The Impacts Of A Novel ATP-sensitive Potassium Channel Opener,Iptakalim,on Restraint-induced Depression-like Behavior In Mice

Depression is a mood disorder,characterized by low mood,slow thought,hypobulia,cognitive impairment and somatic symptoms.Depression gives damage to people and brings heavy burden to society.At present,depression has become the world's fourth largest disease.The World Health Organization predicts that by 2020,the global incidence of depression will be second only to coronary heart disease,leapt to second place.The pathogenesis of depression is complex,including monoamine neurotransmitter hypothesis,neurotrophic hypothesis,immune inflammation hypothesis,neuroendocrine hypothesis and nerve regeneration hypothesis.Currently,drugs for the treatment of depression include tricyclic antidepressant(TCA),monoamine oxidase inhibitor(MAOI),selective serotonin reuptake inhibitors(SSRIs),selective 5-HT reuptake inhibitors(SSRIs),Serotonin and noradrenaline reuptake inhibitors(SNRIs)and other antidepressants.The existing antidepressants are only effective to parts of patients and have side effects with slow onset of treatment,incomplete treatment,long-term medication,high recurrence rate and so on.Therefore,it is urgent to elucidate the pathogenesis of depression and find new targets and develop new drugs for depression.ATP-sensitive potassium channels(K-ATP)are a kind of special non-voltage-dependent potassium channels that couple cell metabolism and electrical activity.It expressed in the cortex,hippocampus,hypothalamus,and neural basal ganglia.K-ATP channel opener has a therapeutic effect on myocardial ischemia,hypertension and diabetes.In recent years,the regulation of K-ATP channels on the central nervous system has also attracted increasing attention.Vivo and vitro studies have shown that,K-ATP channel opener have neuroprotective effect on ischemic brain injury and also have a therapeutic effect on Parkinson.In addition,studies have shown that K-ATP channel is not only as important endogenous protective mechanisms participate in acute ischemia,hypoxia and oxidative stress,but also involve in the pathogenesis of stress-induced depression,Iptakalim is a new class of drugs with independent intellectual property rights in China,Which is a new structural type of fatty amine structure K-ATP channel opener,it is listed as the ninth class of potassium channel opener.Iptakalim is a lipophilic para-amino compound with low molecular weight and can cross the blood-brain barrier freely,this drug was originally designed and developed as an antihypertensive drug.Recent years,a large number of experiments have shown that iptakalim have neuroprotective effect.Our previous studies have shown that iptakalim relieves depression in mice by regulating nerve regeneration and relieves neurological inflammation,suggesting that iptakalim has a certain antidepressant effect.OBJECTIVE:We employed long-term and severe short-term stress to induce depression-like mouse models,and to study the effects and the involved mechanisms of iptakalim on depressive mice.METHODS:1.Mice were placed into 50 ml conical centrifuge tubes with 60 holes for ventilation.In acute restraint stress(ARS),mice were injected with saline or iptakalim(i.p.10mg/kg).Then the mice were restrained for 2 hours after the injection.They were then returned to the home cages for 30 minutes and prepared for molecule detection;In chronic restraint stress(CRS),the restraint time was 6 hours daily for 8 weeks.During the last 4 weeks,mice were injected with saline,iptakalim or fluoxetine(i.p.10mg/kg)before restraint.After cessation of the final restraint,the CRS mice were given behavioral tests,including open field test and forced swimming test(FST),and then euthanized for moleculer detection.2.Blood samples were collected and keep at 4? overnight,then centrifuge for 30 minutes at 1500rpm.We use enzyme-linked immunosorbent assay(ELISA)kits detect the concentration of CORT and ACTH in supernatant.3.The hypothalamus was separated and total RNA was extracted by using a trizol reagent and prepared for quantitative reverse transcriptase PCR by using Master Mix.Real-time PCR was carried out in QuantStudio 5 instrument.The cycling conditions were:denaturation at 95? for 30 s,40 cycles of DNA synthesis at 95? for 5 s and 60? for 34 s.4.The proteins were separated on 10%SDS-polyacrylamide gels,and transferred onto PVDF membrane.After the membranes were then blocked and incubated with specific primary antibodies:PKA and p-PKA(1:500),CREB and p-CREB(1:1000),GAPDH(1:6000),at 4? overnight.After a wash in TBST,the membranes were incubated with HRP conjugated secondary antibody(1:8000).After a total of 4 washes in TBST,protein bands were visualized with an enhanced chemiluminescence.5.The cerebral tissues were fixed with 4%paraformaldehyde at 4? for 24 hours.Sections of brain(5?m)were incubated in 3%H2O2,then washed by a phosphate buffered solution(PBS).After blocking,the sections were then incubated in primary antibodies overnight at 4?.Then washing with PBS,the sections were incubated with Alexa Fluor 488 and 555 donkey anti-mouse or anti-rabbit secondary antibodies(1:1000).Images were captured by a fluorescence microscope after incubation in Hoechst 33258(1:1000).RESULTS:1.Iptakalim reversed long-term stress induced weight loss and depressive behaviorThe weight gain of mice in CRS was significantly lower than that of control mice.Iptakalim reversed this trend and returned the rate of weight gain to normal;In open field test,CRS reduced spontaneous activity of mice;In FST,more immobility behavior and less escape behavior was observed in CRS experienced mice,which indicates they were in a depressive state.Iptakalim and fluoxetine treatment clearly improved depressive state in CRS mice with a significant antidepressant effect.2.Iptakalim ameliorated stress induced disorder of the HPA axisThere was higher CRH expression in ARS exposed mice,however,mice of CRS presented with a higher level of AVP.The concentration of ACTH and CORT was influenced by CRS and ARS,their levels in plasma were reduced after CRS,but elevated after ARS.Iptakalim reverse all changes of the HPA axis.Therefore,ARS remarkably up-regulated p-PKA and p-CREB levels in the hypothalamus,the levels are restored to normal levels by pre-treatment of iptakalim.However,no significant changes of p-PKA and p-CREB after CRS.3.Iptakalim alleviates inflammation and oxidative stress in hypothalamusWe detected the mRNA expression of pro-inflammatory cytokines(such as TNF?,IL-6 and IL-1?)and TLR4 in hypothalamus tissue,and found that both CRS and ARS could elevate these inflammatory markers,except IL-6 in the CRS group.To further determine the changes in hypothalamus and to confirm that the microglia is activated,we used the immunofluorescence method,selecting TNF? and CD11b to detect the activated microglia in hypothalamus.After stress,there was a significantly higher level of co-location of TNF? and CD11b protein expression in hypothalamus,and iptakalim and fluoxetine treatment clearly decreased the microglial TNF? over-expression after stress exposure.Next,we used similar methods to explore the role of oxidative stress in hypothalamus after stress.Compared with control groups,stress induced higher mRNA levels of those indicators,and that higher levels were prevented by iptakalim and fluoxetine treatment.Then,Immunofluorescence analysis was used to confirme the expression of gp91phox protein,and after stress gp91phox significantly increased in neurons of hypothalamus,which was marked by co-location of gp91phox and NeuN.CONCLUSIONS:Our current study demonstrates that both long-term and severe acute stress induce heightened levels of inflammation and oxidative stress in the hypothalamus,which further leads to a disruption of the HPA axis.Moreover,we found that depressive behaviors were generated after long-term stress.Iptakalim,a new K-ATP channel opener,could alleviate stress related inflammation and oxidative stress and normalize disorders of the HPA axis and abnormal behavior.With a similar therapeutic effect to fluoxetine,iptakalim has the potential to be developed as a new antidepressant.