Antidepressant Effects And Mechanisms Of Xiaochaihutang And Main Active Constituent In A Neuroendocrine Mouse Model Of Anxiety/depression

Xiaochaihutang(XCHT),a famous Chinese herbal formula,was first recorded in“Shang Han Lun" writen by Zhang zhongjing two thousand years ago.XCHT has been used clinically in depressive disorders in China.Our previous studies have demonstrated that XCHT improved depressive-like behavior in CUMS model in rats and mechanisms might be associated to maintenance of neurotransmitter homeostasis,enhancing the neurotrophy,regulating of female hormones.Morever,our studies also found Scutellaria baicalensis Georgi,Panax ginseng C.A.Mey.,Bupleurum chinense DC.and Scutellaria baicalensis Georgi compatibility could decrease the immobility time in tail suspension test(TST)and forced swimming test(FST).To further evaluate the therapeutic basis and targets of XCHT,in this study,taking XCHT,core prescription and main active constituent baicalin as points of contact,we used corticosterone(CORT)-induced mouse model of depression to investigate the antidepressant effects and mechanisms of XCHT by behavior tests,neurobiology,protein and chemical composition electropherogram.This findings wish to reveal antidepressant-like effects and mechanisms of XCHT.Firstly,we used chronic CORT(40 mg/kg/day)-induced mouse model of anxiety/depression to investigate antidepressant-like effects of XCHT by several physical and behavioral testing.Our results showed that oral administration of XCHT(2.3,7 and 21 g/kg)for 30 days remarkably normalized chronic CORT-induced the slowness in weight gain,the deterioration in coat state,the escape behavior in open field test and elevated plus maze(EPM),and the increase of immobility time in TST and FST.Moreover,the potential mechanism was explored by investigating hippocampal neurogenesis and the integrity of negative feedback function on HPA axis.The results showed XCHT significantly reversed chronic CORT-induced the reduction of Ki-67/DCX positive cells and DEX-induced plasma corticosterone/c-Fos suppression,suggesting the antidepressant-like effects of XCHT were probably attribute to promoting hippocampal neurogenesis and remodeling the integrity of the negative feedback loop on HPA axis.Secondly,the core in compatibility of XCHT on antidepressant therapy was assessed by the method of orthogonal array design.The results showed that Huangqin(Radix scutellariae),Renshen(Ginseng)and Gancao(Radix glycyrrhizae),defined as HRG,might be the core in compatibility of XCHT on antidepressant therapy.The comparative evaluations on antidepressant effects of XCHT and HRG were executed by TST,FST,novelty suppressed feeding test(NSFT),reserpine-induced hypothermia and palpebral ptosis.The results showed that oral administration of HRG(0.9,2.8 and 8.4 g/kg/day)for 15 days significantly reduced immobility duration in TST and FST without affecting locomotor activity.Both HRG and XCHT increased immobility latency in FST,decreased the latency in NSFT,reversed reserpine-induced hypothermia and palpebral ptosis.Moreover,the potential mechanism was explored by investigating levels of monoamine neurotransmitters in hypothalamus and striatum and neurogenesis in hippocampus.The results showed that both HRG and XCHT significantly increased levels of 5-HT and DA in hypothalamus and promoted hippocampal neurogenesis,suggesting antidepressant-like effects of HRG were probably attribute to increasing the levels of neurotransmitters and promoting hippocampal neurogenesis.To further study the therapeutic basis of XCHT and HRG,Chemical profile of active constituents in plasma and brain after oral administration of XCHT and HRG was revealed by ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS).The results showed that a total 26 active constituents(15 prototype components and 11 metabolites)in plasma and 13 active constituents(10 prototype components and 3 metabolites)in brain were identified after oral administration of XCHT.A total 25 active constituents(14 prototype components and 11 metabolites)in plasma and 16 active constituents(10 prototype components and 6 metabolites)in brain were identified after oral administration of HRG.Active prototype constituents in brain were all from active constituents of plasma.These findings suggested the constituents identified in plasma and brain after oral administration of XCHT and HRG might be the potential active ingredients for the treatment of depression.Based on the results of core prescription and baicalin was the prototype component in plasma and brain,we used the chronic CORT-induced mouse model of anxiety/depression to assess the antidepressant-like effects of baicalin and illuminate the possible molecular mechanisms of baicalin on GR-mediated hippocampal neurogenesis.The results showed that oral administration of baicalin(40,80 or 160 mg/kg)for 4 weeks markedly alleviated several chronic CORT-induced anxiety/depression-like behaviors,including slow body weight gain,increased escape behavior in the open field test and elevated plus maze,and increased immobility time in the tail suspension test and forced swimming test.Baicalin also significantly increased the number of Ki-67and DCX-positive cells to restore chronic CORT-induced suppression of hippocampal neurogenesis.Moreover,baicalin also normalized the chronic CORT-induced decrease in glucocorticoid receptor(GR)protein levels,the increase in GR nuclear translocation and the increase in GR phosphorylation at Ser203 and Ser211.Finally,chronic CORT exposure increased the level of FK506-binding protein 51(FKBP5)and of phosphorylated serum-and glucocorticoid-inducible kinase 1(SGK1)at Ser422 and Thr256,whereas baicalin normalized these changes.Taken together,our findings suggest that baicalin improves anxiety/depression-like behaviors and promotes hippocampal neurogenesis.We propose that baicalin may normalize GR function through SGK1-and FKBP5-mediated GR phosphorylation.Finally,we used iTRAQ to quantitative analysis differential protein between control and CORT model and found a total 105 differential proteins,after XCHT,HRG and baicalin treatment,66 common differential proteins were recovered.GO functional annotation analysis found differential proteins mainly participate in biological process on cellular process and biological regulation,in molecular function on binding and catalytic activity,in cellular component at cell and organelle.A total 10 critical protein hubs were found in Protein-protein interaction network of differential proteins,protein gene named Ywhae,Pgls,Actb,Hsd3b4,Ywhaz,Hsd3b7,Gapdh,Ywhah,Hmga2 and Hist1h3b.Above proteins were involved in the development of hippocamp and adrenal gland,phosphorylation and dephosphorylation,cell proliferation and apoptosis and the regulation of glucocorticoid pathways.These findings suggested the targets of XCHT,HRG and baicalin might be related to above proteins.In conclusion,this study firstly demonstrated XCHT could promote hippocampal neurogenesis and remodel the integrity of the negative feedback loop on HPA axis in CORT-induced mouse model of anxiety/depression.Then,our results firstly showed HRG might be the core in compatibility of XCHT on antidepressant therapy by the method of orthogonal array design and verified the antidepressant efficacy.Chemical profile of active constituents in plasma and brain was detected by UPLC-MS/MS after oral administration of XCHT and HRG to reveal the potential therapeutic basis of XCHT and HRG.Moreover,this study firstly demonstrated baicalin improved anxiety/depression-like behaviors and promoted hippocampal neurogenesis,which might involved in normalizing GR function through SGK1-and FKBP5-mediated GR phosphorylation.Finally,our results firstly showed 66 common differential proteins and 10 critical protein hubs in proteomics of XCHT,HRG and baicalin.Our findings revealed potential therapeutic basis and mechanisms of XCHT and provided scientific basis for XCHT on depression.