Effect Of Dexmedetomidine Pretreatment On HMGB1 Expression In LPS-stimulated Astrocytes

Background:Neuroinflammation,as an important pathological feature of neurodegenerative diseases,mainly involves the release of inflammatory cytokines after activation of glial cells in the central nervous system?central nervous system,CNS?,which triggers an inflammatory cascade amplification effect.Presently,researches on neuroinflammation are mainly concentrated on microglia.Astrocytes?AS?,the main immune cells in CNS,are in close contact with other tissues in the brain and plays a key role in the neuroimmunological system.It is reported that dexmedetomidine?DEX?reduces the level of pro-inflammatory cytokines in endotoxemia model and ischemia-reperfusioninjury possibly through alpha 7 nicotinic acetylcholine receptor??7nAChR?.Objective:To investigate the effect of DEX pretreatment on HMGB1expression in LPS-stimulated AS,and to preliminarily explore the underlying mechanisms concerning with?7nAChR.However,whether DEX could affect HMGB1 expression in LPS-activated AS remains unknow.Methods:1.Separation,culture,purification and identification of astrocytes from both sides cerebral cortexes of SD rats.?1?Astrocytes separation and culture.Cell cultures were prepared from cerebral cortex of 1-day-old SD rats.The tissues were obtained under aseptic conditions and completely dissected the meninges and blood vessels under an anatomical microscope.The single cell suspension was gained by digestion with0.25%trypsin-0.02%EDTA solution for 15 min at 37?combining mechanically repeated pipetting.Cells were resuspended in DMEM containing10%FBS and antibiotics.Cells were cultured in a humidified 5%CO₂,95%air incubator.The culture medium was changed every 2³ days.?2?Astrocytes purification.A differential attachment method was utilized to remove the fibroblasts in the single cell suspension.When the cells reached confluence,the flasks were shaken at 170 rpm overnight at 37?to segregate the microglial and oligodendrocytes.?3?Immunohistochemical detection of glial fibrillary acidic protein?GFAP?.The immunofluorescence staining protocol was used to determine the expression of glial fibrillary acidic protein?GFAP?in astrocytes.2.The influence of DEX pre-treatment on HMGB1 expression of LPS-stimulated astrocytes.Cells were pre-treated with 1?mol/L DEX and/or10 nmol/L?-BGT??-bungarotoxin,specific antagonist of?7nAChR?for 30min,then incubated with different concentrations of LPS?0.1,1,10?g/mL?for24 h.MTT method was applied to detected cell viability.Expression levels of hmgb1 mRNA were measured by real-time polymerase chain reaction?RT-PCR?.Western blot was carried out to detect HMGB1 protein expression level.Results:1.The purity of SD rat cerebral astrocytes identified by immunofluorescence staining with anti-GFAP antibody showed that the proportion of astrocytes in the second generation reached above 95%.2.The influence of DEX pre-administration on the expression of HMGB1.?1?The MTT assay results showed that cell relative viability was not affected by 1?mol/L DEX and/or various concentrations of LPS?0.1,1 and 10?g/mL?treatment for 24 h.?2?hmgb1 mRNA expression level detected by RT-PCR.It showed that LPS?high than 0.1?g/mL?markedly induced hmgb1 mRNA expression in astrocytes.The level of hmgb1 mRNA peaked at 12 h after 1?g/mL LPS incubation for 24 h.hmgb1 mRNA expression level was obviously increased after LPS?1?g/mL?administration for 12 h,which was significantly alleviated by DEX pretreatment for 30 min.While?-BGT,an antagonist of?7nAChR,abolished the effect of DEX pre-administration?DEX pretreatment group VS?-BGT pretreatment group,P<0.05?.?3?HMGB1 protein expression level detected by western blot.Compared with the control group,the expression level of HMGB1 protein in LPS group was significantly increased?P<0.05?.DEX pretreatment significantly reduced HMGB1 protein level?DEX pretreatment group VS LPS group,P<0.05?,which was abolished by?-BGT pretreatment?DEX pretreatment group VS?-BGT pretreatment group,P<0.05?.Conclusion:DEX pretreatment mediated decreased level of HMGB1 via?7nAChR is one of the mechanisms underlying DEX anti-inflammation.