The Influence Of Orexin-A On Hippocampus Neurons And The Mechanism

ObjectiveOrexin-A influence on apoptosis of hippocampus and mechanism of action.Observed in rat hippocampal nerve cells,the Orexin-A combined with its receptors,can increase or decrease apoptosis,the damage to cells play A role or protection.Whether through OX1R/OX2R-Gq-PLC?-1 cell signaling pathways or OX1R/OX2R-Gq-PLC?-4,signaling pathways,activate the extracellular signal regulating kinase phosphorylation of ERK1/2.Methods1.The cultivation of the nerve cells In the hippocampus in 36 born within 24 h of Wistar rats,disinfection,beheaded,acute hippocampal tissue separation.Take meninges and blood vessels,cut up,trypsin digestion,termination of digestion,percussion,filtration,centrifugation,planting,cell count.2.SiRNA design According to the reported in Genbank rats of PLC?-1,PLC?-4mRNA sequence,design for PLC?-1 and PLC?-4 gene sequence of siRNA template.3.Slow virus transfection In 3 days after nerve cells,machine is divided into normal control group(CON),empty virus group(LV-control-siRNA),siRNA1,siRNA2 and siRNA3 group.For 95% of the nerve cells together,join the LV-control-siRNA respect ively,LV-PLC?-1-siRNAl,LV-PLC?-1-siRNA2,LV-PLC?-1-siRNA3;LV-control-siRNA respectively,LV-PLC?-4-siRNAl,LV-PLC?-4-siRNA2,LV-PLC?-4-siRNA3.Transfection ratio(MOI)of 5.Three days later,use the inverted fluorescence microscope to observe the green fluorescent protein expression.4.rt-PCR to detect PLC?-1,PLC?-4 RNA interference efficiency.Detecting the gene expression of every group,choose a quiet efficiency highest siRNA,respectively for subsequent formal experiment.Synthesis cDNA extracted total RNA,reverse transcription,remove genomic DNA,the reverse transcription reaction,PCR.5.Orexin-A intervention Three days after slow virus transfection intervention,eachgroup is divided into with Orexin-A subgroup(n=6)and withnot Orexin-A subgroup(n=6).A total of six subgroups.Join Orexin-A,and the final concentration is 100 nmol/L.6.Flow cytometry technology to detect the apoptosis rate of nerve cells After Orexin-A intervention for 48 h,detect the apoptosis rate.7.Western Blot to determinate the expression of phosphorylated extracellular signal regulating kinase 1/2(p-ERK1/2)After Orexin-A intervention for 48 h,extract total protein,BCA kit to detect protein concentration.Gel preparation polypropylene phthalein amine(sds-page),transfer membrane,wash membrane,sealing,wash membrane,a fight incubation,wash membrane,second incubation,wash membrane,enhanced chemiluminesc ence ECL,compare the quantity of protein expression.Results1.The cultivation of the nerve cells in the hippocampus and transfection: of three grou ps of cells in the hippocampus nerve cell purity>90%,three groups of cells transf ectioneff iciency>90%.2.rt-PCR showed: the PLC?-1 and PLC?-4 siRNAl,siRNA2 and siRNA3 all can be reached the standard of inhibition of gene expression.Among them,the siRNAl in PLC?-1,the siRNA3 in PLC?-4,the inhibition of gene expression,the most efficient to use both for follow-up experiments of transfection.3.Flow cytometry showed: empty virus within the group with Orexin-A subgroup of hippocampal nerve cells apoptosis rate is lower than that without Orexin-A subgroup(P <0.001);the PLC?-1 with Orexin-A subgroup of hippocampal nerve cells apoptosis rate and without Orexin-A subgroup have no significant statistical difference(P>0.05);the PLC?-4groups with Orexin-A subgroup of hippocampal nerve cells apoptosis rate is lower than that of without Orexin-A subgroup(P<0.01).4.Western blot showed: empty virus within the groups with Orexin-A subgroups of the expression of ERK1/2 are significantly higher than those without Orexin-A subgroups(P < 0.001);the expressions of ERK1/2 of PLC?-1 groups with Orexin-A subgroup and without Orexin-A subgroup have no significant difference(P>0.05);the expressions of ERK1/2 of the PLC?-4 groups with Orexin-A subgroup are significantly higher than those without Orexin-A(P<0.001).Conclusion1.Orexin-A in 100nmol/L concentrations can reduce the apoptosis of nerve cells in the hippocampus of hippocampal nerve cells.It has a certain protective effect for hippoc ampal nerve cells.2.Orexin-A can significantly activated phosphorylation of nerve cells in the hippocampus of ERK1/2.Orexin-A rely mainly on the function of nerve cells in the hipp ocampus OX1R/OX2R-Gq-PLC?-1 signaling pathway,while the OX1R/OX2R-GqPLC?-4 signali ng pathways is not the main way.Blocking OX1R/OX2R-Gq-PLC?-4 sign aling pathway will affect the role of Orexin-A in a cell.It provides a new therapeutic target to treat sleep disease such as insomnia and narcol epsy and the damage of learning and memory from them.In the future,by interve ning the signal path,we can influence the role of Orexin-A.