Study On The Effect And Mechanism Of MiRAN-29b-3p/GRM4 Pathway In The Antidepressant Of Ketamine

Objective:Depression is a mental disease characterized on low mood,lack of pleasure,and sleep disorder.According to the World Health Organization,the number of people who suffer from the disease were approximately 350 million.Now,the treatment of depression is mainly traditional monoamine antidepressants,which have slow onset of treatment,long treatment period and inaccurate results.In recent years,a large number of clinical studies found that ketamine has a rapid onset of antidepressant effect,but its antidepressant mechanism remains unclear.In our previous study,our group found that the antidepressant effect of ketamine was related to regulation of the micro RNA-29b-3p(miR-29b-3p)and its target gene GRM4.Then in this study,we further investigated the effects of ketamine on the expression of miR-29b-3p/GRM4 in prefrontal cortex neurons,the effects of miR-29b-3p on neurons,and the effects of high expression of miR-29b-3p on the behavior of depression rats.The results may open up a new avenue and provide us potential therapeutic targets for depression treatment.Methods:1.We used quantitative polymerase chain reaction and western blot to examine the alteration of miR-29b-3p and GRM4 m RNA and protein in the primary rat prefrontal cortical neurons of 50 ?Mol ketamine treatment.2.After infecting the primary prefrontal cortical neurons by lentivirus overexpressed or inhibitied the expression of miR-29b-3p,we used MTT to test the activity of neurons,used the fluorescence microscope to detect the growth of dendrites in neurons,used Tunnel to detecte the apoptosis of neurons,used the patch-clamp to detect the influx of calcium ions in neurons and examined the concentration of glutamate in the intracellular and external fluid of neurons.3.We established chronic mild unpredictable model of depressive rats and constructed adeno-associated viruses(AAV)of overexpressed miR-29b-3p(AAV-miR-29b-3p)and control(AAV-GFP).We locally injected AAV-miR-29b-3p or AAV-GFP or NS into prefrontal cortex of depressive rat by stereotactic.After 28 days,we observed the behavioral change of depressive rat and the expressions of miR-29b-3p and GRM4 in the prefrontal cortex.Results:1.The expression of miR-29b-3p of primary neurons from prefrontal cortex increased significantly with time in 12 hours(P<0.05),while the expression of GRM4 m RNA decreased significantly(P<0.05)at 3h,6h and 12 h after ketamine treatment.The expression of GRM4 protein decreased significantly(P<0.05)at 6h and 12 h after ketamine treatment.2.Compared with low expression of miR-29b-3p,we found that the overexpression of miR-29b-3p of prefrontal cortical neurons could increase the neuron's viability(P<0.05),reduce the neuron's apoptosis(P<0.05),obtain better dendritic growth(P<0.05),decrease the concentration of intracellular excitatory glutamate(P<0.05)and increase the concentration of extracellular excitatory glutamate(P<0.05).3.Compared with the normal neurons from prefrontal cortex,the calcium ion concentration and the inward calcium current of patch-clamp analysis increased significantly(P<0.05).After using the GRM4 blockers(MSOP)or calcium channel blockers(P/Q,N and R),the inward calcium current were significantly reduced(P<0.05).And after using U73122(Phospholipase C inhibitor)or GF109203(Protease C inhibitor),the inward calcium current were also decreased significantly(P<0.05).4.After administration of over-expressed miR-29b-3p AAV into prefrontal cortex of depressive-like rat by stereotactic method,the behavioral of rats on the 28 st day showed that the depressive symptoms of the depression-like rats were significantly relieved(P<0.05).Meanwhile,we found that the miR-29b-3p expression from prefrontal cortex of depression-like rats were increased(P<0.05),but the expression of GRM4 m RNA and protein were decreased(P<0.05).Conclusions:1.Ketamine may increase the expression of miR-29b-3p and decrease the expression of GRM4 in the prefrontal cortical neurons.2.miR-29b-3p in prefrontal cortical neurons plays an important role in maintaining the normal biological function of neurons.3.The overexpression of miR-29b-3p in prefrontal cortex of depressive rat has antidepressant-like effects.