The Effect And Mechanism Of Dexmedetomidine On Mouse Brain-derived Astrocyte Oxygen Glucose Deprivation And Re-oxygenation Injury

Background: Stroke is the second most fatal disease and one of the major causes of long-term disability in the world.Previous studies on stroke have focused on neuronal protection,while astrocyte,which account for 50% of brain volume,has not been fully investigated.Dexmedetomidine（DEX）is a sedative drug commonly used in peri-anesthesia period,and its protective effect on the brain has been reported already.However,there are few studies about its effects on astrocytes in cerebral ischemia reperfusion injury（CIRI）,and its specific mechanism is still not fully understood.Objective: To investigate the role of dexmedetomidine in astrocytes oxygen glucose deprivation/re-oxygenation（OGD/R）injury and its molecular mechanism.Methods: In our experiment,OGD/R model of astrocytes was used to simulate cerebral ischemia-reperfusion injury in vitro,then detect the changes of corresponding indicators.1.Set up the primary astrocytes culture.C57BL/6 mice were decapitated after postnatal day 1-2,then the cerebral cortex tissue was isolated under stereomicroscope,and the primary astrocytes were extracted and cultured.The purity of astrocytes were identified by GFAP immunofluorescence staining.2.To detect the effects of different time point of hypoxia on the viability of primary astrocytes.Primary astrocytes were randomly divided into 5 groups（n=5 for each group）,namely control group,OGD4h/R24 h,OGD6h/R24 h,OGD12h/R24 h and OGD24h/R24 h,hypoxia treatment for 4h,6h,12 h and 24 h separately,with the same re-oxygenation time of 24 h according to published articles.The effect of different periods of hypoxia on the viability of astrocytes was observed by using the CCK-8 assay,then choose the most appropriate time point.3.The effects of different concentrations of dexmedetomidine on the normal growth of primary astrocytes were detected.Primary astrocytes were randomly divided into 5 groups（n=5 for each group）,namely control group and the cells which were treated with dexmedetomidine at the concentration of 0.1,1,10 and 50μM for 24h.CCK-8 assay was used to detect the cell viability.Then we compared the toxicity of different concentrations of DEX to the normal growth of astrocytes,so as to determine the optimum concentration DEX for the subsequent experiment.4.To detect the effects of dexmedetomidine at different concentrations on OGD/R injury of primary astrocytes.Primary astrocytes were randomly divided into 5 groups（n=5 for each group）,namely control group,OGD/R group,dexmedetomidine group with 0.1,1 and 10μM were added at the beginning of re-oxygenation.Cell viability was detected by CCK-8 method.Compared with the control group,the effects of different doses of DEX on astrocyte activity were observed.5.To detect the effects of dexmedetomidine on the expression of inflammatory factors in primary astrocyte OGD/R injury.Primary astrocytes were randomly divided into 3 groups,namely control group,OGD/R group and DEX group.Total RNA was extracted after 24 h re-oxygenation,and the m RNA relative expression levels of IL-6,TNF-α and IL-1β were detected by real-time quantitative polymerase chain reaction（RT-qPCR）assay.6.Effects of dexmedetomidine on expression of Hypoxia inducible factor-1α（HIF-1α）and apoptotic protein in OGD/R injury of astrocytes.Primary astrocytes were randomly divided into three groups,namely control group,OGD/R group and DEX group.When the cellular re-oxygenation was completed the total cellular proteins were extracted and the expression levels of HIF-1α,BAX and Bcl-2 were determined by Western Blot assay.7.Effects of IOX2,the inhibitor of HIF-1α prolyl hydroxylase-2 which can acuumulate HIF-1α in the cytosol,on normal cultured astrocytes and OGD/R astrocytes,as well as the protein level of HIF-1α and apoptotic proteins.Results :1.After 11 days of culture,the growth characteristics under the microscope of the cultured cells were found to conform with the primary astrocyte reported previously.GFAP immunofluorescence staining confirmed that the cultured cells were astrocytes with a purity of 95%,which met the experimental requirements.2.The viability of astrocytes decreased to 86%（v.s CON,p<0.05）after 4h OGD,and to 56.8%（v.s CON,p<0.001）after 6h OGD.The viability of astrocytes decreased time-dependently.After 24 h,the viability had decreased to 28.8%（v.s CON,p<0.001）.Therefore,OGD6 h when half of the cells survived was selected as the optimal hypoxic time point for the subsequent experiment.3.CCK-8 assay showed that dexmedetomidine at 0.1,1 and 10μM had no significant effect on the viability of normal cultured astrocytes（v.s CON,p>0.05）,while dexmedetomidine at 50μM significantly decreased the viability of normal cultured astrocytes（v.s CON,p<0.001）.These results indicated that high concentrations of dexmedetomidine produced cytotoxicity to normal cultured astrocytes,thus it was abolished.4.The cell viability of astrocytes decreased significantly after OGD6h/R24h（v.s CON,p<0.001）,but reversed after been treated with dexmedetomidine at 0.1,1,10μM（v.s CON,p<0.001）,in which 1μM was better compared to 0.1 μM DEX treatment,but no further improvement was observed under 10 μM DEX treatment.Therefore,1μM DEX was selected as the optimal concentration for subsequent administration.5.RT-qPCR results showed that the m RNA levels of TNF-α,IL-6 and IL-1β in astrocytes after OGD6h/R24 h were significantly increased（v.s CON,p<0.001）,while the m RNA levels of these inflammatory factors were significantly reversed after dexmedetomidine treatment at 1μM concentration（v.s OGD/R,p<0.05）.6.Western Blot results showed that HIF-1α protein level was significantly increased after OGD/R（v.s CON,p<0.001）,while dexmedetomidine at 1μM concentration significantly increased its expression level（v.s OGD/R,p<0.001）.7.IOX2 had no distinct influence on the viability of regularly cultured astrocytes（v.s CON,P >0.05）.However,it can reverse the improvement of dexmedetomidine on cell survival rate after OGD/R injury,and can reverse the expression level of HIF-1αand apoptotic protein.Conclusion: Dexmedetomidine at 1μM concentration significantly reduced the injury of primary astrocytes caused by hypoxia re-oxygenation through increasing cell viability after OGD/R and inhibiting inflammatory factor TNF-α The expression of IL-1β and IL-6 m RNA,inhibiting the expression of apoptotic protein BAX and promoting the expression of anti-apoptotic protein Bcl-2,and the protective effect of dexmedetomidine may be related to the inhibition of HIF-1α protein expression,thereby reducing the expression of apoptosis and inflammatory factors.