| Part ?:The expression of GATA-4 in neurons following intracerebral hemorrhage in ratsObjectiveTo investigate the expression of GATA-binding protein-4(GATA-4)in neurons after intracerebral hemorrhage(ICH)in rats.methods1.Group of experimental animals:42 healthy adult male Sprague-Dawley(SD)rats were used and randomly divided into 7 groups:sham group,ICH 6 hours group,ICH 12 hours group,ICH 24 hours group,ICH 48 hours group,ICH 72 hours group and the ICH 7 day group.with 6 rats in each group.2.Establishment of ICH model:Rats were deeply anesthetized,and autologous arterial blood were injected into the right basal ganglia.The rats were euthanized at 6 hours,12 hours,24 hours,48 hours,72 hours,and 7 days according to the experimental group,and the brain was perfused.3.ICH model In vitro:cerebral cortical of rat embryo were collected and the neurons were cultured,then randomly divided into 6 groups:control group,OxyHb 3 hours group,OxyHb 6 hours group,ICH 12 hours group,OxyHb 18 hours group,and ICH 24 hours group.OxyHb solution were added to mimic the ICH.according to the experimental design,collect neurons at predetermined time point.4.The polymerase chain reaction technique(PCR)was used to detect the changes of GATA-4 mRNA levels in rat brain at different time after ICH.Western blotting was used to detect the changes of GATA-4 levels in rat brains and neurons at different time points after ICH.The expression level of GATA-4 in neurons after ICH was analyzed by immunofluorescence.results1.The level of GATA-4 mRNA in brain tissue was increased after ICH in rats.Compared with the sham group,the expression of GATA-4 in brain reached the peak at 24 hours after ICH(P<0.01),and then began to decline,but in 72 hours after ICH,GATA-4 mRNA levels were close to the sha,group.2.Western blot analysis showed that the expression of GATA-4 protein in rats was peaked 24 hours after ICH compared with the sham group.In in vitro,the GATA-4 protein in neurons peaked 6 hours after OxyHb.3.immunofluorescence staining further confirmed that GATA-4 was expressed in neurons and increased in expression after ICH In vivo and vitro.ConclusionsGATA-4 was expressed in neurons and peaks 24 hours after ICH.It is suggested that GATA-4 may be involved in the development of neuronal damage after ICH.Part ?:Changes of GATA-4 effect on neurons after ICH in ratsObjectiveTo investigate the effect of GATA-4 on neurons after ICH in rats,and to determine whether neuronal apoptosis can be alleviated by regulating the expression of GATA-4.methods1.Group of experimental animals:126 health male SD rats were randomly divided into 7 groups(18 rats in each group):sham group,ICH group,ICH+negative control(NC)group,ICH+GATA-4 siRNA group,ICH+empty vector(EV)group,ICH+GATA-4 plasmid group,and ICH+ GATA-4(S105A)mutant group.At 24 hours after ICH,6 rats in each group were euthanized and collected from the cerebral hemisphere on the ICH-injured side for detection.Another 6 rats were randomly selected and euthanized to take paraffin sections.Then the else 6 rats in each group were used for testing neurobehavioral scores.2.ICH model preparation:Rats were deeply anesthetized,and autologous arterial blood were injected into the right basal ganglia.ICH+negative control(NC)group,ICH+GATA-4 siRNA group,ICH+empty vector(EV)group,ICH+GATA-4 plasmid group,and ICH+GATA-4(S105A)mutation group.ICH model were completed after 48 hours performed ventricle Transfection.3.The brain edema after SAH was measured and analyzed by dry and wet method.The neurobehavioral damage was detected by water maze.Fluoro-Jade B(FJB)staining was used to analyze the neuronal cell degradation after different groups of ICH.The apoptosis of rat cerebral cortex was analyzed by TUNEL staining.Immunofluorescence staining(IF)was used to investigate the expression of GATA-4 in neurons.Western-blot method was used to detect the content of GATA-4 in brain tissue.results1.Compared with the sham group,the brain edema and neurological behavior of rats after ICH was severely impaired.In the ICH+GATA-4 siRNA group,this edema and neurobehavioral defect was significantly attenuated after down-regulating the expression of GATA-4.In the ICH+GATA-4 group,the expression of GATA-4 was up-regulated,and the edema and neurobehavioral damage defects in rats were obvious significantly.In the ICH+GATA-4(S105A)mutant group,the expression of GATA-4 protein was up-regulated,but its phosphorylation of amino acid at position of 105 was prevented,and the edema and neurobehavioral impairment defect in rats was also significantly alleviated.2.TUNEL and FJB staining showed that neurons in the brain after ICH had apoptosis.In the ICH+GATA-4 siRNA group,neuronal apoptosis was significantly attenuated.In the ICH+GATA-4 plasmid group,neuronal apoptosis was significantly increased.However,in the ICH+GATA-4(S105A)mutant group,neuronal apoptosis was also significantly attenuated.3.Immunofluorescence of brain sections showed increased expression of GATA-4 in neurons after ICH,and decreased expression in ICH+GATA-4 siRNA group;in ICH+GATA-4 group and ICH+GATA-4(S105A)group,The expression of GATA-4 is increased.4.Western-blot analysis showed that in the ICH+GATA-4 siRNA group,the expression of GATA-4 was decreased;the expression levels of cleaved-caspase-3 and Bax protein were decreased.In the ICH+GATA-4 plasmid group,the expression of GATA-4 was significantly increased,and the expression levels of activated caspase-3 and Bax protein were also significantly increased.In the ICH+GATA-4(S105A)mutant group,the expression of GATA-4 was significantly increased,but the phosphorylation level of the protein was decreased,and the expression levels of cleaved-caspase-3 and Bax protein were decreased.ConclusionsKnockdown of GATA-4 protein expression by small Interfering RNA(siRNA)of GATA-4 protein can decrease Caspase-3 protein activation and Bax protein expression,and reduce neuronal apoptosis and degradation in rats;Overexpression of GATA-4 protein can increase the activation of Caspase-3 protein and the expression level of Bax protein,aggravate the apoptosis and degradation of neurons.After inhibiting the phosphorylation of serine at position 105 of GATA-4 protein,it can reduce Caspase-3 protein activation and Bax protein expression levels,reducing neuronal apoptosis and degradation.It is suggested that GATA-4 protein regulates Bax/Caspase-3 through its phosphorylation,thereby playing a role in neuronal apoptosis after ICH.Part ?:The Mechanism of GATA-4 involved in neuronal apoptosis after ICHObjectiveTo explore the molecular pathway and mechanism of GATA-4 regulation of neuronal apoptosis after ICHmethods1.ICH model In vitro:cerebral cortical of rat embryo were collected and cultured,transfected with siRNA and plasmid for 48 hours,and ICH in vitro model was established by treatment with oxyhemoglobin(OxyHb).Neurons were then collected and analyzed.2.Experimental grouping:First,the cultured neurons were divided into 6 groups:control group,OxyHb,OxyHb+ NC,OxyHb+GATA-4 siRNA,OxyHb+EV and OxyHb+GATA-4 plasmid.Next,the cultured neurons were divided into 5 groups:a control group,an OxyHb group,an OxyHb+ EV group,an OxyHb+GATA-4 plasmid,and OxyHb+GATA-4(S105A).3.The changes of GATA-4 and the expression of NF-?B/Bax in neurons were analyzed by Western-blot method.Cell apoptosis was measured by flow cytometry.results1.After knockdown or up-regulation the GATA-4,the expression of NF-?B,Bax and activated Caspase-3 protein in neurons decreased or increased.Neuronal apoptosis is also reduced or increased accordingly.2.The expression of GATA-4 protein is increased,but after inhibition of GATA-4 protein phosphorylation,the expression of NF-?B,Bax and cieaved-Caspase-3 protein in neurons is decreased,and neuronal apoptosis is also reduced.conclusionsGATA-4 induces neuronal apoptosis via the GATA-4/NF-?B/Bax/Caspase-3 pathway.The effect of GATA-4 on neurons depends on its phosphorylation.Decreasing GATA-4 expression or inhibiting its phosphorylation can reduce neuronal apoptosis. |
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