Mechanism Of Calprotectin 8 Regulating Autophagy-Dependent Ferroptosis In Microglia After Experimental Subarachnoid Hemorrhage

Objective: Subarachnoid hemorrhage(SAH)is an acute cerebrovascular accident with high mortality and morbidity,accounting for 5%-10% of all strokes.Early Brain Injury(EBI)has received great attention because it is thought to contribute to the poor prognosis of SAH.EBI involves a variety of complex pathological processes such as blood-brain barrier(Blood–Brain Barrier,BBB)disruption,brain edema,neuroinflammation,oxidative damage,autophagy,ferroptosis,and neurodegeneration.S100 calcium-binding protein A8(S100A8)is a calcium-zinc-binding protein that is thought to be involved in the regulation of inflammation and immune responses,and has also been shown to mediate cell death by enhancing autophagy and oxidative stress.S100A8 has been found to be involved in the pathological process of various central nervous system(Central Nervous System,CNS)injuries and diseases,and also plays an important role in SAH,but the regulatory mechanism is not clear.Previous in vitro experiments have confirmed that its exogenous stimulation can promote the activation of TLR4 in microglia and play an important role in the proinflammatory response.Interestingly,microglia,as innate immune cells in the central system,are also one of the main expressors of S100A8,and the regulatory mechanism of endogenous S100A8 in EBI has not yet been explored.In this study,we aimed to explore the potential mechanism by which endogenous S100A8 expression in microglial cells participates in early brain injury by regulating the level of microglial autophagy after SAH.Methods: Experiment in mice: The SAH model was established by puncturing the right internal carotid artery with a modified single-clip technique.Western Blot(WB)was used to explore the expression of endogenous S100A8 protein in brain tissue during EBI after SAH.Immunofluorescence(IF)was used to observe its localization in microglia.WB after 24 hours of SAH modeling was used to verify the S100A8 knockdown efficiency.Modified Garcia scale and Beam balance tests were used to evaluate the neurological function of mice.In situ Td T-UTP Nick End Labeling(TUNEL)staining and neuron co-staining were used to observe the apoptosis of neurons in brain tissue.2.Cell experiments: knockdown of S100A8 expression in BV2 microglia cells by lentivirus and stimulation of cells with Oxyhemoglobin(Oxy Hb)mimics SAH injury in vitro.A co-culture model of BV2 and HT22 was constructed,and the changes of HT22 apoptotic protein in the control virus group(BV2-NC group)and experimental group(BV2-KD group)under the stimulation of oxyhemoglobin were compared.Then,WB,quantitative reverse transcription PCR(RT-q PCR),IF and other methods were used to observe the changes of autophagy and ferroptosis in BV2 microglia after S100A8 knockdown.Results:1.The endogenous S100A8 protein in mouse brain tissue gradually increased after SAH and reached a peak at 24h;S100A8 was observed to colocalize with microglia in mouse brain tissue.2.S100A8 knockdown inhibited the activation of autophagy in BV2microglia:(1)Compared with the control group,BV2-KD cells autophagyrelated genes(ATG12,ATG4 C,ATG5,BECN1,LC3 B,VPS34)transcription levels decreased,while P62 and LC3 BII protein was significantly increased;(2)m RFP-EGFP-m LC3 B double fluorescence showed that autophagic flow in BV2 microglia was blocked after S100A8 knockdown.3.S100A8 knockdown inhibited the ferroptosis of BV2 microglia stimulated by oxyhemoglobin:(1)CCK8 indicated that cell viability increased after S100A8 knockdown;(2)ferroptosis-related genes(ALOX5,ACSL4,PTGS2,NOX2,NOS2)expression was significantly inhibited after S100A8knockdown;(3)S100A8 knockdown could increase the protein level of Glutathione Peroxidase 4(GPX4),a key protein in the regulation of ferroptosis;(4)S100A8 knockdown The content of MDA and ROS in post-BV2 microglia decreased significantly,while the total antioxidant capacity increased.4.S100A8 may regulate BV2 microglia autophagy-dependent ferroptosis through the NCOA4 pathway:(1)WB and immunofluorescence results of NCOA4 and ferritin heavy chain showed that S100A8 inhibition significantly reduced the expression of NCOA4 and increased ferritin The protein level of the heavy chain;(2)the reduction of Fe2+ content after S100A8 inhibition also supports that S100A8 knockdown reduces ferritin degradation.5.In the co-culture model of BV2 microglia and HT22 neurons,the apoptosis of HT22 neurons in the BV2-KD group was significantly lower than that in the control group: the expressions of Cleaved capase-3 and Bax proteins,which promote apoptosis,were reduced,while the expression of anti-apoptotic BCL2 Protein expression was significantly increased.6.Using adeno-associated virus to specifically knock down the mouse microglia S100A8,compared with the control group(NC group),the neuronal apoptosis in the microglia S100A8 knockdown group(c KD group)was reduced,and the neuronal apoptosis was reduced.Functional deficits were improved:(1)After SAH modeling,the neurological function evaluation of mice in c KD group was significantly improved compared with NC group;(2)After SAH modeling,there were fewer TUNEL-positive neurons in c KD group than in NC group.Conclusion: This study found that: 1.Targeting microglia S100A8 inhibition can reduce neuronal apoptosis and improve neurological deficits after experimental subarachnoid hemorrhage in mice;2.S100A8 may induce autophagy-dependent ferroptosis in BV2 microglia through m TOR/NCOA4 pathway.These results suggest that microglial endogenous S100A8 is involved in autophagy and ferroptosis in the process of EBI after SAH,and targeting microglial S100A8 inhibition may be a new idea to rescue EBI injury after SAH.