| Objective:Alzheimer’s disease(AD)is a degenerative disease of the central nervous system with progressive clinical memory loss and cognitive dysfunction.Although domestic and foreign scholars have done a lot of research,but so far they have not found an effective treatment.Phosphodiesterase 4(PDE4)is an in vivo enzyme that specifically hydrolyzes cyclic adenosine monophosphate(cAMP).Studies have confirmed that PDE4 inhibitors such as rolipram,rofluopram,and GEBR-7b have the effect of improving AD,but PDE4 inhibitors have a strong side effect of vomiting,which greatly limits its application in the clinic.Roflumilast(RFM)is a long-acting inhibitor of PDE4 with little side effects and is a drug approved by the US FDA for the treatment of chronic obstructive pulmonary disease.In recent years,Vanmierlo et al.found that roflumilast improved cognitive function in mice at non-vomiting doses;Jabaris et al.found that roflumilast also had significant improvement in cognitive function decline induced by hypertension..This suggests that Roflumi last may also have the potential to improve AD,but there are no detailed reports on its detailed role and mechanism.Therefore,this project intends to use APP/PS1 double transgenic induced AD model to observe the effect of roflumilast on cognitive function,anxiety,depression and other behavioral features in AD mice,and from the PDE4/ cAMP/PKA/CREB signaling pathways Stuart’s anti-AD mechanism was studied in order to provide a theoretical basis for the development of AD therapeutic drugs using PDE4 as a target.Methods:1.Establishment of AD model and group administration:APP/PS1 double transgenic male mice were selected as AD mice,and C57BL/6J male mice with the same genetic background were tested as control mice.Roflumilast was administered in two dose groups,the low dose group(5 mg/kg)and the high dose group(10 mg/kg),administered orally for 30 days and administered once a day.2.Observe the effect of Roflumilast on the behavior of mice:The following behavioral tests were performed 20 days after intragastric administer ation.The behavioral tests were performed after intragastric administration for 2 hours.(1)Open-field trials and an elevated plus-maze to evaluate the effect of roflumilast on anxiety and anxiety behaviors in AD mice.(2)New object recognition and water maze to evaluate the effect of roflumilast on learning and memory ability of AD mice.(3)Tail suspension test and forced swim test to evaluate the effect of roflumilast AD on depression in mice.3.HE staining:After the behavior was completed,the mouse brain tissue was taken,fixed in 4% paraformaldehyde,embedded in paraffin,and stained with hematoxylin-eosin to observe the protective effect of roflumilast on AD from the pathology.4.To explore the mechanism of Roflumilast’s influence on behavior in mice: After the behavioral experiment was completed,the mice were sacrificed and the brain tissue was taken.The cortex and hippocampus were isolated and their tissue homogenates were prepared.Through the detection of the following proteins,a preliminary exploration of the role of roflumilast on behavioral mechanisms.(1)Elisa test detected cerebral cortex and hippocampal cAMP protein levels.(2)The expression of CREB,pCREB,BDNF,NPY,Bcl-2,Bax and PDE4 isoforms were detected by western blot,and the mechanism of action of roflumilast was initially explored.5.Experimental data using SPSS16.0 software for statistical comparison between groups of samples using a one-way analysis of variance(ANOVA).When P<0.05,the difference was statistically significant.Results:1.Roflumilast improves cognitive function in AD mice(1)New Object Recognition Experiment: In this experiment,the cognitive index is an index reflecting the cognitive function of the mouse,and the cognitive index = the time of exploring a new object/[exploring a new object time + exploring an old object].This experiment found that the cognitive index of AD mice was significantly lower than the blank control group,and roflumilastcan improve the decline of cognitive index of AD mice,especially in the high dose group of roflumilast has statistically significant differences.(2)Water maze experiment: It is divided into positioning navigation experiment and space exploration experiment.In the positioning navigation experiment,the latency of the search platform was used as the index for evaluating the cognitive function.The results showed that as the number of training days increased,the incubation period for each group of mice to find the platform gradually decreased.From the second day,compared with the blank control group,the latency period of the platform was significantly prolonged in AD mice,and from the third day,roflumilastcould significantly improve cognitive impairment in AD mice and shorten the incubation period of the search platform.In the space exploration experiment on the fifth day,the time when the mice passed the platform for the first time was longer in the model group than in the control group,and there was a significant difference;the time of administration of the low-dose group was shorter than that of the model group,and there was a significant difference;The time of the high-dose group was shorter than that of the model group,and there was a significant difference.In the space exploration experiment,the number of passages through the platform within 1 min was significantly reduced in the model group compared to the control group.There was a significant difference in the number of times that the model group compared with the control group;There was a significant increase in the number of high-dose groups passing through the platform compared with the model group,with significant differences.2.The effect of Roflumilast on the anxiety of AD mice(1)In the open field experiment,the time for mice to explore in the surroundings was longer in the model group than in the control group,and there was a significant difference;the administration of the high dose group was significantly shorter than the model group,with significant differences.The number of mouse arrivals in the central area was significantly higher than that in the model group,with significant differences.The time spent in the central area was significantly longer than that of the model group.(2)In the elevated plus maze,the time that the mice stayed in the open arms was significantly shorter in the model group than in the control group,and there was a significant difference;the high dose group was significantly prolonged compared with the model group.difference.3.The effect of Roflumilast on the depression of AD mice(1)In the tail-suspended experiment,the immobile time in the model group was significantly longer than that in the control group,and there was a significant difference.The administration of the high-dose group was significantly shorter than the model group,with significant differences.(2)In the forced swimming experiment,the immobile time in the model group was significantly longer than that in the control group,and there was a significant difference;there was no significant difference between the low dose group and the model group;the high dose group was significantly shorter than the model group.There are significant differences.4.Roflumilast can improve pathological damage and cell apoptosis in AD mice(1)HE staining.The number of cells in the control group was large and closely arranged.The cells in the model group were disordered,the number was decreased,the gap was large,and the administration group was improved relative to the model group.(2)Roflumilast modulates apoptotic protein expression in AD mice.In the cortex of mouse brain tissue,compared with the control group,the Bcl-2/Bax of the AD mice was significantly decreased.Compared with the AD mice,the Bcl-2/Bax was significantly higher in the roflumilast group.In the hippocampus,compared with the blank control group,the Bcl-2/Bax decreased significantly in the AD mice;compared with the AD mice,Bcl-2/Bax increased significantly in the high dose of roflumilast group.5.Roflumilast can improve the possible mechanism of cell ethology in AD mice(1)Roflumilast modulates PDE4/cAMP/PKA/CREB signaling pathway proteins.Compared with the control group,the PDE4 D content in the cortex of AD mice did not change significantly;compared with the AD mice,the PDE4 D content in the roflumilast group was significantly reduced.In the mouse hippocampus,compared with the control group,the PDE4 D content in the AD mice increased significantly;compared with the AD mice,the PDE4 D content in the roflumilast group was significantly reduced.Compared with the control group,there was no significant difference in PDE4 B content in the cortex of AD mice.Compared with AD mice,the PDE4 B content in the high-dose roflumilast group was significantly reduced.In the mouse hippocampus,the PDE4 B content in the AD mice was significantly increased compared to the control group,and the PDE4 B content in the roflumilast group was significantly reduced compared with the AD mice.In the cortex of mouse brain tissue,compared with the blank control group,the ratio of pCREB/CREB in AD mice was significantly lower;compared with AD mice,the ratio of pCREB/CREB in the roflumilast group was significantly higher;in the mouse hippocampus Compared with the control group,the ratio of pCREB/CREB in AD mice was significantly lower.Compared with AD mice,the ratio of pCREB/CREB in the high dose of roflumilast group was significantly higher.In the cerebral cortex and hippocampus of mice,the content of cAMP in AD mice was significantly higher than that in the control group.Compared with AD mice,cAMP content in the high dose of roflumilast increased significantly.(2)Roflumilast can regulate the expression of BDNF.In mouse hippocampus,the expression of BDNF protein in AD mice was significantly decreased compared with the control group.Compared with AD mice,the expression of BDNF protein in Roflumilast group was significantly increased.In the cortex of mice,compared with the control group,the expression of BDNF protein in AD mice was significantly reduced.Compared with AD mice,the expression of BDNF protein was significantly increased in Roflumilast group.(3)Roflumilast can regulate the expression of APP and BACE1.Compared with the control group,the APP content in the AD mice was significantly increased in the cortex of the mouse brain tissue.Compared with the AD mice,the APP content in the high dose of Roflumilast was significantly reduced.In mouse hippocampus,the APP content in AD mice was significantly increased compared with the control group.Compared with AD mice,the APP content in the high-dose Roflumilast group was significantly reduced.In the cerebral cortex of mouse brain,compared with the control group,the expression of BACE1 protein in AD mice was significantly increased.Compared with AD mice,the expression level of BACE1 in the roflumilast group was significantly lower;in the hippocampus,compared with the control group Compared with AD mice,the expression of BACE1 protein was significantly increased.Compared with AD mice,the expression of BACE1 was significantly decreased in roflumilast group.Conclusion:1.Roflumilast can improve cognitive function,anxiety behavior and depression in APP/PS1 double transgenic AD mice.2.Roflumilast can improve the behavior of APP/PS1 double-transgenic AD mice by inhibiting the expression of PDE4 B and PDE4 D,and then increasing the level of cAMP,promoting the phosphorylation of CREB,promoting the expression of BDNF,and reducing the number of cells.Apoptosis is related to this study found Roflumilast can also reduce APP,BACE1 expression. |
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