| Obejective:Depression, a prevalent psychiatric disorder with symptoms of persistent depressive state, impaired thinking, little words, insomnia and recurrent thoughts of death or suicide, is considered as one of the leading public health problems and has a lifetime prevalence of approximately15～17%in the world today. Current most widely used antidepressant drugs are chemical synthetic drugs including monoamine oxidase inhibitors (MAOIs), selective serotonin reuptake inhibitors (SSRIs), selective noradrenaline reuptake inhibitors (SNRIs) and tricyclic antidepressants (TCA) with characteristics of more side-effects and onset delay, which can only ameliorate some depressive symptoms. Exploring novel, efficient and low side-effects potential drugs from traditional Chinese medicines has generated considerable interests for more and more investigators. Recently, envidences found that traditional Chinese medicine Xiao Bu Xin soup flavonoid extracts, Betel nut, Banxia Houpu decoction, Fructus Akebiae extract (FAE) can produce antidepressant effects. It is recorded in the Compendium of Materia Medica that FAE is the major ingredient in some complex prescriptions for treating mental disorders and cognitive and behavioral deficits, including insomnia and dreaminess, loss of memory, paraphasia, phobia, and depressive disorder. Moreover, a study reported that FAE has antidepressant activity, with dominant ingredient of Hederagenin (HG). The previous studies from our laboratory confirmed that FAE produces antidepressant activity associating with augmenting5-HT and DA nervous system, with HG enriching to approximately70%purity. On the base of previous studies, the aims of the study are to assess the antidepressant effects of HG by cellular and various animal models of depression including behavior despaired model, neonate clomipramine (CL) and chronic unpredictable mild stress (CUMS) and further explore its potential mechanisms through detections of monoamine neurotransmitters levels by HPLC-ECD and BDNF,5-HT1A,5-HTT mRNA expression in hippocampus and cortex of CUMS rats respectively. The studies may lay theoretical and experiment basis foundation for exploring novel, efficient and low side-effects natural antidepressants.Methods:We compared the antidepressant effects of FAE (50mg·kg-1) with HG (10mg·kg-1) to primarily make sure whether FAE or HG that plays the important role on antidepressant effects with consecutive14days administration by Elevated plus maze (EPM) and21days by Tail suspension test (TST) and Forced swimming test (FST). On the base of above results, the antidepressant-like effects of Hederagenin in a series of concentrations (4,8,16μmol·L-1) in vitro were explored by Methyl Thiazollyl Tetrazolium (MTT) assay in corticosterone-induced cytotoxicity on PC12cells to observe the effects of cell protection and mophology. The primary screen tests were carried out to assess the antidepressant effects of different doses of HG (10,20,40mg·kg-1) with consecutive14days administration by behavior despaired mouse model. Further, the activity of SOD and content of MDA in mouse hippocampus were detected by colorimetric Assay Kit. Furthermore, the antidepressant effects of HG were evaluated through endogenous and exogenous depressive models respectively, namely eonatal clomipramine (CL) and chronic unpredictable mild stress (CUMS) animal model, which are widely used rodent models sensitive to the effects of antidepressant agents. Animals were divided into four groups including vechiel group (0.5%CMC-Na), model group (CL+Veh and CUMS+Veh), ESC group (5mg·kg-1) and HG group (5mg·kg-1) when depressive models established successfully. The antidepressant effects of HG in different models were evaluated after consecutive28days administration by Open field test (OFT), EPM and FST. In addiction, BDNF proteins were measured in CL model by immonuhistochemical stain and the detection of monoamine neurotransmitters levels and BDNF,5-HT1A,5-HTT mRNA expression in hippocampus and cortex of CUMS rats were conducted respectively.Results:1. Comparision of antidepressant activities between FAE and HG in behavior despaired model experiment.Mice were received drug administration for21days, subsequently behavious tests were carried out. Compared with Veh group, both FAE (50mg·kg-1) and HG (20mg·kg-1) did not significantly increase percent time spent in open arms of EPM. On the other hand, HG could significantly reduce immobility time in TST and FST, whereas FAE had no significant difference compared with Veh group.2. The antidepressant effects of HG on PC12cell and behavior despaired model in primary screen test.Cell model was established in corticosterone-induced cytotoxicity on PC12cells. MTT results showed that cell viability significantly decreased exposure to corticosterone. Normal PC12cells exhibited adherent growth with spindle or fibrous shape, distinct morphology and intact structure by morphological observation. PC12cells became shrinking to circle shape accompanying with float state and more cells fragments exposured to corticosterone. In contrast, cells posttreated with ESC and HG (4,8μmol·L-1) inhibited the morphological changes that corticosterone induce and alleviated cells damage. However, HG in16μmol·L-1alleviated cells damage with lower cells fragments to some extent without significant difference.Results from behavior despaired model indicated that Compared with vehicle treatment, immobility time was significantly reduced by HG(10mg·kg-1) in TST after single administration. Immobility time in TST and FST were significantly decreased by HG at low dosage without any effects on locomotor activity with consecutive two weeks administration. Moreover, the increase of SOD activity and significant decrease of MDA content in hippocampus were also observed by HG treatment.3. Endogenous neonate clomipramine (CL) rats model experimentA repeated measure of ANOVA was performed for the analysis of body weight changes. Rats were weighted every week after postweaning exhibiting a tendency of gradual increases in body weight changes before drug treatment. Compared with CL+Veh group, Veh, CL+ESC (5mg·kg-1) and CL+HG (5mg·kg-1) group showed higer body weight increases, but there were no significant differences between each group post-treatment. Behavior tests were performed after28days drugs treatment. Results from OFT showed that compared with Veh group, CL+Veh rats exhibited a tendency of reduction in ambulation activity and ESC or HG could increase the ambulation activity, but no significant differences in rearing activity. Compared with Veh group, the percent time spent in open arms significantly decreased in CL+Veh rats, while ESC and HG treatment had a tendency to increase the percent time spent in open arms in EPM. Moreover, immobility time was significantly increased in CL+Veh rats and HG could significantly decreased immobility time in FST.Results from immunohistochemical stain image showed that the numbers of positive cells expressing BDNF decreased compared with Veh group. In contrast, both ESC and HG could increase the numbers of positive cells expressing BDNF.4. Exogenous chronic unpredictable mild stress (CUMS) rats model experimentA repeated measure of ANOVA was performed for the analysis of body weight changes. Rats were weighted every week when experiment started.Rats in the vehicle group without stress treatment showed a significant increase in body weight and also exhibited significant difference compared with CUMS+Veh group from the fifth week. Compared with Veh group, CUMS+Veh, CUMS+ESC and CUMS+HG group showed significant decrease in body weight with significant difference. On the other hand, CUMS+Veh group was higher decrease in body weight than CUMS+ESC and CUMS+HG group even though there were no significant differences. Rats were divided into four groups when this model was established successfully by behavior test assessement. OFT were conducted on day14and28post-treatment respectively. Results from OFT of day14showed that there were no significant differences both in ambulation and rearing activities between each group, but the results from OFT of day28indicated that HG (5mg·kg-1) had a tendency to increase ambulation activity and rearing activity. Compared with CUMS+Veh group, the percent time spent in open arms significantly increased in CUMS+HG group with significant differences in EPM. In addition, immobility time was significantly increased in CUMS+Veh rats and ESC or HG could significantly decreased immobility time in FST showing significant difference.Monoamine neurotransmitters levels and BDNF,5-HT1A,5-HTT mRNA expression in hippocampus and cortex of CUMS rats were measured respectively when behavior tests ended. We found that significant decreases in NA and5-HT levels in hippocampus of CUMS rats and chronic ESC or HG treatment restored both NA and5-HT levels to nomal. Nevertheless, there were no significant differences but ESC and HG had tendencies to improve NA and5-HT levels in cortex. Results from RT-PCR in hippocampus revealed that BDNF mRNA expression decreased in CUMS+Veh group but ESC or HG showed no changes on BDNF mRNA expression compared with CUMS+Veh group. Compared with Veh group,5-HT1A mRNA expression in CUMS+Veh group was decreased and HG could improve5-HT1A mRNA expression to some extent.5-HTT mRNA expression significantly increased in CUMS+Veh group compared with Veh group, and ESC or HG could restore5-HTT mRNA expression. However, there were no significant differences of BDNF,5-HT1A,5-HTT mRNA expression between each group in cortex.Conclusions:In the present studies, data showed that HG exhibited more effective antidepressant activity than FAE. Treatment with HG could ameliorate depressive behaviors in various animal models and protect PC12cells from corticosterone-induced cytotoxicity indicating that HG could produce good antidepressant activities to some extent. The antidepressant effects of HG may be related to regulation of oxidation-oxidase activity for organism, decreased oxidative damage and protection of hippocampus for free oxidative stress damage in behavior despaired model. In addition, we speculated that the restoration of HG to behavior deficit in CL and CUMS models may be associated with significant increases of neurotransmitter levels and decrease of5-HTT mRNA in the hippocampus.In conclusion, we demonstrated that HG, the main ingredient of FAE, produced good antidepressant effects and revealed its potential mechanisms primarily. The results of this study indicated development value of HG as natural drug for antidepressant treatment. |
