Expression And Mechanism Of CGAS Signaling Pathway In Acute Phase Of Mice Intracranial Hemorrhage (ICH) Model

Background: Intracranial hemorrhage(ICH)as a central nervous system(CNS)vascular disease has a high incidence and disability rate around the world especially in developing countries.Even today,there are no effective medicine approaches to treat ICH.ICH brings out irreversible injuries to the whole brain which is even besides the hematoma lesion sites.ICH induces two phases of injuries of brain,one is the immediate injury as a result of direct oppression of hematoma;another is the redundant material of hematoma and inflammation lead secondary injury.There are evidences showed the modulation of secondary injuries and intracranial inflammation can ameliorate the progression of ICH.Cyclic-GMP-AMP synthesis(c GAS)is a significant receptor recognizing cytosolic double strand DNA(ds DNA)danger signals,which gets involved in the type Ⅰ IFN inflammation response of multifarious pathophysiological disorders.The expression profile and mechanism of c GAS influencing brain resident cells is largely unknown.Methods: This research used type Ⅳ collagens to induce ICH model in mice.By using western blot and RT-q PCR methods,transcription and expression of the c GAS,c GAS downstream factors and type Ⅰ IFN in the acute phase(the first and third day)of ICH mice brain were studied.Levels of phosphorylation of c GAS pathway signaling were tested.By using immunofluorescence,the cell-specific expressions of c GAS within mice ICH lesion were studied.By applying microglia BV2 cells line and hippocampus neuron HT22 cells line to substitute brain resident cells,and using hemin for simulating the environment of ICH in vitro,the research used immunofluorescence colocalization analysis and PCR methods to study the ds DNA damage and cytosolic ds DNA releasing in neuron.Furthermore,by flow cytometry or PCR analysis of the neuron and microglia coculture the relationship of neuron with microglia and if DNA can transfer between neuron and microglia were confirmed.Results: in vivo studies showed that transcription and expression of c GAS,STING,IRF3,IFN-β were up-regulated in brain tissue of ICH mice in the first day and more in the third day compared with controls mice.However the phosphorylation of STING and IRF3 had no time-dependent change and was same with the control mice.Surrounding the hematoma of ICH mice,there were plenty of c GAS-positive cells and they were mainly Neu N-positive neuron while a few Iba1-positive cells.In contralateral brain tissue,there were no cells strongly expressing c GAS.In vitro studies showed HT22 cells treated by hemin had more γH2AX location and the colocalization level of TOMM20 and ds DNA was decreased.The cytosolic DNA is from genome and mitochondria.Hemin could induce the up-regulated transcription of c GAS,STING and IFN-β in HT22 cells but not BV2 cells.Coculture of BV2 cells with HT22 cells treated by hemin could enhance the transcription of three factors mentioned above.It was also showed hemin could accelerate the uptake of neuron-derived ds DNA into microglia.Conclusion: Hemin in ICH hematoma could cause genomic DNA injuries,and also the releasing of mitochondria and genomic DNA releasing,which could be recognized by c GAS.The level of ICH expression was particular increased in dying neuron closely to the hematoma;nevertheless it was also increased in a little microglia/macrophages.The expression and transcription of c GAS along with its downstream factors was gradually elevated dependent on time.However,c GAS pathway in neuron was inhibited for some reasons.Hemin had no effect on c GAS pathway of microglia,meanwhile it can promote the c GAS expression in neuron and accelerate the microglia phagocytosis of neuron derived ds DNA,which exactly enhance c GAS pathway expression and type Ⅰ IFN producing in microglia.