Roflupram Supresses HMGB1 And NLRP3-mediated Neuroinflammation In CUMS-exposed Mice

Objective:Depression is a chronic mental disorder characterized with persistently depressed mood and high suicidal tendency.The drugs used in clinical treatment have many disadvantages,so it is of great significance to explore the pathogenesis of depression and find potential target and treatment strategy.As previous studies have shown that PDE4 inhibitors have an antidepressant effect by inhibiting the inflammation response,however the mechanism by which PDE4 inhibitors have a strong anti-inflammatory effect and produce antidepression effects is unclear.Inflammation,microglial activation and depression are closely related to each other.The current experiment was designed to investigate the relationship between the PDE4 inhibitor as an antidepressant and the anti-inflammation effect.Methods:(1)6-week-old mice were subjected to daily stress(not repeat in consecutive three days)to establish a chronic unpredictable mild stress(CUMS)model,and the sucrose preference test(SPT)and forced swimming test(FST)were used to evaluate the behavior performance and eliminate mice with unsuccessful behavior.Roflupram(0.5 mg·kg-1,ig;1 mg·kg-1,ig)and the positive control escitalopram(10 mg·kg-1,ig)were administered once a day until 4 weeks by intragastric administration.Behavioral tests were performed after the compound treatment:open field test(OFT),SPT,tail suspension test(TST)and novelty-suppressed feeding test(NSFT).(2)The hippocampus and cerebral cortex of mice were collected for biological samples followed by behavioral tests:?The level of inflammation factors(IL-1?,TNF-? and IL-6)in the hippocampus and cerebral cortex was detected by ELISA.?Western blot method was used to detect the expression of Iba-1,HMGB1,RAGE,TLR4,NLRP3,Caspasel,ASC,p-ERK1/2,ERK1/2,p-p38,p38,p-NF-?B,NF-?B,PSD95 in hippocampus and cerebral cortex of mice.?The expression of RAGE and TLR4 in the microglia(CDllb as the microglial marker)and the expression of microglial marker(Iba-1)in hippocampus and cerebral cortex were analyzed by immunofluorescence.?Immunofluorescence was used to label microglia(Iba-1)and then for microglia 3D reconstruction,and the morphological parameters of microglia were measured with Imaris software.Results:(1)In the OFT test,there was no statistical difference in the horizontal score(shown as number of crossing),vertical score(number of rearing),travelled distance and averaged speed of mice in each group;compared with the CUMS group,roflupram could increase the sucrose preference ratio in SPT preference,and reduce immobility time in TST and latency time in NSFT.(2)Roflupram could reduce the level of inflammatory factors IL-1?,TNF-? and IL-6 in hippocampus and cerebral cortex compared with the CUMS group.(3)Rofluopram reduced the total length,area,volume,number of branching points,number of terminal points and sholl intersections of microglia,and supressed expression of microglial activation marker Iba-1.(4)Roflupram decreased the expression levels of HMGB1,RAGE,TLR4,NLRP3,Caspase1,ASC,p-p38,p-NF-?B,Iba-1 and increased the levels of p-ERK1/2,PSD95.The treatment with roflupram reduced the ratios of RAGE+/CD11b+ and TLR4+/CD11b+ in hippocampus and cerebral cortex of miceConclusion:Roflupram improved depressive-like behavior in CUMS mice and down-regulated the expression of inflammatory factors.The possible mechanism is that PDE4 inhibitor blocked the microglia activation,and supressed HMGB 1/RAGE&TLR4,downstream p-p3 8,p-NF-?B protein expression,and increased the expression of p-ERK1/2 and reduced NLRP3/ASC/Caspase1.