The Antidepressant-like Effects Of Leonurine In Chronic Mild Stress-Induced Mice And Its Potential Mechanisms

Depression is a prevalent and serious psychiatric disorder,characterized by hopelessness,anhedonia and cognitive deficits,generally leads to suicide.Statistics predicted that depression would become the leading public health problem and highest societal burden disease by 2030.Although the clinical commonly used antidepressants have a certain effect,but there are few rapid,narrow spectrum,adverse reactions used clinically.In addition,medication resistance in patients with depression reached 30%.Thus,there is an urgent need for finding novel efficacious antidepressant drugs with fewer side effects.The pathogenesis of depression is complex,including monoamine neurotransmitter hypothesis;hypothalamus-pituitary-adrenal cortex(HPA)axis hyperactivity hypothesis;brain neurotrophic factor deficiency hypothesis;oxidative stress hypothesis.Over the past few decades,monoaminergic hypothesis such as serotonergic,noradrenergic and dopaminergic has paid more attention in neurobiological studies of depression and action as the basis for antidepressant drugs.Monoamine neurotransmitters disorder were widely found in the pathophysiology of depression.Clinical antidepressants mainly increased the concentration of 5-hydroxytryptamine(5-HT),dopamine(DA),and norepinephrine(NE)neurotransmitters in synaptic cleft to mitigate the symptoms of depression.However,there has no effect on approximately one-third of depression patients in clinic therapy,suggesting the complexity of the pathogenesis of depression.Increasing evidence indicates that inflammation plays a crucial role in the development of depression,resulting in damage in different parts of brain.Psychosocial stressors could increase the levels of pro-inflammatory cytokines,including interleukin(IL)-1β and IL-6 in both systemic and CNS,leading to sickness behavior syndrome.Nuclear factor kappa beta(NF-κB),a transcription factor is involved in CNS functions that regulates many immune and inflammatory genes and plays an important role in depression.Neuroinflammation could induced multiple pathological processes of depression,including monoamine neurotransmitters,glutamate neurotransmission,and glucocorticoid receptor tolerance.Neuroinflammation can also induced neurons damage in the brain regions(such as hippocampus and prefrontal cortex).Neuroimaging consistently reveals atrophy hippocampal volume in major depressive disorder(MDD),which is an important emotional integration area involved in emotional control and response.Atrophy of hippocampus is one of the important pathological features of depression.This is also the key to the pathology of depression.Leonurine,also named SCM-198(4-guanidino-n-butyl syringate),is a chemically synthesized compound based on a bioactive alkaloid extracted from Herba leonuri.Previous studies reported that leonurine exert several biological effects,such as antidiabetic efficacy,cardiovascular and bovine mastitis protection.lt has been reported that leonurus could calm pregnant women suffering from anxiety.Many pathologic mechanisms of anxiety are similar with depression,such as inflammation.It is worth noting that leonurine also beneficial for nervous system disease,including PD,AD and ischemic stroke.Leonurine treatment significantly rescued behavioral deficit of animals,promoted neuronal survival and defended inflammation.It remains unknown whether leonurine exerted a therapeutic effects on depression,which has the highest incidence in psychiatric disorders.To verify this speculation,chronic mild stress(CMS)model was used in this study to investigate the antidepressant-like effects of leonurine and its underlying mechanisms.Our study demonstrates that leonurine alleviated depression-like behaviors,recovered of monoamine neurotransmitters and axnino acid neurotransmitters;improved hippocampus pathologic damage of CMS mice.Furthermore,leonurine increased the levels of neurotrophic factor GDNF and BDNF,and inhibited inflammation via suppressed the activation of NF-κB signaling pathways in the hippocampus of CMS mice.Our findings suggest leonurine may be a promising drug for depression therapy.AIM:C57BL/6J mice were used to investigate the antidepressant-like effects of leonurine in chronic mild stress induced depression model mice and its potential mechanisms.METHODS:We used C57BL/6J(20-25 g)to execute chronic mild stress(CMS)on them.Following CMS modeling,leonurine(30 and 60 mg/kg)was intragastric administration to mice once daily for four consecutive weeks.Fluoxetine(20 mg/kg)was used as positive antidepressant drug.1.We used behavioral tests including sucrose preference test(SPT),forced swimming test(FST)and tail suspension test(TST)to evaluate the effect of leonurine on depressive-like behaviors.2.The contents of monoamine neurotransmitters 5-Hydroxytryptamine(5-HT),Norepinephrine(NE),Dopamine(DA)and its metabolites 5-OH-IH-Indole-3-acetic acid(5-HIAA),Homovanillic acid(HVA),3,4-Dihydroxyphenylacetic acid(DOPAC),and Glutamic acid(Glu),aspartic acid(Asp),gamma-aminobutyric acid(GABA),glycine(Gly),taurine(Tau),serine(Ser),glutamine(Gin)in the hippocampus and prefrontal cortex were measured by high-performance liquid chromatography with electrochemical detection.3,The expression of astrocytes of hippocampus were detected by immunofluorescence.4.The number and morphology of neuronal in hippocampus were detected by by Nissl staining,Tunel staining and Transmission Electron Microscopy.5.Pro-inflammatory cytokines Interleukin(IL)-1β,Interleukin(IL)-6 and1 tumor necrosis factor(TNF)-a levels,the phosphorylation levels of IKKp and P65,BDNF and GDNF in hippocampus were determined by western blot.RESULTS:1.Leonurine ameliorated depressive-like behaviors of CMS miceC57BL/6 mice were executed chronic mild stress(CMS).Leonurine(30 or 60 mg/kg)were administered by oral gavage for 4 weeks after the establishment of CMS model.After leonurine(60 mg/kg)administration four weeks,the sucrose constmption significantly increased(P<0.05),the immobility time in the FST and TST markly declined(P<0.05),respectively,compared with the CMS model group.2.Leonurine improved the levels of monoamine neurotransmitter in CMS MiceThe levels of 5-HT,NE and DA were significantly decreased in the hippocampus and prefrontal cortex of CMS(P<0.05),and the level of 5-HIAA was significantly increased(P<0.01)in the hippocampus of CMS mice,the levels of HVA and DOPAC were significantly increased(P<0.05)in the prefrontal cortex of CMS mice.Leonurine(60 mg/kg)treatment for 4 weeks significantly increased 5-HT,NE and DA levels in the hippocampus(P<0.05)and prefrontal cortex(P<0.05),the level of DOPAC was significantly decreased(P<0.05),respectively,compared with the CMS group.Furthermore,the concentration of Asp significantly increased(P<0.01)5 and the concentration of GABA significantly decreased(P<0.05)in the hippocampus of CMS mice;the concentrations of Tau,Gin were significantly decreased(P<0.05),and the concentration of Ser and Asp were significantly increased(P<0.05),respectively,in the prefrontal cortex of CMS mice.Compared with CMS group,leonurine(60 mg/kg)treatment for 4 weeks significantly decreased the concentration of Asp(P<0.05),increased the concentration of GABA(P<0.05)in the hippocampus,and increased the concentration of Tau(P<0.05),decreased Ser concentration(P<0.05)in the prefrontal cortex of mice.3.Leonurine increased the number of astrocytes in the hippocampus of CMS miceHippocampus were immunostained with GFAP.After six weeks of CMS,the number of glial fibrillary acidic protein(GFAP)-positive cells was significantly decreased in the hippocampal dentate gyrus(DG)compared to the control group.Leonurine(60 mg/kg)administration significantly increased the number of GFAP-positive cells in the hippocampus(P<0.05).4.Leonurine increased the levels of BDNF and GDNF in the hippocampus of CMS mice.Western blotting showed that the levels of neurotrophic factor BDNF and GDNF in hippocampus were significantly decreased(P<0.01)compared with the control group,and leonurine(60 mg/kg)administration significantly increased the levels of BDNF and GDNF(P<0.05).5.Leonurine ameliorated hippocampal nerve damage in CMS miceWith the establishment of CMS model,hippocampal granular cell and pyramidal cell layer thinning and many of those cells in mice appeared smaller,neuronal apoptosis and ultrastructUral damage represented by mitochondria vacuolated and swollen in CMS mice.Furthermore,myelinated axons in CMS model group displayed thicker myelin sheaths compared with the control group,indicated increased of g-ratio(P<0.01).Leonurine(60 mg/kg)treatment for four weeks,the granular and pyramidal cells were significantly increased,neuronal apoptosis was reduced and ultrastructural improvement compared to the CMS group.Compared with the CMS group,the morphology of hippocampal neurons improved,the g-ratio of myelinated axons significantly decreased after four weeks of treatment with leonurine(60 mg/kg)(P<0.01).6.Leonurine suppressed neuroinflammation in the hippocampus of CMS micePro-inflammatory cytokines were analysised by western-blotting.Compared to the control group,the levels of pro-inflammatory cytokines IL-1β,IL-6,TNF-α and the phosphorylation expression of IKKp and P65 in hippocampus were significantly upregulated in CMS group(P<0.01).Leonurine(60 mg/kg)administration dramatically down-regulated the expression of pro－inflarmmatory cytokines IL-1β,IL-6 and TNF-α(P<0.01),suppressesed the activation of NF-κB signaling pathway.CONCLUSIONS:1.Leonurine ameliorated depressive-like symptoms of CMS mice.2.These findings demonstrate that leonurine exerts antidepressant-like effects,which may be mediated,at least in part,by attenuating neuroinflammation and increasing the levels of neurotransmitters of in the hippocampus CMS mice.The major contributions of the present study lie in:1.Leonurine exerted antidepressant effects in CMS mice,which reveal a new opinion for understanding the pharmacological effects of leonurine.Our study provides an insight into the potential therapeutic value of leonurine for depression treatment.