| Objective: Explore the role of DREAM in cerebral ischemia-reperfusion injury and the underlying mechanism of DREAM-mediated modulation of TRPM7 in the process of cerebral ischemia-reperfusion injury.Methods:1)The model of SD rat with a cerebral ischemia-reperfusion injury(I/R)was established;2)Adult SD rats were randomly divided into Normal group and I/R group.The I/R group were further randomly divided into 6 groups,which were 0h,4h,24 h,48h,72 h and 1w group.RT-PCR and Western blotting were used to detect the expression of DREAM mRNA and protein in the infarct core area and surrounding penumbra tissue;3)Adult SD rats were randomly divided into Normal group,I / R group,I / R + Repaglinide(RP)0.5mg/kg group,and I / R + RP 1mg/kg group.They were treated with saline,Vehicle,RP(0.5 mg/kg),RP(1 mg/kg)by intraperitoneal injection respectively,Neurobehavioral function was evaluated at 1st day,3nd day,5th day,7th day after operation with modified Garcia scoring criteria.The rats were sacrificed and their brains were processed on the 7th day after operation,TTC staining was used to evaluate cerebral infarction volume.The expressions of DREAM and apoptosis associated factor Bcl2 and Bax were detected by RT-PCR and Western blot respectively;4)In vitro,immunofluorescence staining was applied to detect expression and localization of DREAM protein in primary neurons;5)We established the model of primary cortical neurons with oxygen and glucose deprivation and reoxygenation(OGD/R)in vitro and detected expression of DREAM by RT-PCR and Western blotting;6)Normal primary neuron and OGD/R neuron were treated by RP in vitro and the expression of DREAM was detected by RT-PCR and Western blotting respectively;7)DREAM + TRPM7,DREAM + TRPM7-N-terminus and DREAM + TRPM7-C-terminus were transient transfected into the HEK293 system in vitro,and coimmunoprecipitation technology was used to investigate whether DREAM binds to TRPM7 and where is binding site.Results: 1)In the model of SD rat with a cerebral I/R,the DREAM expression significantly decreased during 24h~1week after I/R(p <0.05);2)In vivo compared with the I/R model group,the I/R+RP 1mg/kg group had a reduced infarct volume(p<0.05),a significant improvement of neurobehavioral score(p <0.05),a significantly increase of DREAM expression(p <0.0001),and a significantly increase of the value of Bcl2/Bax(p<0.05);3)In vitro DREAM is stably expressed in neurons and astrocytes,and its distribution was mainly in cytoplasm and dendrite of neuron;4)In vitro the expression of DREAM in primary neuron significantly decreased cerebral at 24 h after OGD/R(p <0.0001);5)Small molecule,repaglinide,upregulated the expression of DREAM in normal neurons and OGD/R neuron in vitro;6)Co-immunoprecipitation experiments in HEK293 cell line in vitro confirmed that DREAM interacted with TRPM7 and the binding site was located on the N-terminal and C-terminal of TRPM7 in the neuron cytoplasm.Conclusion: 1)The expression of DREAM significantly decreased during 24 h ~ 1w after cerebral ischemia-reperfusion,which demonstrated that DREAM was involved in the process of cerebral ischemia-reperfusion injury;2)Repaglinide upregulated the expression of DREAM in vitro and played neuroprotective effects on cerebral ischemia-reperfusion injury in vivo;Co-Immunoprecipitation confirmed that DREAM interacted with TRPM7.All above results indicated the possible mechanism of DREAM mediated modulation of TRPM7 in the process of cerebral ischemia-reperfusion injury. |
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