The Mechanism Of Fluoxetine Improving Behavioural Deficits Induced By Alcohol Treatment Via Alleviating RNA Editing Of 5-HT_2C Receptors

Objective:Alcohol addiction is a kind of mental disorder caused by long-term drinking.Alcoholism and major depression are often co-morbid and also increase each other susceptibility,but the mechanism is still unclear.It has been reported that selective serotonin re uptake inhibitors（SSRIs）have therapeutic effects on depressive symptom and reducing alcohol intake of alcoholism.Other researches indicated that reduction of ATP release from astrocyte in brain can aggravate depressive-like behaviors of mice.But,the impact of alcohol on ATP release from astrocytes is unclear.In this study,we investigated the effects of alcohol treatment on protein expression of astrocytes,the effects of alcohol exposure on ATP release from astrocytes,and mechanisms of regulation of fluoxetine to ATP release from astrocytes through RNA editing of 5-HT_2CR on astrocytes,to explore pharmacological mechanism of fluoxetine on alleviating physical and mental symptoms caused by alcohol.Methods:1.Extraction and culture of primary astrocytes.2.Western blot was used to detect the expression of 5-HT_2CR,5-HT_2BR,cFos and ADAR2.3.Selective inhibitors were used to detect the signal transduction pathway of fluoxetine on astrocytes.4.The effect of alcohol and fluoxetine on the rate of 5-HT_2CR RNA editing was detected by gene sequencing.5.The effects of alcohol and fluoxetine on ATP release of astrocytes were detected by using 5-HT_2CR specific agonist MK212 and ATP detection kit.6.CD1mice were randomly divided into two groups---alcohol model group and control group.After the alcohol model was finished,the mice were randomly divided into four groups---control group,alcohol group,fluoxetine treatment group and alcohol plus fluoxetine treatment group.7.The effects of alcohol and fluoxetine on the behavior of mice were tested by behavioral experiments.Results:1.Alcohol can increase the expression of cFos and ADAR2 in a dose-dependent way.2.4 mg/ml alcohol and 1μm fluoxetine did not affect the expression of5-HT_2CR and 5-HT_2BR in astrocytes.Fluoxetine could inhibit the expression of cFos and ADAR2 in astrocytes of the control group and alcohol treated group.3.Fluoxetine inhibited cFos and ADAR2 through 5-HT_2BR/Src/EGFR/Akt pathway.4.Alcohol can increase the editing rate of D site on 5-HT_2CR mRNA in astrocytes and mice,fluoxetine can reduce the editing rate.The effect of alcohol and fluoxetine on 5-HT_2CR mRNA editing disappears after ADAR2 si RNA interference.5.5-HT_2CR specific agonist MK212 can stimulate astrocytes to release ATP.After alcohol treatment,the effect of MK212 is weakened,and fluoxetine restored the effect of MK212 on ATP release.6.Alcohol induced increase of alcohol preference in mice,increase of anxiety like behavior in field test,depression like behavior in tail suspension test and decrease of motor ability in rotating rod test.Fluoxetine treatment restored behavioral abnormality of mice.Conclusion:In Astrocytes,activation of 5-HT_2CR can cause ATP release specifically,which can be reduced by alcohol via upregulated expression of ADAR2 and its RNA editing rate.Fluoxetine can reduce ADAR2 expression and recover function of 5-HT_2CR.These results may imply that the symptoms of alcohol addiction may be caused by upregulation of 5-HT_2CR RNA editing rate and reducing of ATP release from astrocytes.