Anxiolytic Effect Of Koumine And Its Mechanisms

Anxiety disorders are currently the most prevalent psychiatric diseases in the world, and as such represent a huge burden in terms of both their economic cost and their social impact. While benzodiazepines and selective serotonin re-uptake inhibitors(SSRIs) were popular as major anxiolytic drugs, their side effects,including sedation and behavioral changes, tolerance, and dependence issues have led to the pharmaceutical industry to seek out mechanisms, etiological factors and safer alternatives of anxiety disorder.Noticeably, koumine, previous work has shown that it may have therapeutic effect toward anxiety disorder with the most abundant molecule and relatively low toxicity among the alkaloids of G. elegans Benth. It is clear that more behavior evaluation methods and animal models are needed for preclinical development of koumine. Meanwhile, the underlying mechanisms of koumine need further explore.Due to the practical and ethical limitations of applying psychological disorder on human beings, animal models are of great importance in studying the effects of anxiety disorder. Over the past years, predators and their odors might induce anxiety-like behaviors. Meanwhile, acoustic stimulation also might result anxiety.However, predator and their odors are difficult to apply in laboratories within strict management, and the latter needs to choose more appropriate sound to avoid the physiological function damage of animals. Take the two factors into consideration,the predator voice might induce animals anxiety model.During the last decades, the down-regulation of neurosterois biosynthesis has been implicated as a possible contributor to the underlying mechanisms of anxiety disorder. Previous work has shown that effect of koumine possible involved in neurosteroids, especially allopregnanolone. Further studies reveal allopregnanolone modulate anxiety via HPA axis. On the basis of these considerations, it is worth to study the relationship between the anxiolytic effects of koumine and neurosteroids.1 The effects of koumine on state anxiety-like behavior1.1 Effects of koumine on a Functional Observational Battery(FOB) in Mice Mice were randomly divided into normal group, koumine groups in different does. NS, koumine 1, 4 and 16 mg/kg were administered i.g., respectively. The following functional domains: automomic effects, central neural system(CNS)activity, CNS excitability, muscle tone/equilibrium and sensorimotor reactivity were examined in the test. Results showed mice treated with koumine(4, 16mg/kg), the inhibitor effect was characterized by a statistically significant decrease(P<0.05) in response to grabbing, head touch and fear at 1 h post-dose, decrease in the mount of urination at 1 h post-dose(P<0.01) and defecation at 4 h(P<0.05) and24 h(P<0.01) post-dose(1, 16 mg/kg) achieving statistical significance. 4mg/kg express decrease, but no statistically significant difference(P>0.05). Meanwhile,koumine has no effect on automomic effects and muscle tone/equilibrium.1.2 Effects of Koumine in Vogel conflict drinking test Rats were water-deprived before the experiment, after the first 24 h deprivation,rats were randomly divided into normal group, koumine group in different does and diazepam group. Another Twenty-four hours deprivation later,NS, koumine 0.167,0.5 and 1.5 mg/kg or diazepam 1mg/kg were administered s.c. 1h before test,respectively. The result showed koumine(0.167-1.5 mg/kg) having statistically increased the drinking time(P<0.01), the number of shocks accepted(P<0.01) and the number of licks(P<0.01), during the Vogel conflict drinking test of 3 min.However, the first drinking latency between groups was not significantly different.These data suggest that koumine, exertes anxiolytic-like effects in the Vogel conflict drinking test.1.3 Effects of Koumine on mice in OFT Mice were randomly divided into normal group, koumine groups in different does and diazepam group. NS, koumine 0.25, 1and 4mg/kg or diazepam 1mg/kg were administered s.c.1h before test, respectively. Open field behavior and plasma ACTH and CORT were examined in the test. When compared with negative control, the effect of koumine 0.25,1 mg/mg significantly increased the percentage of permanence in the center zone(P<0.01) and the number of entries into center zone(P<0.05). The percentage of time spent in center zone(P<0.05) trend to increase, but no statistically significant difference. Koumine 4 mg/kg does-dependently increased the percentage of permanence in the center zone(P<0.01) and the percentage of time spent in center zone(P<0.01). These data suggest that koumine, exertes anxiolytic-like effects in OFT. But the interactions between koumine and HPA axis are necessary to further study.2 Predator voice is applied as a new pathological anxiety-like model2.1 Effects of predator voice on mice and rats in OFT and EPM ICR mice or SD rats were randomly divided into three groups, background group,acute predator voice groups of 60 d B and 80 d B. The rats in the predator voice groups were treated with predator voice, and background group without it. The result showed that anxiety-like behavior was not affected by the applied acute predator voice in mice. However, compared with background group, acute predator voice groups of 60 d B and 80 d B significantly decreased the percentage of permanence(OFT, P<0.05; EPM, P<0.05), the number of entries(OFT, P<0.05;EPM, P>0.05) and the percentage of time spent(OFT, P<0.01; EPM, P<0.01) in center zone or open arm.but no statistically significant difference in automomic effects. Our results suggest that predator voice, as a novel pathological anxiety model, might induce anxiety.2.2 Effects of diazepam, a classic anxiolytic, on predator voice-induced pathological-like anxiety.Wister rats were randomly divided into four groups, normal group, acute predator voice group and diazepam 0.5, 1mg/kg groups. NS, diazepam 0.5, 1mg/kg were administered s.c.1h before OFT and EPM, respectively. The result showed 0.5, 1mg/kg diazepam significantly increased the percentage of permanence(OFT,P<0.05; EPM, P<0.05), the number of entries(OFT, P>0.05; EPM, P<0.01)and the percentage of time spent(OFT, P<0.05,1 mg/kg DZP P>0.05; EPM, P<0.05,0.5mg/kg DZP P>0.05) in center zone or open arm. The results of our behavioral experiments suggest that the predator voice-induced anxiety model might be applied in developing new anxiolytic drugs.3 Anxiolytic effects of koumine and its mechanisms on pathological anxiety-like model3.1 Effects of koumine on predator voice-induced pathological-like anxiety Wister rats were randomly divided into six groups, normal group, acute predator voice group and koumine groups in different does and diazepam group. NS,koumine 0.167, 0.5 and 1.5 mg/kg or diazepam 1mg/kg were administered 1 h before OFT and EPM. The result showed compared with background group, acute predator voice group trends to decrease in center zone exploration, and compared with acute predator voice group, koumine 1.5 mg/kg trends to increase in center zone exploration, but no statistically significant difference in OFT. The date showed compared with background group, acute predator voice group significantly decreased the percentage of permanence(P<0.01), the percentage of entries(P<0.01) and the percentage of time spent(P<0.01) in open arm. Compared with predator voice group, koumine significantly increased the percentage of permanence(P<0.01), the percentage of entries(P<0.001) and the percentage of time spent(P<0.05) in open arm These data indicate that koumine might possess the properties to ameliorate pathological anxiety-like behavior.3.2 Effects of koumine on progesterone and allopregnanolone in rats prefrontal cortex, hippocampus and amygdala Wister rats were randomly divided into five groups, normal group, acute predator voice group and koumine groups in different does. After behavior test above, the levels of progesterone and allopregnanolone were detected via ELISA. The results showed that acute predator voice cause a significant decrease in progesterone in rats prefrontal cortex(P<0.05), hippocampus(P<0.001) and amygdala(P<0.001)and in allopregnanolone in rats prefrontal cortex(P<0.05), hippocampus(P<0.05)and amygdala(P<0.01). Administration of koumine increases the level in progesterone in rats prefrontal cortex(P<0.05), hippocampus(P<0.001) and amygdala(P<0.001) and in allopregnanolone(P<0.05). These data indicate that koumine might possess the properties to ameliorate pathological anxiety-like behavior may involved activating progesterone and allopregnanolone synthesizing in rats prefrontal cortex, hippocampus and amygdale.3.3 Effects of koumine on plasma ACTH and CORT Wister rats were randomly divided into five groups, normal group, acute predator voice group and koumine groups in different does. After behavior test above, the levels of plasma ACTH and CORT were detected via ELISA, The results showed that acute predator voice cause a significant increase in plasma ACTH(P<0.05) and CORT(P<0.05), administration of koumine increase the level in plasma ACTH(P<0.05) and CORT(P<0.05). These data suggested that koumine ameliorate pathological anxiety-like behavior and the mechanisms may involved in suppress activity of hypothalamus-pituitary-adrenal cortex(HPA) axis.In summary:1. Koumine may possess potent anxiolytic-like profile in state anxiety with high efficiency and low toxicity.2. Predator voice could be easy to apply as a new pathological anxiety model3. Koumine may possess potent pathological-anxiolytic effects,the effect might be related to increase the synthesis of progesterone and allopregnanolone in rats prefrontal cortex, hippocampus and amygdale and suppress activity of hypothalamus-pituitary-adrenal cortex(HPA) axis.