ACSS2 Involved In The Regulation Of Autophagy And Alleviate Early Brain Injury After Subarachnoid Hemorrhage

Background:Subarachnoid hemorrhage(SAH)is a critical neurological emergency with high morbidity and mortality,accounting for about 5-7%of all stroke types and seriously affecting the health of population.Early brain injury is an important part of the pathological process of cerebral injury after SAH,which is associated with delayed brain injury after SAH and is an important factor in the prognosis of patients with SAH.Acetyl-coenzyme A synthetase 2(ACSS2)is a protease involved in mammalian energy metabolism and lipid synthesis,and in recent years it has been reported to act as a transcriptional regulator affecting long-term memory and autophagy gene transcription.Autophagy,as a process of cellular selfdegradation of damaged and non-essential organelles under stress,provides cells with sufficient energy and necessary materials to resist stress,and has a protective effect on a variety of neurological diseases such as traumatic brain injury,neurodegenerative lesions,and stroke.However,there are no studies on the effect of ACSS2 in regulating early endogenous protection after subarachnoid hemorrhage,and its mechanism is not clear.Therefore,the aim of this study was to investigate the role of ACSS2 on neuroprotection in early brain injury after subarachnoid hemorrhage in mice and the related mechanisms.Methods:1.1.To investigate the changes of ACSS2 after SAH,we examined the expression of ACSS2 at different time points after SAH by dual models in vivo and in vitro.They are divided into the following groups according to time points:Sham,6h,12h,24h and 72h.ACSS2 expression and neuronal status at different time points after injury were detected by Western blot,immunohistochemistry and Nissl staining methods.2.To investigate whether the neuroprotective effect in ACSS2 is related to cell autophagy and apoptosis,we regulated the expression of ACSS2 in the brain tissue of mice after subarachnoid hemorrhage by lateral ventricular injection of adeno-associated virus.C57BL/6 mice and primary neurons were randomly divided into sham group,SAH group,SAH+NC group and SAH+ACSS2 group.The regulation of ACSS2 was examined by Western blot and qPCR.expression of autophagy-related proteins(ATG5,LC3Ⅰ,LC3Ⅱ)and apoptosis-related proteins(Bcl-2,Caspase3,Bax)in the above groups was examined by Western blot and immunohistochemistry.Nissl staining was used to observe the changes in neuronal morphology after modulation of ACSS2 expression.In addition,neuronal apoptosis was detected by TUNEL staining in different groups.3.To investigate the effect of ACSS2 on brain edema and neurological function after SAH,C57BL/6 mice were randomly divided into sham group,SAH group,SAH+NC group,and SAH+ACSS2 group.Neurological function was scored for each group of mice 24 hours after the in vivo SAH model was constructed,and brain tissue was subsequently taken to measure brain tissue edema.The changes of cortical neurons were observed by Nissl staining.Results:1.ACSS2 expression was downregulated in the cerebral cortex after SAH and in OxyHb-treated primary neurons in a time-dependent manner.ACSS2 started to decrease at 6 h after SAH and continued throughout the early brain injury period.Compared with the Sham group,ACSS2 protein expression started to decrease at 6h after SAH and reached a low value at 24h after SAH.2.Immunohistochemistry and immunofluorescence double staining showed that ACSS2 protein was expressed in the cytoplasm and nucleus of neurons,localized mainly in the nucleus.3.ACSS2 alleviates SAH-induced neurological deficits,intracranial edema,and neuronal degeneration.4.Compared with the Sham group,autophagy-related proteins(ATG5,LC3Ⅰ,LC3Ⅱ)and pro-apoptotic proteins(Caspase3,Bax)were significantly increased in brain tissues of mice after SAH,while anti-apoptotic protein(Bcl-2)expression was decreased and cell morphology was significantly shrunken.The expression of autophagy-related proteins(ATG5,LC3Ⅰ,LC3Ⅱ)was further increased after upregulation of ACSS2 expression by virus.Meanwhile,the expression of pro-apoptotic proteins(Caspase3,Bax)was decreased,the expression of anti-apoptotic proteins(Bcl-2)was increased,as well as the cell morphology was improved.Conclusion:Through double verification by in vivo and in vitro experiments,our results suggest that ACSS2 upregulates ATG5-induced autophagic pathway after subarachnoid hemorrhage and alleviates neuronal apoptosis in early brain injury in SAH.In addition,ACSS2 may exert neuroprotective effects by alleviating brain edema and improving neurological function.