODIPY 581/591 C11.4.BRD4 knockdown promotes OxyHb-induced HT22 ferroptosis: lentiviral knockdown obtains cell lines with satisfactory knocking effect.BRD4 knockdown could decrease cell viability and significantly increased ROS levels of reactive oxygen species.The lipid peroxide indexes MDA/MDA/BODIPY581/591 were significantly increased.The expression of GPX4,a key protein against ferroptosis,was decreased,and all the above indicators changed significantly in the OxyHb-HT22-KD group compared with the OxyHb-HT22-NC group,and there were statistically significant differences.5.BRD4 regulates ferritinophagy process and autophagy-related genes: BRD4 knockdown leads to increased intracellular iron,and intracellular iron colocalization with lysosome.The expression of FTH1 protein and the expression of autophagy-related protein SQSTM1/p62 decreased,indicating that the autophagy process of ferritin increased.Meantime,the expression of autophagy-related genes(LC3B,ATG4 D,ATG16L,ATG101)were increased.Conclusion: 1.SAH induced a decrease in BRD4 expression in cortical neurons.2.BDR4 may promote neuronal ferroptosis by regulating ferritinophagy.The results show that neuronal endogenous BRD4 is involved in the EBI process after SAH throughing regulates ferritinophagy.