Crocin Alone And Combined With Fluoxetine Mediates Antidepression-like Effect Via Regulation Of MTOR Signaling Pathway

Objective:To observe the protective effect of crocin alone and combined with fluoxetine on corticosterone-induced PC12 damaged cells and the behavioral effects on acute and chronic depression mice,and to explore the role of mTOR signaling pathway in the anti-depression molecular mechanism of crocin alone and combined with fluoxetine.Methods:The cell injury model was established by using CORT to induce nerve cell injury.The protective effect of crocin alone or combined with fluoxetine on CORT-induced PC 12 damaged cells by the CCK-8 method,and to explore the protective effect of crocin on PC 12 damaged cells before and after rapamycin(rapa)pretreatment.Cell morphology was observed under a 20x microscope.Acute depression model and Chronic Unpredictable Mild Stress(CUMS)model were used to detect the therapeutic effect of crocin alone and combined with fluoxetine on depressive-like behavior in mice.Western blot(WB)was used to observe the therapeutic effects of crocin alone or combined with fluoxetine on the relative expression of p-mTOR,PSD95,BDNF and p-ERK in cells and mouse cerebral cortex,so as to further explore the effects of rapamycin pretreatment,a specific inhibitor of mTOR signaling pathway,on the expression of related proteins during the antidepressant effect of crocin.Results:Experimental results of crocin alone:500μmol·L-1 CORT could damage cell survival rate to 50%(p<0.001)in cell experiments,which could be used for subsequent experimental modeling.Crocin administration of 10μmol·L-1 and 30μmol·L-1 significantly increased cell survival rate(p<0.001),and significantly improved cell neuron injury(1μmol·L-1,p<0.01;10μmol·L-1,30μmol·L-1,p<0.001),Western blot showed that Corticosterone can reduce the relative expression levels of BDNF and p-mTOR,crocin alone could improve the expression of BDNF,but there was no obvious effect.preadministration of rapamycin can block the protective effect of crocin on injured nerve cells(p<0.05).In animal experiments,crocin at different concentrations could significantly improve the immobile time in the forced swimming(p<0.001)and tail suspension test(30mg·kg-1,p<0.05)in mice with acute depression,exerting a rapid improvement of depressive behavior.Crocin also improved the bodyweight of chronic stress mice(10mg·kg-1,p<0.05;30mg·kg-1,p<0.01),improved the number of horizontal climbing(30mg·kg-1,p<0.01)and upright times(30mg·kg-1,p<0.05),and reduced the number of forced swimming(30mg·kg-1,p<0.01)and accumulated immobility time of suspended tail test(30mg·kg-1,p<0.05),promoted the expression of BDNF,p-mTOR,p-ERK,and pSD95 proteins in the cerebral cortex of chronic stress and depression mice(30mg·kg-1,p<0.01,p<0.05,p<0.05,p<0.05).After treatment with rapamycin,the antidepressant effect of crocin was inhibited.The expression levels of BDNF,p-mTOR,p-ERK,and PSD95 in the cerebral cortex of mice were significantly decreased(p<0.05,p<0.05,p<0.05,p<0.05,p<0.01).Experimental results of crocin combined with fluoxetine:In cell experiments,crocin combined with fluoxetine increased cell viability(p<0.001),and repair cell neuron damage(p<0.001)compared with the corticosterone group.Compared with single drug,the effect of combination therapy was more significant.Western blot results showed that crocin combined with fluoxetine could increase the relative expression levels of BDNF,p-mTOR and p-ERK compared with the single drug group,but the effect was not obvious.In animal experiments,after 14 days of continuous administration,crocin combined with fluoxetine could improve the bodyweight of chronic stress mice,increase the number of horizontal crawls(p<0.05)and upright times(p<0.05)in the open field test of chronic stress mice,and reduce the immobility time of mice in forced swimming(p<0.05)and tail suspension test(p<0.01).Conclusion:Crocin has rapid antidepressant effects,but these effects can be suppressed by rapamycin.Therefore,it is speculated that the mechanism of its anti-depression action may be related to the mTOR signaling pathway.In addition,crocin and fluoxetine combined medication has the effect of enhancing curative effect.