The Anxiolytic-like Effect Of ZL006-05 And Its Mechanisms

Anxiety is defined as a way of controlling an animal response to threatening or potentially threatening stimuli,whereas excessive anxiety will become pathological anxiety.According to the world health organization,about one hundred million people may suffer from anxiety and depression in Europe in 2030,this will be a leading cause for decreased quality of life worldwide.For decades,despite anxiety disease has brought heavy burden to both individual and society.The global annual expenditure for the treatment of anxiety disorders continues to grow.Classifications of anxiety add to the difficulty of individualized treatment.Our therapeutic methods are still limited.The treatment options currently available for anxiety are selective serotonin-reuptake inhibitors(SSRIs)and the benzodiazepines.These drugs have certain therapeutic effect,however,but also lots of adverse reactions.Therefore,to find anxiolytics with better treatment,higher safety and less side effect is imminent.Studies showed that blocking N-methyl-D-aspartic acid(NMDA)receptors(ketamine,for example)would have great anti-depressive and anxiolytic effect,and its rapid efficacy may depend on the fast synthesis of brain derived neurotrophic factor(BDNF).Although the effect of ketamine continues seven days after single administration.Therefore,how to block NMDA receptors indirectly has become a hot topic.Neuronal nitric oxide synthase(n NOS)and nitric oxide(NO)are associated with neuronal death induced by glutamate,NMDA receptors can activate n NOS to produce NO.The postsynaptic density protein(PSD95)is a bridge protein which connects n NOS and NMDA receptor.Peptides,or small molecule inhibitors can disrupt n NOS-PSD95 coupling,and uncoupling n NOS-PSD95 can inhibit the activation of p38 to reduce cell death.Whether or not disrupting n NOS-PSD95 coupling can have anxiolytic effect similar to NMDA receptor antagonist.IC87201 and ZL006 are two small molecules which can disrupt n NOS-PSD95 coupling,and results showed that single administration have obvious antidepressant effect after 24 h.The focus of our study is ZL006-05,which made modifications with(+)-borneol on the basis of ZL006.In recent years,reports suggested that ZL006 has many pharmacological effects,including stroke repairment,brain protection and rapid antidepressant effect.In addition,(+)-borneol can activate GABA receptor directly.Preliminary study of laboratory showed that ZL006 is extremely unstable in the artificial gastric liquid,its content decreases to twenty percent in just two hours,which limits its use through oral.Then we designed new agent ZL006-05 which has double targets: n NOS-PSD95 uncoupling and GABA receptor agonism.ZL006-05 combines ZL006 with(+)-borneol through esterification,and its stability greatly increases.ZL006-05 may have potential treatment in emotional disorders.In present study,we examined whether or not can ZL006-05 produce anxiolytic effects,and explored its potential mechanisms.Part Ⅰ To evaluate anxiolytic-like effect of ZL006-05,ZL006-05 or vehicle was given in mice through intragastric.Then anxiety-related behaviors tests were examined 24 hours later.Results showed that ZL006-05 caused the mice to display significantly decreased latency to feed in a novel environment,and spend more time in the open arms and in the lit compartment of a light-dark box,without impact on spontaneous activity in open field.Then,we infused ZL006-05 into the hippocampus,prefrontal cortex and amygdala of mice and assessed anxiety-related behavirors 24 h after microinjection.The activity of mice in light box through hippocampus,prefrontal cortex and amygdala infusion was increased significantly by ZL006-05.Therefore,the first part of results suggested that ZL006-05 has anxiolytic-like effect, and limbic regions such as hippocampus,prefrontal cortex and amygdala may mediate this process.Part Ⅱ To verify the role of ZL006 and(+)-borneol in anxiolytic-like effect of ZL006-05,ZL006 or(+)-borneol was administrated in mice.To further validate the effect of n NOS-PSDP95 uncoupling and GABA recetptor agonism,ZL006-05 or ZL006 was given to n NOS knockout mice(n NOS KO)or wild-type(WT)mice,then anxiety-related behaviors were tested.Results showed that ZL006 significantly reduced the latency in novel environment,increased time in open arms.Notably,the anxiolytic-like effect disappeared in n NOS KO mice,explaining dependence of uncoupling n NOS-PSD95.And(+)-borneol significantly increased time in open arms.In addition,ZL006-05 significantly increased activity distances in open arms in both WT and n NOS KO mice,and the latency in novel environment declined in WT mice.So,there might be existing other mechanisms beyond disruption of n NOS-PSD95,such as agonism of GABA receptor in(+)-2-borneol.Therefore,the second part of results showed that the mechanisms underling the anxiolytic-like effect of ZL006-05 may involve both disruption of n NOS-PSD95 and agonism of GABA receptor.Part Ⅲ Changes of BDNF expression in some brain areas are considered as one of the pathogenesis related to neural circuits dysfunction induced by stress.c AMP-reponsive element-binding protein(CREB)is well known for its role in activity-dependent gene regulation and participates in many neuronal processes,such as synaptic plasticity.CREB is also engaged in emotional behaviors in several paradigms including the elevated plus maze,light-dark box and open field.To determine how ZL006-05 regulated anxiety-related behaviors,we examined BDNF and phosphorylation of CREB.BDNF expression in hippocampus,amygdala and prefrontal cortex was tested by westen blot after 24 h ZL006-05 or vehicle administration in mice.Also,we detected BDNF and CREB phosphorylation after 24 h vehicle or ZL006-05 incubation in primary cultured hippocampal neurons.Results showed that ZL006-05 significantly increased BDNF protein expression in hippocampal cortex neurons and limbic regions such as hippocampus,prefrontal cortex,amygdala of mice.In addition,pretreated neurons with ZL006-05,CREB phosphorylation significantly decreased in hippocampal cortex neurons.Therefore,the third part of results showed that BDNF may be the essential molecule mediating the effect of ZL006-05.Summary: Firstly,ZL006-05 has anxiolytic-like effect.Secondly,n NOS-PSD95 uncoupling and GABA receptor agonism may play roles in its effect.Thirdly,ZL006-05 may do anxiolytic-like effect through up-regulation of BDNF and CREB phosphorylation.Therefore,ZL006-05 may become one of novel anxiolytics.