Malva parviflora extract ameliorates the deleterious effects of a high fat diet on the cognitive deficit in a mouse model of Alzheimer’s disease by restoring microglial function via a PPAR-γ-dependent mechanism
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Alzheimer’s disease (AD) is a neuropathology strongly associated with the activation of inflammatory pathways. Accordingly, inflammation resulting from obesity exacerbates learning and memory deficits in humans and in animal models of AD. Consequently, the long-term use of non-steroidal anti-inflammatory agents diminishes the risk for developing AD, but the side effects produced by these drugs limit their prophylactic use. Thus, plants natural products have become an excellent option for modern therapeutics. Malva parviflora is a plant well known for its anti-inflammatory properties.
The present study was aimed to determine the anti-inflammatory potential of M. parviflora leaf hydroalcoholic extract (MpHE) on AD pathology in lean and obese transgenic 5XFAD mice, a model of familial AD. The inflammatory response and Amyloid β (Aβ) plaque load in lean and obese 5XFAD mice untreated or treated with MpHE was evaluated by immunolocalization (Iba-1 and GFAP) and RT-qPCR (TNF) assays and thioflavin-S staining, respectively. Spatial learning memory was assessed by the Morris Water Maze behavioral test. Microglia phagocytosis capacity was analyzed in vivo and by ex vivo and in vitro assays, and its activation by morphological changes (phalloidin staining) and expression of CD86, Mgl1, and TREM-2 by RT-qPCR. The mechanism triggered by the MpHE was characterized in microglia primary cultures and ex vivo assays by immunoblot (PPAR-γ) and RT-qPCR (CD36) and in vivo by flow cytometry, using GW9662 (PPAR-γ inhibitor) and pioglitazone (PPAR-γ agonist). The presence of bioactive compounds in the MpHE was determined by HPLC.
MpHE efficiently reduced astrogliosis, the presence of insoluble Aβ peptides in the hippocampus and spatial learning impairments, of both, lean, and obese 5XFAD mice. This was accompanied by microglial cells accumulation around Aβ plaques in the cortex and the hippocampus and decreased expression of M1 inflammatory markers. Consistent with the fact that the MpHE rescued microglia phagocytic capacity via a PPAR-γ/CD36-dependent mechanism, the MpHE possess oleanolic acid and scopoletin as active phytochemicals.
M. parviflora suppresses neuroinflammation by inhibiting microglia pro-inflammatory M1 phenotype and promoting microglia phagocytosis. Therefore, M. parviflora phytochemicals represent an alternative to prevent cognitive impairment associated with a metabolic disorder as well as an effective prophylactic candidate for AD progression.
KeywordsAlzheimer’s Disease obesity inflammation Malva parviflora microglia PPAR-γ/CD36
Amyloid precursor protein
Area under the curve
Dulbecco’s Modified Eagle Medium
Glial fibrillary acidic protein
Glucose tolerance test
Interleukin 1 beta
Insulin resistance test
- M. parviflora
M. parviflora leaf hydroalcoholic extract
Nicotinic ACh receptors
Phosphate buffer saline
Peroxisome proliferator-activated receptor gamma
Reactive oxygen species
Embryonic alkaline phosphatase
Scavenger receptor A
Tumor necrosis factor
Alzheimer’s Disease International Organization has predicted that 81.1 million people aged 60 years or more will be declared with dementias worldwide by the year 2040 . Alzheimer disease (AD) is the main cause of dementia characterized by a progressive impairment in cognitive function (i.e., memory) and premature death . Dystrophic neurites, synapses, and neuron loss result from the twisted strands of the protein tau (tangles) inside neurons and the progressive accumulation of Aβ peptides, which are produced by the consecutive proteolytic cleavage of the amyloid precursor protein (APP) by the β- and γ-secretases [3, 4, 5]. Adjacent to the amyloid core, microglial cells are frequently found, whereas astrocytes usually surround the plaques. Both cells types produce pro-inflammatory molecules like cytokines and oxygen-free radicals, among others . The presence of elevated pro-inflammatory cytokine concentrations (interleukin (IL) 1 beta (IL-1β), interleukin 6 (IL-6), tumor necrosis factor (TNF)) in the AD brain has been associated with neuronal toxicity and cell death . IL-1β is produced as a biologically inactive pro-form and requires to be processed by caspase-1 for its activation. Caspase-1 activity is controlled by inflammasomes, which are sensors of danger signals. The relevance of the inflammatory process in memory loss and neurodegeneration resulting from Aβ accumulation was recently shown in AD animal models lacking caspase-1 or other inflammasome components [8, 9]. Accordingly, distinct factors that play a role in the onset and progression of AD, including type II diabetes mellitus, hypertension, obesity, and dyslipidemia, have been associated to chronic inflammation [10, 11, 12]. In fact, type II diabetes is a major risk factor for dementia and has been directly associated with the Aβ accumulation in the brain, due to deficient brain Aβ clearance resulting from the competition between insulin and the Aβ for binding to the insulin-degrading enzyme . Therefore, obesity and high-fat diet are apparent risk factors for dementia associated with brain insulin resistance. The excess of adipose tissue in obesity regulates adipokines (e.g., adiponectin, omentin, leptin, resistin) critical to metabolism as well as inflammatory cytokines (e.g., IL-1β, TNF, and IL-6) that correlate with insulin resistance and hyperinsulinemia [14, 15]. Thus, chronic inflammatory process in the periphery might result in dementia.
Consequently, the long-term use of non-steroidal anti-inflammatory agents in humans, such as indomethacin, sulindac, and PPAR-γ agonist, such as ibuprofen, rosiglitazone, and pioglitazone, has been directly linked with a low risk for developing AD and decreased Aβ deposition in vitro and in vivo [16, 17]. Unfortunately, the side effects produced by these drugs limit their prophylactic use. Nevertheless, there are natural products that have been widely used for a long time as a remedy for treating diseases with an inflammatory pathophysiological origin . Experimental evidence indicates that polyphenolic compounds, like flavonoids, phenylpropanoids, and terpenes, among others, counteract inflammation (reviewed in ). Recent studies have shown that Malva parviflora (M. parviflora), a plant widely used in traditional medicine in America and Africa, has hypoglycemic, anti-inflammatory, and anti-oxidant effects [20, 21]. Here we evaluated the long-term effects of a M. parviflora hydroalcoholic extract (MpHE) on the development of AD pathology in obese 5XFAD mice. We show that obesity further promoted Aβ plaques formation in the 5XFAD mice. However, administration of the MpHE reversed obesity-mediated deleterious effects of Aβ deposition on memory and learning. Interestingly, this correlated with microglia-enhanced phagocytic capacity and reduced microglia M1 phenotype activation. Importantly, we demonstrated that MpHE increases microglia phagocytic activity via a PPAR-γ/CD36-dependent mechanism. The phytochemical analysis of the MpHE showed the presence of two active compounds with anti-inflammatory activity, scopoletin and oleanolic acid. Together, our results indicate that the MpHE is a promising candidate as an effective prophylactic for AD development.
Preparation of the M. parviflora hydroalcoholic extract
The leaves of M. parviflora were collected in Ozumba, state of Mexico. One specimen was deposited in the herbarium of Ethnobotanical Garden of Museum of Traditional Medicine and Herbal medicine of INAH Morelos. The species was identified by Margarita Aviles and Macrina Fuentes with the registration number 2048. Plant material was dried in dark conditions at room temperature (RT). Once dried, the plant was milled, by means of a 5HP equipment, until obtaining < 5-mm particles. Plant material (3 kg) was extracted by infusion process at 60° C with an ethanolic solution (60%). The extraction volume used was 2 L of solvent per kilogram of plant material. Liquid extract was filtered and concentrated by low-pressure distillation. Semisolid extract was finally dried by lyophilization process, obtaining 11.11% yield. This hydroalcoholic extract (HE) was submitted to liquid-liquid separation. Water and ethyl acetate phases were obtained with 100 mL of these solvents for each 5 g of the crude extract. Samples of 3 mg/mL of these fractions were used for HPLC analysis. When used for intragastric administration, aqueous suspension of the MpHE (5 mg/mL sterile water) was prepared, stored at 4° C and protected from light.
Analysis of compounds in the hydroalcoholic extract of M. parviflora
Chromatographic analysis was developed by means of Waters equipment constituted by a 2695 separations module and a 2996 photodiode array detector. Separation was carried out by using an RP-18 Supersphere (Merck) column (125 × 4 mm, 5 μm). The mobile phase consisted of a gradient system with water (solvent A) and acetonitrile (solvent B). Initial conditions started with 100% of solvent A (0–1 min), followed by different proportions of solvent A:solvent B = 90:100 (2–4 min), 80:20 (5–7 min), 70:30 (8–14 min), 60:40 (15–18 min), 20:80 (19–22 min), 0:100 (23–26 min), and 100:0 (27–28 min). The sample injection volume was 10 μL with a 1 mL/min flow rate. The detection wavelength was scanned at 190–600 nm. Scopoletin quantification was achieved using a calibration curve, which was constructed by three injections of a solution prepared with a commercial reference of the coumarin standard (7.812, 15.625, 31.25, 62.5, and 125 μg/mL) (Sigma Aldrich, St Louis, MO, USA). Values of the area under the peak of chromatograms recorded at 345 nm were used to obtain the equation: Y = 84,221X − 188,219 R2 = 0.9995. The presence of oleanolic acid in the MpHE was established by comparison of the peak at 25.85 min with a commercial reference of this triterpene (Sigma Aldrich, St Louis, MO, USA).
Macrophage reporter cells and NF-κB/AP-1 activation
We used a commercially available reporter macrophage cell line (RAW-Blue cells, InvivoGen, San Diego, CA, USA), which expresses the secreted embryonic alkaline phosphatase (SEAP) gene under the control of a promoter inducible by the transcription factors NF-κB and AP-1. Briefly, cells were seeded in 24-well plates in Dulbecco’s Modified Eagle Medium (DMEM), 4.5 g/l glucose, 10% heat-inactivated FBS (30 min at 56 °C), 2 mM L-glutamine, 100 mg/mL Normocin, and 200 mg/mL Zeocin. Then, cells were treated with lipopolysaccharide (LPS; 100 ng/mL) alone or together with three different concentrations of MpHE (0.1, 0.5, and 1.0 mg/mL), oleanolic acid (15, 26, 40 μg/mL) or scopoletin (3, 8, 24 μg/mL) for 12 h. In addition, cells were pre-treated with LPS or MpHE for 30 min, and then MpHE or LPS was added for 12 h. NF-κB and AP-1 activity was evaluated indirectly by quantifying the SEAP activity in the culture supernatants. Briefly, 50 μL from each sample were added to 150 μL of the QUANTI-Blue assay buffer (InvivoGen, San Diego, CA, USA) and incubated at 37 °C for 30 min. PBS or the used vehicle to dissolve oleanolic acid or scopoletin were used as negative control. Absorbance at 650-nm wavelength was measured and the SEAP activity was calculated as fold change for each sample (SEAP activity in medium from treated cells minus background (medium without cells) over SEAP activity in medium from untreated cells minus background). In parallel, trypan blue staining was used to determine the cell viability after MpHE treatment.
The 5XFAD transgenic mice, expressing the human APP gene with three familial AD mutations (Swedish (K670N, M671L), Florida (I716V), and London (V717I)) and the human presenilin 1 carrying two mutations (M146L and L286V) were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). Mice were housed in groups of five per cage, in a temperature- (22 ± 1 °C) and humidity-controlled room, with a 12-h light/dark cycle. Food and water were available ad libitum. All tests were performed during the light cycle. Male 5XFAD mice and male wild type (Wt) littermates were used (C57BL/SJL genetic background). Transgenic and Wt mice (6 weeks old) were fed with normal diet (ND) (caloric percentage, 18%, 3.1 kcal/g; Teklad, Harlan Laboratories), or with a high-fat diet (HFD) (caloric percentage, 60%, 5.24 kcal/g; Research diets. Inc, USA). These groups of mice received 50 mg/kg/day of the MpHE or water (vehicle) intragastrically during 8 months. Two additional groups from Wt ND and Wt ND treated with MpHE (C57BL genetic background) were included to determine spatial learning.
For the dose-response experiments, a LPS-mediated neuroinflammation model was used. CD1 male mice were intraperitoneally injected with 125 μg/kg of LPS or vehicle as control for 7 consecutive days. LPS injected mice then received 25, 50, or 100 mg/kg/day of the MpHE or water (vehicle) intragastrically during 7 days. Spatial learning and memory were determined. The Institutional Bioethical Committee approved all animal experiments described in this study.
Food intake and body weight were measured weekly. After 7 months of treatment, glucose tolerance test (GTT) was performed after 6 h food deprivation by intraperitoneal (i.p.) glucose administration (1.8 g/kg body weight). Blood glucose concentrations were measured before glucose administration (t = 0) and 15, 30, 60, and 120 min after injection. One month after, insulin resistance test (IRT) was performed after 6 h food deprivation by i.p. injection of insulin (Humulin® R 1 mU/gram of body mass). Blood glucose concentrations were measured before insulin administration (t = 0) and 15, 30, 60, and 120 min after insulin administration using a Glucometer (Accucheck; Roche) and the data were reported as milligrams per deciliter. Area under the curve (AUC) for glucose concentration in both cases was calculated as reported previously .
Mice were anesthetized with ketamine/xylazine (90 mg/kg/9 mg/kg, i.p.) and perfused transcardially with isotonic saline followed by 4% paraformaldehyde (PFA) in phosphate buffer saline (PBS; pH 7.4). Brains were post-fixed in 4% PFA overnight, followed by 30% (w/v) sucrose solution at 4 °C. Immunofluorescence was performed by the free-floating antibody staining method, using 30-μm-thick sagittal cryostat slices. The slices were washed three times with PBS-T (0.25% Triton X-100 in PBS) for 5 min each and incubated in blocking buffer (PBS-T 0.1% bovine serum albumin) for 1 h at RT. Then, the slices were incubated with mouse monoclonal antibody against Glial fibrillary acidic protein (GFAP) (AB_561049; 1:250) overnight at 4 °C, washed with PBS-T two times for 5 min each and incubated with secondary antibody goat anti-mouse Alexa Flour 488 (AB_2534069; 1:400) in blocking buffer for 2 h at RT. After 2 washes with PBS-T, free-floating brain slices were then mounted with Fluoroshield (Sigma Aldrich Inc) and sealed with a coverslip. Additionally, goat antibody anti-Iba1 (AB_2224402; 1:200) and the secondary antibody donkey anti-goat Alexa Fluor 568 (AB_175704; 1:200, Abcam Inc) were used to identify microglial cells in the brain sections. Images were captured using the Zeiss Axioskop Observer Z1 inverted fluorescence microscope.
PFA-fixed brain tissues of each group were stained in 1% aqueous Thioflavin-S for 5 min. After this, slices were washed sequentially in 80% ethanol, 70% ethanol, and distilled water, then dried and mounted as above described. The brain slices stained with Thioflavin-S were analyzed in a Zeiss Axioskop microscope using an emission spectrum of 550 nm. Images were captured using the Zeiss Axioskop fluorescence microscope. Two or three slices were analyzed in four animals/group. Analyses were performed across the dentate gyrus using a × 5 objective and the automated target detection mode from Image J software (National Institutes of Health, Bethesda, MD, USA). Image size was 1800 × 1800 μm and the percentage of load area was calculated (load area/total area). Additionally, to determine the fraction of amyloid plaques overlapping with microglia in the hippocampus and cortex from 5XFAD mice administered with vehicle or MpHE, the Manders’s coefficient was determined using the plugin JacoP  in Image J software (National Institutes of Health, Bethesda, MD, USA).
Behavioral assessment of cognitive functions by Morris water maze test
Spatial learning memory was assessed by the Morris Water Maze behavioral test. Mice were tested in a 1.83 m circular pool filled with water mixed with nontoxic white paint (titanium oxide). The tank was divided into four quadrants (NW, SW, SE, and NE) of equal size. Mice were trained to mount a hidden/submerged (1.0 cm below water surface) escape platform (15-cm diameter) in a restricted region of the pool (NW). Mice were trained for 5 consecutive days from the four quadrants of the tank; they were placed into the water facing the wall and were allowed to swim for a maximum of 90 s. The trial ended when a mouse climbed onto the platform or after the 90-s interval had elapsed. If a mouse did not locate the platform during a trial, it was placed on the platform by the experimenter during 20 s, and then moved to a holding cage. Escape latencies to the hide platform during the learning phase were analyzed using the integrated time traveled and time needed to reach the platform (AUC) for each group. After two days, a memory test was performed in absence of the platform. Mice were released from the four start points to the platform location and allowed to swim freely in the pool for 90 s. Escape latency and swimming paths were recorded.
Microglial cell culture
Primary cultures of microglial cells were prepared from neonatal (postnatal days 1-3) CD1 mice or from 8- and 10-months-old Wt and 5XFAD mice. Briefly, neonatal brain tissues were mechanically dissociated, treated with Trypsin (0.25 mg/mL, Sigma Aldrich Inc) and DNase I (0.28 mg/mL) at 37 °C for 30 min. After extensive washing, dispersed brain cells were plated as mixed glia cultures (enriched with microglial and astroglia cells) at the density of two brains per dish in DMEM/F12 medium supplemented with 10% FBS, 2 mM glutamine, and antibiotics onto 100-mm Petri dishes at 37 °C and 5% CO2. Eight days after plating, the microglia cells were separated from astrocytes by shaking for 5 h at 250 rpm at RT. The neonatal microglial cultures purity was analyzed by flow cytometry, RT-PCR, and immunofluorescence assays using specific markers for microglia (F4/80), CD11b, astrocytes (GFAP), and neurons (neurofilament M). The antibody anti-F4/80 (AB_1140040; 1.200), anti-CD11b (AB_389305; 1:2000), and anti-GFAP (AB_561049; 1:200) were used for this purpose.
Alternatively, adult brain tissue from wild type and 5XFAD mice was mechanically dissociated and the Neural Tissue Dissociation Kit (Miltenyi Biotec, USA) was used to obtain single-cell suspension. The myelin debris was removed with the Myelin Removal Beads II (Miltenyi Biotec, USA), and the microglia were isolated by positive selection using the CD11b (microglia) microbeads (Miltenyi Biotec, USA), according to the manufacturer’s instructions. The adult microglial cultures purity was analyzed by flow cytometry assays using anti-CD11b antibody (AB_389305; 1:2000).
Microglia classical activation
Neonatal primary microglial cultures (1 × 105 cells per well) were plated onto Lab-Tek coverglass chamber wells (Nalge Nunc International, Rochester, NY, USA). After 1 day, the cells were stimulated with 100 ng/mL LPS in the presence or absence of MpHE (1 mg/mL) for 24 h. Control cells were treated with PBS or MpHE alone. Subsequently, the cells were fixed in 4% PFA for 30 min at RT and permeabilized with PBS containing 0.1% Triton TX-100 for 30 min at RT. F-actin was visualized by incubating cells for 1 h in the dark with Alexa Fluor 488-conjugated phalloidin (AB_2315147; 1:500). Cell nuclei were labeled with 4′, 6-diamidino-2-phenylindole (DAPI, 1:5000, Molecular probes) for 15 min. Microglial cells were imaged using the Olympus FluoView 1000 confocal multiphoton microscope. The different microglial cell morphologies (amoeboid, ramified, and intermediate shapes) were quantified in each treatment from seven random fields per group. The length of the protrusions of the microglial cells was determined using Neurite Tracer plugin from ImageJ software (National Institutes of Health, Bethesda, MD).
In vitro phagocytosis assay
Eight days after separating astroglial cells, microglia from eight and 10-months-old Wt and 5XFAD mice were plated onto Lab-Tek coverglass chamber wells (1 × 105 cells per well) (Nalgene Nunc International, Rochester, NY, USA) and treated with MpHE (1 mg/mL) for 24 h. The medium was then replaced with fresh medium containing 1 × 106 fluorescent E. coli bacteria  or unlabeled latex beads (0.8 μm diameter) (Calibrite, Becton Dickinson), and incubated for 1 or 4 h, respectively. The medium was removed, the microglial cells were washed extensively with PBS, treated with Geneticin (20 μg/mL) for 30 min, and fixed in 4% PFA (Sigma Aldrich Inc) for 30 min at RT. Then, cells were permeabilized with PBS containing 0.1% Triton TX-100 for 30 min at RT. Actin and the nucleus were visualized as described above. The percentage of cells capable of internalizing E. coli bacteria and the average of E. coli ingested by cells were determined. The phagocytosis index was determined by multiplying the percentage of microglial cells with internalized bacteria by the average number of internalized E. coli bacteria  or unlabeled latex beads for each sample. Data were collected from seven random fields per group. Microglial cells were imaged using the Olympus FluoView 1000 confocal multiphoton microscope.
PPAR-γ protein levels and ex vivo phagocytosis assay
Neonatal primary microglial cultures (2.5 × 105 cells) were left untreated or pre-treated with 100 μM GW9662, a specific PPAR-γ inhibitor. After 1 h, cultures were treated with MpHE (0.1 and 1 mg/mL) or with pioglitazone (10 μM; a PPAR-γ agonist) or with oleanolic acid (4.5 μg/mL and 45 μg/mL) for 24 h. The PPAR-γ levels from the cell extracts were determined by immunoblot. Additionally, PPAR-γ levels were also determined in the frontal cortex cell extracts of 5XFAD and Wt mice fed with either ND or HFD non-treated (vehicle) or treated with MpHE.
Ex vivo Aβ phagocytosis assays were performed as previously described . Briefly, adult 5XFAD transgenic mice were perfused with saline solution. Brains were removed and snap frozen in dry ice. After, 10-μm-thick sagittal cryostat slices were obtained and mounted onto poly-D-lysine-coated glass coverslips. Brain slices were dried for at least 2 h at RT and then washed twice with PBS and DMEM serum-free media (Gibco). Each brain slice was cultured with neonatal primary microglial cultures (2.5 × 105 cells) previously untreated or treated with MpHE (1 mg/mL) for 12 h or 100 μM of GW9662 (Sigma Aldrich) for 1 h in FBS-free DMEM medium. After 12 h, the cells were washed and the medium was replaced by DMEM containing 5% FBS and 20% L929 supernatant for 24 h at 37 °C with 5% CO2. Sections were then washed twice with PBS and fixed with 4% PFA for subsequent histology. The brain slices were stained with Thioflavin-S and the plaque’s number and size were quantified.
In vivo phagocytosis assay
Male 5XFAD mice and male Wt littermates were used (C57BL/SJL genetic background). Transgenic and Wt mice (8 weeks old) received 50 mg/kg/day of the MpHE or water (vehicle) intragastrically during 2 months. Then, mice were intraperitoneally injected with the PPARγ inhibitor GW9662 (5 mg/kg) or vehicle (5% DMSO/95%PBS) for the last 3 days before sacrifice. For the in vivo Aβ phagocytosis assays, mice were intraperitoneally injected with 10 mg/kg methoxy-X04 (Tocris) 6 h before sacrifice. Brains were removed and cell suspension was obtained by enzymatic (0.2 mg/mL papain) and mechanical dissociation. Myelin debris was removed by centrifugation (10 min at 700 g) using 30% Percoll (Sigma). The cells present in the pellet were suspended, immunolabeled for microglia specific markers using the antibody anti-CD11b (AB_389305; 1:2000) and the anti-CD36 (AB_2072639;1:200) and analyzed by flow cytometry using a FACSCanto II (BD Biosciences). The CD36 expression analysis was done on the CD11b+ cells while the methoxy-X04 staining analysis was performed on the CD11b+/CD36+ population. Wt unstained and methoxy-X04 stained cells were used to determine background fluorescence and the threshold for non-phagocytic cells, respectively.
PCR and qPCR
Total RNA was isolated from primary cultures (neonatal microglia) or from tissue (cortex) as previously described . Reverse transcription was performed using M-MLV reverse transcriptase (Invitrogen, ThermoFisher Scientific) and oligo (dT) primer. Neonatal microglial cultures purity was determined by semi-quantitative PCR using the following forward (F) and reverse (R) primers: For F4/80: F 5′-GTG CCA TCA TTG CGG GAT TC-3′ and R 5′-GAC GGT TGA GCA GAC AGT GA-3′, for Neurofilament-M: F 5′-GAA CCA CGA GAA GGC TCA AG-3′ and R 5′- CCT CCT CTT CGT GAT TGC TC-3′, for GFAP: F 5′-CAG TTC AGC TGC CAG CGC CT-3′ and R 5′-GCA GAG GCA GGG CAG GAT GG-3′, and for Actin: F 5′-GGG TCA GAA GGA CTC CTA TG-3′ and R 5′-GGT CTC AAA CAT GAT CTG GG -3′. Quantitative real time PCR (qPCR) was carried out on ABI PRISM 7500 sequence detection system (Applied Biosystems, Foster City, CA, USA) using cDNA from Wt or 5XFAD lean or obese mice cortex treated or not treated with MpHE in combination with Maxima SYBR Green/ROX qPCR Master Mix (ThermoFisher Scientific) and specific primers. For TREM-2: F 5′-CTG GAA CCG TCA CCA TCA CTC-3′, and R 5′-CGA AAC TCG ATG ACT CCT CGG-3′; for Mgl1: F 5′-TGA GAA AGG CTT TAA GAA CTG GG-3′, and R 5′-GAC CAC CTG TAG TGA TGT GGG-3′; for CD86: F 5′-GAC CGT TGT GTG TGT TCT GG-3′, and R 5′-GAT GAG CAG CAT CAC AAG GA-3′; for TNF: F 5′-CAG GCG GTG CCT ATG TCT C-3′, and R 5′- CGA TCA CCC CGA AGT TCA GTA G-3′; for CD36: F 5′-GAG CAA CTG GTG GAT GGT TT-3′, and R 5′-GCA GCA GAA TCA AGG GAG AG-3′; for GAPDH: F 5′-GGG AAG CTC ACT GGC ATG G 3′, and R 5′-CTT CTT GAT GTC ATC ATA CTT GGC AG-3′; and for Ndufa-10 (UBC): F 5′-CCG CCT TCT TCA GTA TGC AGA-3′, and R 5′- TGC TTT CGG ATA TAG CCC TGG. Relative expression was calculated by normalizing to Ndufa-10 or GAPDH using the 2−∆∆CT method .
Neonatal microglial culture homogenates were obtained by scraping with ice-cold lysis buffer (20 mM Tris pH 7.4, 137 mM NaCl, 2 mM PPiNa, 2 mM EDTA, 1% Triton X-100, 10% glycerol, 0.5 mM DTT, 25 mM β-glycerophosphate, 200 mM Na3VO4, 1 mM PMSF, and complete protease inhibitor (Roche)). Lysates were incubated 10 min at 4 °C, then centrifuged at 14,000×g for 15 min at 4 °C, and stored at − 70 °C until use. Protein concentration was determined using the Bradford reagent (Bio-Rad). Total cell extracts (20 μg) were resolved by SDS-PAGE and transferred onto nitrocellulose membranes (GE Healthcare). Membranes were blocked with 5% BSA in Tris-buffer saline (TBS, 10 mM Tris pH 7.5, 150 mM NaCl) with 0.1% Tween-20 (Sigma) (TBS-T) and incubated with the primary antibody anti-PPAR-γ (AB_2283990; 1:5000, Santa Cruz Biotechnology), anti-actin (AB_2223041; 1:8000, Millipore), or anti-GAPDH (AB_10622025; 1:6000, Cell Signaling) overnight at 4 °C. After three washes with TBS-T, membranes were incubated with secondary antibody anti-rabbit-HRP (1:10000).
Microglia from 8-month-old Wt and 5XFAD mice were plated onto Lab-Tek coverglass chamber wells (1 × 105 cells per well) (Nalgene Nunc International, Rochester, NY). After one day, cells were stimulated with 100 ng/mL LPS in the presence or absence of MpHE (1 mg/mL) for 24 h. Control cells were treated with PBS or MpHE alone. TNF and IL-6 levels were quantified from the primary microglial cultures supernatants using a commercial ELISA Kit (Biolegend, Cat. 430904 and 431304) following the manufacturer’s instructions.
Adipose tissue histochemistry
Adipose tissue was fixed in 10% paraformaldehyde and embedded in paraffin. 5-μM-thick sections were prepared, deparaffinized in xylene, rehydrated in a graded ethanol series, and then stained with hematoxylin and eosin. Analysis of adipocyte histology was performed using the ImageJ software according to the manual procedure (http://rsbweb.nih.gov/ij/). Immune cells infiltration in adipose tissue was quantified by calculating the ratio of infiltration on 10 fields (× 40), of 3 slides for each individual mice, using 3 mice for each group. Light microscopic images were acquired using a Zeiss LSM510/UV Axiovert 200M confocal microscope with a Nikon COOLPIX 5000 color camera.
Results presented correspond to at least three independent experiments. No sample size calculation was performed. Data are shown as the mean ± SEM or SD. Data were analyzed by one-way ANOVA followed by Tukey’s post hoc test, two-way ANOVA followed by Dunnet’s or Bonferroni’s multiple comparison test, and three-way ANOVA followed by Tukey’s multiple comparison test. Differences were considered statistically significant with a p value < 0.05. Statistical analyses were performed in Prism 7.0b or Prism 8.0 (Graphpad Software, Inc.).
M. parviflora hydroalcoholic extract inhibits LPS-induced NF-κB activity in mouse RAW-Blue macrophages
M. parviflora hydroalcoholic extract improves glucose homeostasis in 5XFAD mice fed with a high fat diet
The treatment with the MpHE delayed the increase in body weight in the 5XFAD animals fed with HFD observing significant difference since the seventh week (p = 0.0267) between both groups (Fig. 2b). Accordingly, with previously published data indicating that M. parviflora regulates glucose metabolism in a type 1 diabetes animal model , no significant differences were observed between animals fed with HFD + M. parviflora extract respect to ND-fed animals at all time points assayed (p = 0.4719 at the week 28) (Fig. 2b). After eight months of HFD feeding and MpHE treatment, the glucose baseline levels (Fig. 2c), the glucose tolerance (Fig. 2d), and insulin sensitivity (Fig. 2e) were similar to those transgenic mice fed with ND. Moreover, in the case of insulin resistance test, the AUC value obtained in HFD transgenic mice treated with MpHE was twofold lower than in the HFD transgenic group without treatment (p < 0.001) (Fig. 2e, lower panel). These results showed that MpHE prevented the systemic insulin resistance and glucose intolerance produced by HFD in the 5XFAD mice.
M. parviflora hydroalcoholic extract reduces amyloidosis and astrogliosis in the 5XFAD mice fed with a high fat diet
Astrocyte activation (astrogliosis) in response to Aβ accumulation is a characteristic of AD development. GFAP staining of sagittal sections from 9-months-old 5XFAD mice showed that long-term treatment (8 months) with the MpHE reduced the astrogliosis throughout the hippocampus of HFD-fed 5XFAD mice (Fig. 3b).
Together, these results point out that MpHE attenuates the inflammatory process in the brain triggered from Aβ accumulation resulting from APP and Presenilin 1 mutations and that MpHE also attenuates the effect of altered glucose metabolism on β-amyloid plaque formation and astrogliosis.
M. parviflora hydroalcoholic extract ameliorates impairments in spatial memory in 5XFAD mice fed with a high fat diet
M. parviflora hydroalcoholic extract regulates microglia activation in vitro
M. parviflora hydroalcoholic extract stimulates the microglia phagocytic activity from 5XFAD mice
M. parviflora hydroalcoholic extract reduces the M1 phenotype in the microglial cells from 5XFAD mice
To further corroborate the anti-inflammatory effect of MpHE, primary microglial cultures from 5XFAD mice (8 months) were treated con LPS (100 ng/mL) in the absence or presence of MpHE (1 mg/mL) for 24 h. Interestingly, although LPS promoted higher production of both TNF and IL-6 in microglia from the 5XFAD mice than that from wild type mice, the increased levels of TNF and IL-6 released to the supernatant in response to LPS (TNF: Wt Ctrl vs Wt LPS p = 0.0434; 5XFAD Ctrl vs 5XFAD LPS p < 0.0001; Wt LPS vs 5XFAD LPS p = 0.0015; and IL-6: Wt Ctrl vs Wt LPS p = 0.0001; 5XFAD Ctrl vs 5XFAD LPS p < 0.0001; Wt LPS vs 5XFAD LPS p < 0.0001) was significantly reduced in response to MpHE (TNF: 5XFAD LPS vs 5XFAD LPS treated with M. parviflora, p = 0.0001; and IL-6: 5XFAD LPS vs 5XFAD LPS treated with M. parviflora, p < 0.0001) (Fig. 8e, f). According with this, the in vivo treatment with the MpHE also reduced the inflammatory process in the adipose tissue of wild type and 5XFAD mice fed with the HFD, since there was a clear reduction in the infiltration of immune cells into the adipose tissue of mice fed with HFD and treated with the MpHE compared with vehicle-treated animals (Additional file 2: Figure S2). Together these results indicate that (1) by regulating microglia phagocytic activity, (2) by preventing the pro-inflammatory M1 phenotype, M. parviflora reduces β-amyloid plaque load and improves learning and memory in lean and obese mice and (3) M. parviflora attenuates the inflammatory process in the adipose tissue of obese mice.
Scopoletin and oleanolic acid are present in the M. parviflora hydroalcoholic extract
These data indicate that the anti-neuroinflammatory effect of the MpHE, observed in this study, might result from the presence of oleanolic acid and scopoletin.
M. parviflora hydroalcoholic extract regulates the phagocytic capacity of microglial cells via the PPARγ-CD36 pathway
Since CD36 is a scavenger receptor involved in Aβ phagocytosis , whose expression is controlled by PPAR-γ , we tested the idea that the MpHE via PPAR-γ regulates CD36 expression. Microglia primary cultures were left untreated or pre-treated with GW9662 for 1 h, then incubated with MpHE (1 mg/mL) for 24 h and CD36 mRNA levels were measured. CD36 mRNA levels were upregulated in response to MpHE (p = 0.04) but prevented by GW9662 (p = 0.04) (Fig. 10b). The CD36 expression levels induced by the MpHE were similar to those resulting from exposing microglial cell to pioglitazone (p = 0.002) (Fig. 10b).
As described above, MpHE contains oleanolic acid, a natural agonist of PPAR-γ (43, 44). Therefore, we tested whether the improved microglial phagocytic activity resulting from MpHE treatment involved oleanolic acid-mediated PPAR-γ activation. In accordance with this idea, oleanolic acid increased PPAR-γ levels specifically in microglia (Ctrl vs 4.5 μg oleanolic acid, p = 0.0414; Ctrl vs 45 μg oleanolic acid, p = 0.0608). Congruently with the fact that PPAR-γ induces its own expression through a positive feedback loop , the PPAR-γ specific inhibitor GW9662 (100 μM) prevented the increase in PPAR-γ levels resulting from the oleanolic acid treatment (4.5 μg/mL oleanolic acid vs GW9662 + 4.5 μg/ mL oleanolic acid (p = 0.0116); 45 μg/mL oleanolic acid vs GW9662 + 45 μg/mL oleanolic acid (p = 0.0079)) (Fig. 12b). Together, these results indicate that the MpHE promotes microglia phagocytosis and degradation of amyloid β plaques by inducing PPAR-γ mediated CD36 expression (Fig. 12c).
Plant extracts have been used as an alternative therapy to revert the AD marks. It is thought that the mixture of compounds with synergistic activities as anti-oxidant and anti-inflammatory activities favors Aβ reduction, acetylcholinesterase inhibition, and monoamines modification [48, 49]. Previously, the M. parviflora extract has been shown to present hypoglycemic, anti-oxidant, and anti-inflammatory activities [20, 21, 50] and even it has been reported to have a beneficial effect against Aβ-injected mice . In the present study, we demonstrate the neuroprotective effect of a long-term treatment with MpHE in the 5XFAD mouse model of AD. This model has been widely used to examine the therapeutic value of candidate agents for AD treatment [52, 53, 54], displaying age-dependent neurological and motor deficits that mimic common phenotypes seen in AD patients, without the presence of neurofibrillary tangles . In addition, we also provide evidence indicating that the MpHE attenuates the negative effect of chronic peripheral inflammation on the development of AD pathological hallmarks. The MpHE reduced adipose tissue inflammation and insulin resistance in obese mice fed with a HFD; a well-established experimental approach to induce insulin resistance . Together, our results show for the first time that a long-term treatment with a MpHE attenuates both peripheral and central inflammation resulting in improved memory and learning abilities in a familial Alzheimer’s disease model. The molecular mechanism activated by the MpHE involves the activation of the PPAR-γ pathway and increased scavenger receptor CD36 expression which improves microglia phagocytic activity thus reducing Aβ load and inflammation in the CNS. Therefore, our data uncover the signaling pathway regulated by the MpHE resulting in neuroprotection.
Our phytochemical analysis identified two compounds in the MpHE, oleanolic acid and scopoletin. Interestingly, both compounds have hypoglycemic, anti-inflammatory, and anti-oxidant properties. A proposed anti-inflammatory mechanism for oleanolic acid involves the upregulation of the transcription factor Nrf2, which inhibits the expression of NF-κB . As oleanolic acid, the scopoletin anti-inflammatory effect also involves the inhibition of the NF-κB and p38 MAPK pathways . Congruent with oleanolic acid and scopoletin anti-NF-κB activity, here, we demonstrate that the MpHE decreases the NF-κB activation in a concentration-dependent manner. Furthermore, our ex vivo experiments show that this treatment effectively prevents microglia activation by inhibiting the morphological changes typically induced by LPS, thus confirming the anti-inflammatory effects of MpHE previously described using ear edema and vascular permeability models . Additionally, it has been shown that synthetic triterpenoid analogs of oleanolic acid attenuate microglia activation and the pro-inflammatory phenotype .
In contrast with previous studies showing that a HFD had no effect on the Aβ plaque burden in the hippocampus from old 3xTgAD mice , we found that HFD increases the deposition of hippocampal β-amyloid plaques in the 5XFAD mice model. This correlated with the fact that these animals were glucose intolerant and showed insulin resistance, which is in agreement with experimental evidence indicating that the energy metabolism in the brain plays a key role in AD development [60, 61, 62]. This is consistent with previous reports that confirm impairment in hippocampal synaptic plasticity in animals fed with HFD [63, 64, 65].
Recent studies showed that a short-term HFD feeding (16 weeks) to 5XFAD mice does not induce metabolic disorder, including blood glucose levels, and does not increase body weight, although the HFD had a notorious impact on the cognition, thus considering these effects as independent phenomena . However, insulin released by the pancreas crosses the blood-brain barrier and binds to and activates its specific receptor in the CNS, including those expressed in the hippocampus. Accordingly, insulin enhances memory in healthy humans and rodents [67, 68]. Thus, impaired insulin receptor signaling contributes to the development of hippocampal insulin resistance (IR). Clinical and preclinical studies suggest that hippocampal IR leads to the neuroplasticity deficits observed in both type II diabetes and AD. Recently, it has been proposed that neuroinflammation may serve as a link between aging, type II diabetes, and AD by causing IR [14, 69]. Pro-inflammatory molecules (e.g., TNF, IL-6, IL-1β) are increased in type II diabetes and AD, which may compromise the hippocampal insulin receptor expression and/or signaling. For instance, TNF may cause hippocampal IR by increasing serine phosphorylation of IRS1 . Although further studies are needed to establish whether IR in the CNS regulates memory by itself or in combination with peripheral IR, experimental data support that enhancement of hippocampal insulin receptor signaling could reduce or reverse the memory deficits observed in type II diabetes and AD . These observations point out the importance of establishing pharmacological and/or preventive strategies to improve cognitive deficits in individuals suffering metabolic syndrome. Interestingly, here, we show that the MpHE abolished the increase in TNF levels in the cortex of lean and obese 5XFAD transgenic mice.
Our results show also that the long-term treatment with MpHE significantly reduces the body weight gain, the impairment in the glucose metabolism in transgenic and non-transgenic mice and surprisingly, reduced the Aβ deposition in transgenic mice. Furthermore, we observed that MpHE significantly decreases the escape latency in the learning and memory tests, in accordance with a previous study where an ethanolic M. parviflora extract increased the learning and memory along with reduced reactive oxygen species (ROS) levels after injection of Aβ in mice . These data are consistent with oleanoic acid and scopoletin neuroprotective effects. The oleanolic acid isolated from Aralia cordata confers neuroprotection in rat cortical neuronal cultures by inhibiting neuronal death and the generation of ROS induced by Aβ. Equally, the ethanolic extract of Aralia cordata possesses antidementia activities in mice injected intracerebroventricularly with Aβ peptides .
Additionally, scopoletin increases presynaptic activity-dependent acetylcholine (ACh) release, enhances hippocampal long-term potentiation (LTP), and improves cognition in cholinergically impaired and in age-impaired mice. Scopoletin neuroprotective effect might result from interacting with nicotinic ACh receptors (nAChRs) enhancing NMDA-dependent LTP . Activated nAChRs can promote neuronal survival and may also decrease the synthesis and deposition of Aβ peptides . If the neuroprotective effects of Malva parviflora involves the interaction of the scopoletin present in the extract with the nAChRs is yet to be determined.
During the development and in adulthood, the microglial cells maintain the brain homeostasis perceiving their microenvironment. The healthy state of the microglial cells is altered by the presence of soluble and aggregated Aβ, by the synaptic degeneration observed in the early AD, among other signals. This causes an increase in the production of inflammatory molecules from the microglia leading to reprograming the expression of receptors involved in the AD progression [32, 75], for example, TNF decreased the expression of scavenger receptor A (SRA) and CD36 resulting in reduced Aβ uptake . Here, we identified the neuroprotector effect of MpHE against the AD progression by the ability to regulate the microglia activation and its phagocytic capacity, increasing the accumulation of ramified microglia with long and thin processes around the Aβ plaques in the cortex and hippocampus. Previous work has demonstrated that microglial primary cultures from adult mice are not compromised in their ability to recognize Aβ fibrils but have deficient phagocytic capacity when compared to that of early postnatal microglial cultures . Interestingly, the MpHE increases the phagocytic index in primary microglial cultures from early postnatal and even from 8- and 10-months-old 5XFAD mice. This effect correlated with a reduction in the M1 pro-inflammatory phenotype since treatment with MpHE clearly promoted a reduction in CD86 and TNF expression levels in both lean or obese 5XFAD mice. Thus, MpHE may alleviate the negative effect of the inflammatory pathways on the insulin receptor signaling and in this way, reverse the memory deficits observed in type II diabetes and AD.
TREM-2 is a molecule directly related to the phagocytic capacity of microglial cells  and to the reduction of the neuronal damage produced by Aβ plaques . According with the fact that TREM-2 expression is upregulated in different mice models of AD in a time-dependent manner , here, we observed that M. parviflora treatment tended to maintain high levels of TREM-2 expression in the cortex of 5XFAD mice at 9 months of age. Recently, it has been described a novel microglia subpopulation associated with neurodegenerative diseases (DAM) that is beneficial for AD . The DAM program is activated in two-step checkpoints where microglia switch from homeostatic to stage 1 DAM (TREM2 independent) and to stage 2 DAM (TREM2 dependent) at late stages of the AD (8 months). Interestingly, the TREM2-dependent stage is associated with phagocytic activity which correlates with previous observations showing that absence of TREM2 in microglia at the late stage of AD, but not at the early stages, exacerbates the disease . Whether M. parviflora accelerates the DAM phenotype (TREM2 dependent) to increase the ability of microglia to digest amyloid plaques that results in neuroprotection warrants additional investigation. Also, our results point out at CD36 as the scavenger receptor mediating microglia phagocytosis of amyloid β plaques in response to MpHE. This involves PPARγ activation, probably mediated by oleanolic acid [43, 44] present in the M. parviflora HE and the subsequent induction of CD36 expression. In agreement with previous results , microglia exposure to pioglitazone resulted in increased PPARγ protein levels. Interestingly, M. parviflora HE had the same effect, suggesting that pioglitazone and the M. parviflora HE activate PPARγ by a similar mechanism. Although PPARγ activation, resulting from pioglitazone exposure, promoted CD36 expression to a similar extent as that resulting from the MpHE treatment, the phagocytosis and clearance activity of pioglitazone-treated microglia towards amyloid β plaques was reduced when compared with that of microglia treated with the MpHE. This suggests that the MpHE through its anti-inflammatory effects might also activate autophagy, a mechanism that is also critical for amyloid β plaques clearance and impaired by the inflammatory environment elicited by amyloid β peptides .
Individuals with type II diabetes have increased risk for developing AD by at least twofold . However, the precise mechanism for this association is not known. Here, we showed that alterations in glucose metabolism (i.e., IR) resulting from obesity exacerbate the memory loss induced by Aβ accumulation. Treatment with the MpHE restores insulin sensitivity and glucose tolerance in obese mice, which correlated with a diminished inflammatory environment and increased cognition in the 5XFAD mice. Additionally, we found that MpHE regulates PPAR-γ activity and mediates microglial amyloid β plaques phagocytosis. Thus, by regulating the microglia phagocytic activity via a PPAR-γ-dependent mechanism and preventing the pro-inflammatory M1 phenotype, M. parviflora reduces β-amyloid plaque load and improves learning and memory in lean and obese mice. Additionally, we do not discard the possibility that MpHE through the activation of PPARγ, prevents IKK activity  and therefore IRS phosphorylation on serine residues, thus improving insulin signaling directly on hippocampal neurons reversing the memory deficits observed in type II diabetes and AD. Therefore, M. parviflora-derivative compounds represent an alternative to prevent cognitive impairment associated with a metabolic disorder as well as an effective prophylactic candidate for AD progression.
Peripheral inflammation resulting from obesity has been shown to exacerbate learning and memory loss in humans and in animal models of AD. Here, we show that obesity further promoted Aβ plaque formation in the 5XFAD mice. However, administration of the MpHE reversed obesity-mediated deleterious effects of Aβ deposition on memory and learning. The MpHE attenuated pro-inflammatory M1 microglia activation while sustaining anti-inflammatory M2 microglia, since CD86 and TNF expression levels observed in the cortex of 5XFAD mice fed with ND and HFD were reduced in the cortex from 5XFAD mice treated with MpHE. In parallel, MpHE tended to increase the levels of Mgl1 and from TREM2 in the cortex of lean and obese 5XFAD mice. In accordance with this, MpHE reduced the astrogliosis throughout the hippocampus of HFD-fed 5XFAD mice. In addition, MpHE increases microglia phagocytic activity via a PPAR-γ/CD36-dependent mechanism. Our data indicate that by regulating microglia phagocytic activity and by preventing the microglia pro-inflammatory M1 phenotype, M. parviflora reduces β-amyloid plaque load and improves learning and memory in lean and obese mice. Our studies also demonstrate that MpHE can prevent not only HFD-induced neuroinflammation, but also systemic inflammation since it reduces the infiltration of immune cells to the adipose tissue and delays the increase in body weight and prevents the systemic insulin resistance and glucose intolerance produced by HFD in the 5XFAD mice. Thus, by regulating the microglia phagocytic activity via a PPAR-γ/CD36-dependent mechanism and preventing the pro-inflammatory M1 phenotype, MpHE reduces the levels of pro-inflammatory molecules (e.g., TNF, IL-6), thus improving learning and memory in obese mice, which could result from restored insulin receptor signaling in hippocampal neurons. Strategies to prevent obesity-associated brain dysfunction are limited. M. parviflora-derivative compounds offer a novel adjunct therapeutic approach to ameliorate obesity-associated peripheral inflammation and neuroinflammation. Therefore, M. parviflora may be a safe and effective alternative to the conventional NSAIDs used for the treatment of neurodegenerative diseases as AD.
We thank Oswaldo López-Gutiérrez for technical support and E. Melchy for the flow cytometry analyses. The authors are also grateful to the M. Sci. Andrés Saraleguí Amaro, to Dr. Jaime Arturo Pimentel Cabrera, and to Q.F.B. Xochitl Alvarado Affantranger, at the Laboratorio Nacional de Microscopía Avanzada (UNAM), for imagen acquisition presented in this study; to S.D. Rodríguez at the Unidad de Histología (Instituto de Fisiología Celular-UNAM) for tissue histological processing; to P. Gaytan for kindly donating the fluorescent E. coli used in this study; and also to G. Cabeza, R. Blancas, and E. Mata for animal care.
This work was performed in fulfillment of the requirements for a PhD degree of Elisa Medrano Jimenez who is enrolled in the Programa de Doctorado en Ciencias Biomédicas (PDCB) at the Universidad Nacional Autónoma de México. The student received fellowship (576297) from CONACYT.
EM-J designed and performed experiments, analyzed and interpreted the data, and wrote the manuscript. IJ-FC conceived the study, performed experiments, and analyzed and interpreted the data. MP-E designed and performed experiments, analyzed and interpreted the data, carried out the statistical analyses, and wrote the manuscript. CER-S performed the in vivo dose-response experiments, analyzed and interpreted the data, and helped to draft the final version of the paper. LA-A and JC-M carried out experiments. AZ performed experiments, analyzed data, and provided infrastructure to carry out some experiments. MH-R, EJ-F, and JT analyzed data and provided infrastructure to carry out some experiments. GP-A designed, analyzed, and interpreted the data and wrote the manuscript. LP-M conceived, coordinated, and supervised the study; designed, analyzed, and interpreted the data; and wrote the manuscript. All authors read and approved the final version of this manuscript.
This work was supported by the CONACyT (grant numbers 155290 and IFC 2016-2282 to L.P.-M. and 154542 to G.P.-A.) and by the DGAPA-PAPIIT (grant numbers IN209212 and IN213316 to L.P.-M., IN227510 and IN212316 to G.P.-A., IA203416 and IA203318 to M.P.-E). We also thank the IMSS support (FIS/IMSS/PROT/G11-2/1018).
The Institutional Bioethical Committee approved all animal experiments described in this study.
Consent for publication
The authors declare that they have no competing interests.
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