Mechanism Of AIM2 Inflammasome-mediated Pyroptosis In Early Brain Injury After Subarachnoid Hemorrhage

Background and Objective:Spontaneous subarachnoid hemorrhage(SAH)is one of the most common critical cerebrovascular diseases with high disability and mortality.With the development of technology and materials,high intracranial pressure,low perfusion,rebleeding has been controlled to some extent,however,the therapeutic effect of many SAH patients is not satisfactory,and the prognosis has not been significantly improved.Early brain injury(EBI)is an important cause of poor clinical prognosis in patients with SAH.The inflammatory response following SAH is one of the important pathophysiological mechanisms leading to EBI.The inflammasome is a cytosolic multimeric signalling complex,which is a component of the innate immune system.More and more studies have shown that inflammasome is involved in the EBI after SAH.Recently,pyroptosis is an inflammatory programmed cell death triggered by inflammasomes.The activation of inflammasomes not only causes rapid cell death,but also further aggravates the inflammatory response by releasing pro-inflammatory mediators.Absent in melanoma 2(AIM2)inflammasome is mainly expressed in neurons and is the only known inflammasome that recognizes double-stranded DNA in cytoplasm.Activation of AIM2 inflammasome could induce neuronal pyroptotic cell death.Therefore,this study intends to explore the role of GSDMD-induced pyroptosis mediated by the AIM2 inflammasome,AIM2/Caspase-1/GSDMD pathway,in EBI after SAHMethods:First part:Cerebrospinal fluid(CSF)was collected from patients undergoing hip arthroplasty,and from patients with SAH within 72 hours following SAH.The level of AIM2 protein in all CSF samples was analyzed by Western blot.The correlation between the level of AIM2 protein and the severity of SAH was observed and analyzedSecond part:The models of SAH in vivo and in vitro were established,and the role of AIM2/Caspase-1/GSDMD pathway in EBI after SAH was explored by Western blot,immunohistochemistry,Nissl staining,immunofluorescence,flow cytometry,and scanning electron microscopeThird part:After interfering with the activation of AIM2 inflammasome through knocking down AIM2 and caspase-1 by a lentivirus,the role of GSDMD-induced pyroptosis mediated by the AIM2 inflammasome,AIM2/Caspase-1/GSDMD pathway,in EBI after SAH was re-detected after SAH in vivo and in vitroResults:First part:Western blot showed that the level of AIM2 protein in CSF of SAH patients was significantly higher than that of hip arthroplasty patients;the higher the Hunt-Hess grade,the higher the level of AIM2 proteinSecond part:In the mouse SAH model in vivo,Nissl staining showed that a large number of many damaged neurons with shrunken cell bodies,condensed nuclei,and dark cytoplasm were observed in the temporal cortex of mice 24 h after SAH Compared with post-SAH 24 h,damaged neurons in the temporal cortex alleviated post-SAH 72 h.Immunohistochemical staining showed that a large number of AIM2,GSDMD,caspase-1 and ASC immunoreactive cells were found in the temporal cortex after SAH.Western blot showed that GSDMD-induced pyroptosis mediated by the AIM2 inflammasome,AIM2/Caspase-1/GSDMD pathway,was significantly activated at 12 h after SAH,reached the peak at 24 h,and decreased at 72 h.In the SAH model of neurons in vitro,scanning electron microscopy revealed that the neurons showed pyroptotic changes after the neurons were stimulated by oxyhemoglobin(OxyHb)Immunofluorescence staining showed that the immunofluorescence of AIM2,GSDMD,caspase-1,and ASC proteins enhanced after the neurons exposed to OxyHb,and AIM2 protein was expressed in the cytoplasm and nucleus,while GSDMD,caspase-1 and ASC proteins were mainly expressed in the cytoplasm.Western blot showed that GSDMD-induced pyroptosis mediated by the AIM2 inflammasome increased significantly at 6 h and continuously increased after the neurons were stimulated by OxyHb.Flow cytometry showed that GSDMD-induced pyroptosis mediated by the AIM2 inflammasome significantly increased at 6 h after the neurons exposed to OxyHb.Third part:After inhibiting the activation of AIM2 inflammasome by knocking down AIM2 and caspase-1,both in vivo and in vitro SAH models showed that GSDMD-induced pyroptosis mediated by the AIM2 inflammasome was significantly reduced,which improved the brain injury after SAH.At the same time,knocking down caspase-1 also alleviated apoptosis via inhibiting the activation of caspase-3.Conclusion:This study confirmed that GSDMD-induced pyroptosis mediated by the AIM2 inflammasome,AIM2/Caspase-1/GSDMD pathway,is involved in EBI after SAH.Inhibiting the activation of AIM2 inflammasome by knocking down AIM2 and caspase-1 can alleviate GSDMD-induced pyroptosis mediated by the AIM2 inflammasome.At the same time,knocking down caspase-1 can also inhibit the activation of caspase-3 and reduce neuronal apoptosis after SAH.Caspase-1 could be an important target for the treatment of EBI after SAH.