Ethics statement
The animal protocol for the present study was approved by the local Institutional Animal Welfare Committee (Permission No. SUDA20211206A03). All experimental schemes were conducted in accordance with the Institutional Guidelines in the Care and Use of Experimental Animals of the Ministry of Science and Technology (Beijing, China). All experimental protocols were made to minimize the sample size and to relieve the suffering of mice.
Animals Processing
Male LDLR−/− mice and C57BL/6 wild-type mice with the same genetic background, seven-week-old and weighting 20 ± 2 g, were purchased from GemPharmatech Co., Ltd. (Nanjing, China) and raised in a barrier facility with a 12/12 h light/dark period at room temperature (22 ± 2℃). After arriving at Laboratory Animal Research Centre, the animals were acclimatized for one week with free access to sterile water and a standard laboratory diet with 10% calories from fat. Then, sixteen LDLR−/− mice were given an atherosclerotic diet with 1.25% cholesterol and 40% calories from fat (XT108C, Jiangsu Xietong Pharmaceutical Bio-engineering Co., Ltd., Nanjing, China) for an 8-week-long feeding period, and were randomly divided into two groups: a model group receiving saline solution (Mod, n = 8) and a treatment group receiving sodium butyrate (SB, n = 8). The animal model of atherosclerosis with cognitive impairment has been successfully established in our laboratory [2]. C57BL/6 mice continued to be fed with a standard laboratory diet, which served as a normal control in behavioral analysis and other examinations (NC, n = 8). Animals from the SB group were treated by gavage with 400mg/kg/d sodium butyrate (Sigma-Aldrich, St. Louis, MO, USA) five days a week according to Du’s report and our pre-experiment [21], and mice from the Mod group and NC group were treated by gavage with the same concentration of normal saline. During the eight-week feeding, all mice had free access to food and sterile water. The formulation and composition of diets were summarized in Supplementary Table S1. The time schedule for the animal experimental study was shown in Fig. 1.
Behavioral Test
After feeding for 7 weeks, a MWM test was performed for examining butyrate on cognitive behavior. During the entire experimental test, the lights were turned off to minimize the light stimuli on the activity performance of mice. Briefly, in a continuous 5-day oriented navigation test, mice were put into a circular black pool to find a hidden platform (1.0 cm below the water surface), which was fixed in one of the four quadrants during the training. The trial was terminated once the animals arrived at the hidden platform within 60 s; otherwise the mice would be artificially taken to the platform to stay for 20 seconds and then removed from the pool. The time that a mouse reached the submerged platform was recorded through a video camera to evaluate spatial learning ability. On the sixth day, the platform was taken away for the spatial probe test. The number of mice crossing the platform, the swimming time spent in the target quadrant and the swimming distance were recorded and analyzed to evaluate the spatial memory ability. Throughout the experiment, Supermaze tracking software (Shanghai Xinsoft Information Technology Co., Ltd., Shanghai, China) was performed for data collection and analysis.
Samples Collection
At the end of the experiments, blood samples from fasting mice were collected; serum was separated for the measurement of circulating LPS level. The length of colon tissue was measured using the same straightedge after dissecting the mice. Subsequently, purpose samples from sacrificed mice were immediately harvested for subsequent tests. Briefly, after washing with ice phosphate buffer saline, the parietal-temporal cortex and hippocampus from the fresh hemi-brain, and colon tissues were dissected and stored in an ultralow temperature refrigerator. The other hemispheres and a part of distal colon tissue fixed in 4% paraformaldehyde were used for immunofluorescence staining or hematoxylin and eosin (H&E) staining. Cecum content was taken in a sterile environment for further tryptophan metabolism via untargeted metabolomics analysis.
Colonic Histopathological Analyses
The distal colon tissues fixed in 4% paraformaldehyde, were dehydrated and embedded in paraffin. Then a series of slices were stained by the H&E staining method and examined under light microscopy (Olympus BX-50, Olympus Optical, Tokyo, Japan). Five discontinuous sections were randomly selected from colon tissue (n = 5 in each group) for histopathological analysis and scoring.
Serum LPS Analyses
The concentration of circulating LPS was measured by an Elisa method following the manufacturer’s guidelines (Nanjing SenBeiJia Biological Technology Co., Ltd., Nanjing, China). The coefficient of variation (CV) for intra-assay precision was less than 9%.
Measurement Of Colonic Tight Junction Proteins And Inflammatory Cytokines
Concentrations of tight junction proteins including zonula occludens-1 (ZO-1), claudin-3 and occludin, and inflammatory cytokines including tumor necrosis factor-alpha (TNF-ɑ), interleukin (IL)-1β and IL-10 from colon tissues were determined by commercial Elisa kits (Nanjing SenBeiJia Biological Technology Co., Ltd., Nanjing, China; Elabscience Biotechnology Co., Ltd., Wuhan, China). In brief, fresh colon samples were homogenized and the supernatants were separated via centrifugation at 10,000g for 15min. The protein concentration of the supernatant was quantified through the BCA protein assay (Beyotime Institute of Biotechnology, Nantong, China). An equal concentration of the supernatant was detected through the Elisa method. The coefficient of variation (CV) for intra-assay precision was less than 9%-11%.
Western Blot Analyses
First, colon tissues, isolated parietal cortex and hippocampus were cleaved in tissue lysate. Protein concentration from tissue supernatant was quantified through the BCA protein assay (Beyotime Institute of Biotechnology, Nantong, China). Second, equal protein (30 µg) was separated via sodium dodecyl sulfate-polyacrylamide gel and then transferred to a polyvinylidene difluoride membrane by electrophoretic transfer method. Third, the membrane was blocked with 5% skim milk at room temperature and then incubated overnight at 4 oC with tight junction proteins including ZO-1, claudin-3 and occludin, toll-like receptor 4 (TLR4) signaling pathway including TLR4, myeloid differentiation primary response 8 (MyD88), TNF receptor-associated factor 6 (TRAF6), TIR-domain-containing adapter-inducing interferon-β (TRIF), receptor-interacting protein (RIP) and IκB kinase β (IKKβ), NLRP3 inflammasome signal factors including nuclear factor-kappa B (NF-κB) p65, NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), cysteinyl aspartate specific proteinase-1 (Caspase-1), IL-1β and IL-18, SCFA receptors including GPR41, GRP43 and GPR109A, Aβ1–42 and p-Tau ser 396/ser 404, and neuroinflammation-related markers including glial fibrillary acidic protein (GFAP) and ionized calcium-binding adaptor molecule 1 (IBɑ1). The primary antibodies mentioned above were purchased from Abcam (Cambridge, MA, USA), ImmunoWay Biotechnology Company (Plano, TX, USA), or Cell Signaling Technology (Danvers, MA, USA). Final, membranes were incubated with secondary antibodies for 1 h at room temperature. And the protein bands were detected using an enhanced chemiluminescence ECL Detection Systems (EMD Millipore). β-actin or tubulin were used as the internal control.
Immunofluorescence Double Staining And Analyses
The paraffin-embedded brain tissues were sliced and stained via immunofluorescence double labeling of GFAP and IBɑ1 according to previous description by the authors.2 Briefly, after blocking in 3% BSA solution for 30 min, the embedded sections were incubated with a primary antibody mixture including mouse anti-GFAP (1:500, Servicebio) and rabbit anti-IBɑ1 (1:500, Wako) overnight at 4 oC. After removing excess primary antibodies with PBS solution, sections were incubated with the respective secondary antibodies (for GFAP, Streptavidin Alexa Fluor 555 conjugate, 1:1000, Invitrogen; for IBα1, Alexa Fluor 488 goat anti-rabbit, 1:500, Invitrogen) for 1 h at room temperature. The sections were counterstained with DAPI (Invitrogen) and images were obtained with Pannoramic Midi (3D Histech Ltd, Budapest, Hungary). The positive area (µm²) and average fluorescence intensity of the target area of each section were quantified with the Area Quantification FL V2.1.2 module in Halo V3.0.311.314 (Indica Labs, USA)) software.
Tryptophan Metabolism Via Untargeted Metabolomics Analyses
Approximately 48 metabolites from mice cecum content were detected and quantified via ultra-performance liquid chromatography (Agilent 1290 II, Agilent Technologies, Germany) coupled to Quadrupole-TOF mass spectrometry (5600 Triple TOF Plus, AB SCIEX, Singapore) (UPLC-MS) according to the reported reference [22]. Briefly, samples mixed with ice-cold 80% methanol, were ground using an abrader at 5000 rpm and centrifuged for 10 min at 12000 rpm. The supernatants were dried using SpeedVac (Genevac miVac, Tegent Scientific Ltd.) and the dried extracts were redissolved with 1% acetonitrile for UPLC-MS analysis. Information-dependent acquisition mode was used for MS/MS analyses of the metabolites, and data acquisition and processing were performed using Analyst® TF 1.7.1 Software (AB Sciex, Concord, ON, Canada). All detected ions were collected using MarkerView 1.3 (AB Sciex, Concord, ON, Canada) into Excel database in the format of two-dimensional matrix. This determination was performed by LipidALL Technologies Co., Ltd. (Beijing, China), and data of the secondary mass spectrometry were extracted via PeakView 2.2 software (AB Sciex, Concord, ON, Canada) and compared with HMDB, Metabolites database, METLIN and standard substance. The identified metabolomics data were analyzed using R language method.
Statistical Analyses
All data were presented as the mean ± standard error of the mean (SEM). The significance of variables among different groups was carried out with one-way ANOVA followed by a Tukey’s post hoc test. Statistical analysis was performed using SPSS 22.0 statistical software (IBM, New York, USA) and GraphPad Prism (version 6.03, GraphPad Software, San Diego, CA). A p value < 0.05 was considered at statistical significance.