Anxiolytic Effect Of Koumine And Its Preliminary Mechanisms In Brain

The incidence of anxiety disorders has increased for years, which seriously affectsour quality of life. Pharmaceutical therapy, in which benzodiazepines （BZDs） andselective5-serotonin reuptake inhibitor （SSRI） are most commonly used, is the maintreatment of anxiety disorders, but they have been limited in clinic due to their sideeffects and the slow onset of effectiveness. Accordingly, it is expected that a brandnew kind of effective anxiolytic drugs owning new action targets are studied.Gelsemium elegans Benth belongs to Strychnos nux-vomica, has been used forcenturies to treat mental diseases, including anxiety disorder. Its active ingredients,alkaloids are proven to be anxiolytic. Koumine is one compound with highest contentand lower toxicity in Gelsemium alkaloids. Previous work has shown that kouminemay have therapeutic effect toward anxiety disorders. However, whether koumine hasanti-anxiety properties needs to be confirmed f on more animal models.Neurosteroids play an important role in the aetiology and treatment of anxietydisorder. Allopregnenolone （3α,5α-THP） is a representative neurosteroid possessinganxiolytic and analgesic effects. Our previous work showed that koumine couldimprove allopregnanolone level in spinal cord, and TSPO [translocator protein （18kDa）] antagonist PK11195significantly inhibited the analgesic effect by koumine in apostoperative pain rat model. TSPO is the rate-limiting step in the synthesis ofallopregnanolone. Therefore, the analgesic effect of koumine is connected toneurosteroids. Meanwhile, it is worth to study the relationship between the anxiolyticeffects of koumine and neurosteroids.In the present project, the anxiolytic effect and properties of koumine isconfirmed. Further, the relationship between koumine and neurosteroids is analysed inorder to elucidate its mechanism of anxiolytic effect.This research contains sections as follows: 1. Anti‐anxiety effect of koumine and its properties1.1Effect of acute s.c. administration of koumine on behavior in mice EPM test.Mice were randomly divided into normal group, diazepam group and kouminegroup in different doses. Mice received an s.c. injection of NS, koumine0.4,2and10mg/kg, or diazepam1mg/kg s.c.30min before testing, respectively. Result indicatedthat diazepam group showed a significant anxiolytic effect in mice EPM. Comparedto saline-treated group, koumine （0.4,2and10mg/kg） in s.c. exhibited significantincreases in OE%and OT%, and2mg/kg dose group optimal, which indicated thatacute s.c. administration of koumine may have potential anti-anxiety effect in mice.1.2Effect of acute s.c. administration of koumine on behavior in rat EPM test.Rats were randomly divided into normal group, diazepam group and kouminegroup in different doses. Rats received an s.c. injection of NS, koumine0.75,1.5and3mg/kg, or diazepam0.5mg/kg s.c.1h before testing, respectively. Results showedthat koumine （1.5and3mg/kg） in s.c. exhibited significant increases in OE%（bothkoumine1.5mg/kg and koumine3mg/kg） and OT%（koumine1.5mg/kg alone）indicating that acute s.c. administration of koumine may have potential anti-anxietyeffect in rats. Diazepam significantly increased the rats’ locomotor activity.1.3Effect of consecutively s.c. administration of koumine on behavior in rat EPM testRats were randomly divided into normal group, diazepam group and kouminegroup in different doses. Koumine group was treated s.c. with koumine0.1875,0.75,1.5and3mg/kg respectively. Normal group was treated s.c. with normal saline.Diazepam group was treated s.c. with diazepam0.5mg/kg on the7^thday and the1-6^thwith saline. All treatment was processed for7consecutive days. The EPM test started1h later after the last administration. Result showed that diazepam exhibited asignificant anxiolytic effect in the test. Compared with normal group, koumine （0.75,1.5and3mg/kg） in s.c. exhibited significant increases in OE%and OT%, indicating asignificant anti-anxiety effect. 2. Mechanism analysis of anti‐anxiety effect of koumine2.1Effects of consecutively s.c. administration of koumine on the levels ofallopregnanolone and pregnenolone in rat hippocampus.Rats were randomly divided into normal group and koumine group in differentdoses. Koumine group was treated s.c. with koumine0.75and1.5mg/kg, respectively.Normal group was treated s.c. with normal saline. All treatment was processed for7consecutive days. The EPM test started1hour after the last administration.4hoursafter the EPM test, the hippocampus tissues from the rats were obtained to detect theneurosteroids by using the liquid chromatography mass spectrometry. Result showedthat koumine-treated groups tended to increase the content of allopregnanolone inhippocampus, but no statistically significant difference. However, koumine1.5mg/kgin s.c. exhibited significant increases in the content of pregnenolone, compared withnormal group.2.2Effect of consecutively s.c. administration of koumine on TSPO immunohistochemical staining in rat hippocampus.Rats were randomly divided into normal group and koumine1.5mg/kg group. Alltreatment was processed for7consecutive days. The EPM test started1hour after thelast administration.4hours after the EPM test, the hippocampus tissues from ratswere immunohistochemically stained to detect the TSPO. The result showed thatTSPO in rat hippocampus was expressed in both normal group and koumine group,but the expression intensities between both groups were not significantly different（P>0.05）.2.3Effect of strychnine on anxiety behaviour in EPM test performed by micetreated with koumine.Mice were randomly divided into normal group, koumine group andstrychnine-koumine co-treated groups. Mice were injected with saline （s.c.） orkoumine （2mg/kg s.c.）30min before testing, and strychnine （0.25,1and4μg i.c.v.）was administered5min before koumine administration. Result showed that koumine 2mg/kg in s.c. exhibited significant increases in OE%and OT%compared to thesaline-treated group. The strychnine-koumine co-treated groups （strychnine4μg+koumine2mg/kg and trychnine1μg+koumine2mg/kg） exhibited significantdecreases in OE%and OT%compared to the koumine-treated group. Thestrychnine-koumine co-treated group （strychnine0.25μg+koumine2mg/kg） failedto inhibit the anxiolytic effect by koumine.In summary：1. Koumine possessed potential anxiolytic effects with its effective doses far awayfrom LD50, which is expected to become a new type of anxiolytics with highefficiency and low toxicity.2. The anxiolytic effect of koumine might be ralated to increase the synthesis ofneurosteroids in rat hippocampus.3. The anxiolytic effect of koumine might be ralated to glycine receptor in brain.