Effect Of Glycogen Synthase Kinase 3? Overexpression In Hippocampus On Antidepressant Activity Of Ammoxetine And Total Flavoids From Xiaobuxin-tang

Objective:Department of Neuropsychiatry,Institute of Pharmacology and Toxicology,Academy of Military Medical Sciences,had been engaged in the research and development of antidepressant drugs and related basic research.Ammoxetine(AMX),a novel compound which was synthesized using duloxetine as the lead compound,had been confirmed to have significant antidepressant effects,and it was far less toxic than duloxetine.Xiaobuxin-Tang,a traditional Chinese herbal,was originally recorded in a book called "Fuxingjue Zangfu Yongyao Fayao",which was found in Mogao Caves of Dunhuang in the northern and Southern Dynasties,XBXT was composed of 4 Chinese herbalmedicinal ingredients,Haematitum,Flos Inulae,Folium.Phyllostachydis Henonis and Semen Sojae Preparatum.We previously confirmed that the total flavonoids(XBXT-2)isolated from the extract of XBXT showed significant antidepressant effect in various animal depression models.In recent years,it had been found that glycogen synthase kinase 30(GSK3?)and its related signaling pathways were closely related to the occurrence and treatment of depression.Studies demonstrated that inactivation of GSK3?(increase its phosphorylation)will produce antidepressant effect.Our previous studies showed both AMX and XBXT-2 could increase the level of GSK3P phosphorylation and activate its signaling pathways.In this study,we used gene technology,making GSK3?overexpression or hyperactive in mice hippocampus.,to explore the influence of AMX and XBXT-2 on the signal pathway mediated by GSK3?,and to clarify whether GSK3? and its related signaling pathways play a key role in the antidepressant effect of AMX and XBXT-2.Methods:We used adeno associated virus as vector,conclude GSK3? wild type or GSK3?S9A gene,making GSK3? overexpression or hyperactive in mouse hippocampus by microinfusion.A series of behavioral and molecular biology experiments were ued to investigate whether GSK3? is the key molecular target of AMX and XBXT-2 to play the role of antidepressant and anxiolytic effect.(1)The effect of GSK3? overexpression and hyperactive in hippocampus on the antidepressant effect of AMX:Making GSK3? overexpression or hyperactivity in mouse hippocampus by microinfusion,using tail suspension test(TST)?forced swim test(FST)to investigate its influence to antidepressant effects of AMX,then use western blot to investigating the changes of brain derived neurotrophic factor(BDNF)and protein content of PI-3K/Akt/GSK3 beta signaling pathway in hippocampus.(2)The effect of GSK3phyperactive in hippocampus on the antidepressant effect of XBXT-2:Making GSK3? hyperactive in mouse hippocampus by microinfusion,then use TST?FST?elevated plus maze test(EPMT)?staircase test(ST)to investigating its influence to antidepressant effect and anxiolytic effect of XBXT-2.Making GSK3?hyperactive in mouse hippocampus by microinfusion,then use learned helpless model to investigating whether GSK3? lose its inhibition would increase the susceptibility to depression.Western blot to investigating the changes of brain derived neurotrophic factor(BDNF)and protein content of PI-3K/Akt/GSK3 beta signaling pathway in hippocampus.Results:(1)AMX(2.5mg/kg)significantly reduced immobility time in AAV group in TST,AMX(5mg/kg)significantly reduced immobility time in AAV group in FST,but GSK3? overexpression or hyperactive in hippocampus cancelled this effect;Neither drugs nor GSK3? overexpression or hyperactive in the hippocampus had no effect on the locomotor activity of mice;Chronic administration of AMX(5mg/kg)increased level of BDNF and p-Akt?p-GSK3??p-CREB,but GSK3? overexpression or hyperactive in hippocampus both cancelled this change.(2)Chronic administration of XBXT-2(100mg/kg)reduced the immobility time in mice in AAV group in TST and FST,but GSK3? hyperactive cancelled this effect.Chronic administration of XBXT-2(100mg/kg)significantly increased the percentage of entrances and time into open arms in AAV group in EPMT,GSK3? hyperactive reduced the percentage of entrances into open arms and cancelled XBXT-2 induced anxiolytic-like effects.Chronic administration of XBXT-2(100mg/kg)significantly reduced the number of rearings in AAV group in ST,while GSK3? hyperactivity cancelled XBXT-2 induced anxiolytic-like effects in this test.In LH model,escape failures and escape latency significantly increased in GSK3? hyperactive mice compared to AAV mice,In sucrose preference test,sucrose preference significantly decreasewd in GSK3? hyperactive mice compared to AAV mice,and XBXT-2 could not reverse it.On the basis of LH model,western blot showed Chronic administration of XBXT-2(100mg/kg)significantly increased level of BDNF and Akt?GSK3??CREB phosphorylation in AAV mice,but GSK3P hyperactivity cancelled this change,and the level of BDNF and Akt?CREB phosphorylation was significantly decreased in GSK3? hyperactive mice.