Actions And Mechanisms Of Hydrogen Sulfide On Phenotypic Transformation Of Astrocytes After Cerebral Ischemia/reperfusion Injury

Background:Ischemic stroke is one of the leading causes of death and disability.During the pathological process of ischaemic stroke,astrocytes are activated and produce morphological and functional changes,named’reactive astrocytes’.The activated astrocytes are divided into the"A1"and"A2"phenotypes.Hydrogen sulfide（H₂S）is a widespread gaseous signalling molecule in the body that is not only involved in physiological functions but can also influence pathological processes.Our previous study at found that H₂S inhibited the reactive proliferation of astrocytes after cerebral ischaemia.However,whether H₂S affects astrocyte phenotypic transition has not been clearly reported.The large conductance Ca²⁺-activated K⁺channels(BK_Ca)consist of theαsubunit,which forms the core pore,and the auxiliary subunitsβandγ.BK_Cachannels are important players in neural activity,including regulation of neural firing,control of neurotransmitter release,and mediated microglia phagocytosis.Our group has found that H₂S activation of BK_Cachannels protects neurons from ischemic/hypoxic injury,but whether H₂S activation of BK_Cachannels affects astrocyte phenotype conversion has not been reported.Therefore,in this study,we investigated the effects of H₂S and lipopolysaccharide（LPS）-based neuroinflammation on astrocyte phenotype conversion after brain R by establishing an ischemia/reperfusion（I/R）model in mice.A model of astrocyte oxygen and sugar deprivation/reoxygenation（OGD/R）was constructed to investigate whether activation of BK_Cachannels is involved in H₂S-mediated astrocyte phenotypic transition.Purpose:1.to investigate the role of lipopolysaccharide（LPS）-based neuroinflammation on the conversion of reactive astrocytes after brain I/R and to further explore its relationship with endogenous H₂S.2.to explore the effect of H₂S on the phenotypic conversion of reactive astrocytes in vitro by culturing astrocytes in the OGD/R model.3.to explore the relationship between H₂S-mediated phenotypic transition in reactive astrocytes and BK_Cachannel activation.Method:1.A mouse I/R model was constructed using bilateral common carotid arteries（BCCAs）ligation.A lipopolysaccharide-based neuroinflammation model was also constructed by intraperitoneal injection of LPS（1 mg/kg/day）.1.1 Experimental grouping:Wild type C57BL/6（wild type,WT）and CSE knock out（knock out,KO）mice were divided into the following groups（n=7 groups each）:WT mice sham-operated group,WT mice brain I/R group,WT mice brain I/R+Na HS（4.8 mg/kg）group,WT mice brain I/R+LPS（1 mg/kg）group,WT mouse brain I/R+LPS（1 mg/kg）+Na HS（4.8 mg/kg）group,KO mouse sham-operated group,KO mouse brain I/R group,KO mouse brain I/R+Na HS（4.8 mg/kg）group.1.2 Absence field experiments to detect spontaneous behaviour and motor exploration ability of mice.1.3 Morris water maze experiment（MWM）to test the spatial learning and memory ability of mice.1.4 The neuronal damage in the hippocampal region of mice was observed by HE staining.1.5 Western Blot to detect the expression of GFAP,S100A10 and C3.1.6 Immunofluorescence assay was used to detect the number of Neu N,GFAP,S100A10 and C3 positive cells.2.Construction of astrocyte OGD/R model.2.1 Experimental groups:control group,OGD/R group,OGD/R+Na HS（200umol/l）group,OGD/R+IBTX（100nmol/l）,OGD/R+IBTX（100nmol/l）+Na HS（200umol/l）,and OGD/R+Fasudil（5umol/l）.In subsequent experiments,we validated the four groups,control,control+BK_Caopener BMS-191011（10umol/l）,OGD/R,and OGD/R+BMS-191011（10umol/l）.2.2 Cells in the OGD/R treated group were replaced with sugar-free medium and placed in a three-gas incubator（94%N₂-5%CO₂-1%Q₂）for 3.5 h in hypoxia,then replaced with high-sugar medium in a constant temperature incubator for 24 h for reoxygenation to establish the OGD/R model.2.3 The cck-8 kit was used to detect cell viability.2.4 Detection of neuron-specific enolase（NSE）activity and H₂S content in mouse serum and cultured astrocyte supernatant using ELISA kits.2.5 Western Blot to detect the expression of GFAP,S100A10,C3,PKA and BK_Caalpha subunits.Result:1.The results of the open field and water maze experiments showed that mice in the brain I/R group exhibited significant impairments in exploratory behaviour and spatial memory compared to the sham-operated group.In contrast,LPS-treated mice or CSE KO mice showed more severe impairment in place navigation ability and spatial exploration ability.Exogenous H₂S donor Na HS attenuated the behavioral deficits and cognitive dysfunction induced by brain I/R in LPS-treated or CSE KO mice.2.HE staining,NSE assay and Neu N immunofluorescence staining results showed that compared with the sham-operated group brain I/R induced neuronal damage in the hippocampal tissue of mice.In LPS-treated mice,neuronal damage induced by brain I/R was more severe and could be ameliorated by Na HS.3.Western Blot results showed that brain I/R induced increased expression of GFAP,C3 and S100A10 in mouse hippocampal tissue（P<0.01,compared with the WT sham-operated group）;LPS treatment in mouse hippocampal tissue promoted brain I/R-induced increase in GFAP and C3 proteins and decreased S100A10expression（P<0.05,compared with the brain I/R R group）;whereas H₂S had an inhibitory effect on brain I/R-induced GFAP and C3 expression in hippocampal tissue,with or without LPS treatment.In addition,brain injury-induced increases in GFAP and C3 proteins were more significant in CSE KO mice than in WT mice（P<0.05）;Na HS treatment inhibited the expression of GFAP and C3 but promoted the expression of S100A10 after brain injury in CSE KO mice.4.Immunofluorescence staining showed that LPS upregulated brain I/R-induced increase in the number of GFAP-positive cells and C3-positive cells in hippocampal tissues of mice.Na HS treatment inhibited brain I/R-induced upregulation of GFAP-positive cells and C3-positive cells in hippocampal tissues of mice and promoted the upregulation of positive cell number of S100A10.5.In cellular experiments,Western blot results showed that OGD/R upregulated GFAP and C3expression in astrocytes,which could be inhibited by the exogenous H₂S donor Na HS.H₂S treatment also promoted S100A10 expression and increased astrocyte activity and H₂S content in the culture supernatant after OGD/R injury.Although 100 nmol/L IBTX did not affect OGD/R-mediated proliferation of reactive astrocytes,nor did it affect the effect of Na HS on C3,S100A10 and GFAP expression in astrocytes.However,OGD/R reduced the expression of the BK_Cachannel alpha subunit in astrocytes,while supplementation with H₂S promoted its expression,and the positive control drug Fasudil had similar efficacy.In addition,H₂S upregulated the expression of BK_Cachannel alpha subunit,protein kinase A（PKA）,while IBTX had no effect on this.6.In cell experiments with the addition of the BK_Cachannel opener BMS-191011,OGD/R-induced upregulation of GFAP and C3 expression in astrocytes could be inhibited by BMS-191011.BMS-191011 had no significant effect on altered astrocyte viability after ODG/R,but upregulated S100A10 in OGD/R astrocytes and control cells.cells and control cells.Conclusion:1.H₂S inhibits the proliferation of A1 astrocytes induced by neuroinflammation after brain I/R and promotes the conversion of reactive astrocytes to the A2phenotype.2.The mechanism of H₂S-mediated phenotype conversion of reactive astrocytes may be related to the upregulation of BK_Cachannels.