The Therapeutic Effects Of Icariin On The Aggravated Symptoms And Pathological Changes Of Alzheimer’s Disease Induced By Psychological Stress

1.BackgroundAlzheimer’s disease(AD)is a neurodegenerative disorder characterized by progressive cognitive impairment.Prominent neuropathological features of AD areβ-amyloid(Aβ)plaques and neurofibrillary tauopathy consisting of threads and tangles,which are observed throughout the brain,including the areas critically involved in memory formation and emotional regulation.Inflammation has a causal role in the pathogenesis of AD.There has been considerable recent research to identify the pathways of neuroinflammation and the cell types involved.It has become clear that microglial cells are a major contributor to the inflammatory process in the brain.Microglia are now a major focus of neurodegenerative disease research and defining the physiological properties of microglia is crucial to understanding their potential role in neuronal loss and AD.Microglia are involved in Aβ clearance,which is both beneficial,to inhibit Aβ build up,and deleterious,when levels of Aβ are elevated,as prolonged inflammation will result.Mounting evidence shows that psychological stress is involved in AD pathogenesis,as patients with posttraumatic stress disorder or depression often develop dementia and even clinical AD.The elevated level of cortisol,a stress hormone,found in AD patients plays a crucial role in contribution to the comorbidity.Studies in animal models further demonstrated the association of hypothalamic-pituitary-adrenal axis activation with AD pathogenesis in the frontal cortex and hippocampus.Chronic unpredictable stress(CUS)significantly increased both serum corticosterone levels and amyloid precursor protein(APP)fragments.Aβinfusion triggered APP misprocessing in the hippocampus and prefrontal cortex(PFC)of rats,which was further exacerbated by stress.Furthermore,chronic stress including restraint/isolation stress in particularly elevated Aβ40 and Aβ42 levels of brain,accelerated amyloid plaque formation and impaired learning and memory in mice.More importantly,a recent study demonstrated that prenatal stress induced a constant neuroinflammatory response which contributes to a more vulnerable profile;the later can subsequently lead to an aberrant response to accumulating Aβ peptides,and ultimately modify the extent of Aβ neuropathology.Microglial activation is often classified into classical(M1)and alternative(M2).M1 microglia may contribute to dysfunction of the neurotrophic system by expressing pro-inflammatory cytokines,such as tumor necrosis factor(TNF)-α,interleukin(IL)-1β and IL-6.In the Ml phenotype of the microglia,the activation of nuclear factor κB(NF-κB)may play a critical role in the production of pro-inflammatory cytokines,leading to neurotoxic outcomes.The M2 phenotype,also known as the neuroprotective microglial phenotype,releases different mediators including IL-4.IL-10 and transforming growth factor(TGF)-p to antagonize inflammation-induced damage in the central nervous system(CNS),and is associated with enhanced phagocytosis of deposited amyloid.Stress paradigms,including CUS and chronic social defeat stress,are among the factors that could induce M1 microglia polarization.However,the effect of microglial activation induced by psychological stress on clearance ability of Aβ remains unknown.Peroxisome proliferator-activated receptors(PPARs)are a group of nuclear receptor proteins regulating gene expression as ligand-activated transcription factors.Three closely related PPAR isoforms have been identified(alpha,beta and gamma).Among the three isoforms.PPARy has the highest expression in the CNS,where it has been identified in neurons,astrocytes,and glial cells.Specifically,PPARγ appears to be linked to stress modulation and to mediate the conversion between microglia phenotypes.PPARy induced the M2 phenotype microglia under A(3 toxicity via adiponectin treatment through its effects on the anti-inflammatory response.Moreover,pioglitazone which was used to treat depressive-like behaviors in a chronic mild stress mouse model is associated with PPARy-mediated M2 activation of the microglia.Icariin,a natural flavonoid compound extracted from Epimedium brevicornum Maxim(a traditional Chinese herb),has been proven to have a wide range of effects including anti-oxidant,anti-bacterial and anti-inflammatory properties.Previous studies investigated the pharmacological properties of icariin in the CNS and showed that it can markedly attenuate cognitive deficits in several models of AD and alleviate the neuronal injury induced by ischemia.It was also reported that icariin can attenuate the inflammatory response through PPARy activation in rats.But,whether icariin could attenuate AD pathogenesis by up-regulating PPARy needs to be clarified.2.Objectives2.1 To investigate the effects of chronic unpredictable mild stress(CUMS)on microglia M1 phenotype markers such as IL-1β,IL-6,TNF-α and TLR4-NF-κB signal in the hippocampus and prefrontal cortex of mice.2.2 To investigate the effects of restraint/isolation stress(RIS)on learning and memory function as well as the accumulation of Aβ and the phenotype of microglia activation in the hippocampus and prefrontal cortex of APP/PS1 mice,and whether icariin could attenuate AD pathogenesis by up-regulating PPARγ.2.3 To establish the lipopolysaccharide(LPS)and icariin treated BV2 microglia model and investigate the effects of LPS stimulation and icariin treatment on the phenotype and the expressions of PPARy of BV2 microglia.3.Materials and methods3.1 Experimental animals,cell line and grouping3.1.1 Study 1:30 male C57BL/6 mice,7 to 8 weeks old,weighing 18-22 grams after acclimatization(7 days)were divided into 3 groups(n=10 each group)randomly:control group(Ctr),CUMS group(CUMS),CUMS+TAK-242 group(CUMS+TAK).Mice in CUMS group were subjected to CUMS for 21 days while mice in CUMS+TAK group were intraperitoneally injected TLR4 antagonist TAK-242 30 min before CUMS.3.1.2 Study 2:30 male APP/PS1 mice,aged 3 months,weighing 24-30 grams after acclimatization(7 days)were divided into 3 groups(n=10 each group)randomly:control group(Ctr),restraint/isolation stress group(RIS),restraint/isolation stress+icariin group(RIS+ICA).Mice in RIS group were subjected to restraint/isolation stress for 28 days while mice in RIS+ICA group were treated with icariin by oral administration after RIS for 6 months.3.1.3 Study 3:BV2 cells are maintained in Dulbecco’s modified Eagle medium(DMEM)with 10%fetal bovine serum(FBS),2 mM L-glutamine,100 U/ml penicillin and 100 mg/ml streptomycin in a 5%C02 incubator.There are totally 4 groups:control group(Ctr),LPS group(LPS),LPS+icariin 5 μM group(LPS+ICA5)and LPS+icariin 10 μM group(LPS+ICA10).3.2 Chronic unpredictable mild stressThe stressors were included as follows:8 hours food deprivation,8 hours water deprivation,45°lean cage,white noise(1500 Hz,92 PB,1 h),day and night confused,foot shock 15 min(50 mV,10 s duration,average 1 shock/min)and horizontal oscillation 20 min.The stress process lasted for 21 days.The time and form of stressor daily were at random and each type of stressor was guaranteed to be performed three times during the CUMS period.3.3 Restraint/isolation StressRestraint/isolation stress is a paradigm used to build depressive animal models.Each mouse was individually restrained in restraint tubes for 6 hours per day,and the mice were deprived of water and food during the restraint/isolation stress.3.4 Drug treatments3.4.1 Study 1:Mice in CUMS+TAK-242 group were received both CUMS procedure and a daily intraperitoneal injection of TLR4 antagonist TAK-242(3 mg/kg,freshly suspended in 80%polyethylene glycol 400)at 30 min prior to stress exposure.The control groups and the CUMS groups were given 80%polyethylene glycol 400(vehicle)to balance the systematic error.3.4.2 Study 2:Icariin was orally administered daily to the mice in the RIS+ICA group(60 mg/kg,freshly suspended in ddH2O)from 4 to 10 months of age.The mice in the control group and restraint/isolation stress group were given ddH2O(vehicle)in the same volume to balance the systematic error.3.4.3 Study 3:Cell culture medium are changed into DMEM/F12 1:1 medium without serum 1 hour before LPS(1 ug/ml)stimulation in LPS,LPS+ICA5 and LPS+ICA10 groups.And the cells in LPS+ICA5 and LPS+ICA10 groups are co-treated with icariin in 5 and 10 μM for 6 hours.3.5 Behavioral testsThe sucrose preference test was conducted to evaluate an anhedonia state,one of the core symptoms of depression.The open field test was used to measure spontaneous activity and exploratory behaviors.The elevated plus maze test was performed to assess anxiety-like behaviors.The Y maze test and Morris water maze test were used for evaluating spatial learning and memory.3.6 RadioimmunoassaySerum samples were obtained from C57BL/6 mice.Serum corticosterone(CORT)was measured using a commercially available radioimmunoassay(RIA)kit.The RIA was performed according to manufacturer’s instruction.3.7 Immunohistochemistry3.7.1 Study 1:After the behavioral tests,4 C57BL/6 mice in each group were randomly selected to be anesthetized and transcardially perfused.The brain was taken out and embedded in paraffin block.The paraffin-embedded hippocampus and prefrontal cortex were sliced into 5 μm coronal sections to detect the distribution and expression of NF-κB by immunohistochemistry.3.7.2 Study 2:After the behavioral tests,4 APP/PS1 mice in each group were randomly selected to be anesthetized and transcardially perfused.The brain was taken out and embedded in paraffin block.The paraffin-embedded hippocampus and prefrontal cortex were sliced into 5 μm coronal sections to detect the distribution and expression of Aβ1-42,Iba-1 and iNOS by immunohistochemistry.3.7.3 Study 3:Microglial cells are plated on glass coverslips overnight.After drug treatment they are fixed in 1%paraformaldehyde.The distribution and expressions of iNOS is detected by immunocytochemistry.3.8 Enzyme-Linked Immunosorbent AssayThe total protein was isolated from tissues or cells.The protein concentrations were measured.The levels of IL-1β,IL-6,TNF-α,IL-4,IL-10,TGF-β1,NF-κB component p65,and AP1-42 were measured using commercially available enzyme-linked immunosorbent assay(ELISA)kits according to the instructions of the manufacturer.Serial dilutions of protein standards and samples were added to 96-well ELISA plates followed by HRP labeled antibodies for IL-1β,IL-6,TNF-α,IL-4,IL-10,TGF-β1,NF-κB p65,and Aβ1-42 to form antibody-antigen-enzyme labeled antibody complexes.After complete washing with wash solution,TMB substrate solution was added,which under the catalysis of HRP was converted to blue.The reaction was stopped using the stop solution.Optical density at 450 nm was detected using the iMark Microplate Absorbance Reader.The concentration of each sample was calculated from the linear equation derived from the standard curve of known concentrations of NF-κB p65,AP1-42 and the cytokines-3.9 Western blottingThe total protein was isolated from tissues or cells.The protein concentrations were measured.After denaturation,protein samples were separated by electrophoresis and transferred to polyvinyl idene difluoride(PVDF)membranes.The membranes were incubated with appropriate primary antibodies overnight at 4℃ after blocked.On the following day,the PVDF membranes were washed and incubated with secondary antibodies.The PVDF membranes were washed again and developed by using a chemiluminescent method.The grey value of stripes was analyzed with the use of Image J 14.0 software.4.Results4.1 The results of study 1:Effect of chronic unpredictable mild stress on the behavioral tests,phenotype of microglia activation and neuroinflammation in the hippocampus and frontal cortex in C57BL/6 mice4.1.1 C57BL/6 mice that exposed to CUMS manifested obvious depressive-like behaviors and memory impairment.4.1.2 CUMS resulted in elevated expression of microglia M1 phenotype markers such as IL-1β,IL-6,TNF-α and NF-κB in the hippocampus and prefrontal cortex of C57BL/6 mice.4.1.3 The TLR4 antagonist,TAK-242,could effectively ameliorate the depressive-like behaviors and memory abnormalities,and normalize the expressions of IL-1β,IL-6,TNF-α and NF-κB.4.2 The results of study 2:Effect of restraint/isolation stress on the symptoms and pathological changes of AD in the hippocampus and prefrontal cortex of APP/PS1 mice and the therapeutic effects of icariin4.2.1 APP/PS1 mice that exposed to RIS manifested obvious depressive-like behaviors at 4 months of age and memory impairment at 10 months of age.4.2.2 RIS resulted in elevated expression of microglia M1 phenotype markers,accumulation of Aβ and decreased expression of PPARy in the hippocampus and prefrontal cortex of APP/PS1 mice at 10 months of age.4.2.3 Icariin could effectively ameliorate the memory impairment and elevated expression of microglia M1 phenotype markers,accumulation of Aβ and decreased expression of PPARy in the hippocampus and prefrontal cortex of APP/PS1 mice at 10 months of age.4.3 The results of study 3:Effect of icariin on the expression of PPARy and phenotype of BV2 microglia activation induced by lipopolysaccharide4.3.1 1 ug/ml LPS increased levels of IL-6 and TNF-α significantly and the decreased level of TGF-β1.4.3.2 Compared with the LPS group,10 μM icariin treatment could decrease the elevated levels of IL-6,TNF-α and iNOS and increase the decreased level of TGF-β1 induced by LPS stimulation,in particularly,up-regulate the expression of PPARγsignificantly.10 μM icariin treatment also up-regulated the expression of PPARγsignificantly compared with the control group while 5 μM icariin treatment only decreased the elevated levels of IL-6,TNF-α and iNOS and increased the decreased level of TGF-β1 induced by LPS stimulation compared with the LPS group.5.Conclusions5.1 Psychological stress could result in depressive-like behaviors,memory impairment and elevated expression of microglia M1 phenotype markers in the hippocampus and prefrontal cortex of C57BL/6 mice by activating TLR4-NF-κB pathway.5.2 Psychological stress could result in depressive-like behaviors,worse memory impairment and higher expression of microglia M1 phenotype markers and accumulation of Aβ in the hippocampus and prefrontal cortex of APP/PS1 mice by suppressing the expression of PPARy.5.3 Icariin could attenuate M1 activation of microglia and Aβ plaque accumulation in the hippocampus and prefrontal cortex by up-regulating PPARy in psychological stressed model of Alzheimer’s disease.5.4 10 μM icariin treatment could up-regulate the expression of PPARγ significantly and attenuate Ml activation induced by LPS in BV2 microglia in vivo study.