2.1 Animals and Diets
Six-week-old female C57BL/6J mice, obtained from Animal experiment center of Ningxia Medical University and were placed in a polycarbonate cage with a temperature control chamber (ambient temperature 22 ± 2 ℃, air humidity 40%-70%), 12 h dark/light cycle in Animal Experiment Center of Ningxia Medical University. Lieber-DeCarli liquid feed including alcohol feed (product number: TP4030M3) and non-alcoholic feed (product number:TP4030M3C) were purchased from TROPHIC Animal Feed High-tech Co., LTD., Nantong, China. Sodium butyrate was obtained from Shanghai Beinuo Biotechnology Co., LTD (V900464-25G).
2.2 Experimental Design
After a 1-week period of acclimation of the control liquid diet, female C57BL/6J mice (n = 60, 6 weeks old) were fed the modified Lieber-DeCarli liquid diets as previously described. Briefly, mice were randomly allocated into 4 groups (15 mice/group): (a) pair-fed (PF) group (PF/CON), mice were fed ethanol (EtOH)-free modified Lieber-DeCarli liquid diets as PF control (CON); (b) alcohol-fed (AF) group (AF/CON), mice were fed EtOH-containing modified Lieber-DeCarli liquid diets as AF control; (c) PF with sodium butyrate (NaB) group (PF/NaB), mice were fed EtOH-free modified Lieber-DeCarli liquid diets containing sodium butyrate as sodium butyrate control; and (d) AF with sodium butyrate group (AF/ NaB), mice were fed EtOH-containing modified LieberDeCarli liquid diets with sodium butyrate as sodium butyrate intervention group. Mice in AF groups were fed the modified Lieber-DeCarli liquid diets containing EtOH with an energy composition of 18% protein, 19% carbohydrate, 35% fat, and 28% EtOH, whereas mice in PF groups (PF/CON and PF/NaB) received an equal amount of calories as AF groups with the alcohol-derived calories substituted with isocaloric maltose–dextrin (carbohydrate). Components of the liquid diets were shown in Table S1. Groups (a) and (c) were the pair-fed controls for groups (b) and (d), respectively. Liquid diets were freshly prepared from powder daily according to the manufacturer’s instruction. Average daily volume of liquid intake per mouse was monitored and calculated in AF groups. Mice in PF groups consume equal amounts of diets. During the study, body weight was recorded weekly. After 6 weeks of feeding, mice were sacrificed and the associated indicators were investigated.
2.3 Behavioral analysis
2.3.1 New object recognition (NOR)
NOR experiments were used to evaluate the ability of mice to recognize new objects in the environment. First, mice were habituated in an empty white chamber box for 5 min. After 10 min, each mouse was returned to the same chamber with two identical objects placed in the corners and allowed to freely explore for 5 min. The time spent exploring each object was recorded. Exploration was defined as reaching the object, sniffing it from a distance of < 2 cm, and/or touching it with the nose. After 5 min, one object was replaced with a novel one, the mice were allowed to explore for another 5 min. The time spent by the mice exploring the novel and old objects was recorded and was used to calculate a recognition index as follows: (time novel) / (time novel + time old).
2.3.2 Morris water maze (MWM)
Spatial learning and memory were analyzed using the MWM task. The MWM was divided into four equal quadrants, and a platform was hidden 1 cm below the surface of the water. One day before starting training, for acclimation, the animals were made to swim freely in the swimming pool for 60 s without an escape platform. The training session consisted of 6 days, four trials per day, and the time spent for finding the hidden escape platform was recorded as escape latency for each mouse. If mice could not find the escape platform within 60 s, they were directed to land on the platform and allowed them to stay for 20 s. To test how well the animals had learned the position of the escape platform, we gave a probe trial at the end of training day 6 and mice were allowed to swim freely for 60 s with no escape platform. Video tracking was used to automatically measure how well the mice remembered the previous location of the escape platform.
2.3.3 Y- maze
Y-maze apparatus was used to assess spatial working memory. Y-maze consists of three long, wide, and high arms of 34, 10, and 10 cm, which are the starting arm, novel arm, and known arm, respectively. The experimental animals were placed in the test room to adapt to the environment 10 min before the experiment. During the first stage, the novel arm was blocked by a white baffle, allowing mice to only move freely in the other two arms for 5 min, and the mouse movement track and time were recorded in the starting arm and known arms. Then the mice were placed in the starting arm, the novel arm was opened, the exploration was performed for 5 min, and the mouse movement track and time were still recorded. Memory performance was given by the percentage of time spent in the novel arm over the time spent exploring all arms.
2.3.4 Shuttle Box
Shuttle box, consisted of two equal-sized compartments with divided with a central inverted U-shaped gate, was used to evaluate the learning and memory ability of rodents. Each testing session began with acclimatization to the chambers for 2 min, followed by 30 trails with an inter-trial interval of 30 s. A tone (60 dB) and light (8 W) were co-administered as conditioned stimuli for 10 s in each assay. An electrical foot shock (0.3 mA) was used as the unconditioned stimulus and administered for 5 s following the presentation of the conditioned stimuli. The procedure was performed for a total of 5 days and the active evasions were monitored continuously.
2.3.5 Open field test (OFT)
The OFT was performed to evaluate anxiety and locomotor activity in mice. Mice were moved into the behavioral test room at least 1 h before the experiment. The test consisted of 10 min trial in a white opaque 40, 40, and 30 cm arena and the center zone defined as a square covering 25% of the total area and allowed to explore it freely for 10 min. Their ambulatory and rearing activities over the last 5 min were recorded using an automatic video tracking system (Smart version 3.0; Panlab, S.L.U., Barcelona, Spain). Moreover, the area was cleaned with 75% ethanol and allowed to dry completely between each test. The total travelled distance was considered as an index of locomotor activity. Rearing and defecation were considered indices for ‘anxiety levels’, while increased proportion of distance or time spent in the center indicated decreased anxiety.
2.3.6 Forced swim test (FST)
The FST was used to assess depressive behavior in rodents. Briefly, mice were placed into plastic buckets with 19 cm in diameter and 23 cm deep, filled with water at 25°C. The duration of mobility and immobility phases was recorded using an automatic video tracking system (Smart version 3.0; Panlab, S.L.U., Barcelona, Spain) within 5 min.
2.4 Hematoxylin and eosin staining (HE staining)
After the mice were anaesthetized, they were perfused intracardially with PBS and then fixed with 4% paraformaldehyde. Then, the brain and liver were extracted and fixed in 4% formaldehyde solution again for 24 h, washed with running water, dehydrated in ethanol, and paraffin-embedded, then sliced (5 µm thickness) and placed onto glass slides. HE staining of the brain tissues was conducted according to standard protocols. Briefly, after deparaffinization with xylene and rehydration with graded alcohol solutions, tissue sections were stained with hematoxylin solution (ZSGB-BIO, China) for 10–15 min followed by 2 dips in 1% acid ethanol (1% HCl in 75% ethanol) and rinsed in distilled water for 5 min. Then the sections were stained with eosin solution (ZSGB-BIO, China) for 3 min and rinsed again in distilled water for 3 min. Ultimately, dehydration with graded alcohol solutions and clearing with xylene, the slides were sealed by neutral balata. The mounted and dried slides were then examined and photographed using an Olympus microscope (Aomori Olympus, BX51, Japan).
2.5 Nissl staining
Nissl staining was performed to analyze the degree of morphological changes in chronic alcohol exposed group and chronic alcohol exposed plus sodium butyrate -treated group. Sections (5 µm) were cut from each block on coated slides and dried overnight at 37°C. After that, tissue sections were deparaffinized with xylene followed by graded rehydration in ethanol (100%, 95%, 80%, and 70%) and distilled water. Then, sections were stained for 1h in the 0.5% cresyl violet solution (Nissl staining kit, Solarbio Technology, Beijing) at 60°C and washed with distilled water. Next, hydrochloric acid and ethanol were used for differentiation. The differentiation was controlled under endoscopy and terminated when the background was clean and Nissl bodies were clearly visible. Finally, the sections were dehydrated, cleared in xylene, and used neutral resin to seal the slices. The morphological changes of neurons in various hippocampal regions (CA1, CA3, and DG) and prefrontal cortex areas were observed under a microscope (Bar = 20 µm).
2.6 Immunohistochemistry staining (IHC staining)
IHC staining was performed using 5 µm paraffin-embedded brain tissue sections. The slides were dewaxed and rehydrated. Subsequently, the slides were washed 3 times with PBS, antigen retrieval was performed with sodium citrate in microwave oven at 100°C for 17 min, blocked in 20% normal goat serum, and incubated with rabbit antibody against Ibal (1:8000; ab178847, Abcam, MA, USA) at 4°C overnight in a humidified container. After washes with PBS, the slides were incubated with horseradish peroxidase (HRP) -conjugated goat anti-rabbit immunoglobulin-G (IgG) secondary antibody (1:1000 dilution, Abbkine, No. A21020, China) for 30 min at 37°C. After 4 min of reaction with substrate-chromogen 3, 3′-diaminobenzidine, the slides were counterstained with hematoxylin after the IHC protocol. Images were captured with a microscope. Positive areas in 40 × optical fields of the prefrontal cortex, hippocampal DG, CA1, and CA3 regions. Quantification was done using Image J software.
2.7 Immunofluorescence staining (IF staining)
IF staining was performed at room temperature. In brief, mice were euthanized and perfused with PBS (0.01 M, pH 7.40, 4°C) transcardially first and then perfused with 4% paraformaldehyde (PFA) solution (pH 7.40, 4°C). The cerebrums were dissected before soaked in the same PFA solution for fixation (24 h, 4°C). The samples were transferred to a 30% sucrose solution for dehydration (48 h, 4°C). After that, the brain samples were imbedded and frozen (− 80°C), and then sliced into coronal slices (20 µm thick) which were mounted on glass slides. Before staining, the slides were washed with PBS and processed with a mixture of Triton X-100 (0.3%) and 3% BSA (Sigma A7906) for 1 h. Next, the slides were incubated with primary antibodies overnight (24 h, 4°C). Then, the corresponding secondary antibodies were added and allowed to co-incubation (1 h, 24°C). Afterwards, the slides were washed and sealed with a DAPI reagent (Thermo Fisher, MA, USA) and subsequently examined under a fluorescence microscope (Leica, Germany). Primary antibodies used were anti-Iba-1 (1:100; ab-178847, Abcam, MA, USA), anti-Arg1 (1:100; DF6657, Affinity, MA, USA) and anti-NOS2 (1:100; sc-7271, SANTA CRUZ, MA, USA). Secondary antibodies used were anti-rabbit (1:100; ab150077, Abcam, MA, USA) and anti-mouse (1:100; ab150117, Abcam, MA, USA).
2.8 Isolation of brain microglial cells
Microglial cells, major inflammatory innate immune cells in the CNS, were isolated by collagenase digested from the mice brain tissues as previously described. Briefly, brain tissue was minced with scalpels prior to digestion with 10ml of 0.25% (weight by volume) collagenase IV (Sigma, San Francisco, USA) and 20µL DNase Ⅰ (28 U/mL Sigma, D5025, San Francisco, CA, USA) for 20 min while rotating at 37°C. Subsequently, the digested tissues were collected, mashed, and filtered through a 200-mesh nylon membrane (Sigma-Aldrich, S3895, Oakville, ON, Canada). The homogenate was centrifuged for 7 min at 421 × g, and then the supernatant was carefully poured off and discarded. Myelin was removed by centrifugation of homogenates resuspended with 37% Percoll (Solarbio Technology, Beijing, China) for 10 minutes at 500 × g, without brakes. The myelin layer and the supernatant were aspirated and the pellet was kept. Following this, the cells were washed with 1× Hank’s Balanced Salt Solution (HBSS) and centrifuged for 10 min at 550 × g. The resulting cell pellet was washed twice with Dulbecco modified Eagle medium (DMEM) before resuspension. Finally, cells were then resuspended at a concentration of 1 × 107 per ml and subjected to staining for flow cytometry.
2.9 Flow cytometry analysis
All flow cytometry staining steps were performed at 4°C in the dark. To prevent non-specific binding, cells were incubated for 20 min with 1 µL CD16/CD32 (No. 214228, BD Biosciences, USA) to block Fc receptors. Subsequently, 100 µL of suspended cells were stained with PE-conjugated anti-mouse CD45 antibody and APC-conjugated anti-mouse CD11b antibody (No. 553081, BD Biosciences, No. 553312, USA) for 30 min. Meanwhile, cells were stained with corresponding isotype control antibodies. Finally, the prepared samples were analyzed using Cyto FLEX flow cytometer (Beckman Coulter, USA).
2.10 Enzyme-linked immunosorbent assay (ELISA)
TNF-α, IL-1β, IL-6, IL-18 and IL-10 were measured by ELISA (R&D Systems, Minneapolis, MN) in the hippocampus and prefrontal cortex. Briefly, isolation of hippocampus and prefrontal cortex from fresh mice brain tissue rinsed with cold saline. Then, they were weighed, and homogenized on PBS. To obtain the cytokine supernatant, the homogenates were centrifuged for 5 min at 10,000 × g. Cytokine levels in the hippocampus or prefrontal cortex were determined by ELISA with commercial reagent kits following the manufacturer’s instruction (Cloud-Clone Corp, China). The absorbance at 450 nm was determined using a microplate reader (Varioskan LUX, Thermo Fisher Scientific, USA), and the OD value was recorded for further evaluation.
2.11 Reverse transcription‑quantitative (RT‑q) PCR
Total RNA was extracted from the hippocampus and cortical tissue using an RNA simple Total RNA kit (Servicebio, Wuhan, China), and the SweScript RT I First Strand cDNA Synthesis Kit (Servicebio, Wuhan, China) was used to synthesize cDNA.Then, RT-qPCR was performed using the SYBR Green RT-qPCR system (SYBR-Green; Servicebio, Wuhan, China) according the manufacturer's protocol. The sequences for the primers were as follows:
NF-κB p65-F: 5′-CGAGTCTCCATGCAGCTACG-3′,
NF-κB p65-R: 5′-TTTCGGGTAGGCACAGCAATA-3′;
All primers were purchased from Servicebio. For relative quantification, the reaction conditions were in accordance with the manufacturer's instructions: initial denaturation at 95°C for 3 min, followed by 40 cycles of denaturation at 95°C for 10 sec, annealing at 60°C for 40 sec and extension at 72°C for 30 sec; iQ™5 software (Bio-Rad Laboratories, Inc.) was used to conduct PCR amplification. The RNA expression levels were evaluated using the 2−ΔΔCq method. PRISM 8 soft-ware (GraphPad Software, Inc.) was used to evaluate the relative RNA level. All measurements were carried out 3 times (three independent experiments).
2.12 Western Blot
Mice hippocampus and cortical tissue were lysed by RIPA buffer (125 mM NaCl, 25 mM TRIS-Cl pH 7.4, 1 mM EGTA-TRIS pH 7.4, 1% Triton-X100, 0.5% sodium deoxycholate, 0.1% SDS and Complete EDTA-free protease inhibitor mixture (Roche Applied Science)) on ice for 30 min and blow and beat the spearhead every 10 min during this period. The protein concentrations were determined by BCA Protein Assay Kit (Pierce) and denatured by boiling for 5 min. Equal amounts of protein (40‑60 µg per lane) were separated by 10% SDS‑PAGE and gels were transferred onto 0.45 µm nitrocellulose membranes (Bio-Rad, Richmond, CA) under wet transfer conditions using 1× Transfer Blotting Buffer (Boston Bio Products) with 30% methanol for 40 min at a constant 220 MA. After transfer, the membrane was blocked with 0.1% BSA in TBST for 1 h and then probed with the rabbit monoclonal PPAR-γ antibody (1:1000, ab272718, Abcam), the mouse monoclonal NF-κB p65 (1:1000, sc-8008, Santa Cruz), the mouse monoclonal GPR109A (1:1000, sc-377292, Santa Cruz) and mouse monoclonal β-Actin (1:1000, ab8226, Abcam) in the blocking solution (PBS at 0.1 M with 5% w/v non-fat dry milk) overnight at 4°C. After three washes with PBS/0.05%-Tween 20, the membrane was incubated for 1.5 h in the blocking solution at room temperature with the goat anti‑mouse IgG 800RD (1:5000, cat. No. 926‑32210, BD Biosciences) or goat anti‑rabbit IgG 680RD (1:5000, cat. No. 925‑68071, BD Biosciences). Finally, Odyssey CLX software (9141‑00; BD Biosciences) was used for imaging and the lanes were analyzed by Image J.
2.13 Statistical analysis
GraphPad Prism version 8 (GraphPad Software Inc., La Jolla, CA, USA) and the statistical package for the social sciences (SPSS) 23.0 software (IBM Inc., Armonk, NY, USA) were used for statistical analyses. All data were checked for normality and homogeneity of variance using the Shapiro-Wilk and Levene tests, respectively. Data were expressed as mean ± standard error. The differences among multiple comparisons were analyzed using two-way analysis of variance followed by the Tukey multiple comparison test. Difference between the two groups was assessed using student’s t test (two-tailed). Body weight, MWM test and shuttle-box test recorded consecutively were analyzed using repeated measure ANOVA following Tukey’s multiple comparisons test. For data analysis that did not meet the normality and homogeneity of variance tests, nonparametric tests were used. P < 0.05 was considered a statistically significant difference.