The Expressions Of Cation-chloride Cotransporters KCC2 And NKCC1 In The Brainstem Of Insomnia Model Rats Induced By PCPA

BackgroundsInsomnia is one of the most common sleep disorder,and it also is a very common clinical manifestation of many physical,mental and behavioral diseases;however,its mechanism is not clear enough so far.As for anatomic structure of sleep,it is widely accepted that ascending reticular inhibitory system(ARIS) of brainstem is closely related to wakefulness-sleep since ARIS performs its sleep-inducing function.GABA_AR in brain is a kind of ionotropic receptor which consists of recognition site of GABA,benzodiazepine and chloride ion channel. Classical sedative and hypnotic drugs like diazepam excite GABA_AR, open Cl~- channel to make Cl~- influx,then synaptic membrane hyperpolarize to form quick postsynaptic inhibitory potential.Cation-Chloride Cotransporters(CCCs) is just a key family which plays very important role in maintaining the balance of concentration between intracellular chloride ion and extracellular chloride ion.Among seven types of CCCs,only KCC1,KCC2,KCC3 and NKCC1 are expressed in central nerve system.KCC2 is in charge of neuronal Cl~-extrusion while NKCC1 is responsible for neuronal Cl~- intrusion in order to form a pair of substances to maintain balance of concentration of Cl~-.A lot of researches have proved that dysfunction of CCCs induce many nerve system diseases related to abnormality of concentration of Cl~-,like epilepsy and lasting pain.All of these give us a hint that CCCs may interrupt GABA function.And insomnia is such a kind of disease induced by neuron excitation/inhibition dysfunction.As for classical drug for insomnia,diazepam,a kind of benzodiazepine receptor,is able to promote GABA to connect to GABA_A receptor,increase the frequency of chloride ion channel opening,and make more chloride ion influx so that synapse membrane hyperpolarize and form inhititory postsynaptic potential.In this process,what kind of change will happen to KCC2 and NKCC1 simultaneously? There is no report pertinent to it so far in the world.ObjectivesTo make PCPA-induced experimental insomnia model,and design normal saline contral group and diazepam interference group at the same time,in order to investigate the possible roles of KCC2 and NKCC1 in mechanism of insomnia.Methods1.Grouping and building model:18 male adult Sprague-Dawley (SD) rattus were divided into three randomized groups.Model group:PCPA was injected intraperitoneally by 300mg/kg/d 8:30-9:00 AM for 2 days;Control group:NS was injected intraperitoneally instead of PCPA at the same time;Interference group:diazepam was administrated intragastricly by 0.92mg/kg/d for 5 days at 9:00-9:30 AM after making model.2.The expressions of KCC2mRNA and NKCC1mRNA in the brainstem were detectec by RT-PCR.3.The concentration of intracellular Cl~- in brainstem was detected by fluorescence probe MQAE with the help of laser confocal microscopy.Results1.Comparing with control group,the expression of mRNA of KCC2 in model group was down-regulated(p＜0.01);however,it is up-regulated in interference group comparing with model group(p＜0.01),and still down-regulated comparing with control group(p＜0.01).2.Comparing with control group,the expression of mRNA of NKCC1 in model group was a little up-regulated,but there was no statistical difference;however,it is down-regulated in interference group comparing with model group(p＜0.01) and control group(p＜0.01).3.Comparing with control group,the concentration of intracellular Cl~- in model group is up-regulated(p＜0.01);it is down-regulated in interference group comparing with model group(p＜0.01),and higher than control group without statistical diference(p＞0.05).Conclusions1,Insomnia may have something to do with the down-regulation of KCC2 mRNA and rise of intracellular concentration of Cl~-.2,The mechanism of sedative-hypnotic diazepam may have something to do with the up-regulation of KCC2 mRNA, down-regulation of NKCC1 mRNA,and decrease the intracellular concentration of Cl~-.