| Objective:Glutamate induced neuronal excitotoxicity may contribute to the formation and development of Sepsis-associated encephalopathy(SAE),also known as sepsis encephalopathy(SE).The aims of the study are to investigate the protective effect of microRNA-219(miR-219)on the hippocampal neurons against glutamate induced excitotoxity injury,and to explore preliminarily its mechanisms via target gene of miR-219.The research may provide new ideas and basic experimental data for the future exploration of complex pathogenesis of SAE.Part One:The protective effect of miR-219 on the hippocampal neurons injuried by glutamate in ratMethods:(1)The primary hippocampal neurons were obtained from E18-E19 SD rat.The cultured neurons were identified by immunofluorescence cytochemistry with anti-NF-200 antibody.(2)The in vitro cell model of glutamate-induced hippocampal neuron injury was established.The groups were decided as different glutamate concentrations or action time.(3)MTT assay was used to analyze the cell viability of the cultured hippocampal neurons induced by glutamate.(4)qRT-PCR(Quantitative Real-time PCR)was applied to measure the expression of miR-219 in the hippocampal neurons induced by glutamate.(5)After transfection with mir-219 mimic into hippocampal neurons,qRT-PCR was used to analyze the overexpression of mir-219 in hippocampal neurons.And MTT was applied to analyze the effect of overexpression of mir-219 on the cell viability of hippocampal neurons treated with glutamate.(6)Flow cytometry(FCM)was used to investigate the role of miR-219 in the cell apoptosis in the hippocampal neurons treated with glutamate.Results:(1)We successfully cultured primary rat hippocampal neurons.And the identification results showed that the purity of hippocampal neurons was over 90%,which met the requirements of subsequent experiments.(2)After primary cultured hippocampal neurons were damaged by glutamate,the result of MTT showed the activity of hippocampal neurons in all experimental groups decreased,with statistically significant differences from the normal group(p<0.05).And,the decrease of cell viability of hippocampal neurons was presented a dependent relation with glutamate concentration and action time.According to the above results,the glutamate concentration of 125?M and the action time of 15min were selected for the subsequent excitatory toxicity injury model.(3)After primary cultured hippocampal neurons were damaged by glutamate,the result of qRT-PCR showed that the expression of miR-219 in all experimental groups was dropped,with statistically significant differences from the normal group(P<0.05).And,the expression of miR-219 in hippocampal neurons was showed a dependent relation with the dose of glutamate.(4)After transfection with a mir-219 mimic into hippocampal neurons,the result of qRT-PCR showed that the expression of miR-219 in the miR-219 mimic transfection group was increased,with statistically significant differences from the normal control group and negatice control(mimic control)group(P<0.01).(5)After transfection with a mir-219 mimic into hippocampal neurons and treated with glutamate,the result of MTT showed that the cell viability of hippocampal neurons in miR-219 mimic transfection group was increased,with statistically significant differences from the negative control(mimic control)group(P<0.05).(6)After transfection with a mir-219 mimic into hippocampal neurons and treated with glutamate,the FCM results revealed that the percent of apoptitic cell of hippocampal neurons in glutamate treatment group was increased,with statistically significant differences from the normal control group(P<0.01).And,the percentage of apoptitic cell of hippocampal neurons in miR-219 mimic transfection group was decreased,with statistically significant differences from the negative control(mimic control)group(P<0.01).Conclusion:(1)The primary rat hippocampal neurons were cultured successfully,and the in vitro cell model of glutamate-induced hippocampal neuron injury was established.(2)After glutamate-induced injury,the expression of miR-219 was decreased in hippocampal neurons.(3)Transfection with mir-219 mimic into hippocampal neurons can increase the expression of mir-219,And,overexpression of mir-219 alleviated glutamate-induced cell viability decrease and apoptosis increase in hippocampal neurons,which indicated that miR-219 has a protective effect to neuroexcitatory toxic.Part Two:The preliminary study about miR-219 in protecting against glutamate-induced excitotoxityMethods:(1)Targetscan(http://www.targetscan.org/)was used to find target genes of miR-219 and to choose appropriate target gene.(2)The recombined luciferase CAMK?? expression vector and its mutant were constructed.The luciferase reported gene system was applied to identify that CAMK??was a direct target gene of miR-219.(3)Western blot was applied to investigate the effect of miR-219 overexpression on the CAMK?? expression in hippocampal neurons.(4)After glutamate-induced injury,qRT-PCR was used to analyze the expression of CAMK?? mRNA in hippocampal neurons.(5)After glutamate-induced injury,ELISA was used to measure the effect of overexpression of miR-219 on the activity of Caspase-3 in hippocampal neurons.(6)After glutamate-induced injury,Western blot was used to detect the effect of overexpression of mir-219 in hippocampal neurons on the expression of apoptosis-related proteins(Bax and bcl-2).(7)The recombined CAMK?? expression vector(pcDNA3.1-CAMK??)was constructed by molecular clone technology.The Western blot was used to detect the expression of CAMK?? in hippocampal neurons transfected with recombined vector pcDNA3.1-CAMK?? for 48 hours.(8)The rescue experiment was applied to identify that miR-219 has a protective effect to glutamated-induced injury in hippocampal neurons by regulating the expression of CAMK??.(9)Western blot was used to test the cell signal pathway of miR-219 in the protecting against glutamated-induced injury in hippocampal neurons.Results:(1)The predict result of TargetScan showed that there were 212 direct target genes controlled by miR-219.The interesting target gene we selected in these predict target genes was CAMK??.(2)The result of luciferase reporter gene system showed that the activity of luciferase co-transfected with mir-219 mimic and CaMK?? wild type was decreased,with statistically significant differences from the negative control(mimic control)group(P<0.01).The other groups had no change.(3)Western blot revealed that CAMK?? expression in the miR-219 mimic transfection group was dropped,with statistically significant differences from the normal control group and negative control(mimic control)group(P<0.01).(4)After glutamate-induced injury,the result of qRT-PCR showed that the expression of CAMK?? mRNA in low dose(50 ?M)group with glutamate treatment was increased,with statistically significant differences from the normal control group(P<0.05).And,the expression of CAMK?? mRNA in middle dose(125 ?M)group and high dose(250 ?M)group with glutamate treatment was increased more significantly(P<0.01).The expression of CAMK? mRNA in hippocampal neurons was showed a dependent relation with the dose of glutamate.(5)The result of ELISA showed that the activity of Caspase-3 in glutamate treatment group was increased,with statistically significant differences from the normal control group(P<0.01).The activity of Caspase-3 in the miR-219 mimic transfection group was reduced,with statistically significant differences from the negative control(mimic control)group(P<0.01).(6)The result of Western blot showed that the bcl-2/Bax ratio in hippocampal neurons was reduced after 125 M glutamate injury,with statistically significant differences from the normal control group(p<0.01).The bcl-2/Bax ratio in mir-219 mimic transfection group was increased,with statistically significant differences from the negative control(mimic control)group(p<0.05).(7)The result of Western blot in rescue experiment showed that the expression of CAMK?? in the recombined expression vector(pcDNA3.1-CAMK??)was increased,with statistically significant differences from the normal control group and the negative control(vector control)group(P<0.01).However,the expression of CAMK?? in the hippocampal neurons co-transfected with pcDNA3.1-CAMK?? and miR-219 mimic was reduced,with statistically significant differences from the pcDNA3.1-CAMK??transfection group(P<0.01).(8)The result of MTT assay in rescue experiment showed that the cell viability of all glutamate treatment groups was reduced,with statistically significant differences from the normal control group(P<0.01).However,after glutamate treatment,the cell viability of co-transfection group(pcDNA3.1-CAMK?? and miR-219 mimic)and vector transfection group(pcDNA3.1)was increased with statistically significant differences from the pcDNA3.1-CAMK?? transfection group(P<0.05).(9)The result of Western blot showed that the p-Akt/t-Akt in the hippocampal neurons treated with glutamate was increase,with statistically significant differences from the normal control group(p<0.05).After glutamate and PI3K/Akt inhibitor treatment,the expression of p-Akt was significantly decreased(p<0.01).After glutamate treatment,the expression of p-Akt in miR-219 mimic transfection group was increased,with statistically significant differences from the negative control(mimic control)group(p<0.01),and there was no significant change in the expression of t-akt(p>0.05).Conclusion:(1)The CAMK?? was the target gene of miR-219 and the expression of CAMK??was inhibited by miR-219 in hippocampal neurons.(2)After glutamate-induced injury,the expression of CAMK?? mRNA was increased significantly in hippocampal neurons.In hippocampal neurons,miR-219 played a role of protecting against glutamated-induced injury via regulating the expression of CAMK??.(3)After glutamate-induced injury,mir-219 can inhibit the activity of caspase-3 enzyme and increase the bdl-2/Bax ratio to reduce the cell apoptosis induced by glutamate.(4)MiR-219 played a protective role against glutamated-induced injury in hippocampal neurons via the PI3K/Akt cell signaling pathway. |
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