A Preliminary Study On The Antidepressant Effect And Molecular Mechanism Of Dl-3n-butylphthalide

BACKGROUNDMajor depressive disorder(MDD)is a common mental disorder characterized by remarkable and persistent lower mood and there were more than 350 million patients with depression globally.It was reported by World Health Organization that MDD has become the leading cause of disability in the worldwide,which brings heavy burden for the family of patients and our society,and it is estimated to be the leading cause of disease burden by 2030.Current first-line pharmacotherapies for MDD are based on enhancement of monoaminergic neurotransmission,but these antidepressants are still insufficient and produce significant side-effects.Consequently,the development of novel antidepressants and therapeutic targets are desired.The new use of old drugs is to find new indications for clinical existing drugs,which can significantly shorten the time and cost of drug development,hence it has become an important focus for researching novel antidepressants.Dl-3-n-butylphthalide(NBP)is a compound with proven efficacy in treating ischemic stroke through improving the energy metabolism and neuroprotection of brain cells,yet its therapeutic effects and mechanisms for MDD remain unexplored.Our study was to investigate the antidepressant effect of NBP,detect the alteration of the metabolites involved in energy metabolism and explore the candidate molecular mechanism.Our study will help find the new antidepressant target and study novel antidepressant drugs.OBJECTIVEFirstly,based on the depression model with chronic NBP intervention,we evaluate the antidepressant effect of NBP.Secondly,we test the metabolites of hippocampus(HP)and prefrontal cortex(PFC)involved in the glycolysis,tricarboxylic acid(TCA)cycle and purine metabolism pathways and perform the correlation analysis between the differential metabolites and behavior to clarify the effect of NBP on energy metabolism.Thirdly,we detect the gene and protein level of key enzymes of TCA cycle and key molecules associated MDD and candidate pathway to further explore the molecular mechanism of NBP affecting the energy metabolism.METHODS1.Intragastric administration of placebo(CON+PLA group and CSDS+PLA group)or NBP(60mg/kg,diluted in soybean oil,CSDS+NBP group)were applied throughout the duration of the experiment,and after two weeks' drug intervention,the chronic social defeat stress model was constructed to perform 10 days physical and psychological stress.A series of related behavioral experiments were carried out to evaluate the effect of NBP on the depression-like phenotype.2.We employed a targeted LC-MS/MS-based metabolomic approach to investigate the level of 29 metabolites involved in energy metabolism and analyzed the differential metabolites in the HP and PFC.Correlation analysis between the differential metabolites and behavior was performed to find the behavioral phenotype-related metabolites.3.We applied RT-PCR and western blot to detect the gene and protein level of key enzymes of TCA cycle,purine receptor,key molecules related to MDD and key proteins of AKT/CREB pathway(AKT,p-AKT,CREB and p-CREB).RESULTS1.Compared to the CON+PLA group,the CSDS+PLA group performed social deficits,anxiety-like and despair-like behavior.It was reflected that the SI ratio,numbers of interaction zone entries and time in interaction zone in social interaction test in the CSDS+PLA group were decreased.Numbers of entering into three chambers in three-chambered social test and numbers of center zone entries and rears in open field test in the CSDS+PLA group were also significantly decreased.Immobility time in the tail suspension test of CSDS+PLA group mice was increased.There were significant differences between the CSDS+PLA group and CSDS+NBP group.Compared with the CSDS+PLA group,the body weight in CSDS+NBP group was increased,the SI ratio and numbers into interaction zones in the social interaction test were increased,and the number of entering into three chambers in three-chambered social test and numbers of center zone entries in open field test were increased in CSDS+NBP group.Immobility time in the tail suspension test of CSDS+NBP group mice was decreased2.As shown in the results of targeted metabolism,compared with the CON+PLA group,five metabolites(TPP,DHAP,GDP,GTP and succinate)were significantly increased and one metabolite(D-G-6-P)decreased in the CSDS+PLA group in the HP.Meanwhile six metabolites(GTP,succinate,NAD,PEP,3-P-D-G,oxaloacetate)were significantly decreased and one metabolite(GMP)increased in the CSDS+NBP group compared with the CSDS+PLA group.In the PFC,compared with the CON+PLA group,two metabolites(citrate,isocitrate)were decreased and two metabolites(NAD,NADP)increased in the CSDS+PLA group.The differentially altered metabolites mainly involved in glycolysis and TCA cycle pathways and showed significant brain region specificities.Correlation analysis showed that the level of succinate,GMP,3-P-D-G,PEP and oxaloacetate in the HP and citrate and isocitrate in the PFC significantly correlated with observed behavior.3.As revealed in the results of RT-PCR,compared with the CSDS+PLA group,the gene expression of SDHc and P2rx1 were significantly increased and Sucla2-GDP was reduced in the CSDS+NBP group in the HP.And the level of CS was decreased in the CSDS+NBP group in the PFC.The result of western blot showed increased expression of p-AKT,p-AKT/AKT and p-CREB in the CSDS+NBP group compared with the CSDS+PLA group in the HP.In the PFC,p-CREB levels were increased in the CSDS+PLA group compared with the CON+PLA group,while the CSDS+NBP group showed a marked reduction in p-CREB levels compared with the CSDS+PLA group.There were no statistically significant changes in other proteins.CONCLUSIONNBP could effectively attenuate CSDS-induced social deficits,anxiety-like and despair-like behavior.Targeted metabolomics profiling of the HP and PFC revealed that NBP alters metabolite levels of glycolysis and TCA cycle components and differential metabolites showed significant brain region specificities.Correlation analysis revealed that differential metabolites in the HP and PFC correlate with observed behavior.NBP also activated AKT and CREB phosphorylation to affect energy metabolism.Altogether,our results show that NBP can exert antidepressant effects by regulating energy metabolism via the AKT/CREB signaling pathway.Our findings provide the evidence that NBP has antidepressant effects and yield important insights into the molecular energy metabolism changes underlying the therapeutic actions of NBP,and further aid research of novel antidepressant drugs.