?-hydroxybutyrate Alleviates Depressive Behaviors In Mice Possibly By Increasing The Histone3-lysine9-?-hydroxybutyrylation

Objectives:To explore the role of histone3-lysine9-?-hydroxybutyrylation(H3k9bhb)in depression,we detecte the protein level of H3k9bhb in mouse model of depression and observe its changes after treatment.Methods:1.Two different mouse models of depression were established.The spatial restraint stress model:adult male ICR mice,weighted 20-25 gram(g),were randomly assigned into 2 groups,control group(Con)and the spatial restraint stress group(Str).The Str group mice were individually confined in a well-ventilated centrifuge tubes(50 ml)for 2 hours(09:00 AM-11:00AM)per day for consecutive 21 days.The Dexamethasone-induced mouse model:adult male ICR mice,weighted 20-25 gram(g),were randomly assigned into 2 groups:control group(Con)and the Dexamethasone-induced group(Dex).Mice were given intraperitoneal injection of dexamethasone(1mg/kg)every day(08:00 AM-9:00 AM)for consecutive 21 days.2.The assessment of depressive mouse models:Tail suspension test(TST),Sucrose preference test(SPT)and Forced swimming test(FST)were used to assess the success of depressive mouse models and behavior response to treatment.3.The spatial restraint stress model of depression were uesed for the assessment of treatment response.Both Con group and Str group were randomly assigned into three groups respectively,control group(Con + NS,Con + IMI,Con + BHB),stress and treatment group(Str + NS,Str + IMI,Str + BHB).Mice of different groups were given intraperitoneal injection of normal saline(NS),imipramine(IMI,20mg/kg)and ?-hydroxybutyrate(BHB,300mg/kg)once a day respectively for consecutive 21 days.4.The level of?-hydroxybutyrate of the brain in different groups were detected by an assay kit(?-Hydroxybutyrate Assay Kit).5.Western blot was used to detect the changes of expression of H3k9bhb,H3K9ac and BDNF in hippocampus,hypothalamus,prefrontal cortex,amygdala and cultured primary neurons respectively.6.Primary cortical neurons were cultured and deal with Dex and BHB.The neurons were arranged for four groups,Control group(Con,Con+ BHB),stress and treatment group(Dex,Dex+9BHB).Dexamethasone(20uM for 48h)was given at the 5th day in vitro,?-hydroxybutyrate(10mM,overnight)was given at the 6th day in vitro.7.Primary cultured neurons were exposed to different doses of ?-hydroxybutyrate(0mM,5mM,10mM,20mM)for about 24 hours at the 5th day and the protein level of H3k9bhb and H3k9ac was detected at the 6th day.8.To explore whether elevated endogenous ketone can improve the depression like behavior,another group of spatial restraint stress mice were arranged into four groups,control group(Con,Con + DR,Con + Ket,Con + Exe),stress and treatment group(Str,Str + DR,Str + Ket,Str+Exe).Normal diet(Con),Dietary restriction(DR),Ketogenic diet(Ket)and exercise(Exe)were geven respectively.TST,FST were used to assess the therapeutic effect of above methods.Results:1.Both of spatial restraint stress model of depression and Dexamethasone-induced mouse model were successfully established.2.The protein level of H3k9bhb of hypothalamus was reduced significantly in both of depressive models(n=5,5;p<0.05).2.?-hydroxybutyrate treatment improved the depressive like behavior of mice(n=8,8;p<0.05),and alleviated the decrease of protein level of H3k9bh(n=5,5;p<0.05).3.The level of p-hydroxybutyrate of hippocampus tissue was reduced in stress group compared to control group(n=5,5;p<0.05),and showed an increased trend after treatment by BHB and IMI(n=5,5;P=0.18).4.The downregulation of protein level of BDNF was reversed by BHB in Stress mouse model(n=5,5;p<0.05).5.The increase of protein level of H3k9bhb not H3k9ac was dose-dependent with?-hydroxybutyrate.p-hydroxybutyrate reversed the decrease of protein level of H3k9bhb and BDNF induced by dexamethasone in cultured primary neurons(n=3,3;p<0.05).6.?-hydroxybutyrate reversed the downregulation of mRNA level of BDNF induced by dexamethasone in cultured primary neurons(n=3,3;p<0.05).7.Such influential factors as dietary restriction,ketogenic diet and exercise that can elevated endogenous ?-hydroxybutyrate,significantly improved the depressive behavior of mice(n=8,8;p<0.05).Conclusions:These results revealed that H3K9bhb was involved in occurrence and modulation of depression.The regulation mechanism of H3K9bhb is possiblly associated with activation of BDNF.