Molecular Basis And Mechanism Of Action Of Albizzia Julibrissin In Depression Treatment

Albizia julibrissin Durazz.,a leguminous deciduous shrub,is one of the most common herbs used for depression and anxiety treatment.Its bioactivity includes relieving depression,calming nerves,regulating qi and activating collates,promoting blood circulation and reducing swelling.It is often used for the treatment of restlessness,depression,insomnia and forgetfulness.Clinical and preclinical studies have shown that Albizia julibrissin Durazz.extracts possess sedative,hypnotic,antidepressant and other pharmacological effects.However,the bioactive constituents and molecular mechanism of Albizia julibrissin Durazz.remain unclear.In this study,two compounds,（-）-syringaresin-phenol-4-0-β-d-furanosyl-（1→2）-β-D-glucopyranose and（-）-syringaresin-4,4’-bis-β-D-glucoside were isolated and identified from the extract of the dried bark of Albizia julibrissin Durazz.by the separation and identification procedures.Their effects on cell viability,SERT activity,SERT cell surface expression,and conformation of SERT were investigated.These studies could provide experimental evidence to support the development of new lead compounds for novel antidepressant drugs.The main experimental results are shown below:（1）Two compounds that inhibit SERT activity were separated from 70%ethanol extract of the bark of Albizia julibrissin Durazz.,syringaresinol-4-O-β-D-Apiofuranosyl-（1→2）-β-D-glucopyranoside and syringaresinol-4,4’-bis-O-β-D-glucopyranoside.The separation procedure was used:70%ethanol→macroporous resin→high-speed countercurrent chromatography→preparative column.This procedure produced SAG 120.78 mg/5 kg and SBG 217.86 mg/5 kg.（2）A SERT assay platform was established to investigate the inhibitory effects of Albizia julibrissin Durazz.extracts on SERT transport activity.The fluorescent substrate of SERT,4-（4-（dimethylamino）phenyl）-1-methylpyridine（APP⁺）,was used to detect SERT activity.The amount of fluorescence transported into the cells expressing SERT refers SERT activity.The experimental results showed that SAG and SBG strongly inhibited APP⁺transport into the cells.Their IC₅₀were 5.25±0.30μM and 8.51±0.50μM.The inhibitory effects of SAG and SBG on NET and DAT were also investigated.The results showed that the two compounds showed weakly inhibitory effects on NET and DAT,respectively.The IC₅₀values for NET were 17.36±1.94μM and 30.05±3.61μM,for DAT were 20.37±2.13μM and32.28±2.85μM,respectively.（3）To study the cytotoxicity of SAG and SBG on cell proliferation.The number of living cells was tested by CCK8 with the addition of SAG or SBG at a concentration of 10 x IC50.The results showed that SAG and SBG had little effect on cell proliferation.（4）Addition of SAG or SBG to the reaction solution,decreased V_maxwith little change in K_mfor APP⁺,indicating that both compounds inhibited SERT noncompetitively.（5）The effects of SAG and SBG on the cell surface expression of SERT were investigated.After treatment with SAG and SBG,the membrane proteins were labeled with NHS-SS-Biotin,and then the labeled SERT was examined by Western Bolt.The results showed that SAG and SBG did not alter the cell surface expression of SERT.（6）To investigate the effects of SAG and SBG on the conformation of SERT,a SERT mutant,Y107C,was used for measuring changes in its accessibility to the cysteine reagent,MTSET,after treatment with these compounds.The experimental results showed that SAG and SBG induced a conformational shift of the extracellular substrate pathway toward to the outward-closed state of SERT,which differs from the action of conventional antidepressant drugs.