Ettcheto et al. Cell Biosci (2021) 11:141 https://doi.org/10.1186/s13578-021-00646-w Cell & Bioscience RESEARCH Open Access Dexibuprofen ameliorates peripheral and central risk factors associated with Alzheimer’s disease in metabolically stressed APPswe/PS1dE9 mice Miren Ettcheto1,2,3,12* , Elena Sánchez‑Lopez2,4,5, Amanda Cano2,4,5,6, Marina Carrasco1,2,3,7, Katherine Herrera3,8, Patricia R. Manzine9, Triana Espinosa‑Jimenez1,2,3, Oriol Busquets10, Ester Verdaguer2,3,8, Jordi Olloquequi11, Carme Auladell2,3,8, Jaume Folch2,7 and Antoni Camins1,2,3 Abstract Background: Several studies stablished a relationship between metabolic disturbances and Alzheimer´s disease (AD) where inflammation plays a pivotal role. However, mechanisms involved still remain unclear. In the present study, we aimed to evaluate central and peripheral effects of dexibuprofen (DXI) in the progression of AD in APPswe/PS1dE9 (APP/PS1) female mice, a familial AD model, fed with high fat diet (HFD). Animals were fed either with conventional chow or with HFD, from their weaning until their sacrifice, at 6 months. Moreover, mice were divided into subgroups to which were administered drinking water or water supplemented with DXI (20 mg ­kg−1 ­d−1) for 3 months. Before sacrifice, body weight, intraperitoneal glucose and insulin tolerance test (IP-ITT) were performed to evaluate periph‑ eral parameters and also behavioral tests to determine cognitive decline. Moreover, molecular studies such as Western blot and RT-PCR were carried out in liver to confirm metabolic effects and in hippocampus to analyze several path‑ ways considered hallmarks in AD. Results: Our studies demonstrate that DXI improved metabolic alterations observed in transgenic animals fed with HFD in vivo, data in accordance with those obtained at molecular level. Moreover, an improvement of cognitive decline and neuroinflammation among other alterations associated with AD were observed such as beta-amyloid plaque accumulation and unfolded protein response. Conclusions: Collectively, evidence suggest that chronic administration of DXI prevents the progression of AD through the regulation of inflammation which contribute to improve hallmarks of this pathology. Thus, this com‑ pound could constitute a novel therapeutic approach in the treatment of AD in a combined therapy. Keywords: APPswe/PS1dE9, Alzheimer´s disease, Dexibuprofen, Unfolded protein response, neuroinflammation, Synapsis, Cognitive deficits, High fat diet, Metabolic alterations, βA plaques Background Alzheimer´s disease (AD) is a progressive neurodegen- erative disease characterized by severe impairments of *Correspondence: mirenettcheto@ub.edu 1 Department of Pharmacology, Toxicology and Therapeutic Chemistry, cognitive function leading to development of drastic Faculty of Pharmacy and Food Science, University of Barcelona, Barcelona, dementia. For years, this pathology has been defined by Spain the combined presence of amyloid-β (Aβ) plaques and Full list of author information is available at the end of the article © The Author(s) 2021. 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The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Ettcheto et al. Cell Biosci (2021) 11:141 Page 2 of 17 neurofibrillary tangles composed by hyperphosphoryl- for this reason non-steroidal anti-inflammatory drugs are ated Tau. However, the continuous increase of cases being one the research focus concerning to AD therapeu- worldwide together with the failure of proposed treat- tic strategy. ments until now have located this pathology as one of the Ibuprofen (IBU) is one of the most used nonsteroidal great health-care challenges of the twenty-first century, anti-inflammatory drugs (NSAIDs) which has demon- promoting new approach about its etiology and, there- strated promising results in decreasing hallmarks of AD fore, about its therapeutic strategies. [24], by contrast, its chronic intake has demonstrated In this regard, in the recent years, several studies have multiple adverse effect such as gastric damage. For these focused on the potential relationship between AD and reasons, our study has focused in the evaluation of dex- metabolic disorders [1, 2], considering AD as a multifac- ibuprofen (DXI), the active enantiomer of IBU [25, 26], torial disease. In fact, longitudinal studies have indicated which has been demonstrated that it has not only a worsening performance of specific abilities including more powerful anti-inflammatory and analgesic effect working memory in patients with metabolic disorders [3, compared to IBU but also less ulcerogenic side effects 4]. In line with this hypothesis, obesity and type 2 diabe- [27–30]. Therefore, taking these previous findings into tes mellitus (T2DM) also have been associated to cogni- account, and according to the Herrup hypothesis which tive decline in several studies considering them as key suggests that peripheral inflammation could be one of contributors not only in cognitive alterations [5–8] but the responsible of neuroinflammation associated to neu- also in the development of AD [9, 10]. As a result, a new rodegeneration, the aim of our study is to investigate the concept have been introduced by scientific community to central and peripheral effects of DXI in the progression define AD named “type 3 diabetes” [11, 12]. of AD and describe the involved mechanisms in APPswe/ In this context, the association between T2DM and AD PS1dE9 (APP/PS1) female mice, familial AD mice model, is very complex where several molecular pathways are fed with high fat diet (HFD). interlinked such as insulin resistance, peripheral inflam- matory response, and endoplasmic reticulum stress, among others [13]. Likewise, insulin resistance is one of Methods the main hallmarks of obesity and T2DM which plays an Animals and treatment essential role in the maintenance in the cell energy home- In this study, six-month old female APP/PS1 double ostasis. Surprisingly, the appearance of similar abnor- transgenic mice were used. Female sex was chosen due to malities in both, peripheral tissues of T2DM patients and the fact that it has been reported that they produce a sig- AD brains has been demonstrated [14–17]. Moreover, nificantly higher amyloid burden and subsequent plaque Wu and coworkers have proven the existence of periph- deposition than their male counterparts [31]. Moreover, eral metabolic changes in plasma and liver of AD mice these transgenic mice express a Swedish (K594M/N595L) models, suggesting that AD development is not only mutation of a chimeric mouse/human APP gene (mo/ caused by alterations in the brain but also that systemic huAPP695swe) together with the human exon-9-deleted impairment plays a key role in the pathology [18]. In fact, variant of presenilin 1 (PS1-dE9). Animals were divided it has also been demonstrated that peripheral alterations into 4 groups: (1) mice fed with conventional chow (APP/ in the insulin pathway observed in patients with T2DM PS1), (2) mice fed with conventional chow and addition- contribute to alterations in brain insulin, leading to an ally treated with DXI (APP/PS1 DXI), (3) mice fed with increase of Aβ accumulation and a decrease of its clear- a palmitic acid-enriched high fat diet (APP/PS1 HFD) ance, which induce neuronal damage and, therefore, cog- containing 45% of fat mainly from hydrogenated coconut nitive decline, creating a vicious cycle of pathogenesis oil (Research Diets Inc, New Brunswick, USA) (APP/PS1 [19]. HFD) and (4) mice fed with HFD and additionally treated As it has been mentioned before, inflammation is with DXI (APP/PS1 HFD DXI). The drug was adminis- involved not only metabolic disorders but also in AD, and tered in drinking water at a dose of 20 mg ­kg−1 ­d−1 for is considered a critical process in this pathology. How- 3 months, from mice aged 3 months old until their sacri- ever, its dual contribution to the disease adds complexity fice at 6 months, as previously described [30]. At least, 10 to the process. Moreover, increasing clinical and experi- animals per group were used for this study. All mice were mental studies have evidenced that acute and chronic given access to water and food ad libitum and kept under systemic inflammatory pathologies may be associated controlled light, temperature and humidity conditions. with the risk and acceleration of AD [20]. In line with this Every possible effort was made to reduce the number of hypothesis, numerous epidemiological studies have dem- animals used and minimize their suffering. Mice were onstrated that anti-inflammatory drugs can reduce the treated in accordance with the European Community risk of development AD in more than 50% fold [21–23], Council Directive 86/609/EEC and the procedures were Ettcheto et al. Cell Biosci (2021) 11:141 Page 3 of 17 established by the Department d’Agricultura, Ramaderia in phosphate-buffered saline (PBS) (pH 7,2) prior to pre- i Pesca of the Generalitat de Catalunya. incubation in a blocking solution (10% fetal bovine serum (FBS), 1% of triton X-100 in PBS at room temperature for Glucose and insulin tolerance test 1 h and then, they were incubated O/N at 4 °C with the Intraperitoneal glucose tolerance test (IP-GTT) and insu- corresponding primary antibody and immediately with lin tolerance test (IP-ITT) were performed in accordance the corresponding secondary antibody for 2 h at room with the previously described guideline [32]. Briefly, mice temperature. were fasted, at least, for 6 h prior to carry out both pro- Enzymatic immunohistochemistry was performed as cedures and at least 10 animals were used per group. Tail previously described by our group [36]. All the antibod- blood was collected at multiple time points. Blood glu- ies used for these experiments have been described in cose levels were assayed with Accu-Chek Aviva glucom- Table 1. eter (Roche; Mannheim, Germany). Those animals in All the samples were mounted onto gelatinized slides which blood glucose concentration reached values below with Fluoromount G (EMS). Image acquisition was per- 20 mg/dl were treated with glucose at a dose of 1 g/kg. formed with an epifluorescence microscope (BX41, Laboratory Microscope, NY-Olympus America Inc.). Cognitive test For plaque quantification, similar and comparable histo- Morris water maze logical areas, particularly from the cortex, were selected. APP/PS1 mice were subjected to Morris water maze Immunohistochemistry labeling was analyzed through (MWM) test in order to assess the spatial memory and ImageJ software. learning abilities as previously described by our group [33]. Acquired data were analyzed using SMART V3.0 Immunoblot analysis (Panlab Harvard Apparatus, Germany) video tracking For Immunoblot, 5–6 mice from each experimental system, calculating results individually for each animal. group were sacrificed by cervical dislocation and their At least, 10 animals per group were utilized. hippocampus and liver were dissected. Total protein extraction was carried out by homogenizing tissue sam- Novel object recognition test ples in lysis buffer (50 mM Tris HCl pH: 7.4, 150 mM The novel object recognition test (NORT) was used for NaCl, 5 mM EDTA, 1%Triton X-100) containing a pro- assessing the hippocampal-dependent recognition mem- tease and phosphatase inhibitor mixture (Complete, ory of mice as it has been previously detailed [34]. At Roche Diagnostics, Barcelona, Spain). Protein concen- least, 10 mice per condition were used. Data were ana- tration was determined by Pierce BCA Protein Assay lyzed by discrimination index (DI) which was calculated Kit (Pierce Company, Rockford, MI, USA) and aliquot using the following equation: of samples containing 10 μg were used for perform- ing western blot procedure as previously described by New object exploration time − old object exploration time DI = our group [34]. The antibodies employed in the experi- total exploration time ment are listed in Table 2. Finally, blots were exposed doc™ XRS + Molecular Imager detection system (Bio- Data measured and represented in seconds. to Chemiluminescence-based detection kit in a Chemi- Immunofluorescence, S‑thioflavin staining and enzymatic Rad), through Image Lab™ image analysis software immunohistochemistry (version 5.2.1). Obtained results were normalized to the To perform these 3 techniques, 4–5 mice were anesthe- tized by i.p. injection of ketamine (100 mg/kg) and xyla- cine (10 mg/kg) and perfused with 4% paraformaldehyde Table 1 Primary and secondary antibodies for immunohistochemistry (PFA) diluted in 0.1 M phosphate buffer (PB). Brains and immnunofluorescence were removed and stored in the same solution over- Protein Reference night (O/N). 24 h later, they were cryoprotected in 30% sucrose-PFA-PB solution for at least 1 day and samples DBN1 ABN 207 (Merck Millipore) were kept at − 80 °C until their use. Coronal sections of GFAP Z0334 (Dako) 20 μm of thickness were obtained using a cryostat (Leica IBA1 O19-19,741 (Wako) Microsystems, Wetzlar, Germany). Synaptophisin MO776 (Dako) To perform the experiments, free-floating technique 2nd-ary Anti-rabbit IgG Biotin antibody B8895-1ML (Sigma Millipore) was used for all of them. Immunoshistochemistry and produced in goat S-thioflavin protocols were carried out as previously 2nd-ary AlexaFluor 488 (Goat anti-mouse) A11001 (Life Technologies) described [35]. Briefly, free-floating sections were rinsed 2nd-ary AlexaFluor 594 (Goat anti-rabbit) A11080 (Life Technologies) Ettcheto et al. Cell Biosci (2021) 11:141 Page 4 of 17 Table 2 Primary and secondary antibodies for Western Blotting triplicate and data were normalized to Gapdh. Primers of Protein Reference detected genes are detailed in Table 3. ADAM10 ab39177 (Abcam) Statistical analysis AKT #9272 (Cell Signaling Technology) Differences between animals/samples were analyzed by pAKT (S473) #4060 (Cell Signaling Technology) two-way ANOVA, comparing two variables (treatment eIF2α #9722 (Cell Signaling Technology) and diet). In this test, when the interaction between 2 peIF2α (S51) #9721 (Cell Signaling Technology) factors was not significant, statistical values were rep- GAPDH MAB374 (Millipore) resented through # where # p < 0.05, ## p < 0.01, ### GSK3β #9315 (Cell Signaling Technology) p < 0.001 and #### p < 0.0001). When significant inter- pGSK3β (S9) #9336 (Cell Signaling Technology) action was obtained, Tukey´s post-hoc test was per- IREα Sc 390,960 (Santa Cruz Biotechnology) formed to compare the different groups where significant pIREα (S724) NB100-2323 (Novusbio) values were represented by * (* p < 0.05. ** p < 0.01, *** IRS2 Ab134101 (Abcam) p < 0.001 and **** p < 0.0001). All data are presented pIRS2 (S731) Ab3690 (Abcam) as mean ± SEM, in the Graph Pad 6.0 Prism software 2nd-ary Goat anti-rabbit 31,460 (Invitrogen) (Graph Pad Software Inc., San Diego, CA, USA). 2nd-ary Goat anti-mouse 31,430 (Invitrogen) Results DXI treatment ameliorates HFD‑related peripheral alterations in APP/PS1 mice corresponding glyceraldehyde 3-phosphate dehydroge- In order to evaluate the effect of DXI treatment on nase (GAPDH) and expressed in arbitrary units (a.u). metabolic parameters after HFD intake in APP/PS1 mice, firstly, body weight measurement and IP-GTT Real time PCR and IP-ITT were performed and immediately protein Samples were obtained as described in the 2.5 Immu- level of molecules involved in insulin signaling path- noblot test section where 4–6 animals were used. RNA way were evaluated. Two-way ANOVA of the weight extraction was performed by TRIsure-based extraction gain revealed a significant effect of diet (p < 0.0001) (Bloline GmbH) as previously described by our group and treatment (p < 0.05) with interaction between both [34]. The RNA pellet was reconstituted in RNAse free variables (p < 0.05). As expected, animals fed with HFD water and RNA concentration was measured using a showed a significant increase of body weight com- NanoDropTM 1000 Spectrophotometer (Thermo Scien- pared to those fed with conventional chow (p > 0.0001 tific, MA, USA). 2 μg of RNA was reversely transcribed for non-treated animals; p < 0.05 for treated animals). with the High-Capacity cDNA Reverse Transcription Surprisingly, mice fed with HFD and treated with DXI Kit, according to manufacturer’s protocol (Applied Bio- significantly reduced this parameter reaching val- systems). To perform the real time PCR (RT-PCR), equal ues similar to animals fed with normal chow (p > 0.05) reagent SYBR Green® qPCR Master Mix (K0253, Pierce, cDNA amounts of each animal were selected using as a (Fig. 1A). Moreover, the weight gain observed in APP/ Thermo Fisher Scientific) in a Step One Plus™ Real-Time PS1 HFD mice was accompanied by alterations in peripheral glucose metabolism in both, IP-GTT and IP- PCR System (Applied Biosystems). Samples were run by ITT. As previously described by our group and also by Table 3 Genes analyzed by RT-PCR Gene Forward Reverse Atf3 CTG​GAG​ATG​TCA​GTC​ACC​AAG​TCT​ TTT​C TC​GCC​GCC​TCC​T TT​ Atf4 AGC​AAA​ACA​AGA​CAG​CAG​CC ACT​C TC​T TC​T TC​CCC​C TT​GC Atf6 TTT​CAG​GGC​AGG​GCC​ATT​ CCC​GGG​ACA​AAC​AGG​TCT​T Bip CAG​ATC​T TC​TCC​ACG​GCT​TC GCA​GGA​GGA​ATT​CCA​GTC​AG Chop CGA​AGA​GGA​AGA​ATC​AAA​AAC​C TT​ GCC​C TG​GCT​CCT​C TG​TCA​ Gapdh TCT​ACC​CAC​GGG​CAA​GTT​CAA​ GGT​T TC​TCC​AGG​CGG​CAT​GT Ir TGT​CCC​CAG​AAA​AAC​C TC​T TCA​ AAG​GGA​TCT​TCG​C TT​TCG​GG Irs1 ACG​AAC​ACT​T TG​CCA​T TG​CC CCT​T TG​CCC​GAT​TAT​GCA​GC Ettcheto et al. Cell Biosci (2021) 11:141 Page 5 of 17 Fig. 1 Body weight comparison of the different experimental groups (n = 8–10 mice per group) B IP-GTT and D IP-ITT experimental profiles (n = 8–10 independent samples per group). AUC was calculated from the timepoint 0 until the end of the experiments for both test C IP-GTT and E IP-ITT. Statistical analysis was performed through two-way ANOVA and Tukey´s post-hoc where * denotes p < 0.05; ** denotes p < 0.01 and **** denotes p < 0.0001 other authors [32, 37], two-way ANOVA showed a sig- compared to their controls (p < 0.05; p < 0.01), achiev- nificant effect of diet in both test, IP-GTT and IP-ITT ing glucose concentrations similar to those animals fed respectively (p < 0.05; p < 0.0001)), and in this last test with conventional chow (Fig. 1B–E). also in the treatment factor (p < 0.05) with interaction At molecular level, proteins involved in the insu- between both factors in both test (p < 0.05; p < 0.05). lin signaling pathway in liver were analyzed to confirm Specifically, Tukey’s post-hoc revealed that HFD intake peripheral metabolic alterations. In line with observed induced a significant increase of fasting glucose levels previous data in this study, two-way ANOVA dem- in response to glucose and insulin intraperitoneal injec- onstrated a significant effect of treatment variable tion in comparison to mice fed with conventional chow (p < 0.0001) in phospho protein kinase B (p-AKT) protein (p < 0.01; p < 0.01, respectively). By contrast, the results level. In the case of phospho glycogen synthase kinase 3 obtained in APP/PS1 HFD after DXI treatment were beta (p-GSK3β), our data also showed a significant effect intriguing. While the glucose levels observed in APP/ of treatment (p < 0.001) with interaction between both PS1 HFD group pointed out metabolic disturbances, variables (p < 0.01). Specifically, our data demonstrated animals fed with the same diet but with an additional a significant increase in APP/PS1 mice fed with HFD DXI treatment showed a significant decrease of fast- and treated to DXI group in comparison to non-treated ing glucose levels in both test, IP-GTT and IP-ITT, group (p < 0.001). However, although upward trend was Ettcheto et al. Cell Biosci (2021) 11:141 Page 6 of 17 observed in animals fed with conventional chow after analyzed variables, treatment and diet (p < 0.0001; DXI treatment, no significant differences were observed. p < 0.05) respectively, demonstrating that DXI treat- Lastly, AKT and GSK3β did no show differences among ment improves cognitive decline previously described experimental groups (Fig. 2). by our group not only in transgenic animals fed with conventional chow [30] but also in those fed with HFD DXI treatment improves cognitive decline observed (Fig. 3C). No significant differences were observed in both APP/PS1 mice fed with conventional chow on the speed among any of the groups of the test day. and obesogenic diet Therefore, results of scape latency were not due to dif- In previous studies, it has been demonstrated that ferences in the swimming speed of the animals. These APP/PS1 mice showed cognitive decline in terms of results are attached as supplementary material. spatial learning and memory [30, 38, 39]. Therefore, we In order to support these results, an immunofluores- evaluated whether DXI was able to rescue the cogni- cence assay against synaptophysin was carried out. In tive impairment observed in this mice model through line with the results observed in the behavioral test, our both tests, MWM and NORT. Regarding MWM, as it results showed that the DXI treatment induced a sub- is shown in the Fig. 3A, B, the training performed by stantial qualitative increase of this protein in the CA3 different groups showed an improvement of learning of the hippocampus compared to non-treated groups ability in those animals treated with DXI compared to (Fig. 4A, B). In agreement with these results, the quan- non-treated animals. In line with these results, 2-way titative analysis of these images showed the significant ANOVA analysis showed a significant effect of the effect of treatment variable (p < 0.005) after the applica- treatment variable in the test day (p < 0.01) independ- tion of two-way ANOVA. Moreover, these data were ent of the diet that they had consumed (Fig. 3A, B). corroborated with an enzymatic immunohistochemis- Moreover, results observed in the NORT after two- try against drebrin (DBN1). As it can be observed in the way ANOVA indicated a significant effect of both image, mice treated with DXI showed a clear increase in Fig. 2 A–C Immunoblot representative images and GAPDH-normalized quantifications of molecules related to insulin signalling (pAKT/AKT and pGSK3β/GSK3β in liver extracts of APP/PS1, APP/PS1 HFD, APP/PS1 DXI and APP/PS1 HFD DXI experimental groups (n = 4–6 independent samples per group). Statistical analysis was performed through two-way ANOVA. Significant differences were found treatment variable: #### denote p < 0.0001 Ettcheto et al. Cell Biosci (2021) 11:141 Page 7 of 17 Fig. 3 Behavioural test A Scape latency of training in MWM and B Scape latency of probe trial. All data were measured in seconds. Statistical analysis was performed through two-way ANOVA. Significant differences were found in treatment variable: ## denote p < 0.01 C NORT, DI expressed in seconds. (n = 10 independent samples per group. Two-way ANOVA statistical analysis was performed (Treatment variable: #### denote p < 0.0001) (Diet variable: # denote p < 0.05) DBN1 protein level in CA3 and gyrus dentatus (GD) of [42]. In correlation with previously showed data, two- the hippocampus (Fig. 4C). way ANOVA of the results of pro-disintegrin and metal- loproteinase domain-containing protein 10 (ADAM10) DXI reduces Aβ‑plaque burden in APP/PS1 mice fed and ADAM10 protein levels revealed a significant effect with conventional chow and obesogenic diet of diet (p < 0.001) and treatment (p < 0.001), confirming It is widely demonstrated that APP/PS1 mice develop that both factors alter the protein levels of this protein time-dependent accumulation of Aβ plaques in the brain, (Fig. 5B–D). which is enhanced with the obesogenic diet intake [40, 41]. To evaluate the effect of DXI on this process, S-thi- DXI treatment reduces the neuroinflammatory response oflavin staining was performed for plaque detection. As in APP/PS1 fed with conventional chow and obesogenic expected, two-way ANOVA showed a significant effect diet of the diet (p < 0.0001) in the quantification of cortical Aβ It is well known that neuroinflammation plays a key plaques deposition in APP/PS1 mice, demonstrating an role in the development of AD [43, 44], process which increase of these plaques caused by HFD intake. Moreo- is exacerbated by the HFD intake [45–47]. In line with ver, treatment factor also results in a significant effect these previous data, two-way ANOVA of glial fibrillary (p < 0.0001), indicating that long-term administration of acidic protein (GFAP) quantification indicated a signifi- DXI reduced Aβ plaques deposition independent of the cant implication of the treatment variable (p < 0.0001) diet (Fig. 5A). and diet variable (p < 0.05) with interaction between both Likewise, Western blot analysis was carried out to factors (p < 0.05). Moreover, two-way ANOVA for ion- evaluate the effect of DXI in non-amyloidogenic path- ized calcium binding adaptor molecule 1 (IBA1) pro- way, which is considered as the neuroprotective route tein levels demonstrated a significant effect of treatment Ettcheto et al. Cell Biosci (2021) 11:141 Page 8 of 17 Fig. 4 A Immunofluorescence against synaptophysin in the hippocampal CA3 areas and quantification of the intensity staining. Two-way ANOVA analysis was performed (Treatment variable: # denote p < 0.05). B 3D surface mapping analysis. Interactive 3D surface Plot v 2.4, ImageJ. Higher tridimensional relief and colour intensity were correlated with higher protein level. Scale 50 µm. C Immunofluorescence against DBN1 in the hippocampal GD and CA3 areas. Scale 50 µm (p < 0.0001) with interaction between both variables evaluated proteins involved in the main unfolded protein (p < 0.01). Specifically, Tukey´s post-hoc demonstrated response (UPR) cascades such as inositol-requiring enzyme that HFD consumption show a higher reactive profile -1alpha (IRE1α) and eukaryotic initiation factor 2 alpha when they were quantified by their covered area in APP/ subunit (eI2Fα). In this line, statistical analysis of pIRE1α PS1 fed with HFD compared to transgenic mice fed confirm a significant effect of treatment (p < 0.01) previ- with conventional chow (p < 0,05; p < 0,05, respectively). ously described [49] with interaction between both fac- By contrast, observed reactivity in both, astrocytes and tors (p < 0.05). Tukey´s post-hoc demonstrated that DXI microglia, was drastically decreased in response to DXI treatment induced a significant decrease in pIRE1α pro- treatment independently of diet (GFAP: p < 0,001 APP/ tein levels in APP/PS1 fed with obesogenic diet compared PS1 vs APP/PS1 DXI; p < 0.0001 APP/PS1 HFD vs APP/ to their control (p < 0.01). However, in the case of animals PS1 HFD DXI) and (IBA1: p > 0.0001 APP/PS1 vs APP/ fed with conventional chow, although DXI group showed PS1 DXI; p < 0.0001 APP/PS1 HFD vs APP/PS1 HFD a downward trend, the observed changes were not signifi- DXI) (Fig. 6A–D). cant. Regarding to eI2Fα, in line with previous studies [50, 51], two-way ANOVA revealed a significant effect of diet DXI reduces endoplasmic reticulum stress and treatment in peI2Fα protein levels in hippocampus Taking into account the involvement of endoplasmic reticu- (p < 0.0001) and p < 0.0001) respectively with interaction lum stress in multiples pathologies among them in inflam- between both variables ( p < 0.0001). After Tukey´s post- matory processes [48] and metabolic disorders [49], we hoc analysis, our results confirmed a significant increase Ettcheto et al. Cell Biosci (2021) 11:141 Page 9 of 17 Fig. 5 A Thioflavin staining of βA plaques (Green). Images were obtained from cortex. Scale bar 50 µm. B Representative histogram of the quantification of Aβ deposits (n = 4–6 independent samples per group, with at least 5 slices analyzed per simple). Two-way ANOVA statistical test was performed (Treatment variable: ### denote p < 0.001) (Diet variable: #### denote p < 0.0001). C, D Immunoblot representative images and GAPDH-normalized quantifications of molecules related to non-amyloidogenic pathway (pro-ADAM10 and ADAM10) from hippocampal extracts (n = 4–6 independent samples per group). Statistical analysis was performed through two-way ANOVA. (Pro-ADAM10, treatment and diet variables: ### denote p < 0.001). (ADAM10, treatment variable: ####denote p < 0.0001 and diet variable: ## denote p < 0.01) in the phosphorylated levels of this protein due to obeso- factor 3 (Atf3) expression showed a significant effect of genic intake in comparison to APP/PS1 mice fed with con- diet (p < 0.001) and treatment (p < 0.05) with interaction ventional chow (p < 0.0001), effect which was completely between both variables (p < 0.05). Tukey´s post-hoc mul- reverted due to DXI treatment (p < 0.0001). No significant tiple comparison indicated a significant increase in APP/ effect of either diet nor treatment was observed in IRE1α PS1 HFD DXI group compared to its control (p < 0.05). and eI2Fα protein levels (Fig. 7A, B). No significant difference was observed in binding immu- Regarding to mRNA expression, different transcript noglobulin protein (Bip) expression (Fig. 7C). factors located downstream of IRE1α and eI2Fα in the UPR pathway were analyzed. In this context, no signifi- DXI improves insulin signaling pathway APP/PS1 mice cant effect neither in diet nor treatment variable were hippocampus observed in activating transcription factor 4 (Atf4) and In parallel, we evaluated mRNA expression profile of insu- activating transcription factor 6 (Atf6) mRNA expres- lin repetor (Ir) and insulin receptor substrate 1 (Irs1) in the sion. However, both of them showed interaction between hippocampus due to HFD intake has not been only closely both factors, (p < 0.05) and (p < 0.05) respectively. When related to alterations in UPR pathway but also to insulin sign- analyzing C/EBP homologous protein (Chop, statisti- aling disturbances [49]. Two-way ANOVA analysis revealed cal analysis demonstrated a significant effect of diet in a significant effect not only in diet variable (p < 0.05) but also the expression of this molecule (p < 0.05), confirming the in treatment variable (p < 0.01) with interaction between both alteration of UPR due to high fat diet intake. Finally, two- factors (p < 0.05) in hippocampal Ir mRNA expression. Specif- way ANOVA of the results of activating transcription ically, Tukey´s post-hoc test indicated that Ir transcripts were Ettcheto et al. Cell Biosci (2021) 11:141 Page 10 of 17 Fig. 6 A Immunofluoresce against GFAP (astrocytes in red). B Representative histogram of quantification of astrocytes reactivity. C Immunofluoresce against Iba1 (microglia in red). D Representative histogram of quantification of microglial reactivity. Scale bar: 31,8 µm. Images were taken from the CA1 of hippocampus. (n = 4–6 independent samples per group, with at least 5 slices analyzed per sample). Statistical analysis was performed by two-way ANOVA and Tukey´s post-hoc where * denotes p < 0.05; *** denotes p < 0.001 and **** denotes p < 0.0001 significantly increased in APP/PS1 animals treated with DXI involved in the development of cognitive loss in AD [52– in comparison to mice without treatment (p < 0.05). Despite 55], leading to be considered as a multifactorial disease. this, the effect was blocked by HFD intake (Fig. 8A). Thus, the neuroinflammatory process, associated mainly Regarding protein levels, the immunoblotting analy- with microglial activation, could be the bridge between sis revealed a significant effect in hippocampal phospho the peripheral initiation of a stress stimuli which will insulin receptor substrate 2 (pIRS2) protein level of diet finally end with neuronal loss. In this context, the use of (p < 0.05) and treatment (p < 0.0001) with interaction NSAIDs have been proposed as a possible therapeutic between both factors (p < 0.05). Multiple comparison strategy to delay or stop the progression of this neuro- performed indicated a significant increase in pIRS2 in degenerative disease. Likewise, different epidemiological APP/PS1 mice induced by HFD intake (p < 0.01). By con- studies have pointed out the benefits of chronic admin- trast, these levels were drastically decreased after DXI istration of NSAIDs in terms of reducing the risk for suf- treatment in both cases, HFD and conventional chow- fering late onset AD [56–59]. Nevertheless, they have not fed animals (p < 0.01; p < 0.0001), respectively. In line with been able to fully achieve their objective, probably due to these results, our data demonstrated that DXI treatment the late diagnosis of the pathology. Thus, in spite of being induced a significant increase of pAKT protein levels well known that inflammation plays a key role in the AD, independent of the diet that animals had consumed in its contribution to the pathology has not been fully clari- the 2 way-ANOVA statistical analysis (p < 0.001). AKT fied. Our present study evaluates the effect of a chronic results were not significant (Fig. 8B, C). administration of DXI in the inflammatory process sur- rounded AD development, but also in the metabolic Discussion alterations such as weight gain, hyperglycemia and insu- Several epidemiological studies have demonstrated that lin resistance induced by HFD intake in APP/PS1 mice. HFD intake is related to numerous pathologies such as These alterations directly affect the memory process, Aβ obesity, T2DM and inflammation, all of them strongly Ettcheto et al. Cell Biosci (2021) 11:141 Page 11 of 17 Fig. 7 A, B Immunoblot images and quantification of GAPDH- normalized molecules involved in endoplasmic reticulum stress (pIRE1⍺/IRE1⍺; pEIF2⍺/EIF2⍺) from hippocampal extract (n = 4–6 independent samples per group). C mRNA expression profile (n = 4–6 independent samples per group, with 3 technical replicates per sample) of Bip, Atf3, Atf4, Atf6 and Chop from hippocampal extract. Statistical analysis was carried out through two-way ANOVA (Chop, diet variable: # denotes p < 0.05) and Tukey´s post-hoc where * denotes p < 0.05; ** denotes p < 0.01; *** denotes p < 0.001 and **** denotes p < 0.0001 deposition, glial reactivity and UPR, as well as in insulin the concomitant development of inflammation are the signaling pathway (Fig. 9). main contributors of insulin resistance. Likewise, stud- Several studies have revealed that metabolic alterations ies in human obesity and insulin resistance have revealed constitute a high risk factor for the development of late a clear relation between chronic administration of pro- onset AD [60, 61]. These findings have been the key in inflammatory signaling pathways and decreased insu- the approach of alternative point of view in terms of dis- lin sensitivity [67–69]. In accordance with these results, ease etiology and pathophysiology. In this context, sev- our results confirm that the chronic administration of eral authors have considered AD as a systemic pathology, DXI reduces weight gain, glucose and insulin impaired where brain in not the only responsible of pathological response and insulin signaling pathway alterations in alterations [62–64]. In fact, it has been demonstrated that liver induced by HFD intake in APP/PS1 mice, revert- insulin resistance is a shared hallmark feature of obesity, ing the metabolic alterations tightly associated to AD T2DM and neuropathological processes underlying cog- development. nitive aging and dementia [65], together with inflamma- Conversely, it is well known that brain gliosis activa- tion. The latter, as it has been previously discussed, plays tion plays an essential role in both, the development of a key role in these pathological processes [66]. In line all neurodegenerative disorders and metabolic disorders with this, there are epidemiological evidence suggesting [70]. In fact, several studies have remarked that neuro- a correlation between inflammation and insulin resist- inflammation is a clear feature of the pathology which is ance state, where it has been described that obesity and highly enhanced after HFD consumption as it has been Ettcheto et al. Cell Biosci (2021) 11:141 Page 12 of 17 Fig. 8 A mRNA profile (n = 4–6 independent samples per group, with 3 technical replicates per sample) of Ir and Irs1 from hippocampal extract. B, C Immunoblot images and quantification of GAPDH- normalized hippocampal molecules involved in insulin signalling pathway (pIRS2/IRS2; pAKT/ AKT) (4–6 independent samples per group). Two-way ANOVA was carried out and Tukey’s post-hoc where * denotes p > 0.05; ** denotes p < 0.01; **** denotes p < 0.0001) demonstrated in our study and also by other research At the molecular level, our study shows that chronic groups [71, 72]. Likewise, it has been described that neu- treatment with DXI increases drastically the active roinflammatory process contributes to synaptic dysfunc- ADAM10 protein levels, which could be related with the tion and behavioral deficits in early stages of the disease Aβ plaques lowering and the cognitive improvement. In [73, 74]. In this context, our results demonstrate that fact, ADAM10 has been proposed as a therapeutic tar- the chronic administration of DXI drastically reduced get in terms of treating neurodegenerative conditions, the glial reactivity observed in both, APP/PS1 and APP/ including late onset AD, given that its activity has been PS1 HFD mice, leading to the restorage of synaptophy- associated to neuroprotection through Aβ plaques reduc- sin, DBN1 and behavioral impairments. Regarding to tion [78]. Likewise, ADAM10 is considered as a relevant the accumulation of Aβ oligomers, MacPherson and enzyme in the synapses since previous studies demon- coworkers demonstrated that peripheral administration strated its involvement in the re-modeling of the synaptic of tumor necrosis factor alpha (TNFα) inhibitors modi- spines [79, 80]. Moreover, in accordance with our data, it fied the proinflammatory profile of AD mice, thus rescu- has been suggested that ADAM10 represents a new way ing long term-potentiation together with the decrease of to regulate excitatory neurons in the hippocampus which Aβ plaques [74]. In line with these previous results, our could explain the improvement observed in our mice study confirms that the reduction of the inflammatory after treatment. response also contributes to the reduction of Aβ burden It has been described that neuroinflammation also in the brain. Moreover, altogether confirms the study produces endoplasmic reticulum stress, thus lead- performed by Ho et al. and others, which demonstrated ing to UPR disrupted activation [81]. In fact, Pintado that preclinical AD animal models developed periph- and coworkers demonstrated that lipopolysaccharide- eral insulin resistance which coincide with the onset of induced neuroinflammation produce UPR activation Aβ accumulation in the brain, reinforcing the hypothesis [81], leading to synaptic dysfunction, neuronal plas- that connect peripheral alterations with AD hallmarks ticity decrease and axonal alteration, all of them con- [75–77]. tributing to neurodegeneration [82]. In line with this association, chronic activation of the UPR has emerged Ettcheto et al. Cell Biosci (2021) 11:141 Page 13 of 17 Fig. 9 Schematic diagram of the DXI effects. This drug may be an interest compound in the prevention of cognitive loss in AD due to its ability to act on different risk factors. In the present work, where APP / PS1 female mice suffer a metabolic stress associated with a HFD, we demonstrate that DXI improves peripheral parameters associated with insulin resistance. It also improves the cognitive process related to a decrease in the neuroinflammatory process, activation of the non-amyloidogenic pathway, improves the reticulum stress process in addition to parameters related to the synaptic process. All these results suggest that this drug could be used in a combinatorial therapy, for example associated with memantine and acetyl cholinesterase inhibitors for the prevention of late-onset AD as a conserved feature among various neurodegenera- in pIRE1α. Furthermore, these data corroborate the tive in both, animal models and post-mortem studies of studies performed by Duran-Aniotz et al. [90] which tissues obtained from patients [83], neurodegenerative demonstrated that the ablation of IRE1α reduced the disorders which share an accumulation of specific mis- Aβ deposition and synaptic and cognitive function folded proteins [84–86]. Specifically, in prion-infected restorage in AD mice models. Besides, these studies mice, functional studies have demonstrated that sus- are in line with others which showed the fundamental tained phosphorylation of eIF2α dramatically reduces involvement of this protein in pathological conditions. expression of synaptic proteins [87]. Likewise, its sup- It is involved not only in neurodegenerative diseases pression has been associated with the relief of AD- but also in obesity, inflammation and T2DM, among related plasticity and memory deficits, demonstrating others. Therefore, its inhibition could offer an inter- that the chronic activation of this signaling has adverse esting avenue for AD [91–93]. Going further, ATF3 is effects on synaptic function [88, 89]. These previ- a stress-induced and adaptive-responsive gene located ous data are in accordance with our results, since our downstream of eIF2α which basal levels are low. How- data showed not only a significant increase in p-eIF2α ever, they can rapidly increase under stress [94, 95]. By protein level in APP/PS1 mice fed with an obesogenic contrast, its functions are variable depending on the diet in comparison to APP/PS1 mice but also a sig- context, where evidence demonstrates that NSAIDs nificant reduction after DXI treatment. Moreover, ani- induce ATF3 expression and activation, conferring mals treated with DXI showed a significant decrease neuroprotection through the inflammatory cytokines Ettcheto et al. Cell Biosci (2021) 11:141 Page 14 of 17 downregulation [95, 96] and promoting neural out- Acknowledgements Not applicable growth [97]. In this context, our results supported these previous studies showing a significant increase in Authors’ contributions Atf3 mRNA expression in response to the stress caused ME proposal and schematization of the article, design the experiments, performed experiments, collected and analyzed the data, writing-original by the obesogenic diet intake, which is drastically full draft, editing figures. ESL performed experiments, bibliography search, increased due to NSAIDs effect. writing-original draft section. AM performed experiments, bibliography Finally, our study demonstrates a significant improve- search, writing-original draft section. MC performed experiments, analyzed data, bibliography search, writing-original draft section. KH performed experi‑ ment in the hippocampus insulin signaling pathway, ments, analyzed data, editing figures. PRM performed experiments, bibliogra‑ which is in accordance with this reported by Das- phy search. TE performed experiments, analyzed data. OB manuscript revision, gupta and coworkers, who demonstrated that cates- bibliography search, linguistic correction. EV manuscript revision, experiment supervision. JO manuscript revision, linguistic correction. CA manuscript revi‑ tin improves insulin sensitivity by attenuating UPR sion, experiment supervision. JF bibliography search. AC writing-original draft, through in silico and in vivo validation [98]. proposal and schematization of the article, funding acquisition. All authors read and approved the final manuscript. Funding Conclusions This work was supported by the Spanish Ministry of Economy and Com‑ In summary, the present study confirms for the first petitiveness under the project SAF2017-84283-R, Biomedical Research Networking Centre in Neurodegenerative Diseases (CIBERNED) under project time that DXI chronic administration in a mixed model CB06/05/0024 and the European Regional Development Founds. AC [Amanda of preclinical obesity associated T2DM and familial Cano] is supported by the Spanish Ministry of Science, Innovation and Univer‑ AD, improved-related hallmarks associated to both dis- sities under the Grant Juan de la Cierva (FJC2018-036012-I). PRM is supported by grants 2015/26084–1 and 2017/13224–5, São Paulo Research Foundation eases and modifying both central and peripheral targets (FAPESP)—Brazil. involved in the development of AD. Our data strongly suggest that DXI could be a promising therapeutic Availability of data and materials All data generated or analysed during this study are included in this published strategy for the modification of the AD course. Due article. to the complexity of the disease and the multiple risk factors associated to its development where inflamma- Declarations tory process is key, we propose a combinatory therapy of different drugs considering essential the administra- Ethics approval and consent to participate All the experiment on APP/PS1 transgenic mice were performed in accord‑ tion of NSAIDs with few side effects such as DXI with ance with the European Community Council Directive 86/609/EEC and the the aim to delay AD development (Additional file 1: Fig. procedures were established by the Department d’Agricultura, Ramaderia i S1). Pesca of the Generalitat de Catalunya. Consent for publication Not applicable. Abbreviations Aβ: Amyloid beta; AD: Alzheimer´s disease; ADAM10: Disintegrin and metallo‑ Competing interests proteinase domain -containing protein 10; AKT/ p-AKT: Protein kinase B/ phos‑ The authors declare that they have no conflict of interest. pho- Protein kinase B; APP/PS1: APPswe/PS1dE9; Atf3: Activating factor 3; Atf4: Activating factor 4; Atf6: Activating factor 6; a.u.: Arbitrary units; Bip: Binding Author details immunoglobulin protein; Chop: C/EBP homologous protein; DBN1: Drebrin; 1 Department of Pharmacology, Toxicology and Therapeutic Chemistry, DI: Discrination index; DXI: Dexibuprofen; EI2Fα/p- EI2Fα: Eukaryotic initiation Faculty of Pharmacy and Food Science, University of Barcelona, Barcelona, factor 2 α subunit/ phospho Eukaryotic initiation factor 2 α subunit; FBS: Fetal Spain. 2 Biomedical Research Networking Centre in Neurodegenerative bovine serum; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; GD: Diseases (CIBERNED), Madrid, Spain. 3 Institute of Neuroscience, University Gyrus dentatus; GFAP: Glial fibrillary acidic protein; GKS3β/ p-GKS3β: Glycogen of Barcelona, Barcelona, Spain. 4 Institute of Nanoscience and Nanotechnology synthase kinase 3 β; HFD: High fat diet; IBA1: Ionized calcium binding adaptor (IN2UB), University of Barcelona, Barcelona, Spain. 5 Department of Pharmacy, molecule 1; IBU: Ibuprofen; IP-GTT​: Intraperitoneal-glucose tolerance test; Pharmaceutical Technology and Physical Chemistry, Faculty of Pharmacy IP-ITT: Intraperitoneal-insulin tolerance test; Ir: Insulin receptor; IRE1a/ p-IRE1a: and Food Science, University of Barcelona, Barcelona, Spain. 6 Research Center Inositol-requiring enzyme-1a; Irs1: Insulin receptor substrate 1; IRS2: Insulin and Memory Clinic, Fundació ACE. Institut Català de Neurociències Aplicades receptor substrate 2; MWM: Morriw water maze; NSAID: Nonsteroidal anti- – International University of Catalunya (UIC), Barcelona, Spain. 7 Department inflammatory drug; NORT: Novel object recognition test; O/N: Over/night; PB: of Biochemistry and Biotechnology, Faculty of Medicine and Life Science, Uni‑ Phosphate buffer; PBS: Phosphate-buffered saline; PFA: Paraformaldehyde; versity Rovira I Virgili, Reus, Spain. 8 Department of Cellular Biology, Physiology RT-PCR: Real time PCR; T2DM: Type 2 Diabetes Mellitus; TNFα: Tumor necrosis and Immunology, Faculty of Biology, University of Barcelona, Barcelona, Spain. factor α; UPR: Unfolded protein response. 9 Department of Gerontology, Federal University of São Carlos (UFSCar), São Carlos 13565‑905, Brazil. 10 Dominick P. Purpura Department of Neurosciences, Supplementary Information Albert Einstein College of Medicine, New York City (10461), USA. 11 Labora‑ tory of Cellular and Molecular Pathology, Facultad de Ciencias de La Salud, The online version contains supplementary material available at https://​doi.​ Instituto de Ciencias Biomédicas, Universidad Autónoma de Chile, Talca, Chile. org/​10.​1186/​s13578-​021-​00646-w. 12 Unitat de Farmacologia I Farmacognòsia, Facultat de Farmàcia I Ciències de L’Alimentació, Universitat de Barcelona, Av. Joan XXIII 27/31, 08028 Barcelona, Additional file 1: Fig. S1. Speed on the training day (no statistical differ‑ Spain. ences were obtained). Ettcheto et al. Cell Biosci (2021) 11:141 Page 15 of 17 Received: 7 April 2021 Accepted: 4 July 2021 22. Stewart W, Kawas C, Corrada M, Metter E. Risk of Alzheimer’s disease and duration of NSAID use. Neurology. 1997;48(3):626–32. 23. Ozben T, Ozben S. 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