Ketogenic diet alleviates cognitive dysfunction and neuroinflammation in APP_PS1 mice via the Nrf2_HO-1 and NF-κB signaling pathways.pdf

NEURAL REGENERATION RESEARCHVol 18No.12December 20232767NEURAL REGENERATION RESEARCHwww.nrronline.orgResearch ArticleKetogenic diet alleviates cognitive dysfunction and neuroinflammation in APP/PS1 mice via the Nrf2/HO-1 and NF-B signaling pathways Abstract Alzheimers disease is a progressive neurological disorder characterized by cognitive decline and chronic inflammation within the brain.The ketogenic diet,a widely recognized therapeutic intervention for refractory epilepsy,has recently been proposed as a potential treatment for a variety of neurological diseases,including Alzheimers disease.However,the efficacy of ketogenic diet in treating Alzheimers disease and the underlying mechanism remains unclear.The current investigation aimed to explore the effect of ketogenic diet on cognitive function and the underlying biological mechanisms in a mouse model of Alzheimers disease.Male amyloid precursor protein/presenilin 1(APP/PS1)mice were randomly assigned to either a ketogenic diet or control diet group,and received their respective diets for a duration of 3 months.The findings show that ketogenic diet administration enhanced cognitive function,attenuated amyloid plaque formation and proinflammatory cytokine levels in APP/PS1 mice,and augmented the nuclear factor-erythroid 2-p45 derived factor 2/heme oxygenase-1 signaling pathway while suppressing the nuclear factor-kappa B pathway.Collectively,these data suggest that ketogenic diet may have a therapeutic potential in treating Alzheimers disease by ameliorating the neurotoxicity associated with A-induced inflammation.This study highlights the urgent need for further research into the use of ketogenic diet as a potential therapy for Alzheimers disease.Key Words:Alzheimers disease;APP/PS1 mice;cognitive impairment;ketogenic diet;neuroinflammation;nuclear factor-kappa B pathway;nuclear factor-erythroid 2-p45 derived factor 2/heme oxygenase-1;therapeutic benefitshttps:/doi.org/10.4103/1673-5374.373715Date of submission:February 2,2023 Date of decision:March 8,2023 Date of acceptance:March 10,2023 Date of web publication:May 12,2023 Introduction Alzheimers disease(AD)is a progressive neurodegenerative disease marked by cognitive decline and subsequent mental and behavioral impairments.It is the most frequent form of dementia,with an insidious beginning and sluggish progression(Lane et al.,2018).The pathogenesis of AD remains incompletely elucidated,yet existing literature indicates a multifactorial etiology involving genetic,environmental,and lifestyle factors.The amyloid hypothesis has gained significant traction in the scientific community.This hypothesis proposes that the aggregation of amyloid-beta(A)peptides into plaques impairs synaptic communication and provokes neuroinflammation(Selkoe and Hardy,2016).Despite ongoing efforts,the development of efficacious AD treatments necessitates more comprehensive understanding of the intricate mechanisms underlying the disease.There are currently few medications effective in lowering the risk of developing AD or in slowing disease progression,and the contentious approval of an IgG1 anti-A antibody in 2021 aimed at the removal of A plaques may be the only success to date(Asher and Priefer,2022).As a result,public health is focusing heavily on preventative measures to promote healthy brain aging,with the objective of lowering the burden of cognitive impairment and dementia in the older population.Researchers investigating the neuropathology and origins of AD have uncovered a diverse set of intricate molecular fingerprints.It is generally accepted that the A aggregation and tau hypotheses have slowly come to dominate in AD-related research(Sharifi-Rad et al.,2020;Ashrafian et al.,2021;Ciccone et al.,2021).Moreover,hypometabolism(Szablewski,2021),mitochondrial dysfunction(Atlante et al.,2022;Zeng et al.,2022),inflammation(Bajwa and Klegeris,2022),and oxidative stress(Beura et al.,2022)have all been linked to the etiopathogenesis of AD.New evidence suggests that nutritional and metabolic treatments may be able to address these problems,which have been challenging to tackle owing to a lack of effective prevention and therapeutic strategies(Shohayeb et al.,2018).The ketogenic diet(KD)is a nutritious dietary pattern that is low in carbs,high in fat,and moderate in protein.In a limited sample of patients,Krikorian et al.(2012)reported that 6 weeks on a KD enhanced verbal memory performance versus patients on a high carbohydrate diet.Medium chain triglyceride-containing KDs have been shown to enhance cognitive performance in A-positive individuals,restoring mitochondrial function and decreasing A toxicity(Hertz et al.,2015;Wu et al.,2020).These findings are encouraging,particularly if the benefits are sustained in longer-term research.The mechanism of neuroprotection afforded by a KD is still unknown.However,several studies have shown that a KD is associated with a reduced inflammatory response and oxidative damage(Hersant and Grossberg,2022;Kong et al.,2022;Rojas-Morales et al.,2022).Neuroinflammatory reactions are related to the development of AD in experimental trials.As immune cells of the central nervous system,activation and proliferation of microglia and astrocytes play a critical role in the control and release of inflammatory mediators and are thought to be 1Department of Neurology and Institute of Neurology,Ruijin Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai,China;2Lab of Translational Research of Neurodegenerative Diseases,Institute of Immunochemistry,ShanghaiTech University,Shanghai,China*Correspondence to:Shengdi Chen,PhD,MD,;Yuyan Tan,PhD,MD,.https:/orcid.org/0000-0002-6242-1025(Shengdi Chen);https:/orcid.org/0000-0003-1779-6554(Yuyan Tan)Funding:This work was supported by the National Natural Science Foundation of China,Nos.82171401,81971187(to SC),and 81971183(to YT),grants from Shanghai Municipal Science and Technology Major Project,No.2018SHZDZX05(to SC)and Shanghai Municipal Education Commission,No.2017-01-07-00-01-E00046(to SC).How to cite this article:Jiang J,Pan H,Shen F,Tan Y,Chen S(2023)Ketogenic diet alleviates cognitive dysfunction and neuroinflammation in APP/PS1 mice via the Nrf2/HO-1 and NF-B signaling pathways.Neural Regen Res 18(12):2767-2772.Graphical AbstractFrom the ContentsIntroduction2767Methods 2768Results2769Discussion2769Jingwen Jiang1,Hong Pan1,Fanxia Shen1,Yuyan Tan1,*,Shengdi Chen1,2,*Ketogenic diet has a therapeutic potential in treating AD via the Nrf2/HO-1 and NF-B signaling pathways2768 NEURAL REGENERATION RESEARCHVol 18No.12December 2023NEURAL REGENERATION RESEARCHwww.nrronline.orgResearch Articleessential pathological indicators of neuroinflammation(Guo et al.,2022).Reduced expression of the transcription factor,nuclear factor-erythroid 2-p45 derived factor 2(Nrf2),and its target genes(NQO1,HO-1,and GCLC),as well as modification of Nrf2-related pathways,are reported in the brains of patients with AD(Ballard et al.,2011).The Nrf2 transcription factor is an important regulator of inflammation and can decrease the inflammatory responses generated by inflammatory factors such as nuclear factor-B(NF-B)(Saha et al.,2020).There have been few studies to determine if a KD can improve cognitive function and the underlying mechanisms.Thus,we sought to determine if a KD controls Nrf2 in the amyloid precursor protein(APP)/presenilin 1(PS1)mouse model of AD,and whether it exerts anti-inflammatory effects by reducing activity of the NF-B signaling pathway.Methods Animals and dietAPP/PS1(B6.Cg-TgAPPswe,PSEN1dE9;MMRRC Cat#034832-JAX,RRID:MMRRC_034832-JAX)double-mutant transgenic mice derived from the C57BL/6J strain were kindly provided by University of Tbingen,Tbingen,Germany.Specific pathogen-free C57BL/6J mice were purchased from the Zhejiang Branch of Vital River Laboratory Animal Technology Co.Ltd.,China(license No.SCXK(Zhe)2019-0001).Control mice were age-matched wild-type(WT)littermates without the transgene.In all tests,male mice were used.Mice were kept in groups of five in cages at room temperature(22 1C)on 12/12-hour light/dark cycles.All animal experiments were conducted with the approval of the Ethics Committee at Ruijin Hospital,Shanghai Jiao Tong University School of Medicine(approval No.2018-243)and in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals(8th ed,National Research Council,2011).All procedures were carried out in compliance with the Animal Research:Reporting of In Vivo Experiments(ARRIVE)guidelines(Percie du Sert et al.,2020).Dyets,Inc.(Bethlehem,PA,USA)provided the KD(HF93.5)and control diet(CD)(HF85-C).In terms of calories,the KD had 93.5%fat,4.7%protein,and 1.8%carbohydrate.A typical laboratory diet of 11.5%fat,10.4%protein,and 78.1%carbohydrate(in kcal)served as the CD.Multiple vitamins and minerals were present in both the KD and CD.The specific ingredients are shown in Table 1.Behavioral testingNovel object recognition test A typical behavioral test used to examine hippocampus-dependent recognition memory is the novel object recognition test(NORT).It is based on the fact that animals spend more time analyzing a novel object than a known one(Leger et al.,2013).To test the cognitive ability of the mice,the test is conducted in an open empty box(40 40 40 cm3)with two items that are approximately the same height and volume,yet differ in shape and appearance.During the familiarization phase(Figure 2A),mice were exposed to two identical objects situated at an equal distance in either corner for 10 minutes,then the box was cleaned using 75%ethanol.After 1 hour,the animal was returned to the box and tested again.During the second phase(Figure 2A),one of the initial objects was replaced with a new item of comparable mass,and the animal was allowed to explore freely for approximately 5 minutes.NORT data were evaluated using a video tracking system(Softmaze;Xinruan,Shanghai,China).The recognition index(%)for new object exploration was determined as follows:time spent on novel object/(time spent on novel object+time spent on familiar item)100.Y-maze spontaneous alternation testSpontaneous alternation in the Y-maze(which is used to measure spatial working memory)is based on the natural curiosity of mice(Kraeuter et al.,2019).Testing takes place in a Y-shaped maze with three equal-sized arms constructed of opaque plastic.The arms(35 5 10 cm3)were placed at a 120 angle from each other and labeled A,B,and C(Figure 2C).The mice were placed at the terminal of arm B and permitted to freely explore the three arms for 8 minutes.The proportion of alternation was calculated by recording the number of arm entries and triads.Total arm entries were recorded manually by the observer(who was blinded to the treatment)or automatically by Supermaze(Xinruan).A successive entry in three separate arms was classified as an alternation.An entrance was recorded when all four limbs were within the arm.The proportion of alternations(entry into a different arm from the previous two)was computed using the following formula:alternations/(arm entries 2)100%.Tissue collectionAt 6 months of age,the animals underwent behavioral testing and were then placed in a chamber filled with anesthetic vapor(approximately 0.6 mL liquid isoflurane per liter of chamber volume)(Shanghai Yuyan Scientific Instrument Company,Shanghai,China,Cat#100150)until respiration ceased.This typically occurred within 2 minutes(Overmyer et al.,2015).The cortex and hippocampus of mice(n=6)were dissected into cold phosphate-buffered saline(PBS)(Servicebio,Wuhan,China,Cat#G0002)and immediately stored at 80C for subsequent study.The remaining animals(n=6)were perfused with saline,postfixed in 4%paraformaldehyde(Servicebio,Cat#G1101)at 4C overnight,then dehydrated for 72 hours in 30%sucrose solution.Next,the brains were frozen and embedded in optimal cutting temperature(OCT)compound(Sakura,Torrance,CA,USA,Cat#4583)before being sliced into uniform pieces(12 m)with a microtome(Leica,Wetzlar,Germany).Frozen sections were thawed and placed on positively charged slides(ProbeOn Plus;Thermo Fisher Scientific,Waltham,MA,USA)before storage at 20C.Immunofluorescent stainingAntigen retrieval from frozen brain slices required 2030 minutes of microwaving(Midea,Foshan,Guangdong,China,Cat#M1-211A)in citrate buffer(pH 6.0)(Servicebio,Cat#G1202).A blocking mixture of 10%bovine serum albumin(BSA)(Aladdin,Shanghai,China,Cat#B265994)and 0.3%Triton X-100(Aladdin,Cat#T434386)in PBS was applied after 30 minutes.Sections were incubated overnight at 4C with primary antibodies diluted in normal goat serum solution(2%)(Beyotime,Shanghai,China,Cat#C0265).Rabbit anti-A142 antibody(1:500,Cell Signaling Technology,Danvers,MA,USA,Cat#14974,RRID:AB_2798671),anti-glial fibrillary acidic protein(GFAP)antibody(1:200,Cell Signaling Technology,Cat#12389,RRID:AB_2631098),and anti-AIF-1/ionized calcium-binding adapter molecule 1(Iba1)antibody(1:50,Cell Signaling Technology,Cat#17198,RRID:AB_2820254)were used as primary antibodies at 4C overnight.GFAP is a marker for activated astrocytes,while Iba1 is a marker for activated microglia.After the primary antibody incubation,the slices were rinsed extensively with PBS and then treated with Alexa 488-or Alexa 594-conjugated goat anti-rabbit IgG secondary antibodies(1:500;Jackson ImmunoResearch Laboratories,West Grove,PA,USA,Cat#111-585-003,RRID:AB_2338046 Alexa 488-and AB_2338059 Alexa 594-)for 1 hour at 37C in the dark.Staining of nuclei for 10 minutes in 4,6-diamidino-2-phenylindole(DAPI)(Beyotime,Cat#C1005)was followed by washing in PBS.The Zeiss AxioVert A1 fluorescence microscope(Oberkochen,Table 1 Ingredients in ketogenic and control dietsIngredientg/kgControl dietCaisein100DL-methionine1.5Sucrose0Comstarch0Dyetrose751.5Shortening0Corn oil50Cellulose50Mineral Mix#20000035Vitamin Mix#300050X10Choline bitartrate2Ketogenic dietCaisein45DL-methionine1.5Dyetrose17Cellulose50Lard260Anhydrous Milkfat100Corn oil50Mineral Mix#20000035Vitamin Mix#300050X1Choline bitartrate2Experimental designThe use of 3-month-old male mice was based on the intended aim of observing treatment effects at the early stages of AD progression and amyloid plaque formation(Bilkei-Gorzo,2014,Webster et al.,2014).A previous study found that male and female rodents respond differently to high-fat diet challenge,with female mice showing a higher preference for high-fat diet,dramatic increase in visceral fat,impaired glucose tolerance,and decreased energy expenditure compared to male mice(Maric et al.,2022).Thus,we selected male mice for our experiment to reduce variability and increase the robustness of our statistical analyses.Three-month-old male APP/PS1 mice were randomly assigned to one of two groups(n=12 per group):(1)AD model(APP/PS1)and KD treatment group(APP/PS1+KD);and(2)AD model(APP/PS1)and CD treatment group(APP/PS1+CD).The control group(WT+CD,n=12)consisted of 3-month-old male WT mice treated