The Experimental Research About The Effect Of Ketamine On The Expressions Of Microrna-206and BDNF Protein Of Cultured Hippocampus Neurons

Background and Objective: In recent years, researchersreported that ketamine exerted a quick, remarkable, and persistentantidepressive effect, and found that the expression of brain-derivedneurotrophic factor (BDNF) induced by ketamine was one of mechanismsabout ketamine’s antidepressant-like action, but it was still not clearthat how ketamine to induce the expression of BDNF. Recent studiesfound that microRNAs(miRNAs) bind to the3-untranslated region oftheir mRNA targets and negatively regulate gene expression viadegradation or translational inhibition, and miRNAs are involved in theregulation of almost all major cellular functions including normaldevelopment, physiology and disease. It is unknown that whether theexpression of BDNF induced by ketamine is regulated by miRNAs.Computer analysis and other related researches found thatmicroRNA-206(miR-206) may obviously regulate BDNF proteinexpression, and our former miRNA chip test and animal experimentsindicated that ketamine resulted in the obvious down-regulation ofmiR-206expression and up-regulation of BDNF expression in rathippocampus. Therefore the main purpose of this study is to furtherinvestigate the effect of different concentrations of ketamine on miR-206and BDNF expression in cultured hippocampal neurons. Methods: Thenewborn SD rats’ hippocampus were separated, and primary hippocampalneurons were cultured. Primary hippocampal neurons cultured for7dayswere randomly divided into four groups, including normal control，50，100and300μM ketamine group. The cultured hippocampal neurons inthe50，100and300μM ketamine group were treated with50，100or 300μM ketamine (final concentration), respectively; The RNA wasextracted at0.5，1，3，6，12h after treatment; After ketamine treatmentfor6hours, normal culture solution were used and the total proteins wereextracted at24h and48h after treatment; The changes of neuronal miR-206, BDNF mRNA and BDNF protein relative expression levels in eachgroups were tested by qRT PCR and Western Blot, respectively. Repeatedthree times/time point/group. Results:(1) The relative expression levelsof miR-206and BDNF mRNA within normal control or300μM ketaminegroup and between normal control and300μM ketamine group showedno statistical difference(P>0.05);(2) The relative expression levels ofmiR-206and BDNF mRNA within50or100μM ketamine group hadobvious statistical difference (P <0.05). Further analysis by the S-N-Ktest revealed that the relative expression levels of miR-206reducedgradually at1,3,6,12h after50or100μM ketamine treatment (P <0.05),while the relative expression levels of BDNF mRNA significantlyincreased at3,6,12h (P <0.05) after50or100μM ketamine treatment;(3)The relative expression levels of miR-206and BDNF mRNA at each timepoint between50and100μM ketamine group had no statistical difference(P>0.05).(4) The relative expression levels of BDNF protein at24or48h in the four groups showed statistical difference (P <0.05). Furtheranalysis by the S-N-K test revealed that the relative expression levels ofBDNF protein in50and100μM ketamine groups were obviously higherthan that in normal control group (P <0.05). Conclusions:1.50or100μM ketamine could decrease miR-206, and increase BDNF-mRNAexpression levels within12h, the peak value of miR-206and BDNFmRNA expression levels appeared at6-12h after ketamine treatment，andmiR-206and BDNF-mRNA expression levels had no differences between50and100μM ketamine group.2.50or100μM ketamine induced BDNF protein expression of cultured hippocampal neurons at24and48h aftertreatment.