The Molecular Mechanisms Of The Activation And Cell Death Of Astrocytes In The Neuroinflammation Induced By Bacterial Infection

Aim Infectious disease in the central nervous system(CNS)is one of the common diseases in the department of neurology.Its clinical manifestations are caused by the bacterial invasion in CNS.Bacterial meningitis(BM)needs efficient antibiotic treatments following timely diagnosis,due to its rapid onset.However,in clinical practice,cerebrospinal fluid bacterial culture,the golden criteria of diagnosis,has low positive rate and long-time cost,which is hard to meet the clinical requirement.Besides,BM patients often suffer from sequelae,such as impairment of cognitive function and hearing loss,with no effective treatments at present,which places a huge burden on the patients and society.Therefore,early diagnosis and intervene of the following sequelae are two main problems in the department of neurology.Procalcitonin(PCT)is specifically induced by bacterial infection.Histocyte such as adipocyte are able to upregulate the expression and release of PCT into the blood after treated by bacteria or bacterial components like lipopolysaccharide(LPS).Thus,serum PCT is a perfect diagnostic biomarker for the systemic bacterial infection and sepsis.But whether it is also suitable for brain cells is unknown.It is well known that astrocytes and microglia are remarkably activatated by the bacteria and LPS,with the increased expression of inflammatory factors.So,we first investigated that whether brain cells increased the expression and release of PCT under the treatment of bacteria and LPS,providing the theoretical basis for the PCT as a diagnostic biomarker for CNS bacterial infection.Previous studies have demonstrated that the sequelae of BM patients are related to the overamplification of neuroinflammatory network mediated by the glia cells.As the most abundant cell in CNS,astrocytes quickly proliferate and activate during the bacterial infection,playing a critical role in the overamplification of the neuroinflammatory network.Thus,decreasing the amount of overactivated astrocytes by apoptosis or growth arrest is an effective method to control the neuroinflammatory network.Gas1 is well demonstrated to inhibit the proliferation and induce apoptosis in the field of cancer research.Recently,it was found that Gas1 was abundantly expressed in the astrocytes,with unclear functions.Whether are Gas1 involved in the astrocytic fate regulation? We tried to investigate the role of Gas1 in the astrocytic fate regulation and the underlying signalling pathway.By establishing the neuroinflammation models using LPS,we investigated the astrocytic activation,the expression and release of PCT,as well as the fate regulation of activated astrocytes.Methods We first established the neuroinflammation models by the injection of S.pneumoniae and LPS into the brain.The spatio-temporal expression of PCT and CSF PCT were detected by immunofluorescent staining,western blot and Elisa.Then,we observed the expression of Gas1 and change of apoptosis in activated astrocytes before and after LPS injection into the brain.In vitro,Brdu incorporation assay,flow cytometry,CCK8,LDH release assay,ROS detection and TUNEL were performed after the treatment of LPS in combined with inflammatory factors to primary astrocytes to investigate the relationship between cell cycle,cell viability,apoptosis and Gas1 expression.We further designed si RNA targeted Gas1 to downregulate the expression of Gas1,to study the effect of Gas1 on the survival and cell cycle of reactive astrocytes.Western blot was performed to analyse the signalling pathway of Gas1.Results1.The hippocampal astrocytes were significantly activated in the neuroinflammatory model by the injection of S.pneumoniae and LPS into the brain,accompanied with the increased expression of Gas1 and CSF PCT levels.Besides,hippocampal neurons and infiltrating neutrophils were also positive for PCT.2.PCT was also positively expressed in human hippocampal astrocytes and neurons.3.The amount of apoptosis astrocytes was significantly increased after LPS injection into the brain,with the upregulated expression of astrocytic Gas1.4.LPS treatment had no obvious effects on the survival of astrocytes.However,the combination of LPS,IFN? and TNF? remarkably increased the level of oxidative stress,the amount of apoptosis and cell growth arrest,as well as the expression of Gas1.5.After the expression of Gas1 was downregulated by si RNA transfection,the apoptosis caused by the combination of LPS,IFN? and TNF? was decreased,with no obvious change of the cell growth arrest.6.Gas1 could increase the ratio of Bax and Bcl2,activate caspase-3,leading to the apoptosis of activated astrocytes,with no significant impact on the inflammatory signalling pathway and the expression of proliferating cell nuclear antigen.Conclusion 1.We demonstrated the existence of PCT in the astrocytes of rats and human brain.Furthermore,the expression of PCT and CSF PCT levels were significantly increased under the treatment of LPS and S.pneumoniae,suggesting that PCT might be a candidate diagnostic biomarker for the bacterial infection in CNS.2.We found that neuroinflammation induced by LPS caused increased apoptosis of activated astrocytes,cell growth arrest and upregulated expression of Gas1,suggesting that apoptosis and cell growth arrest were two approaches to decrease the amount of activated astrocytes.3.We revealed the role of Gas1 in the apoptosis of activated astrocytes.Interfering the expression of Gas1 could result in the decreased apoptosis of activated astrocytes,with no obvious influence on the cell growth arrest,suggesting that Gas1 can be a potential target to regulate the apoptosis of reactive astrocytes and the level of neuroinflammatory network,to finally decrease the sequelar such as the impairment of cognitive function of BM patients.4.We disclosed the molecular mechanisms of the apoptosis of activated astrocytes caused by Gas1,that is increasing the ratio of Bax and Bcl2,and activating caspase-3.