Protective Effect Of Flavonoid-rich Extract From The Leaves Of Diospyros Kaki On Aging And APP/PS1 Mice And Its Related Mechanisms

BackgroundAging is a major factor underlying brain dysfunction,which is marked by memory loss,cognitive impairment,and many age-related neurodegenerative disorders,such as Alzheimer’s disease(AD).Multidimensional pathological features,including oxidative stress,neuroinflammation,and cell metabolic imbalance,are associated with aging of the brain,which consequently result in neuronal death and synaptic dysfunction.AD is an age-dependent,irreversible neurodegenerative disease.With the dramatic increase of the world’s population and life expectancy,the prevalence of AD is growing at an ever-increasing rate.One of the major hallmarks of AD is the deposition of extracellular senile plaques composed primarily of amyloid-β peptide(Aβ).Aβ,generated from sequential cleavage of amyloid precursor protein(APP)by β-site APP cleavage enzyme 1(BACE1)and y-secretase,promotes neurotoxicity in AD by directly or indirectly inducing oxidative stress,inflammation,synaptic failure,and cognitive impairment.Aβ leads to the up-regulation of the receptor for advanced glycation end products(RAGE),which mediates entry of peripheral Aβ into the brain,and functions as an"inflammatory" receptor after binding to Aβ.Microglial activation,downstream pro-inflammatory signaling cascades,and oxidative stress were found to be up-regulated by Aβ production.Inflammatory cytokines,such as tumor necrosis factor alpha(TNF-α)and interleukin-1β(IL-1β),may lead to microglial activation,further secretion of pro-inflammatory molecules,Aβ aggregation,neuronal dysfunction,and cell death.Nuclear factor-KB-p65(NF-κB p65),is a subunit of the NF-κB transcription complex that regulates pro-inflammatory pathways and acts as a linker between oxidative stress,Aβ,and inflammation.Environmental insults,including oxidative stress and Aβ plaques,probably via interaction with RAGE,result in the activation of NF-κB,which mediates gene transcription of RAGE and inflammatory cytokines,like TNF and IL-6.Therefore,interventions attenuating Aβ-induced microglial activation,oxidative stress,and inflammation,especially at an early stage,might be beneficial for AD treatment.Although some drugs are available to temporarily alleviate symptoms of AD,there are no effective measures for preventing or delaying the onset or stopping AD progression.Extracts from persimmon leaves,of which flavonoid is the main component,have been widely used in China to treat apoplexy and its sequela.In addition to its well-known antioxidant and neuroprotective effects,this leaf extract also provides a number of other beneficial aspects.Studies reported that a standardized flavonoid extract of persimmon leaves(FLDK)exerted protective effects in vivo in two different brain ischemia and reperfusion injury rat models,as well as protective effects in vitro in hippocampal and primary cortical neurons following glutamate or hypoxia injury.FLDK also protected NG108-15 cells from oxidative injury induced by hydrogen peroxide,possibly by improving the cellular redox state and upregulating Bcl-2 levels.Ethyl acetate extract from persimmon leaves showed a potential protective effect on Aβ1-42-induced cognitive dysfunction in rats,probably via improving the antioxidative defense system and attenuating mitochondrial-mediated neuronal apoptosis.Flavonoids and triterpenoids are likely the major active components in the ethyl acetate extract.However,it is unclear if the flavonoid-rich ethanol extract from the leaves of Diospyros kaki(FLDK)could delay aging.Part I Flavonoid-rich Extract from the Leaves of DiospyrosKaki Attenuates D-galactose-induced Oxidative Stress andNeuroinflammation-mediated Brain Aging in MiceObjectiveTo investigate the protective effects of FLDK on learning and memory impairment,neuroinflammation,oxidative stress,apoptosis,and synaptic dysfunction in D-gal-induced aging mice.Methods1.Isolation,purification and identification of flavonoids from persimmon leavesThe leaves of Diospyros kaki L.were dried at room temperature.The dried and milled leaves were refluxed with 85%(v/v)ethanol three times for two hours each time.The filtrates were combined and concentrated to obtain a crude extract by vacuum evaporation.Then the extract was redissolved in water and added to a a macroporous adsorption resin for purification by washing with 70%alcohol.The filtrates were extracted for five times using n-butanol.The n-butanol solution was combined and concentrated into a thick paste,and then 95%ethanol was added to redissolve.The extract was filtered to remove the residue.The collected liquid was finally concentrated and dried.High performance lipid chromatography(HPLC)analyses were carried out using a Shiseido chromatographic system(Shiseido co.,LTD.,Tokyo,Japan)with a dual pump(F3201),an auto sampler(NASCA 5100),a column oven(F3004),and a 4-flow channel degasser(F3202).FLDK were separated on a CAPCELL CORE C18 column(2.7 μm particle size,100 mm× 2.1 mm i.d.,Shiseido Co.,Ltd.,Tokyo,Japan)with a 10 mm×2.1 mm precolumn(Thermo Accucore C18,2.1 μm,Thermo Fisher Scientific,Co.,USA).The mobile phases were composed of acetonitrile(A)and water with 0.1%formic acid(B)using a multistep gradient elution of 15%A for 0-10 min,35%A for 10-20 min,95%A for 20-45 min,95%A for 45-55 min,15%A for 55-55.1 min,15%A at 55.10-60.0 min with the flow rate kept at 1.0 mL/min.The injected sample volume was set at 5 μL,and the column oven temperature was 20℃.After separation,the HPLC flow was directed to the mass spectrometer(MS)inlet after 1:10 splitting.FLDK analyses were performed on an API 5500 QTRAP mass spectrometer(Applied Biosystems Sciex,Singapore)with an electrospray ionization source(ESI)in negative mode.Nitrogen was used as the carrier,heater,and collision gases.The Applied Biosystems Analyst version 1.5.2 software was used to control for data acquisition and analysis.HPLC-MS verified that the ethanol extract from the leaves of Diospyros kaki contained the following flavonoids:myricetin,quercetin,kaempferol,hyperoside,astragalin,vitexin.After being hydrolyzed to aglycone,the extract was contained more than 79.35%total flavonoids as determined by UV Spectrophotometry.2.GroupingThirty-six 10-week-old Kunming male mice were randomly divided into normal control group(NC group),aging model group(D-gal group),low-dose FLDK intervention group(F1 group),and high-dose FLDK intervention group(F2 group)(n= 9 per group).D-gal group received subcutaneous injections of D-galactose at 100 mg/(kg d)for 10 weeks to mimic aging and were orally gavaged with vehicle.NC group were injected with normal saline and received the same volume of vehicle by oral gavage.The flavonoids F1 and F2 intervention groups were fed 40 and 80 mg/(kg d)FLDK,respectively,by oral gavage after subcutaneous injection of D-gal.3.Behavioral tests3.1 Y-maze testThe Y-maze test was used to determine special working memory performance.After being acclimated in the environment,each mouse was placed at the end of one arm of the maze.The series of arm entries were recorded during three 8-min sessions.Spontaneous alteration behavior was defined as the number of entries into three arms.Alteration behavior(%)was calculated as:(alterations/total entries-2)×100.3.2 Morris water maze testMorris water maze test was used to detect the escape latency(EL),the number of crossing across the platforms and the time spent in target quadrant(the swimming time in the site where the platform had been placed)of each group,and to explore the protective effect of FLDK on the spatial learning and memory ability of aging mice.The experiment was divided into five consecutive days of positioning navigation tests and one day of space exploration experiments.4.Detection of AGEs and RAGE in the brainThe content of AGEs in brain tissue was detected by enzyme-linked immuno sorbent assay(ELISA),and the expression of RAGE protein was detected by western blot.5.Microglia and astrocytesWestern blot and immunohistochemistry were used to detect the expression of microglia marker Ibal and astrocyte marker GFAP.6.NF-κB nuclear translocation and inflammatory factor expressionWestern blot was used to detect the expression of nucleus and cytoplasm NF-κB and TNF-α,IL-1β,COX-2 and iNOS in the of brain tissue.7.Oxidative stress level detectionThe kit detects the antioxidant enzymes SOD,GSH-Px activity and MDA content.8.Detection of neuronal apoptosisWestern blot was used to detect the expression of apoptosis pathway-related proteins PI3K,p-Akt,t-Akt,JNK,Bcl-2 and Bax.9.Detection of synaptic related proteinsThe expression of synaptophysin,synaptotagmin,p-CREB,t-CREB and p-CaMKII was detected by western blot.10.Statistical analysisStatistical analysis was performed using SPSS20.0.Significant differences between treatment groups were analyzed with one-way analysis of variance(ANOVA)followed by the Tukey’s test.Repeated-measures ANOVA followed by the Tukey’s test were used for comparision of escape latency in the MWM test.Data are expressed as means±standard error of the mean(SEM).P values<0.05 were considered statistically significant.Results1.Identification results of flavonoids from persimmon leavesHigh performance liquid chromatography-mass spectrometry confirmed that the extract of flavonoids from persimmon leaves includes quercetin,kaempferol,myricetin,hypericin,astragaloside,vitexin,etc.After the extract was hydrolyzed to aglycon,UV spectrometry was used.The photometric method confirmed that the total flavonoid content was 79.35%.2.Effects of FLDK on memory impairment in D-gal-aged miceThe D-gal group showed a significant decrease in the percentage of spontaneous alteration compared to the NC group(P<0.01).The percentage of spontaneous alteration was significantly increased in FLDK-treated mice at both 40 and 80 mg/kg(P<0.05 and P<0.01,respectively).EL increased in the D-gal group on the second day compared to the NC group(P<0.05).Administration of oral FLDK(40 and 80 mg/kg)significantly reduced EL(both P<0.05).In the probe test,the searching frequency(the number of times that the mice crossed the site where the platform had been placed)and the time spent in target quadrant(the swimming time in the site where the platform had been placed)decreased in the D-gal group compared to the NC group(both P<0.001).FLDK at both dosages(40 and 80 mg/kg)alleviated the decreased number of crossings and the time spent in target quadrant(P<0.01).In addition,the high dose of FLDK(80 mg/kg)showed better effects than the low dose of FLDK(40 mg/kg)on the time spent in the quadrant(P<0.05).3.Effects of FLDK on AGEs and RAGE levels in D-gal-aged miceD-gal significantly(P<0.01)increased AGEs levels in the hippocampus and cortex regions,which was attenuated by FLDK(P<0.05).Our results showed that the interaction of D-gal with its receptor,RAGE’s,significantly increased RAGE expression in the hippocampus and cortex regions compared to the NC group(P<0.01).However,FLDK markedly reversed this upregulation of RAGE compared to the D-gal group(P<0.05).4.FLDK suppressed microglial and astrocytes in D-gal-aged miceWestern blots showed significant increases in GFAP and Ibal protein expression in the D-gal group compared to the NC group(P<0.01),and these expression levels were attenuated by low and high doses of FLDK(P<0.05).Similar to the western blot results,immunohistochemistry showed an increase in activated Ibal+ and GFAP+cells in the cortex and hippocampal dentate gyrus(DG)regions of the D-gal vehicle group compared to the vehicle NC group.FLDK treatment decreased the number of activated microglia and astrocytes in the cortex and hippocampal dentate gyrus regions of D-gal-FLDK-cotreated mice.5.FLDK reduced nuclear NF-κB translocation and various inflammatory markers in D-gal-aged miceSubcutaneous injection of D-gal significantly increased nuclear translocation of NF-κB(cytoplasm:P<0.05;nucleus:P<0.01)compared to the NC group.FLDK administration attenuated nuclear translocation(nucleus P<0.05;cytoplasm P<0.05).Nuclear translocation of NF-κB increased expression of pro-inflammatory cytokines.In this study,the D-gal group mice showed a significant increase in inflammatory biomarkers,including TNF-α,IL-β,COX-2,and iNOS(P<0.01,P<0.001,P<0.01,P<0.01,respectively).Cotreatment with FLDK at both low and high doses attenuated neuroinflammatory cytokine production(TNF-α,IL-1β,COX-2 and iNOS)compared to the D-gal group(P<0.05).6.FLDK attenuates oxidative stress in D-gal-aged miceD-gal markedly increased ROS production compared to the NC group(P<0.001),while cotreatment of D-gal and FLDK significantly alleviated ROS production(P<0.05).MDA is a marker of lipid peroxidation.There was a marked increase in MDA in the D-gal-aged mice(P<0.001),while FLDK treatment significantly reduced MDA levels in the F1 and F2 groups(P<0.01).Additionally,SOD,GSH-Px,and CAT activity were measured to determine the antioxidant capacity of the aged brains.All of the above markers were reduced after D-gal administration(P<0.001,P<0.05,P<0.001,respectively),while FLDK significantly improved SOD,GSH-Px and CAT activity(P<0.05).7.FLDK alleviates neuronal apoptosis in the brains of D-gal-aged miceThe results showed that PI3K and p-Akt decreased markedly in the cortex and hippocampus of D-gal-aged mice compared to the NC mice(P<0.05).However,the reduction of PI3K and p-Akt were largely suppressed in the mice treated with FLDK after D-gal injection(P<0.05).We did not observe any significant changes in total Akt.Our results suggest that activation of p-JNK in the D-gal group was comparable to the NC group(P<0.05).FLDK suppressed p-JNK activation in the F1 and F2 groups compared to the D-gal group(P<0.05).The results also indicate that D-gal reduced anti-apoptotic Bcl-2 and increased pro-apoptotic Bax compared to the NC group(P<0.01 for both).In contrast,treatment with low and high doeses of FLDK reversed the effects on Bcl-2 and Bax protein expression in the cortex and hippocampus of D-gal-aged mice(P<0.05),suggesting that FLDK has anti-apoptotic properties.8.FLDK rescues D-gal-induced reduction of synaptic proteins in the brainThe results indicate that synaptic proteins,including synaptophysin,synaptotagmin,p-CREB,and p-CaMK Ⅱ,were significantly reduced in the D-gal group compared to the NC group(P<0.05).Additionally,FLDK reversed the reduction of synaptic proteins in the F1 and F2 groups compared to the D-gal group(P<0.05).ConclusionFLDK administration alleviated oxidative stress induced ROS production and lipid peroxidation and sustained activity of endogenous antioxidant enzymes.Moreover,FLDK reduced microglia and astrocyte activation,decreased AGEs/RAGE expression,downregulated NF-κB nuclear translocation,inhibited the release of inflammatory mediators,attenuated neuronal apoptosis,and alleviated synaptic dysfunction.These effects partially explain the mechanisms by which FLDK attenuates learning and memory impairment in D-galactose-aged senescent mice.Part II Flavonoid-Rich Extract from the Leaves of Diospyros Kaki Attenuates Cognitive Deficits,Amyloid-Beta Production,Oxidative Stress,and Neuroinflammation in APP/PS1 Transgenic MiceObjectiveTo explore the effect of early intervention of FLDK on learning and memory function,Aβ deposition and metabolism,microglial activation,markers of oxidative stress,and inflammation in the brain of APP/PS1 mice.Methods1.GroupingThis study selected four-month-old APP/PS1 double transgenic AD male mice,and the control group selected C57 wild-type male mice of the same age.They were randomly divided into four groups(n=8):wild-type controls(WT),wild-type+FLDK(WT+FLDK),APP/PS1 mice(APP/PS1),and APP/PS1 mice+FLDK(APP/PS1+FLDK).In WT+FLDK and APP/PS1+FLDK groups FLDK was administered orally(80 mg/kg·day)solubilized in physiological saline solution containing 0.5%DMSO for 12 weeks by gavage.Wild-type controls and APP/PS1 controls received an equal volume of vehicle.2.Behavioral testingAfter the drug intervention in mice,the six-day Morris water maze test was used to detect the escape latency(EL)of each group,the number of crossing platforms and the parade time of the original platform.The protective effect of FLDK on the spatial learning and memory ability of AD mice was discussed.The test is divided into five consecutive days of positioning navigation tests and one day of space exploration tests.3.Detection of Aβ content in the brainThe expression of amyloid Ap in hippocampus and cortex of each group was detected by immunohistochemistry and immunofluorescence.The content of Aβ1-40 and Aβ1-42 in brain tissue was detected by enzyme-linked immuno sorbent assay(ELISA).4.Detection of protein expressionWestern blot was used to detect protein expression and phosphorylation in the brain.Including:①microglia marker Ibal;②APP,PS,BACE1,RAGE,p-AMPK,t-AMPK,NF-κB,p-PERK,t-PERK;③inflammatory factors TNF-α,IL-1β,COX-2,iNOS.5.Oxidative stress level detectionImmunofluorescence was used to detect 8-OHDG,and the antioxidant enzyme SOD,GSH-Px activity and MDA content were detected by the kit.6.Statistical analysisStatistical analysis was performed using SPSS20.0.Significant differences between treatment groups were analyzed with one-way analysis of variance(ANOVA)followed by the Tukey’s test.Repeated-measures ANOVA followed by the Tukey’s test were used for comparision of escape latency in the MWM test.Data are expressed as means ± standard error of the mean(SEM).P values<0.05 were considered statistically significant.Results1.FLDK prevents learning and memory impairment in APP/PS1 mice in the MWM testAPP/PS1 mice spent more time searching for the hidden platform after training compared with WT mice(p<0.001).FLDK ameliorated the time for APP/PS1 mice significantly(p<0.01),indicating that FLDK attenuated memory deficits in AD mice.In the probe trial,the number of crossings and the time spent in the target quadrant were significantly decreased in the APP/PS1 mice compared with WT mice(p<0.001).This effect was improved by FLDK(number of crossings,p<0.05;time in target quadrant,p<0.001).There were little differences in escape latency,the number of crossings,and the time spent in the target quadrant in FLDK-treated WT mice compared with vehicle-treated WT mice.No change of the swimming speed was observed between different groups(p>0.05),so latencies were able to represent differences in performance.These results suggest that FLDK ameliorated cognitive deficits in APP/PS1 mice,while having no cognitive effect in healthy mice.2.FLDK reduces the level of Aβ and microglial activation in the brains of APP/PS1 miceNo senile plaques were detected in the brains of WT mice,while a significant amount of large senile plaques containing Aβ was observed in the cortex and hippocampus of APP/PS1 mice.FLDK-treatment resulted in a reduction in the size and number of senile plaques.Quantification of the Aβ load identified by immunohistochemical staining(brain area of Aβ-positive plaques),and of the Aβ1-40 and Aβ1-42 levels by ELISA revealed that FLDK-treatment led to a significant reduction of Aβ load and Aβ1-40/Aβ1-42 levels in APP/PS1 mice(p<0.01).3.FLDK reduces the activation level of microglia in APP/PS1 miceMicroglial activation was detected around senile plaques in APP/PS1 mice by double-staining with Aβ and Ibal antibodies.FLDK-treatment significantly decreased Ibal-positive staining,and therefore,the number of activated microglial near senile plaques in APP/PS1 mice(p<0.001).Western blot analysis showed that the expression of Ibal in APP/PS1 mice increased significantly in comparison with WT mice(p<0.001).Treatment with FLDK reduced the expression of Ibal in APP/PS1+FLDK mice compared with vehicle-treated APP/PS1 mice(p<0.01).FLDK represented no effect on microglial activation in the brains of WT mice.4.FLDK reduces the expression of BACE1,activates AMPK,and decreases PERK/eIF2a in the brains of APP/PS1 miceFLDK had no significant effect on the expression of APP and PS1 in APP/PS1 mice.BACE1 increased in APP/PS1 mice with hyperphosphorylation of PERK/eIF2a occurred compared with WT mice(p<0.001),whereas little change in p-AMPK was noticed.FLDK decreased the protein level of BACE1(p<0.001),increased phosphorylation of AMPK(p<0.001),and dephosphorylation of PERK/eIF2a(p<0.001).While FLDK showed almost no effect on BACE1,phospho-AMPK or phospho-PERK/eIF2a expression in vehicle-treated WT mice.(?)5.FLDK reduces the expression of inflammatory markers in the brains of APP/PS1 miceThe protein levels of RAGE,nuclear NF-κB-p65,TNF-α,IL-1β,COX-2,and iNOS were low in the brains of WT mice and FLDK-treated WT mice.However,all examined proteins were elevated in APP/PS1 mice.Quantification of Western blots revealed that compared with APP/PS1 mice,RAGE,nuclear NF-KB-p65,TNF-a,IL-1β,COX-2,and iNOS expression were significantly reduced,while cytoplasm NF-κB-p65 increased after chronic treatment with FLDK in APP/PS1 mice(p<0.01).In contrast,FLDK showed negligible effects on protein expression in vehicle-treated WT mice.6.FLDK attenuates oxidative stress in the brains of APP/PS1 miceAPP/PS1 mice showed significantly higher 8-OHDG labeled area than WT mice(p<0.001),and this was significantly reduced in the FLDK-treated APP/PS1 mice(p<0.001).The area of 8-OHDG labelling showed almost no difference between the vehicle-treated WT mice and FLDK-treated WT mice(P>0.05).We also compared the enzymatic activities of anti-oxidant enzymes in the two groups of mice.SOD and GSH-Px activity was considerably lower in APP/PS1 mice compared with WT mice(p<0.001),while a distinct increase in SOD and GSH-Px activity occurred in FLDK-treated APP/PS1 mice when compared with vehicle-treated APP/PS1 mice(p<0.05).However,no difference was seen for both enzymes between vehicle-treated WT and FLDK-treated WT mice.A significant increase of MDA was noted in APP/PS1 mice compared with WT mice(p<0.001).FLDK-treatment reduced this change in APP/PS1 mice(p<0.05),but showed little change in WT mice.Conclusion1.FLDK improves the impairment of learning and memory ability of APP/PS1 mice.2.FLDK regulates Aβ metabolism by decreasing BACE1,increasing p-AMPK,decreasing p-PERK/eIF2α protein expression level,and attenuates Aβ load in APP/PS1 mice brain.3.FLDK alleviates the levels of inflammation and oxidative stress in APP/PS1 mice.