Effect Of Icariin On Ubiquitination Of Amyloid Precursor Protein In Alzheimer’s Disease Mice

Objective: To investigate the effect of icariin(ICA)on the ubiquitination modification of β-amyloid precursor protein(APP)in Alzheimer’s disease mice.Methods: 1.Experiment in vitro:Human embryonic kidney(HEK)293 cells stably overexpressing human wild type APP695(OE-APP)and HEK293 cells(Control)were applied in the present study.Six small experiments were included as fellow:(1)Grouping:Control group,ICA(10,20,40,60,80,100 μM)groups;Processing method: After the treatment of ICA,cell viability was detected by MTT assay.(2)Grouping: Control group+ Cycloheximide(CHX)50 μg/m L(0,0.5,1,2,3,4 h),ICA(60 μM)+ CHX 50 μg/ml(0,0.5,1,2,3,4 h);Processing method: After the treatment of CHX at different time points,APP protein level was detected by western blot.(3)Grouping: Control group,Control +ICA(60 μM)group,Control + proteasome inhibitors MG132(20 μM)group,ICA(60 μM)+ MG132(20 μM)group;Processing method: APP antibody was used for co-immunoprecipitation(Co-IP),and then the levels of ubiquitin(Ub),Ub K48,Ub K63,APP,HRD1 were detected by western blot.(4)Grouping: Control group,Control + ICA(60 μM)group,OE-APP group,OE-APP + ICA(60 μM)group;Processing method: The level of HRD1 protein after ICA treatment was detected by western blot.(5)Grouping:Control-si RNA group,HRD1-si RNA(60,70,80 n M)group;and Control-si RNA group,Control-si RNA + ICA(60 μM)group,HRD1-si RNA(80 n M),HRD1-si RNA(80 n M)+ICA(60 μM)group;Processing method: After transfection of HRD1-si RNA,the silencing efficiency of HRD1 was measured by western blot,and then the levels of Ub,Ub K48,Ub K63,APP,HRD1 were also detected by western blot.(6)Grouping: Control group,Control + ICA(60 μM)group;Processing method: Immunofluorescence double staining was used to detect the co-localization of EEA1 or LAMP2 and APP.2.Experiment in vivo: Six months old male APP/PS1 transgenic mice and wild type(WT)mice were randomly divided into 5 groups: WT control group(n=10),WT + ICA 60 mg/kg group(n=10),APP/PS1 control group(n=10),APP/PS1 + ICA 60 mg/kg group(n=10)and APP/PS1 + DPZ 1 mg/kg group(n=8),treated daily for 3 months by intragastric infusion,while mice in control groups were administered with distilled water.Morris water maze and Y maze tests were used to detect the spatial learning and memory function of mice.Afterwards,brain tissue was collected,total protein was extracted,APP antibody was used for Co-IP,and western blot was used to detect Ub,Ub K48,Ub K63,APP,HRD1 protein levels;immunofluorescence double staining was used to detect the co-localization of EEA1 or LAMP2 and APP.Results: Experiment in vitro showed that the concentration of ICA no more than 80 μM did not affect the cell viability;ICA accelerated APP protein degradation,and this effect was related with ICA enhancing the ubiquitination level of APP,especially the total ubiquitination and K48-linked polyubiquitination.In addition,ICA could promote HRD1 protein level;80 n M HRD1-si RNA could decrease the HRD1 protein level to 73%.After successful transfection,K48-linked polyubiquitination,K63-linked polyubiquitination and total ubiquitination of APP were obviously reduced.ICA obviously decreased the co-localization of EEA1 and APP,however,almost no effect on the co-localization of LAMP2 and APP.The results of experiment in vivo showed that ICA could significantly improve the spatial learning and memory function of mice,and ICA could stimulate the ubiquitination of Ub K48-linked APP in the brain of AD model mice,but had little effect on Ub K63-linked APP ubiquitination;in addition,ICA could decrease the co-localization of EEA1 and APP in AD model mice,however,almost no effect on the co-localization of LAMP2 and APP.This result was consistent with that from in vitro.Conclusion: ICA promotes the ubiquitination and proteasome-dependent degradation of APP by up-regulating HRD1,thereby improving the spatial learning and memory function of AD mice.