Effects Of Quercetin Intervention On Cognition Function In APP/PS1 Mice Under Different Vitamin D Status

Alzheimer's disease(AD),an age-related progressive neurodegenerative disease,is the leading cause of dementia in the elderly.But the etiology of AD remains unknown and there are no effective cures.Thus,it is urgent to find new strategies effective for AD prevention and treatment under the global AD challenge.Quercetin,a natural flavonoid,exhibits various biological function,such as anti-inflammation,anti-oxidation and gut microbiota regulation.Furthermore,it was identified as a promising compound exhibiting neuroprotective potentials against AD.Additionally,vitamin D(VD)has also been widely reported to possess neuroprotective properties.Interestingly,increasing evidence suggested potential crosstalk actions existing between quercetin and VD,and an epidemiological study reported that the beneficial effects of quercetin might be affected by serum vitamin D status.However,whether the beneficial effects of quercetin supplementation on cognitive function were associated with VD status has yet to be elucidated,which led us to explore the underlying probability.Objective:To explore whether the effects of quercetin intervention on cognitive function in APP/PS1 mice will be varied under different vitamin D status;and what are the potential mechanisms involved in.Particularly,we investigated how interventions will affect P-amyloid protein(AP),tau,neuro-inflammation,synaptic function,as well as microRNA(miRNA)and the gut microbiota profiles.Methods:A total of 28 male APP/PS1 transgenic mice were divided into following 4 groups:Control group(CON,fed with VD level=1000 IU/kg AIN-93G diet),Low-VD-level group(LVD,fed with VD level=500 IU/kg modified AIN-93G diet supplemented with 0.08%quercetin),Medium-VD-level group(MVD,fed with VD level=1000 IU/kg modified AIN-93G diet supplemented with 0.08%quercetin)and High-VD-level group(HVD,fed with the same diet as MVD group and given extra 1000IU vitamin D3 by intramuscularly injection every two weeks).At the end of 20 week's intervention we conducted the glucose tolerance test(GTT)and insulin tolerance test(ITT),followed by Morris water maze(MWM)test to assess the memory and spatial learning ability of mice.After that,all mice were fasted overnight.The serum 25(OH)D3 was analyzed using reagent based on the chemiluminescent immunoassay.Hippocampus and cortex from the hemi-brains were dissected.Additionally,the feces of mice were collected.For the pathology of A?,we used western bolt to measure the level of key protein in A? metabolism,including insulin-degrading enzyme(IDE),?-site amyloid precursor protein cleaving enzyme 1(BACE1)and Cathepsin B.Hemisphere of brain was stained with Congo red to observe the A? plaques visually.For the pathology of tau,we used western bolt to measure the level of tau phosphorylation at ser396 and ser404 sites,and the protein related with phosphorylation,such as protein phosphatase-a(PP2A),extracellular signal-regulated kinases 1/2(ERK1/2),AMP-activated protein kinase(AMPK),cell cycle dependent kinase 5(CDK5)and p35/25.Immunohistochemistry was used to detect the p-tau at ser202/thr205.For the level of neuroinflammation and synaptic function,western blot measured the level of protein involved in inflammation including NLR family pyrin domain containing 3(NLRP3),glial fibrillary acidic protein(GFAP)and ionized calcium binding adaptor molecule-1(Iba-1).And protein for synapse brain was detected including brain-derived neurotrophic factor(BDNF),postsynaptic density-95(PSD95)and synaptophysin.Immunofluorescence staining was carried out for GFAP and Iba-1 in hemi-brain.Key miRNAs associated with AD pathology was determined by Real-time PCR,including miR-26a?miR-125b?miR-138?miR-132 and miR-219.And 16S rRNA gene sequencing was used to reveal the profile of microbiota from feces.Results:1.Body weight,food intake and blood biochemical indexesThere was no difference for body weight and food intake among groups.During GTT test,there was no difference for AUC among groups.In ITT test,AUC was significantly increased in LVD group compared to CON group.Compared to CON group,serum level of 25(OH)D3 was significantly decreased in LVD group,while significantly increased in HVD group.2.Cognitive performance via MWMOn the 3rd and 4th day of navigation test,LVD group demonstrated reduced latency to platform.As for spatial probe trials,HVD group has increased time spent in target quadrant;while LVD group had increased number of crossing and swimming distance in target quadrant compared to CON.Mice in LVD group was swimming straight toward the hidden platform and spent more time in 4th quadrant.3.A? and Tau pathology3.1 A? pathology:In hippocampus,compared to CON,both MVD and HVD group showed increased IDE and reduced BACE1 protein expression,and there was no difference for ADAM10 and cathepsin B among groups.In cortex,LVD group had reduced ADAM 10 and cathepsin B,in addition MVD and HVD group had reduced BACE1;and there was no difference for IDE among groups.Via Congo red staining,A? plaques in both hippocampus and cortex were reduced from LVD group.3.2 Tau pathology:Compared to CON,there was significant reduction in the phosphorylation of tau at ser396 and ser404 in both hippocampus and cortex of LVD mice;meanwhile,p-Tau ser396 was also reduced in hippocampus of MVD group.Immunohistochemical staining showed that both hippocampus and cortex displayed reductions of positive staining of p-tau(ser202/thr205)from LVD group;while no difference for p-tau were found from MVD and HVD group.LVD group presented a significantly lower level of CDK5 in hippocampus,and p-ERK1/2 in both hippocampus and cortex compared to CON.There was no difference for PP2A,p-ERK1/2,p-AMPK,CDK5 and p35/25 protein expression from MVD and HVD group relative to CON group.4.Neuro-inflammation and synaptic plasticityIn hippocampus,GFAP and Ibal were significantly reduced from LVD group relative to CON,meanwhile HVD group demonstrated reduced Ibal.In cortex,NLRP3 from LVD group,and Ibal from both LVD and MVD group were also significantly reduced.The results of immunofluorescence staining were in consistent with the western blot.BDNF was only significantly elevated in hippocampus from LVD group;meanwhile,no alteration was observed for PSD95,synaptophysin and BDNF protein expression in cortex among groups.5.miRNAIn hippocampus,the level of miR-26a and miR-125b was significantly decreased,while miR-132 was markedly induced from LVD group compared to CON group.Additionally,miR-26a was down-regulated in hippocampus of HVD group;while there was no difference for miR-138 and miR-219 expression among groups.In addition,no difference for miR-26a,miR-125b,miR-138 and miR-219 expression were observed in cortex among groups.6.Gut microbiota profile and its relationship with ADThe ACE and Chaol was significantly increased from LVD group compared to CON group.At the phylum level,Firmicutes,Bacteroidetes and Proteobacteria dominated the microbiota of all groups.At the genus level,compared to CON,the relative abundance of Glutamicibacter,Facklamia and Aerococcus were only significantly enhanced from LVD;meanwhile,Helicobacter,LachnospiraceaeUCG006 and Staphylococcus were all significantly elevated from the three groups;Additionally,the relative abundance of Roseburia was significantly increased from both MVD and HVD,and Ruminiclostridium 9 was significantly decreased in HVD;while Lactobacillus was not different among groups.Spearman correlation analyses showed that,in hippocampus,p-tau at ser396&404,GFAP,Ib?1,miR-26a and miR-132 were negatively correlated with Aerococcus;and p-tau at ser404 and Ib?l were negatively correlated with Facklamia,Conclusions:1.Under low vitamin D level,APP/PS1 mice supplemented with quercetin had impaired insulin tolerance.2.Under low vitamin D status,APP/PS1 mice supplemented with quercetin had improved cognitive function.While under medium and high vitamin D status,the cognition deficits of APP/PS1 mice with quercetin intervention were not alleviated.3.Improved cognitive function from LVD group might be partially associated with reduced A? plaques,tau phosphorylation and neuroinflammation,as well as upregulated BDNF protein expression post intervention.4.The alterations of miRNA might also be partially involved in improving cognition function from LVD group.Supposedly,reduced miR-125b from LVD group might be associated with improved ERK1/2-CDK5 pathway,consequently resulting in reduction in tau phosphorylation,and elevated miR-132 might led to reduction in neuroinflammation and tau phosphorylation,in addition,elevated miR-26a might be associated with BDNF protein expression.5.The improved cognitive function from LVD group might also be associated with enhanced gut microbiota diversity and alterations in microbial composition(especially the elevation in the relative abundance of Facklamia and Aerococcus at the genus level).