The Role And Mechanism Of Stimulator Of Interferon Genes In Early Brain Injury Following Experimental Subarachnoid Hemorrhage

BackgroundSubarachnoid hemorrhage(SAH),mainly caused by ruptured intracranial aneurysms,is a fatal cerebrovascular disease with a high of mortality and disability.According to epidemiological statistics,the mortality rate of SAH is as high as 30%.Additionally,those who survived from SAH could suffer from chronic neurological impairments such as hemiplegia,aphasia and cognitive dysfunction,causing a heavy burden to their family and to the society.Early brain injury(EBI),which refers to the brain injury within the first 72 hours after SAH,is believed to the important factor accounting for the poor prognosis of SAH patients.The underlying mechanism of EBI involves many complex processes including increased intracranial pressure,brain edema,autophagy,and inflammation.Thus,carrying out studies focusing on the pathophysiological mechanism of EBI and developing novel therapeutic strategy is of great significance to improve the prognosis of SAH patients,which is becoming one of the research hotspots worldwide.Stimulator of interferon genes(STING),a pattern recognition receptor that widely distributed in mammalian immune cells,can activate the immune system and exert many immune defense effects such as anti-virus and anti-tumor immunity.However,the abnormal activation of STING has also been proved to be closely related to the occurrence and development of many inflammation-related diseases and autoimmune diseases such as acute pancreatitis,systemic lupus erythematosus.With the deepening of research,a growing body of studies demonstrated the role of STING in central nervous system diseases.STING was reported to participate in the pathological process following multiple CNS diseases,including traumatic brain injury,Parkinson’s disease and multiple sclerosis,indicating that STING might be an important therapeutic target for a variety of human diseases,especially for central nervous system diseases.However,the exact role of STING in the pathophysiological mechanism of SAH-induced EBI remains unclear.Therefore,the current study was designed to investigate the role of STING in the pathological process of early brain injury following SAH and further explore the underlying mechanism,aiming to provide a novel therapeutic strategy for SAH.MethodsPart 1The SAH model in mice was established through the endovascular perforation technique.Adult male C57BL/6 mice were randomLy divided into seven group as follows:sham group,SAH 3 h group,SAH 6 h group,SAH 12 h group,SAH 24 h group,SAH 48 h group and SAH 72 h group.The neurological deficit score in each group was evaluated using the modified Garcia score.And the expression of STING was assessed by Western blot analysis.Double immunofluorescence staining was introduced to determine the distribution of STING.Part 21.Adult male C57BL/6 mice were treated with STING agonist CMA and STING antagonist C-176 after SAH insult,respectively.The animals were randomLy divided into four groups as follows:sham group,SAH+vehicle group,SAH+C-176 group and SAH+CMA group.2.The mortality,SAH grade,brain water content and neurological behavior test(both in short term and in long term)were evaluated in all groups.TdT-mediated dUTP Nick-End Labeling(TUNEL)staining and Nissl staining were used to measure the neuronal injury.Immunofluorescence staining were used to assess the activation and polarization of microglia.Western blot was conducted to evaluate the expression of TBK1,p-TBK1,iNOS,IL-1β,NLRP3,ASC and caspase-1.qPCR was introduced to measure the mRNA level of CD16,iNOS,IL-1β,IL-6,TNF-α and MCP-1.Part 31.Adult male C57BL/6 mice were treated with corn oil(vehiclel),5%DMSO(vehicle2),STING antagonist C-176 or AMPK inhibitor Compound C,respectively.The mice were randomLy assigned into five groups as follows:sham group,SAH+vehicle1 group,SAH+C-176 group,SAH+C-176+vehicle2 group and SAH+C-176+Compound C group.2.SAH grade and neurological behavior test were measured at 24 hours postmodeling.The neuronal injury was evaluated using TUNEL staining in all groups.The expression of TBK1,p-TBK1,AMPK,p-AMPK,iNOS,IL-1β,NLRP3,ASC and caspase-1 were determined through Western blot analysis.The mRNA level of CD 16,iNOS,IL-1β,IL-6,TNF-α and MCP-1 were evaluated using qPCR.ResultsPart 11.Western blot analysis indicated that the expression of STING was significantly increased after SAH and peaked at 24h post-modeling.Subsequently,the expression of STING decreased gradually.However,the level of STING in SAH 72 group was still higher than that in sham group.2.Immunofluorescence staining showed that the number of STING-positive cell was increased at 24h after SAH,which was consistent with Western blot results.Furthermore,double immunostaining of STING with different cell biomarkers suggested that STING was mainly expressed in microglia.Part 21.Compared with sham group,the SAH mice suffer from an increased brain water content and remarkable neurological impairment,accompanied with significant neuronal injury(evidenced by the increased number of TUNEL-positive neurons and decreased number of Nissl-positive cell)and neuroinflammation(evidenced by increased number of activated microglia and upregulated protein expression of iNOS,IL-1β,NLRP3,ASC and caspase-1,as well as increased mRNA level of CD 16,iNOS,IL-1β,IL-6,TNF-α and MCP-1.2.Compared with SAH+vehicle group,administration of STING agonist CMA significantly increased the ratio of p-TBK1/TBK1 in brain,while administration of STING antagonist C-176 obviously decreased the ratio of p-TBK1/TBK1.3.Compared with SAH+vehicle group,administration of CMA significantly aggravated the mentioned brain injury indicators after SAH,while pharmacological inhibition of STING with C-176 significant alleviated SAH-induced brain edema,neuronal injury and neuroinflammation,resulting in a remarkable improvement in both short term and long term neurological function.Part 31.The ratio of p-AMPK/AMPK was significantly increased at 24h after SAH,and application of C-176 further upregulated the phosphorylation of level AMPK,which was significantly inhibited by Compound C.2.Compared with SAH+vehicle group,administration of C-176 significantly inhibited neuroinflammation,alleviated neuronal damage and attenuated neurological dysfunction.3.Compared with SAH+C-176 group,preconditioning with Compound C obviously increased the protein expression of iNOS,IL-1β,NLRP3,ASC and caspase-1,as well as increased mRNA level of CD16,iNOS,IL-1β,IL-6,TNF-α and MCP-1.Additionally,the decreased number of TUNEL-positive neurons after C-176 treatment was reversed by Compound C.More importantly,the neuroprotective effect of C-176 in attenuating SAHinduced neurological deficits was abolished by Compound C.Conclusion1.STING was activated in the pathological process of early brain injury following SAH.And STING was mainly distributed in microglia,indicating that STING may play an important role in SAH-induced early brain injury through modulating microglial function.2.Activation of STING significantly aggravated brain edema,neuronal injury and neurological impairment in early brain injury after SAH,while pharmacological inhibition of STING with C-176 significantly inhibited neuroinflammation and afforded a robust neuroprotection against SAH by attenuating neuronal injury and neurological deficits,suggesting that STING could exacerbate SAH-induced early brain injury via mediating neuroinflammation.3.Inhibition of STING may have the potential to attenuate early brain injury following SAH through AMPK-related anti-inflammatory pathway.STING might be a novel and promising therapeutic target for SAH.