In this study, 6 weeks-old male C57BL/6J mice, weighing 18–22 g, were obtained from Jiesijie (Shanghai, China). All mice were housed in a room under a 12 h regular light/dark cycle at an ambient temperature 21°C. The animal studies were approved by the Ethics Committee of China and conducted in accordance with ethical standards.

Twenty-five male C57BL/6 mice, aged 6–8 weeks, were fed with water solution and alcohol solution in double bottles. The concentration of alcohol solution was 3%, 6%, and 10% (v/v), and the concentration gradient of alcohol was increased every 4 days to train the alcohol adaptation ability of mice. After 8 days, the alcohol solution concentration was increased to 10% and feeding was continued for another 16 days. After the end of the addiction cycle, the alcohol solution was withdrawn for 24 h, that is, alcohol withdrawal. Five mice in the control group drank water solution, and the other conditions were the same as those in the experimental group. Change the position of the water bottle every day to avoid position preference.

The donor mice (control group, n = 5) was placed in a sterile stool collection box, and immediately after the stool was discharged, the feces were collected with sterilized forceps into a sterile centrifuge tube in an ice box at 4°C. The donor feces were weighed and immersed in sterile PBS for 15 min. The feces were fully shaken into suspension by oscillator, centrifuged at 800 r/min for 5 min at 4 ° C. Transplantation was performed with the supernatant.

Open field test (OFT) is commonly used to measure anxiety-like behavior and motor activity in mice (Resmim et al., 2023). Due to the fear of the open field, mice move less in the central area and mainly move in the peripheral area. However, the exploration characteristics of mice lead to their motivation to move in the central area. The reduction of movement time and distance in the central area indicates that the anxiety of mice increases, which can be observed. The mice were placed in an open area (50 cm × 50 cm × 40 cm, length × width × height) for 1 min to acclimate to the environment and then freely explore for 5 min. A video camera was placed above the center of the open field chamber, and the movement of each mouse was recorded by an automatic video tracking system, which was converted by the video analysis system of animal behavior into the distance and time of movement in the central area, the total distance of movement, and the speed, etc., which were used as dependent variables. During the test, 75% ethanol was used to clean the open field for each new mouse to avoid residual odor information affecting the results of the next mouse. Each mouse was tested three times, the movement time and movement distance were averaged.

The light-dark transition test (LDT) is also a behavioral test to measure anxiety-like behaviors in mice (Rosa-Goncalves et al., 2022). The two sides of the light and dark chambers were equal sized compartments (20 cm × 20 cm × 25 cm, length × width × height), one half of which was light and the other half was dark, and the middle was connected by small holes to allow the mice to walk freely. Rodents have a propensity for darkening and spontaneous exploration of new environments, and the exploratory nature of mice drives them to attempt open-box activity. However, anxious mice quickly moved to the dark chamber in response to the bright light stimulus and rarely left the dark chamber. Therefore, an increase in the time spent in the dark box indicates an increase in anxiety, which can be used to observe the anxiety of the mice. During the experiment, the mice were placed in an open box, acclimated to the environment for 1 min, and then explored freely for 5 min. A video system was installed above the light and dark box, and the retention time and activity of mice in the light and dark box were observed and recorded by an automatic video tracking system. During the test, 75% ethanol was used to clean the open field for each new mouse to avoid residual odor information affecting the results of the next mouse. Each mouse was tested three times, the time spent in the dark box was averaged.

According to the manufacture’s instructions (Elabscience, Wuhan, China), the concentration of 5-HT, LPS, FABP2, TNF-α, IL-4 and IL-18 were determined by ELISA assays. A triplicate of each sample (100 μL) was added to enzyme-linked immunosorbent assays (ELISA) plates. The working curve was established using a reference standard, reaction reagents are added sequentially according to the instructions. Inflammatory cytokine levels were determined by 450 nm absorbance measurements on a microplate reader. Assays were performed in triplicate with three independent experiments.

Mice were deeply anesthetized with pentobarbital sodium (40 mg/kg, i.p.), and subsequently perfused with 0.1 M PBS (pH 7.4, 37°C), followed by fixation in 4% (w/v) paraformaldehyde in 0.1 M PBS. The colon and brain tissue samples were sliced into 5 μm sections using a crystal microtome (Leica RM 2135, Wetzlar, Germany), followed by xylene dewaxing and gradient ethanol hydration as described previously. Tissues on slides were equilibrated in 0.1 M Tris-buffered saline solution for a duration of 10 min and blocked with 10% goat serum for 1 h. Subsequently, tissues of hippocampus were incubated with primary antibodies for 1 h, including anti-5HT1A (1:100, ab85615, abcam), or anti-5HT2A (1:100, ab66049, abcam), and tissues of colon were incubated with primary antibody anti-ZO-1 (1:100, ab216880, abcam) or anti-occludin (1:100, ab216327, abcam) for 1 h, respectively. Following primary antibody incubation, the slides were thoroughly washed with PBS and subsequently subjected to secondary antibody incubation. Finally, the slides were mounted with glycerin-based mounting media and examined using a fluorescence microscope.

Total RNA was extracted from mouse tissues using Trizol (Invitrogen, Carlsbad, CA, United States). The cDNA was produced by using HiScript II QRT Supermix (Vazyme Biotech), according to the manufacturer’s protocol. The mRNA transcription levels were measured according to the instructions of One Step qRT-PCR SYBR Green kit (Vazyme Biotech). The gene β-Actin was chosen as an internal control, and the 2^−ΔΔCt method was used to calculate the mRNA relative expression level. Four repeats were taken independently to determine significance. The primers used are listed in Table 1.

According to the instructions of the E.Z.N.A Stool DNA kit (Omega Bio-tek, Norcross, GA, United States), microbial DNA was extracted from the stool. Next, the purity and concentration of the DNA extraction were determined by a NanoDrop 2000 spectrophotometer, and the quality of the extracted DNA was measured by 1% agarose gel electrophoresis. The V3–V4 region of 16S rDNA gene was selected for PCR using 338F (5′-ACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GGACTACHVGGGTWTCTA-3′) primers. The amplified PCR fragments were purified using Agencourt AMPure XP beads in order to generate a sequencing library. The sequencing was conducted on an Illumina MiSeq platform using the paired-end (PE) method with 300 bp read length and 40% phiX quality control. The sequencing data have been deposited in the GenBank short-read archive (SRA) with accession number: PRJNA720241.

Trimmomatic¹⁹ v0.39 was used to filter low-quality reads with the parameters “TRAILING:20,” “SLIDINGWINDOW:50:20,” and “MINLEN:50.” Following quality control, FLASH²⁰ v1.2.9 was employed to merge paired reads with a minimum overlap length of 10 bp and an allowable mismatch density of 0.2 in the overlapping region. Subsequently, USEARCH²¹ v11.0.667 was used to cluster aligned reads with UPARSE at a similarity threshold of 0.97. For taxonomy assignment of OUT representative sequences, QIIME²² v1.9 was employed via RDP method with reference database SILVA 128²³ and bootstrap value set at 0.7 for enhanced accuracy and reliability of results obtained from this analysis approach. The resulting table contained information on both OUT abundance as well as taxonomic classification data.

Alpha diversity was calculated using Mothur²⁴ v1.4 on rarified OUT tables. The Shannon indices of community biodiversity were assessed based on the abundance of OTUs. Principal coordinates analysis was performed using MicrobiomeAnalyst²⁵.

The data acquired in this study were subjected to analysis utilizing the software package GraphPad Prism 8. The data are presented as the means ± SEM, accompanied by the number of samples (n) in each group. After histogram and Kolmogorov–Smirnov analysis, the obtained data accorded with normal distribution. For comparison between the two groups, an unpaired t-test was used to analyze the statistical significance. For comparisons among more than two groups, one-way analysis of variance (ANOVA), followed by Tukey’s t-test was used. Results with a p-value less than 0.05 were deemed statistically significant.

In addition to the withdrawal symptoms after alcohol withdrawal, alcohol addiction patients also have clinical signs such as gradually increasing alcohol consumption and strong alcohol addiction. After establishing the alcohol addiction model of C57BL/6 mice with double bottle supply of water solution and alcohol solution, the body weight and alcohol preference ratio of mice in the alcohol group and the control group were compared. The alcohol preference ratio was calculated as the ratio (percentage) of the weight of alcohol solution consumed by the mice to the total weight of water and alcohol solution consumed at the same time, and this result was used to compare the degree of alcohol preference of the mice. It can be seen from Figure 1A that from day 0 to day 12, when the concentration of alcohol solution increased from 3% to 6% and then to 10%, the intake of alcohol solution of mice gradually increased, and the alcohol preference ratio increased from 53% to 68% and then to 88%. After day 12, the alcohol solution concentration was maintained at 10%, and the alcohol preference ratio of the drinking mice as a whole was also maintained at 70% and above. During the drinking period, the weight change of mice was shown in Figure 1B. The weight gain of mice in the alcohol drinking group was 6.8% lower than that in the control group. On the 24th day of drinking, the weight change of mice in the two groups was significantly different (p < 0.05).

OFT results showed that the mean movement time of alcohol withdrawal group (13.5 s) was shorter than that of alcohol group (15.6 s) and control group (20.0 s) (Figure 2A). The mean distance in the central region of mice in alcohol withdrawal group (304.0 mm) was lower than that in alcohol group (367.6 mm) and control group (388.2 mm) (Figure 2B). The ratio of distance traveled in the central region to total distance in alcohol withdrawal group (5.3%) was lower than that in alcohol group (6.7%) and control group (8.2%) (Figure 2D). These three sets of data suggest that BALB/c mice develop anxious and depressed mood with reduced exploratory activity in the central region after alcohol withdrawal. However, there was no statistically significant difference compared with control and pre-withdrawal mice. The total distance and average speed of movement were used to analyze the autonomous behavior of mice in the open field box, as shown in Figures 2C,E in control group (p < 0.05). The voluntary behavioral activity of mice increased after abstinence. According to LDT analysis, as shown in Figure 2F, the mean retention time in the dark box of alcohol withdrawal group, alcohol group and control group was 213.4 s, 189.4 s and 171.5 s, respectively. After alcohol withdrawal, the mice spent more time in the dark box, and the mice in the drinking group spent slightly more time in the dark box than the control group. The mice in the drinking group showed a trend of anxiety and depression during drinking and the trend increased after alcohol withdrawal.

The dilution curves of the three groups in Figure 3A tended to be flat with the increase of the number of analyzed sequences, indicating that the amount of data sequenced in this study was reasonable and had covered almost all the bacteria in the sample, and few new species will appear even if the amount of sequencing continues to increase.

The Alcohol_24d group was close to the control group and far from the Alcohol_0d group in the figure, and these three groups were able to separate on the PC1 and PC2 axes (Figure 3B). The results also indicated that the species composition and abundance of GM in mice were changed after alcohol consumption, excluding the influence of other conditions.

Figures 3C,D show a comparison of the alpha diversity indices ACE and Shannon. ACE index showed that compared with the Alcohol_0d group, the GM species richness of the Alcohol_24d and control groups was significantly increased, and the GM species richness of the control group was significantly higher than that of the Alcohol_24d group (p < 0.01). Species diversity assessed by Shannon index showed that compared with the Alcohol_0d group, both the Alcohol_24d and control groups had significantly increased GM species diversity, and the control group had slightly higher species diversity than the Alcohol_24d group. This suggests that alcohol consumption leads to a decrease in GM species richness and diversity in mice.

The species abundance and community composition of the samples were analyzed at different taxonomic levels. The difference of intestinal flora between the alcohol dependent mice and healthy mice was analyzed, at the phylum level (Figure 3E), the top two dominant bacteria in the three groups were Bacteroidetes and Firmicutes, and their proportions in the three groups were different. In order of relative abundance of species, Actinobacteria and Proteobacteria were next in Alcohol_0d group, and Patescibacteria and Proteobacteria were the third and fourth in Alcohol_24d group and control group. At the genus level (Figure 3F), the first two dominant bacteria in the three groups were norank_f_Muribaculaceae and Lactobacillus, and the abundance of Lactobacillus decreased in Alcohol_24d group. According to the relative abundance of species, Streptococcus and Saccharimonas had a higher proportion in Alcohol_0d group, and Turicibacter and Candidatus_saccharimonas had an increased proportion in Alcohol_24d group. The Turicibacter and Prevotellaceae_UCG_001 genera accounted for a higher proportion in the control group. The community composition and species richness of Alcohol_24d group changed compared with Alcohol_0d group and control group.

ACE index evaluated species richness, and the richness of AL_FMT group was higher than that of AL_PBS group. Compared with alcohol group, GM species richness of mice in AL_FMT group was significantly increased (p < 0.001) (Figure 4A). Shannon index showed that GM species diversity of mice in alcohol group, AL_FMT group and AL_PBS group had no significant difference (Figure 4B). These results indicated that FMT to drinking mice would increase the GM species richness of mice, but there was no significant change in species diversity. The difference of intestinal flora between the AL_FMT mice and healthy mice was analyzed, at the phylum level (Figure 4C), the abundance of Verrucomicrobia increased while the abundance of Epsilonbacteraeota decreased in AL_FMT mice. At the genus level (Figures 4D,F), the contents of two genera (Ruminococcaceae UCG-014 and Akkermansia) in AL_FMT group was significantly increased, while the abundance of Helicobacter, Odoribacter, Lachnospiraceae and Alistipes was significantly decreased in AL_FMT group. The species composition of AL_FMT and alcohol group could be separated clearly by PCA, while the AL_PBS group was similar to alcohol group (Figure 4E). It indicated that FMT could alter the GM composition and abundance in alcohol dependent mice.

After 14 days of FMT, alcohol-dependent mice were subjected to behavioral tests before and after alcohol withdrawal. Behavioral tests were performed on alcohol-addicted mice receiving FMT from healthy donors (AL_FMT group), and behavioral tests were performed on AL_FMT mice after 24 h of alcohol withdrawal (AL_FMT_Withdrawal group). Behavioral tests were performed in the AL_PBS group. Behavioral tests were performed in the AL_PBS group 24 h after alcohol withdrawal (AL_PBS_Withdrawal group). The motion trajectories of OFT and LDT are shown in Figures 5A,B, compared with the control group, the alcohol group mice preferred to move around the open field and in the dark box after alcohol withdrawal 24 h, depression-like behavior was improved when they received the microbiome from control mice. There was no significant difference in mice behavior between the alcohol group and the control group without alcohol withdrawal.

As a major neurotransmitter, 5-hydroxytryptamine (5-HT) can regulate the secretion of opioid peptide, gamma-aminobutyric acid, glutamic acid and other transmitters, and plays a key regulatory role in many aspects of emotion, cognition, substance dependence and so on. What’s more notable is that 95% of 5-HT is produced in the enteric nervous system. Therefore, we explored the effect of FMT on 5-HT content, the results showed that the 5-HT content was significantly increased in alcohol dependence mice intestinal and brain tissues after receiving the fecal microbiome from healthy mice (Figures 6A,B). Considering that the abnormal function of some 5-HT receptors is also a cause of substance dependence, we examined a range of receptors (5-HT1A, 5-HT2A, 5-HT1B, 5-HT3, 5-HT6) associated with it. As shown in Figure 6D, the expression of 5-HT1A and 5-HT2A receptors was decreased in alcohol-dependent mice, while FMT significantly increased the mRNA expression level of 5-HT1A and 5-HT2A. We further examined the presence of 5-HT receptors in the hippocampus by immunohistochemical assay, the results were consistent with those of qPCR (Figure 6C). It indicated that FMT may alleviate alcohol withdrawal syndrome possibly through increasing the 5-HT and its receptor 5-HT1A and 5-HT2A.

Alcohol metabolites can lead to the destruction of tight junction proteins in the intestine. The results of fluorescence quantitative PCR showed that, compared with healthy mice, the mRNA transcription levels of ZO-1 and occludin in the intestine of alcohol-dependent mice decreased by 3–4 times, respectively, and after FMT treatment, ZO-1 and occludin transcription levels recovered to healthy levels (Figure 7B). To further confirm the structural integrity of TJs, the immunofluorescence technique was used to detect the protein expression level of ZO-1 and occludin in colon. The results showed that the expression level of ZO-1 and occludin decreased significantly in alcohol-dependent mice, while FMT was able to ameliorate the changes of the two proteins (Figure 7A).

In order to ascertain the impact of FMT on intestinal permeability, the ELISA analysis was performed to determine the serum levels of LPS and FABP2, which served as biomarkers of intestinal barrier permeability, as shown in Figures 8A,B. Compared to the control group, the LPS and FABP2 in alcohol dependence mice serum increased, while FMT significantly decreased the change levels, suggesting the reparative impact of FMT on intestinal leakage.

To further investigate the influence of FMT on the pro-inflammatory factor, a series of ELISA assays were performed. The results revealed that FMT significantly decreased the levels of TNF-α, IL-4 and IL-18 (Figures 8C–E), which indicated that FMT could ameliorate the inflammatory reaction in alcohol dependence mice.