Research On The Antidepressant Effect And Mechanism Of Arctigenin

Objective:Depression is a complex mental disorder,and its pathogenesis remains still to be unclear.Currently,the first-line clinical drugs are only effective for one third of the patients,and they have disadvantages such as large toxic and side effects,high price,and can not be taken for a long time.Therefore,it is of great significance to research and develop effective,low toxicity and low price antidepressants.Arctiin and arctigenin were the main active component of Arctium lappa L.Our previous research found that arctiin regulated high mobility group protein 1(HMGB1)/toll-like receptor(TLR)4/nuclear transcription factor-?B(NF-?B)and tumor necrosis factor-?(TNF-?)/tumor necrosis factor receptor(TNFR)1/NF-?B signaling pathway to inhibit the activation of microglia and the excessive production of neuroinflammation,thereby exerting an antidepressant effect.Arctigenin is a metabolite of arctiin.It has been reported that arctigenin has antidepressant and anxiolytic effects,but its mechanism of action is unclear.Therefore,in this study,we based on the HMGB1/TLR4/NF-?B and TNF-?/TNFR1/NF-?B signaling pathways to explore the potential mechanism of the antidepressant effect of arctigenin by in vivo and in vitro experiments.Methods:(1)Wild-type(WT)and TLR4 gene knockout(TLR4^-/-)C57BL/6 male mice were used as the research object to establish a 6-week CUMS mouse depression model.The tail suspension test(TST)and forced swimming test(FST)were used to investigate whether TLR4 is involved in stress-induced depression-like behavior.(2)C57BL/6 male mice were given arctigenin(25,50 or 100 mg/kg)or sertraline(10mg/kg)by gavage for 2 weeks,and TST,FST and open field test(OFT)were used to preliminarily explore the antidepressant effect of arctigenin.(3)C57BL/6 male mice were used as the research object to establish a 6-week CUMS mouse depression model,and the mice were given arctigenin(25,50 or 100 mg/kg)or sertraline(10 mg/kg)by gavage for 6 weeks.The weight gain,sucrose preference test,the immobility time of TST and FST were used as indicators to clarify the improvement effect of arctigenin on depression-like behavior of CUMS mice.Prefrontal cortex(PFC)and serum were collected The effects of arctigenin or sertraline on neuron injury in mouse PFC induced by CUMS were observed by Nissl staining and immunohistochemistry.The effect of arctigenin or sertraline on the expression of 20 cytokines in mice PFC induced by CUMS was detected by antibody array.Western blotting was used to determine the effects of arctigenin or sertraline on the expression of ionized calcium binding adaptor molecule-1(Iba-1),HMGB1,TNF-?,interleukin(IL)-1?,inducible nitric oxide synthase(i NOS),TLR4,myeloid differentiation primary response gene 88(My D88),tumor necrosis factor receptor(TNFR)1,tumor necrosis factor receptor associated factor 2(TRAF2),receptor interacting protein(RIP),phospho-nuclear factor-?B(p-NF-?B)p65,phospho-inhibitor of NF-?B-?(p-I?B-?),NF-?B p65 and I?B-?protein in mouse PFC induced by CUMS;the effects of arctigenin or sertraline on the levels of TNF-?,IL-1?,nitric oxide(NO),indoleamine 2,3-dioxygenase(IDO),5-hydroxytryptamine(5-HT)and dopamine(DA)in PFC or serum of mice induced by CUMS were determined by enzyme-linked immunosorbent assay(ELISA)or Griess reaction;Immunohistochemical was used to observe the effect of arctigenin or sertraline on the expression of HMGB1,TLR4 and p-NF-?B p65 in mouse PFC microglia induced by CUMS.(4)Molecular docking technology was used to preliminarily observe the binding mode of arctigenin or sertraline with TLR4 or TNFR1,and then local surface plasmon resonance(LSPR)co-immunoprecipitation experiments and were used to detect the effect of arctigenin or sertraline on the interaction between HMGB1 and TLR4 or TNF-?and TNFR1.(5)The primary microglia from the PFC of newborn C57BL/6 mice was isolated,and Iba-1 antibody and DAPI were used to carry out immunofluorescence staining to measure their purity.The best concentration of arctigenin or sertraline in vitro was screened by MTS experiment.Western blotting was used to determine the effects of arctigenin or sertraline on the expression of Iba-1,HMGB1,TNF-?,IL-1?,i NOS,TLR4,My D88,TNFR1,TRAF2,RIP,p-NF-?B p65,p-I?B?,NF-?B p65 and I?B-?protein in primary microglia stimulated by HMGB1 or TNF-?;the effect of arctigenin or sertraline on NF-?B transcription activity in primary microglia induced by HMGB1 or TNF-?was determined by luciferase reporter assay The effect of arctigenin or sertraline on HMGB1 level in supernatant of primary microglia induced by TNF-?was measured by ELISA.Immunofluorescence method was used to observe the effect of arctigenin or sertraline on the expression of Iba-1,HMGB1,TLR4 and the nuclear translocation of NF-?B p65 in primary microglia induced by HMGB1 or TNF-?.(6)BV2 and N2a cells were used to establish a transwell co-culture system,and detect the effect of arctigenin or sertraline on the apoptosis of N2a cells induced by BV2 cells activated by HMGB1or TNF-?.Results:(1)Compared with WT mice,the immobility time of TST and FST in TLR4^-/-mice was significantly shortened,suggesting that TLR4 is involved in stress-induced depression-like behavior(2)Arctigenin significantly reduced the immobility time of TST and FST,but did not affect the spontaneous locomotor activity of mice,suggesting arctigenin has a potential antidepressant effect.(3)Arctigenin could significantly increase the weight gain and sucrose preference and significantly reduce the increased immobility time in TST and FST induced by CUMS procedure in a dose-dependent manner,and the effect was equivalent to the positive drug sertraline.It indicated that arctigenin could improve the depression-like behavior of mice induced by CUMS.In addition,arctigenin or sertraline could restore the degree of neuronal injury in PFC of CUMS depressed mice.The results of antibody array.showed that compared with the model group,the levels of TNF-?,GM-CSF,IL-1?,IL-5,IL-6,IL-7,IL-9,IL-13,and IFN-?in the PFC of CUMS model depression mice increased,compared with the model group,the levels of TNF-?,GM-CSF,IL-1?,IL-6,IL-7 and IL-9 in the PFC of mice treated with arctigenin decreased,and the levels of TNF-?,GM-CSF,IL-1?,IL-5,IL-6,IL-7,IL-9,IL-13 and IFN-?in the PFC of mice treated with sertraline decreased.Western blot results showed that arctigenin or sertraline significantly reversed the expression of Iba-1,HMGB1,TNF-?,IL-1?,i NOS,TLR4,My D88,TNFR1,TRAF2,RIP,p-NF-?B p65,p-I?B?,NF-?B p65 and I?B-?in mouse PFC induced by CUMS.ELISA results showed that arctigenin or sertraline significantly decreased the levels of TNF-?,IL-1?,NO,IDO in the PFC or serum,and significantly increased 5-HT and DA levels in the PFC or serum of mice induced by CUMS.In addition,immunohistochemical results showed that arctigenin or sertraline inhibited the high expression of HMGB1,TLR4 and p-NF-?B p65 in mouse PFC microglia induced by CUMS.(4)Molecular docking results showed that arctigenin or sertraline had good binding to both TLR4 and TNFR1 receptors.In addition,LSPR and co-immunoprecipitation results showed that arctigenin or sertraline had a good blocking effect on the interaction between HMGB1 and TLR4 or TNF-?and TNFR1ligand receptors.(5)The purity of primary microglia isolated and purified was not less than 98%.The in vitro dose of arctigenin was 5?M or 10?M,sertraline was 1?M.Arctigenin or sertraline significantly reversed the activation of primary microglia induced by HMGB1 or TNF-?and the expression levels of Iba-1,HMGB1,TNF-?,IL-1?,i NOS,TLR4,My D88,TNFR1,TRAF2,RIP,p-NF-?B p65,p-I?B?,NF-?B p65and I?B-?protein.At the same time,it inhibited the transcription activity of NF-?B and the nuclear translocation of NF-?B p65 in primary microglia induced by HMGB1 or TNF-?,as well as the translocation of HMGB1 from nucleus to cytoplasm.In addition,arctigeninor sertraline reduced the release of HMGB1 in the supernatant of primary microglia induced by TNF-?.(6)Arctigenin or sertraline inhibits the apoptosis of N2a cells caused by the activation of BV2 cells induced by HMGB1 or TNF-?.Conclusion:Arctigenin attenuates microglial activation and neuroinflammation through HMGB1/TLR4/NF-?B and TNF-?/TNFR1/NF-?B signaling pathways,thus improving CUMS-induced depression-like behavior in mice.It is suggested that arctigenin may become a new lead compound for treating depression.