Involvement Of Synapse-related Proteins And Neuroinflammation In The Antidepressant-like Effect Of FCPR16 In Mice Exposed To CUMS

Objective:Depression is a complex neuropsychosis,characterized by long-lasting dejection and a high suicide risk.Substantial evidence revealed that PDE4 inhibitors exerted antidepressant-like role by up-regulating cAMP level.Rolipram,the first generation PDE4 inhibitor possessed a strong antidepressant-like effect in basic researches,as well as in phase ? clinical trials,but was not approved for clinical use due to the severe emesis effect.However,FCPR16,a structural analogue of traditional PDE4 inhibitor,designed and synthesized in our laboratory.And the previous study showed that it did not cause emesis in Beagle dogs experiment.In this study,we evaluated the antidepressant-like effect of FCPR16 in mice by behavioral despair model and chronic unpredictable mild stress(CUMS)model,and researched its underlying mechanism.Methods:(1)Behavioral despair model was used to screen the antidepressant-like effect of FCPR16.After a single administration of FCPR16(0.35,0.7,1.4,2.8 mg/kg,i.g.)for 45 min,mice were accepted open field test(OFT),tail suspension test(TST)and forced swimming test(FST).(2)CUMS model was used to further confirm the antidepressant-like effect of FCPR16.Mice were divided into control group without stresses,and experimental groups which were subjected to CUMS for 6 weeks,and then received FCPR16(1.5 mg/kg,i.g.qd.),positive control Escitalopram(10 mg/kg,i.g.qd.),or vehicle for 3 weeks.The FST and TST were conducted at the baseline(6 weeks after CUMS and prior to drug treatment)and upon the completion of the 21 days drug treatment.The sucrose preference test(SPT)and novelty-suppressed feeding test(NSFT)were conducted after treatment.(3)After behavior experiments,cerebral cortex and hippocampus of mice were collected to conduct the following molecular biology experiments:?to detect the levels of cAMP and inflammatory cytokines(TNF-?,IL-1?,IL-6 and IL-10)by ELISA;?to measure the proteins expression of CREB,p-CREB,BDNF,ERK1/2,p-ERK1/2,EPAC-2,synapsinl,PSD95 and doublecortin(DCX)by Western blot;?to measure the mRNA levels of microglial Ml markers(TNF-? and iNOS)and M2 markers(Argl and CD206)by RT-PCR;? to measure the numbers of CD206+ cells in cerebral cortex and hippocampus and DCX+ cells in hippocampus by Immunofluorescence.(4)To further confirm the effect of FCPR16 on anti-inflammation and microglial phenotypes shifting,we established BV-2 cells models.? After pretreated with FCPR16,BV-2 cells were incubated with or without LPS for another 24 h.The level of TNF-? in culture medium was measured by ELISA.? After pretreated with FCPR16 or IL-4,BV-2 cells were incubated with or without LPS for another 24 h.The mRNA levels of Ml marker(iNOS)and M2 marker(Argl and Yml)were detected by RT-PCR.Results:(1)In the behavioral despair model,FCPR16(1.4 mg/kg and 2.8 mg/kg)decreased the immobility time in both FST and TST.Neither agent altered the numbers of crossing and rearing in OFT.(2)FCPR16 decreased the immobility time in FST and TST compared with the CUMS group,and improved the sucrose preference ratio in SPT,and reduced the latency in NSFT.(3)Compared with the CUMS group,FCPR16 increased the protein level of DCX and the numbers of DCX+ cells in hippocampus of mice and up-regulated the levels of cAMP,BDNF,EPAC-2,p-CREB,synapsinl and PSD95 in cerebral cortex and hippocampus.Besides,FCPR16 decreased the protein levels of TNF-?,IL-1?and IL-6,and the mRNA levels of TNF-? and iNOS,while increased the protein levels of IL-10,and the mRNA levels of Argl and CD206.The numbers of CD206+cells were increased in mice treated with FCPR16.(4)FCPR16 suppressed TNF-? release in LPS-induced BV-2 cells.In conjunction with IL-4,FCPR16 decreased the mRNA level of iNOS and increased the mRNA levels of Argl and Ym1,Conclusion:FCPR16 possessed an antidepressant-like effect in both Behavioral despair model and CUMS mice.The observed behavioral effects were apparently associated with the up-regulation of cAMP/PKA/CREB and cAMP/EPAC-2 signaling pathways,suppression of pro-inflammatory cytokines release,amelioration of microglial M1/M2 polarizations shifting and improvement of synaptic function and neurogenesis.