Antidepressant Effects And Cognitive Enhancement Of Chlorbipram And The Underlying Mechanisms

Objective:Phosphodiesterase4(PDE4)regulate the level of intracellular cAMP(cyclic adenosine monophosphate,cAMP)through catalyzing the second messenger cAMP.PDE4 inhibitors inhibit the activity of PDE4,increase the intracellular cAMP level,activate the cAMP/protein kinase A(PKA)/cAMP response-element binding protein(CREB)signaling pathway,further affect the downstream signal molecules such as brain-derived neurotrophic factor(BDNF),thus exert various biological effects.Various of studies showed that PDE4 inhibitors has obvious antidepressant and cognition enhancing effects.However,no PDE4 inhibitors have yet been brought to market as antidepressants because of their intolerant side effects,mainly emesis.Our research group designed and synthesized a novel PDE4 inhibitor chlorbipram,and confirmed that it can dose-dependently inhibited PDE4 activity.Also we have confirmed that chlorbipram has good antidepressant activity in the forced swimming test and the tail suspension test without affecting the locomotor activity in mice.Furthermore,we evaluated the emetic potential of chlorbipram in beagle dogs.However,during 120 minutes observation period,chlorbipram did not express emesis profiles suggesting that chlorbipram has litter or no emetic activity.In this present study,we aim toPart 1 Antidepressant effects of chlorbipram,a phosphodiesterase-4 inhibitor investigate the antidepressant effect of chlorbipram and its potential mechanisms in a chronic unpredictable mild stress(CUMS)rat model.Methods:In this research,we use CUMS animal model which is widely used as an animal model of depression which recapitulates a multitude of behavioral characteristics and biochemical states of depression in human.Wistar rats were divided into two groups including normal control group,CUMS model group to accept either control treatment or CUMS for six weeks.Then the CUMS treatment started.The stressed groups were exposed to the following stressors for six consecutive weeks:food or water deprivation;restricted access to food;cage tilt 45°for 24 hours;overnight illumination;soiled cage(200 ml water spilled onto 100 g sawdust bedding)for 24 hours;stroboscopic lighting for 1 hour(100 flashes/min);paired housing;forced swimming(water temperature 25 ℃,15 min);restraint;forced swimming in cold water(water temperature 4 ℃,5 min).Rats were tested for sucrose preference experiments to determine whether the model success respectively after 4 weeks,6 weeks CUMS treatment.If the depressive models established successfully,the CUMS group will devided into four groups according to the test:0.05 chlorbipram group(0.05 mg/kg chlorbipram and CUMS),0.15 chlorbipram group(0.15 mg/kg chlorbipram and CUMS),0.45 chlorbipram group(0.45 mg/kg chlorbipram and CUMS)and ESC group(5.0 mg/kg ESC and CUMS).After 28 days drug treatment,mice were subjected to the following behavioral tests;the open field test(OFT)was explored to the ambulation,rearing and feces;mice were then subjected to the sucrose consumption test(SCT)by recording the sucrose preference;the Forced swim test(FST)was adopted to study the time of immobility and the Elevated plus maze(EPM)was explored to the time in the open arms.Hippocampus of rats were collected for determination of cAMP and the expression of p-PKA、p-CREB.synapsin 1、PSD95 and BDNF mRNA followed by ELISA,Western blotting and RT-PCR respectively after behavioral tests.We also test the effect of chlorbipram on the expression of BDNF、p-CREB protein in hippocampals of depression rats followed by immunofluorescence.Results:After four weeks treatment of chlorbipram,the rat’s depressive and anxiety symptoms were significantly improved,especially the effect of the high dose of chlorbipram.The OFT showed that the feces of CUMS rats were significantly higher than that of normal control rats(P<0.001),and this effect was reversed by 0.45 mg/kg chlorbipram and ESC treatment(P<0.05,P<0.05).We also found that there were no significant differences between each group both in ambulation and rearing activities,which indicated that CUMS and drug treatment didn’t affect the locomotor activity in rats.In FST,the immobility time was significantly increased in CUMS rats compared with normal control rats(P<0.01).ESC or 0.15 and 0.45 mg/kg chlorbipram could significantly decrease the rats’ immobility time(P<0.01,P<0.05,P<0.01).In the EPM test,we found that chlorbipram and ESC could reverse the decrease time in open arms in CUMS rats.In the SCT,the sucrose preference of CUMS rats were decreased by CUMS procedures(P<0.001)and ESC or 0.15 and 0.45 mg/kg chlorbipram could increase the sucrose preference significantly(P<0.01,P<0.01,P<0.01).The ELISA analysis demonstrated that the cAMP level of CUMS rats was significantly lower than that of normal control rats(P<0.05).ESC and 0.45 mg/kg chlorbipram could significantly elevated the level of cAMP(P<0.05,P<0.01).Compared with normal control rats,the protein levels of p-PKA,p-CREB,synapsin 1 and PSD95(P<0.01,P<0.01,P<0.01,P<0.001)in the hippocampus of CUMS rats were significantly decreased.Chlorbipram elevated protein expression level of p-PKA，p-CREB in CUMS rats hippocampus as well as the level of synapsin 1 and PSD95.BDNF mRNA expression in the hippocampus of CUMS rats were measured as well when animals were killed.Results from RT-PCR in hippocampus identified that BDNF mRNA expression were decreased in CUMS rats(P<0.001)and that ESC or 0.15 and 0.45 mg/kg chlorbipram could reverse this decrease significantly(P<0.01,P<0.001,P<0.01).BDNF,p-CREB levels were also detected by immunofluorescence.We found a significant decrease of BDNF,p-CREB levels in neuronal cell bodies in hippocampus after CUMS treatment which was significantly reversed both by ESC and 0.45 mg/kg chlorbipram.Conclusion:In this present study,our results showed that chlorbipram could reverse depressive behaviors in CUMS model and exhibited an effective antidepressant effect which may be related to the effect of regulate the level of cAMP/p-PKA、p-CREB and BDNF as well as synapsin 1 and PSD95 in the hippocampus.In conclusion,we demonstrated that the novel phosphodiesterase-4 inhibitor,chlorbipram,has a good antidepressant effect which is possibly related with the activation of intracellular cAMP/PKA/CREB signal pathway.Our reseach indicated that chlorbipram may be developed as an effective antidepressant.Part 2 The PDE4 inhibitor chlorbipram improves cognitive deficits in aged miceObjective:To explore the protective role of chlorbipram,a new phosphodiesterase 4(PDE4)inhibitor in learning and memory in aged mice.Methods:The aged mice were 20-month-old and the young mice were 6-month-old.Both of them were divided into two groups to receive either vehicle or chlorbipram daily for three weeks.Chlorbipram was dissolved in vehicle at the dosage of 0.5 mg/kg.Open field test(OFT)was carried out to analyze the ambulation,rearing and feces.Novel object recognition(NOR)test was performed to assay the recognition index of identical and novel objects.Morris water maze(MWM)was used to evaluate space learning and memory ability.ELISA was adopted for the content determine of cAMP in the hippocampus of mice and western blotting for the combination level of phosphorylated protein kinase A(p-PKA)and phosphorylated cAMP response-element binding protein(p-CREB).The level of BDNF mRNA were detected by RT-PCR after behavioral tests.Results:The OFT showed that the feces of normal control aged mice were significantly higher than that of normal control young mice(P<0.05),which was obviously reduced after chlorbipram treatment.According to the NOR test,the recognition index in normal control aged mice was significantly lower than that of normal control young mice(P<0.01),which was significantly improved after chlorbipram treatment(P<0.05).Compared with normal control young mice,the normal control aged mice spend more time on reaching the hidden platform in acquisition trial(P<0.01)with less exploration time(P<0.01),less entries(P<0.01)and distance(P<0.01)in the target quadrant in Morris water maze test,while these effects were ameliorated by chlorbipram treatment as well.The ELISA analysis demonstrated that the cAMP level of normal control aged mice was significantly lower than that of normal control young mice(P<0.01).Compared with normal control young,the protein level of p-CREB,p-PKA as well as mRNA level of BDNF in the hippocampus of normal control aged mice were significantly decreased(P<0.01,P<0.01,P<0.01).Chlorbipram both elevated cAMP level and the protein expression of p-PKA(P<0.01)and p-CREB(P<0.01)as well as the mRNA level of BDNF in the hippocampus of aged mice(P<0.01).Conclusion:Chlorbipram appreciably improve the impaired learning and memory behavior in aged mice,which is possibly related with the activation of intracellular cAMP/PKA/CREB signal pathway.