Studies On The Anti-depression And Mechanism Of Radix Angelica Sinensis And Its Ingredients

Background: Depression is a disorder characterized by persistent depression and cognitive dysfunction.Because of the disadvantages of high morbidity,high recurrence rate,high disability rate and high fatality rate,depression has attracted more and more attention in medical field.The pathogenesis of depression is still unclear,and the therapy of depression has not made a fundamental breakthrough yet.Therefore,clarifying the pathogenesis of depression and developing effective antidepressants are the primary tasks of medical workers.Angelicae Sinensis Radix(AS),a well-known traditional Chinese medicine(TCM)herb,has been wildly used for replenishing the blood and promoting circulation in Asia for thousands of years.Related research demonstrated that AS is frequently used in classical anti-depressant formula,such as Xiaoyao San,Wuyou Tang,Danggui Shaoyao San,Yigan San.Furthermore,it has been confirmed that AS also possessed the pharmacological activity of anti-depression.However,these reports just observed the effect of AS on the depressive symptoms,and did not observe the effect of AS on the blood system on the depression model.Besides,the anti-depression mechanism of AS was still unclear.Several reports have demonstrated that some main constituents from AS possessed indeed anti-depression activity,such as,ferulic acid,vanillic acid and butylphthalide.However,there are still no systematic studies on the antidepressant activity of constituents from AS.In the current study,we aim to clarify the effect of AS on the depressive symptoms and haematological anomalies of rats induced by CUMS,and elucidate the correlation between them and the related mechanism.The results not only provides scientific basis for the development of the efficacy of AS,but also provides new strategies for clinical treatment of depression.Another aim is to screen active ingredients with antidepressant effect from AS,and then elucidate the related mechanism of the active ingredients.The results will lay a pharmacological foundation for the development of natural antidepressant products with definite clinical efficacy and controllable quality from AS.Objective:(1)To clarify the effect of AS on the depressive symptoms and haematological anomalies of rats induced by CUMS.To elucidate the correlation between the anti-depression effect and the pharmacological activity of modulating the blood system of AS and the related mechanism from the perspectives of metabonomics and molecular biology.(2)To screen active natural products with antidepressant effect from AS.(3)To confirm the in vivo anti-depressant effect of coniferyl ferulate(CF),and explore the possible mechanisms involved in the anti-depressant effect of CF.Methods(1)The anti-depression effect of AS was investigated via detecting the indicators of body weight,sucrose preference rate,crossing numbers,rearing numbers in OFT and immobility time in the FST on the model of CUMS.Then the improvement effect of AS on the blood system was investigated via detecting the indicators of peripheral blood routine,blood gas and whole blood viscosity on the model of CUMS.Then the correlation between the effect of anti-depression and pharmacological activity of modulating the blood system of Radix Angelicae Sinensis were analyzed.A metabolomic approach based on UPLC-MS/MS combined with1 H NMR was employed to analyze the endogenous metabolites and metabolic pathways in serum and liver samples.Finally,the expression of hypoxia inducible factor-1?(HIF-1?),pyruvate dehydrogenase lipoamide kinase isozyme 1(PDK-1)and lactate dehydrogenase A(LDHA)were further determined by western blotting.(2)The extract of AS was obtained by a supercritical fluid extraction system and then separated via various column chromatographic techniques to obtained compounds.The structures of these compounds were elucidated on the basis of comprehensive analysis of spectroscopic data(NMR?MS).Besides,other compounds from AS were acquired from the markets.Then the compounds obtained through isolation and market purchase were tested on the models of corticosterone-induced PC12,glutamate-induced PC12 and H2O2-induced PC12,and a high-throughput screening model on neurotransmitter transporter inhibitors.(3)Tail suspension test(TST)and forced swimming test(FST)were performed to investigate the anti-depressant effect of CF in vivo.Methyl thiazolyl tetrazolium(MTT)assay and lactate dehydrogenase(LDH)release assay were used to investigate the protective effect of CF against cell damage caused by glutamate.Cell apoptosis,mitochondrial membrane potentials(MMPs),intracellular Ca2+concentration and reactive oxygen species(ROS)were measured on a FACS Culibur flow cytometer.The activity of SOD and the level of malondialdehyde(MDA)were determined by assay kits.The levels of p-NR2 B,p-Ca MK II,p-JNK,p-p38,cytochrome C,Bax,Bcl-2,and caspase-3 was analyzed by Western blot.Results:(1)AS(7.5 g herb/kg,15 g herb/kg)could significantly increase the decreased body weight,sucrose preference and locomotor activity induced by CUMS procedure,and reduce the increased immobility time in FST induced by CUMS procedure.CUMS treatment of rats for 28 days resulted in a significant decreased in mean corpuscular volume(MCV),the level of monocyte proportion(MO%),partial pressure of oxygen(PO2),oxygen saturation(s O2),hydrogen ion concentration(p H),and an increase in the level of red blood cell(RBC),platelet count(PLT),and red blood cell distribution width(RDW),partial pressure of carbon dioxide(PCO2)and blood viscosity.These phenomenon could be reversed after the administration of AS.The analysis of metabonomics demonstrated that 40 metabolites in serum and 47 metabolites in liver were disturbed after the CUMS procedure.A total of 26 metabolites in serum and 27 in liver were significantly intervened by AS treatment.Further results proved that AS modulated could inhibit the expression of HIF-1?,PDK-1 and LDHA in depression rats.(2)12 compounds,including Z-butylidenephthalide(1),ligustilide(2),E-butylidenephthalide(3),palmitic acid(4),cis-Z,Z'-3a.7a',7a.3a'-dihydroxy ligustilide(5),tokinolide A(6),tokinolide C(7),?-sitosterol(8),riligustilide(9),14-Acetoxy-12-senecioyloxytetradeca-2E,8E,10E-trien-4,6-diyn-1-ol(10),falcarindiol(11),and coniferyl ferulate(12),were isolated from AS.Tokinolide C was a new compound.11 compounds from AS,including senkyunolide A(13),senkyunolide H(14),senkyunolide I(15),sedanolide(16),levistilide A(17),ferulic acid(18),5-Hydroxymethylfurfural(19),imperatorin(20),vanillic acid(21),umbelliferone(22),and butylphthalide(23),were acquired from the markets.In vitro experiments demonstrated that senkyunolide A and ferulic acid could protect PC12 cells from corticosterone-induced damage,ligustilide,Z-butylidenephthalide,coniferyl ferulate and tokinolide A could protect PC12 cells from glutamate-induced damage,senkyunolide A,senkyunolide I,butylphthalide could protect PC12 cells from H2O2-induced damage.Beside,5-Hydroxymethylfurfural and coniferyl ferulate showed significant effect on the high-throughput screening model on neurotransmitter transporter inhibitors.(3)The results showed that CF obviously decreased the immobility time in tail suspension test(TST)and forced swimming test(FST).It could significantly attenuate the decrease of cell viability,the release of lactate dehydrogenase(LDH),and the increase of apoptosis rates induced by glutamate.CF could also suppress the influx of Ca2+ and the elevation of p-NR2 B,p-Ca MK II,p-JNK,and p-p38 level induced by glutamate.Besides,CF could also inhibit the generation of reactive oxygen species(ROS),the decrease of SOD activity,the elevation of malondialdehyde(MDA)level,and suppress the loss of mitochondrial membrane potential(MMPs)and the activation Bcl-2/Bax mediated apoptotic pathways induced by glutamate.Conclusion:(1)These results suggested that modulation of the blood system was involved in the anti-depression effect of AS.The mechanism may be associated with the promotion of body's energy metabolism,the stabilizing of cell membrane,the promotion of serum protein synthesis,and the enhancing of immunity.Beside,HIF-1?-mediated energy metabolism pathway was involved in the anti-depression effect of depression.(2)Senkyunolide A,ferulic acid,ligustilide,Z-butylidenephthalide,coniferyl ferulate,tokinolide A,senkyunolide I,butylphthalide and 5-Hydroxymethylfurfural were the active ingredients with antidepressant effect from AS.(3)CF exert indeed anti-depressant effect in vivo and in vitro.The inhibition of NMDAR-Ca MKII-MAPKs signaling pathway,oxidative stress,and mitochondrial apoptotic pathways were involved in the anti-depressant effect of CF.