Sixty C57Bl/6 J male mice (11 weeks old) were obtained from the Experimental Animal Center of Xuzhou Medical University (Xuzhou, China, SCXK (Su)20150009), and housed in environmentally controlled conditions (temperature 22 °C, 12 h light/dark cycle). After a 1week acclimatization period, mice were used for experiments in accordance with the Chinese Council on Animal Care Guidelines and approved by the Institutional Animal Care Committee of Xuzhou Medical University.
Microbiota-accessible carbohydrate rich diet experiment and an antibiotic cocktail administration
The mice were randomly divided into three groups (n = 15): (1) the control group (Con) was fed with a grain-based rodent lab chow (LabDiet 5010, 50 g/kg from fat, (5% fat by weight); plant polysaccharide-rich ranging a diverse source of plants including corn, soybean, wheat, oats, alfalfa, and beet, (15% neutral detergent fiber by weight)); (2) the HF-FD group was fed with a diet with high-fat (315 g/kg from fat: soybean oil 55 g and lard 260 g, (31.5% fat by weight)) and fiber-deficient (50 g/kg cellulose, low accessibility by gut microbiota, (5% fiber by weight)); (3) the group was fed with a diet rich in MACs based on the HF-FD (HF-MAC): mixed with 316 g/kg from fat (soybean oil 56 g and lard 260 g, (31.6% fat by weight)), and LabDiet 5010 powder 634 g/kg (obtained from plant polysaccharide-rich ranging a diverse source of plants including corn, soybean, wheat, oats, alfalfa, and beet, 10% neutral detergent fiber by weight) as previously described  (diet details are outlined in Table S1). In addition, another two groups (n = 12) paralleled with HF-FD group and HF-MAC group were given a cocktail of antibiotics (HF-FD + AB, HF-MAC + AB) in the drinking water to investigate the role of gut microbiota in MAC intervention. Two days prior to HF-FD diet initiation, the HF-FD + AB and HF-MAC + AB mice were given drinking water containing ampicillin (1 g/L), vancomycin (0.25 g/L), neomycin (1 g/L), and metronidazole (1 g/L), which was prepared fresh every 3 days . The HF-FD diet group showed increased body weight compared to the control group from week 4 onwards, in addition to exhibiting a significantly higher final body weight following completion of the 15-week diet (32.6%, p < 0.01). The supplementation of MACs in the HF-FD diet suppressed body weight gain and exhibited a lower final body weight than HF-FD mice (− 8.13%, p < 0.05); however, they were still significantly higher than that of the control group (18.9%, p < 0.05, Fig. S1A). Furthermore, fat accumulation (Fig. S1B) and liver weight (Fig. S1C) were significantly increased in the HF-FD mice compared to control mice. In addition, they displayed adipocyte hypertrophy (Fig. S1D), crown-like structures (CLS) in which macrophages surround dead adipocytes (Fig. S1E), signs of hepatic steatosis (ballooning and steatosis) (Fig. S1F and G), altered glucose metabolism, including hyperinsulinemia, increased homeostatic model assessment-insulin resistance (HOMR-IR), and glucose tolerance (Fig. S1H-K). MAC supplementation to some degree attenuated these metabolic disturbances. All the groups were given intervention for 15 weeks followed by two cognitive behavior tests (described below).three days later, the mice were sacrificed by CO2 asphyxiation and blood, cecum content, colon, and brain tissue were collected for further examination.
The nesting behavior and temporal order memory tests were performed as previously described [26, 27], to evaluate spontaneous rodent behavior and recognition memory, respectively. In brief, during the nesting behavior test, the deacon nest score and untorn nestlet weight were used to evaluate the activities of daily living typically altered in patients with cognitive impairment.
The temporal order memory test comprised two sample trials and one test trial with an inter-trial interval of 60 min between each trial. In each sample trial, the mice were allowed to explore two copies of the same object for 4 min; however, the objects were different between the two sample trials (sample trial 1: object A and A’; sample trial 2: object B and B’). During the test trial, one object from sample trial 1 (A; old familiar) and another object from sample trial 2 (B; recent familiar) were presented, and the animals were allowed to explore the open field for 3 min. A discrimination ratio was calculated by using the formula [(old familiar time − recent familiar time)/total exploration time]. Intact object recognition memory for temporal order was considered if the mice spent more time exploring the old familiar object compared with the recent familiar object.
Microbial DNA extraction, PCR amplification, and Miseq sequencing in cecal contents
Genomic DNA amplification and sequencing were conducted as our previous study . Briefly, microbial DNA was extracted from the cecal contents of mice using the E.Z.N.A. stool DNA Kit (Omega Bio-tek, Norcross, GA, U.S.) according to manufacturer’s protocols. The 16S rDNA V3-V4 region of the eukaryotic ribosomal RNA gene was amplified by PCR (95 °C for 2 min, followed by 27 cycles at 98 °C for 10 s, 62 °C for 30 s, and 68 °C for 30 s and a final extension at 68 °C for 10 min) using primers 341F:CCTACGGGNGGCWGCAG; 806R:GGACTACHVGGGTATCTAAT, where the barcode is an eight-base sequence unique to each sample. PCR reactions were performed in triplicate, with a 50 μL mixture containing 5 μL of 10 × KOD buffer, 5 μL of 2.5 mM dNTPs, 1.5 μL of each primer (5 μM), 1 μL of KOD polymerase, and 100 ng of template DNA. Amplicons were extracted from 2% agarose gels, purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA), and quantified using QuantiFluor-ST (Promega, USA). Purified amplicons were pooled in equimolar and paired-end sequenced (2 × 250) on an Illumina platform according to the standard protocols.
Bacterial quantification in feces
For quantification of total fecal bacterial load, total DNA was isolated from known amounts of feces using the QIAamp DNA Stool Mini Kit (Qiagen). DNA was then subjected to quantitative PCR using the QuantiFast SYBR Green PCR Kit (Biorad) with universal 16S rRNA primers (5′-AGAGTTTGATCCTGGCTCAG-3′ and 5′-CTGCTGCCTCCCGTAGGAGT-3′) to measure total bacteria number. Results are expressed as bacteria number per mg of stool, using a standard curve.
Fecal albumin ELISA
Fecal pellets were collected prior to the behavioral tests, snap frozen in liquid nitrogen, and stored at − 80 °C. Pellets were resuspended at 10 mg/ml in sterile phosphate buffered saline (PBS) and the concentration of albumin determined by ELISA in accordance with manufacturer’s instructions (Elabscience, Cat.E-EL-M0656c, China).
Measurement of serum cytokines and short-chain fatty acids (SCFAs)
Serum TNF-α, interleukin-6 (IL-6), and interleukin-1β (IL-1β) levels were measured using ELISA kits and performed according to the manufacturer’s instructions (Thermo Fisher, USA). GC–MS analysis of short-chain fatty acid composition in the serum was performed as previously described . Briefly, separation was performed on an Agilent HP-INNOWAX capillary column (30 m × 0.25 mm × 0.25 μm). The column temperature was held at 90 °C for 1 min, increased to 120 °C at 10 °C/min, held for 8 min, then increased to 150 °C at 5 °C/min, before being increased to 250 °C at 25 °C/min, and held for 2 min. The injection volume was 1.0 μL with a split ratio 10:1. The carrier gas was high-purity helium with a flow rate of 1.0 mL/min. The mass spectrometer was operated in electron impact mode (70 eV) at 0.2 s/scan and recorded over the mass range of m/z 50–500, a solvent delay time of 2 min. The inlet, interface, and ionization source temperatures were 250 °C, 230 °C, and 250 °C, respectively.
Lipopolysaccharide (LPS) determination
The concentration of circulating serum LPS was measured by ELISA (Limulus assay kit, Cat.18110115, China). All samples for LPS measurements were performed in duplicate.
Intraperitoneal glucose tolerance test (IPGTT)
The IPGTT was conducted as previously described by our laboratory . Briefly, mice were fasted overnight followed by an intraperitoneal injection of glucose (2 g/kg). Blood samples were obtained from the tail vein at 0, 30, 60, 90, and 120 min following the injection of glucose. Blood glucose levels were measured with glucose meter. Total area under the curve (AUC) was calculated using the trapezoidal method.
Measurement of homeostatic model assessment-insulin resistance index
Fasting blood glucose was measured using glucometer strips (Roche, Germany). Fasting insulin was measured using a commercial ELISA kit (Crystal Chem, USA). Homeostatic model assessment-insulin resistance (HOMA-IR) was calculated using the following equation: fasting insulin (mU·L − 1) × fasting blood glucose (mmol·L − 1)/22.5 .
Thickness measurements of the colonic mucus layer
Post Carnoy’s fixation, methanol-stored colon samples were embedded in paraffin, cut into thin sections (5 μm), and mounted on glass slides. Alcian blue staining was performed as previously described , and the thickness of the colonic sections was measured (10 measurements per section/2 sections per animal/5 animals per group) using ImageJ after cross-validation using anti-MUC2 staining.
Bacteria localization by fluorescence in situ hybridization (FISH)
The staining of bacteria localization at the surface of the intestinal mucosa was conducted by FISH as previously described . Briefly, transverse colonic tissues full of fecal material were placed in methanol-Carnoy’s fixative solution (60% methanol, 30% chloroform, 10% glacial acetic acid) for a minimum of 3 h at room temperature. Tissues were then washed in methanol (2 × 30 min), ethanol (2 × 20 min), and xylene (2 × 20 min). Paraffin-embedded tissue was cut into sections (5 μm). The tissue sections were dewaxed by preheating at 60 °C for 10 min, followed by incubation in xylene (2 × 20 min) and 100% ethanol for 10 min. Deparaffinized sections were incubated at 37 °C overnight with EUB338 probe (5′-GCTGCCTCCCGTAGGAGT-3′) diluted to 10 μg/mL in hybridization buffer (20 mM Tris–HCl, pH 7.4, 0.9 M NaCl, 0.1% SDS, 20% formamide). Subsequently, sections were incubated with wash buffer (20 mM Tris–HCl, pH 7.4, 0.9 M NaCl) for 10 min and PBS for 3 × 10 min, before mounted in DAPI containing mounting medium.
Immunohistochemistry and analysis
Mucin 2 (MUC2) localization in the colon was detected by staining the colonic tissue sections (5 μm) with anti-MUC2 antibody (Abclonal, A14659) diluted 1:500 in TBS, followed by incubation with goat-anti-rabbit Alexa-488 conjugated antibody (1:1000) (Invitrogen, A32731) in TBS. Frozen brain tissue (hippocampus) was cut in 20 μm sections using a cryostat from bregma − 3.3 mm to − 4.16 mm according to a standard mouse brain atlas . The brain slices were incubated with 10% goat normal serum for 15 min at room temperature, followed by incubation with the primary antibodies, anti-Iba1 (Wako, 019–19,741), anti-CD68 (BIO-RAD, MCA1957T), and anti-GFAP (abcam, ab7206) at 4 °C overnight. Following primary antibody incubation, sections were washed with PBS and incubated with either Alexa Fluor®-594 (abcam, ab150160) or Alexa Fluor®-488 (abcam, ab150077) at 37 °C for 1 h. Finally, the sections were counterstained with DAPI (Sigma, D9542) and then imaged with microscope (OLYMPUS IX51). Quantification of positively stained cells in the CA1, CA3, and DG regions were counted using ImageJ. Images obtained from the CA1, CA3, and DG regions were thresholded and processed by a cleaning algorithm including size-based particle exclusion and manual pruning of overlapping cell profiles. For each subsequent profile, the morphological parameters of area and perimeter were calculated. Circularity was calculated by the following formula: 4π × (area/perimeter2). Ramification index of Iba1+ and GFAP+ cells were quantified by grid-cross analysis using the ImageJ 1.46r (http://imagej.nih.gov/ij/download.html) [33, 34]. Briefly, a grid was overlaid by images of the CA1, CA3, and DG regions. The number of grid-crossing points per individual cell was counted, and the mean number of grid-crossed points per cell was calculated.
Histological analysis and morphometry
Epididymal adipose tissue was fixed in 10% buffered formaldehyde and embedded in paraffin. Tissue sections (5 μm) were stained with hematoxylin and eosin and imaged at × 100 magnification under OLYMPUS microscope (BX51, Japan). Using the software ImageJ 1.46r, 2 fields per section and 6 sections per fat mass were analyzed to quantify the number of crown-like structures (CLS) consisting of dead adipocytes surrounded by macrophages.
To determine the degree of liver damage, fresh frozen liver sections (10 μm) were stained with hematoxylin and eosin. Three fields per section in 3 sections of each mouse were viewed under an OLYMPUS microscope (BX51, Japan) and images were captured. The histological parameters of steatosis and ballooning were scored as previously described . The steatosis grades were defined as follows: 0 (< 5%), 1 (5–33%), 2 (> 33–66%), and 3 (> 66%). The ballooning classifications were grouped as 0, no ballooning cells; 1, few ballooning cells; 2, many cells/prominent ballooning.
Total RNA was extracted from homogenized tissues in Trizol (Thermo Fisher Scientific, Waltham, MA, USA) under sterile conditions. Purified RNA (1 μg) was used for RT-PCR to generate cDNA with a High-Capacity cDNA Reverse Transcription Kit (Takara, Dalian, China) The resulting cDNA was used for quantitative PCR in a real-time PCR detection system (Bio-Rad, Hercules, CA, USA). The relative mRNA expression level was determined with the 2-ΔΔCt method with GAPDH as the internal reference control. Primer sequences were as follows: mTNFα--forward (F): CTTGTTGCCTCCTCTTTTGCTTA, mTNFα--reverse (R): CTTTATTTCTCTCAATGACCCGTAG, mIL-1β--forward (F): TGGGAAACAACAGTGGTCAGG, mIL-1β--reverse (R): CTGCTCATTCACGAAAAGGGA, mIL-6--forward (F): TCACAGAAGGAGTGGCTAAGGACC, mIL-6--reverse (R): ACGCACTAGGTTTGCCGAGTAGAT, mReg3γ--forward (F): 5′TTCCTGTCCTCCATGATCAAA-3′, mReg3γ--reverse (R):5′CATCCACCTCTGTTGGGTTC-3. mGAPDH--forward (F): AGAAGGTGGTGAAGCAGGCATC, mGAPDH--reverse (R): CGAAGGTGGAAGAGTGGGAGTTG. mCD68--forward (F): TCACCTTGACCTGCTCTCTCTAA, mCD68--reverse (R): GCTGGTAGGTTGATTGTCGTCTG.
Mouse colon and hippocampal tissue were homogenized in ice-cold RIPA lysis buffer, supplemented with complete EDTA-free protease inhibitor cocktail and PhosSTOP phosphatase inhibitor. The homogenate was sonicated 6 times for 4 s with 6-second interval on ice and then centrifuged at 12,000 g for 20 min at 4 °C. The supernatant was collected, and the protein concentration was quantitated using a BCA assay. Equal amounts of protein were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes. Subsequently, membranes were blocked with 5% non-fat milk at room temperature for 1 h and then incubated with the primary antibodies at 4 °C overnight. Primary antibodies included anti-Occludin (Abcam, ab167161), anti-ZO1 (Abcam, ab96587), anti-p-IRS-1 (Ser307) (CST, 2381 T), anti-IRS-1 (CST, 2382S), anti-Iba1 (Wako, 019–19,741), anti-GFAP (Abcam, ab7206), anti-p-GSK-3β(Ser9) (CST, 9322S), anti-GSK-3-β (CST, 12456 T), anti-p-AKT (Ser473) (CST, 4060 T), anti-AKT (CST, 4691 T), anti-Tau5 (Abcam, ab80579), anti-p-Tau (S202 + T205) (Abcam, ab80579), anti-Synaptophysin (Abcam, ab32127), anti-PSD95 (CST, 3450), anti-Synapsin I (Abcam, ab18814), anti-PTP1B (Abcam, ab189179), GAPDH (ABclonal, AC033), and β-Actin (ABclonal, AC026). Following primary antibody incubation, membranes were washed in TBST and incubated with either HRP-linked anti-rabbit IgG secondary antibody (CST, 7074) or HRP-linked anti-mouse IgG secondary antibody (CST, 7076S) at room temperature for 1 h. Protein bands were detected with Clarity™ ECL Western Blot substrate (Bio-Rad, 1,705,060) and visualized using ChemiDoc Touch imaging system (Bio-Rad).
Transmission electron microscope (TEM)
Mice were sacrificed by CO2 asphyxiation and then transcardially perfused with 4% paraformaldehyde. The hippocampal CA1 region was collected and rapidly fixed in a solution composed of 4% paraformaldehyde and 2.5% glutaraldehyde, following a wash with 0.1 M phosphate buffer solution (PBS, pH 7.4) and postfixed with 1% osmic acid for 2 h. Subsequently, the tissue was washed with double distilled water and dehydrated with an ethanol and acetone gradient. Samples were embedded with different concentrations of epoxy resin and polymerized at 37 °C for 24 h, followed by 45 °C for 24 h, and 60 °C for 24 h. The samples were cut into ultrathin sections (70 nm) and stained with uranyl acetate and lead citrate. Two grids per specimen and 10 photographs per grid were randomly taken of the synaptic terminals and viewed on a transmission electron microscope (TEM) (FEI Tecnai G2 Spirit TWIN, America) to estimate synaptic morphometry. Gray type I synapses (asymmetric synapses considered to mediate excitatory transmission) were identified in the micrographs by the presence of synaptic vesicles (SVs) and dense material in postsynaptic axon terminal. The postsynaptic density (PSD) thickness was evaluated as the length of a perpendicular line traced from the postsynaptic membrane to the most convex part of the synaptic complex. The widths of the synaptic clefts (SCs) were estimated by measuring the widest and narrowest portions of the synapse and then averaging these values.
Data was analyzed using the statistical package SPSS (version 20, IBM Corporation, Chicago, IL, USA). Data was firstly tested for normality before differences among the control and HF-FD and HF-MAC groups were assessed using one-way analysis of variance (ANOVA), followed by a post hoc Tukey’s Honest Significant Difference (HSD) test or Kruskal-Wallis test for multiple comparisons among the groups. p < 0.05 was considered to be statistically significant. For 16S rRNA gene sequence analysis, all reads were deposited and grouped into operational taxonomic units (OTU) at a sequence identity of 97% , and the taxonomic affiliation of the OTUs was determined with quantitative insights into microbial ecology (QIIME, version 1.8.0) against the Greengenes database (version 13.8) . Based on Kyoto Encyclopedia of Genes and Genomes (KEGG) functional pathway, the predicted functional composition of the intestinal microbiome was inferred for each sample using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) . Statistical analyses were conducted with STAMP , and functional differences in orthologs among groups were assessed by a one-way ANOVA followed by post hoc Tukey-Kramer or Kruskal-Wallis test for multiple comparisons.