Acetobacter pasteurianus BP2201 alleviates alcohol‐induced hepatic and neuro‐toxicity and modulate gut microbiota in mice

Abstract

The excessive consumption of alcohol results in a dysbiosis of the gut microbiota, which subsequently impairs the gut microbiota‐brain/liver axes and induces cognitive dysfunction and hepatic injury. This study aimed to investigate the potential effect of Acetobacter pasteurianus BP2201 in reducing the negative effects of alcohol consumption on cognitive function and liver health by modulating the gut microbiota‐brain/liver axes. Treatment with A. pasteurianus BP2201 improved alcohol‐induced hippocampal damage, suppressed neuroinflammation, promoted neuroprotein expression in the hippocampus and enhanced cognitive function. At the same time, A. pasteurianus BP2201 can also reduce serum lipid levels, relieve oxidative stress, inhibit TLR4/MyD88/NF‐κB pathway, reduce the secretion of TNF‐α and IL‐1β, so as to improve alcoholic liver injury. Concomitantly, the treatment with A. pasteurianus BP2201 leads to a shift in the intestinal microbiota structure towards that of healthy individuals, inhibiting the proliferation of harmful bacteria and promoting the recovery of beneficial bacteria. In addition, it also improves brain cognitive dysfunction and liver health by affecting the gut microbiota‐brain/liver axes by promoting the synthesis of relevant amino acids and the metabolism of nucleotide base components. These findings demonstrate the potential of regulating the gut microbiome and gut microbiota‐brain/liver axes to mitigate alcohol‐induced disease.

This study demonstrates the potential therapeutic efficacy of A. pasteurianus BP2201 in ameliorating cognitive dysfunction and liver damage induced by alcohol consumption in mice. The findings indicate that treatment with A. pasteurianus BP2201 results in significant improvements in cognitive function, liver health and intestinal microbial community structure. These outcomes suggest that A. pasteurianus BP2201 may regulate the intestinal microbiome, thereby reducing alcohol‐induced damage, and may serve as a potential treatment for alcohol‐related diseases. This study provides a promising avenue for developing interventions aimed at preventing or treating alcohol‐induced brain and liver injuries by modulating the gut microbiota‐organ axes.

INTRODUCTION

Approximately 2.3 billion individuals worldwide partake in alcohol consumption, and of this population, 75 million may encounter an alcohol‐related disorder that can inflict multi‐system impairments, particularly in the liver, intestines and brain (Meroni et al., 2019). In recent years, there has been a mounting fascination in understanding the intricate interplay between alcohol and the gut microbiota‐brain axis and the gut microbiota‐liver axis (Gupta et al., 2021). These vital systems perform pivotal functions in regulating numerous physiological processes, underscoring the imperative need for a more comprehensive understanding of the implications of excessive alcohol consumption on these critical systems.

The gut microbiota‐brain axis is a bidirectional regulatory axis, that facilitates interactions and communications between the intestinal microbiota and the central nervous system (Ding et al., 2020; Morais et al., 2021). Metabolites and signalling molecules produced by gut microbes can impact nerve signalling and brain functions through several mechanisms such as the transportation of neurotransmitters or via circulation within the bloodstream (Wekerle, 2018). The equilibrium of gut microbiota can be disrupted by alcohol consumption, inducing dysbiosis (Brüssow, 2020) and a consequential overgrowth of deleterious microorganisms (Liu, Guo, et al., 2022; Liu, Vigorito, et al., 2022; Liu, Wu, et al., 2022). The toxins these microorganisms release can translocate across the intestinal mucosa, enter the bloodstream and breach the blood–brain barrier (Logsdon et al., 2018). This translocation can lead to the manifestation of neuroinflammation and oxidative stress, both of which can provoke cellular and neural impairment, ultimately impairing cognitive functionality and inducing altered behaviour (Fischer & Maier, 2015; Wang et al., 2021). Alcohol also exerts a profound impact on neurotransmitter dynamics, particularly the production and release of serotonin and dopamine, key molecules with vital roles in cognitive function (Valenzuela, 1997; Yang et al., 2022). Specifically, alcohol intake can result in an eventual reduction in serotonin levels, culminating in the manifestation of symptoms of depression and anxiety both of which exert a deleterious effect on cognitive performance (Marcinkiewcz et al., 2016). Furthermore, the consumption of alcohol leads to heightened dopamine release, thereby increasing the risk of alcohol addiction and consequent cognitive decline (Siciliano et al., 2018).

Both the gut and liver exhibit a tightly interdependent relationship, wherein both nutrients and non‐nutrients assimilated from the gut gain entry into the liver through the hepatic portal vein (Albillos et al., 2020). However, this pathway is not unidirectional but rather operates as a dynamic two‐way communication system, referred to as the gut microbiota‐liver axis (Dehhaghi et al., 2020). The liver assumes a pivotal role in alcohol catabolism, and as such chronic alcohol consumption is known to induce hepatic damage, such as alcohol‐induced liver disease (Liu, Tsai, & Hsu, 2021; Liu, Wang, & Wu, 2021). Furthermore, intestinal dysbiosis following alcohol consumption results in the production of toxic metabolites that gain entry into the gut microbiota‐liver axis circulation via the compromised intestinal barrier, exacerbating the progression of liver disease (Engen et al., 2015). In addition, alcohol consumption impairs intestinal barrier function, augmenting intestinal permeability and facilitating the translocation of gut bacteria and endotoxins into the systemic circulation (Rao, 2009). These deleterious effects culminate in the manifestation of systemic inflammatory responses and immune system dysregulation, eventually culminating in the development of alcohol‐induced liver damage (Nagy, 2015).

The intricate interplay between the gut‐brain and gut‐liver axes and the consumption of alcohol underscores the significance of preserving a healthy gut microbiome as a means of mitigating the deleterious effects of alcohol (Mancini et al., 2018). Supplementation with probiotics presents a potential avenue for upholding the equilibrium of the intestinal microbiota and fostering the proliferation of advantageous microorganisms within the gut microenvironment (Li, Ai, et al., 2020; Li, Niu, et al., 2020). Probiotics, a type of exogenous beneficial microbiota, are capable of flourishing and proliferating within the gut (Sánchez et al., 2017). These microorganisms play a critical role in modulating the composition and diversity of the intestinal microbiome, as well as enhancing the integrity of the intestinal barrier (Liu, Tsai, & Hsu, 2021; Liu, Wang, & Wu, 2021). Numerous studies have demonstrated that a specific dose of probiotics has the potential to impede the proliferation of pathogenic bacteria within the gut, ameliorate intestinal mucosal damage and reinstate the equilibrium of the gut microbiota that has been compromised due to alcohol consumption (Jiang et al., 2020). As such, probiotics represent a prospective therapeutic intervention method for mitigating the detrimental consequences of alcohol on the gut microbiota‐brain and gut microbiota‐liver axes. A. pasteurianus, a member of the Acetobacter genus, has a well‐characterised alcohol degradation ability mediated by two enzymes: ethanol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) (Lynch et al., 2019; Wu et al., 2018). Initially, ADH initiates the conversion of ethanol to acetaldehyde by removing hydrogen atoms, leading to the production of acetaldehyde and the reduction of NAD+ to NADH. Acting as a catalyst, ADH accelerates ethanol oxidation and facilitates NAD+ reduction (Gao et al., 2021; Zakhari, 2006). Subsequently, ALDH plays a crucial role by catalysing the further oxidation of acetaldehyde to acetic acid through hydrogen atom elimination (Miah et al., 2021). Notably, A. pasteurianus effectively executes the degradation of ethanol through a precisely orchestrated series of enzymatic reactions (Covarrubias et al., 2021). Its potent ability to oxidise alcohol to acetic acid and high acetic acid resistance make it the primary industrial strain in vinegar fermentation (Lynch et al., 2019). Recent investigations have revealed that the co‐ingestion of acetic acid bacteria and alcohol results in a reduced alcohol concentration within the human body, modulation of intestinal microbiota dysbiosis caused by alcohol consumption and attenuation of the adverse effects of alcohol on the body (Lin et al., 2020; Qiu et al., 2021). The elucidation of the intricate alcohol degradation pathway employed by A. pasteurianus not only unveils its exceptional capacity for alcohol metabolism but also provides valuable insights for biotechnological applications and potential bioengineering endeavours in the field of microbial alcohol degradation.

This study aimed to investigate the potential effects of A. pasteurianus BP2201 in mitigating alcohol‐induced cognitive dysfunction and hepatic injury through the modulation of the gut microbiota and the gut microbiota‐brain/liver axis, as well as to elucidate the underlying mechanisms behind this apparent therapeutic function.

EXPERIMENTAL PROCEDURES

Strain and culture conditions

The bacterial strain A. pasteurianus BP2201, derived from brewing mass samples, was utilised in the present investigation. The bacterial culture was maintained under aerobic conditions in a YPD liquid medium (10.0 g/L yeast extract, 20.0 g/L peptone, 20.0 g/L glucose) at a temperature of 30 °C for a duration of 24 h. The bacterial biomass obtained after cultivation was subsequently harvested and re‐suspended in phosphate buffer solution (8.0 g/L NaCl, 0.2 g/L KCl, 0.2 g/L Na₂HPO₄, 0.2 g/L NaH₂PO₄ and pH 7.4) at a density of 1 × 10⁹ CFU/mL, for further analysis.

Cellular toxicity test

The cellular toxicity of A. pasteurianus BP2201 cells was measured by haemolytic activity and the lactate dehydrogenase (LDH) assay. A. pasteurianus BP2201 was cultured by streaking on blood agar media containing 5% defibrinated sheep blood and incubated for 24 h at 37°C to evaluate their possible haemolytic activity (Parhamifar et al., 2013). The release of LDH by A. pasteurianus BP2201 cells, which represents the cytotoxicity level, was detected using an LDH cytotoxicity detection assay kit according to the manufacturer's instructions (Beijing Leagene Biotechnology Co., Ltd.). The supernatant was mixed with the LDH reagent, and the relative LDH levels were measured at an absorbance of 490 nm. The released LDH rates are presented according to the manufacturer's instructions.

Alcohol modelling mice experiment

This study selected female C57BL/6J mice (aged 8 weeks, weight 18–20 g), purchased from GemPharmatech, and housed in an SPF‐level environment in the Animal Experimental Center of China Pharmaceutical University. The selection of female mice was based on their documented higher susceptibility to alcohol‐induced damage relative to male mice (Li et al., 2019; Shukla et al., 2019). After undergoing a period of adaptive feeding for a duration of 1 week, forty 8‐week‐old mice were subjected to random assignment into two distinct experimental groups: the short‐term alcohol‐induced cognitive dysfunction group and the long‐term alcohol‐induced liver injury group. The former group comprised 18 mice, while the latter group consisted of 22 mice.

The short‐term alcohol‐induced cognitive dysfunction group was randomly subdivided into three experimental groups: the ConS group (Short‐term control group), the AES group (Short‐term alcohol intake group) and the AcS group (Short‐term alcohol intake treated with A. pasteurianus BP2201 group), consisting of eight mice per group. Each group except the ConS group were treated with 50% alcohol at a dosage of 5 g/kg, once every 12 h, for a total of three doses (Feizolahi et al., 2019). Half an hour after the final alcohol administration, the water maze test was conducted (Van Skike et al., 2012). Nine hours later, the mice were sacrificed and blood, hippocampal and cecum contents were collected. Part of the hippocampus was immobilised in Formalin for histological study, and the rest of the hippocampus and cecum contents were preserved at −80°C. The long‐term alcohol liver injury group was randomly subdivided into three experimental groups: the ConL group (Long‐term control group), the AEL group (Long‐term alcohol exposure group) and the AcL group (Long‐term alcohol intake treated with A. pasteurianus BP2201 group). Except for the ConL group, all mice received 35% alcohol at a dose of 4.8 g/kg daily for a duration of 14 days (Colombo et al., 2007; Xia et al., 2018). Nine hours after the final alcohol administration, liver, colon contents and blood were collected. The liver was weighed, and a portion of it was immobilised in Formalin for histological examination, while the remainder of the liver and colon contents were stored at −80°C. Throughout the study, daily recordings of body weight were taken, and the mice were fed with water and standard feed. Additionally, a bacterial solution containing A. pasteurianus BP2201 (1 × 10⁹ CFU/mL) was administered to the AcS and AcL group half an hour before each alcohol administration.

Morris water maze test

Short‐term alcohol‐induced cognitive dysfunction group mice were trained in the water maze for 4 days, with each mouse trained four times per day (Othman et al., 2022). In each training session, the mice were placed in the water at the midpoint of each quadrant, with a time limit of 60 s per session. During the training, the mice were placed facing the pool wall in the water, and data recording began. After the mice found and climbed onto the platform, the mouse was allowed to stay in place for 15 s. If the mice failed to find or climb onto the platform within 60 s of entering the water, it was placed on the platform and allowed to stay for 15 s before the next training session began. At the end of the modelling, the mice were placed in the water at the midpoint of the first quadrant, and their latency period, total swimming distance and swimming speed while searching for the platform were recorded.

Organ damage score

After the mice in the group of short‐term alcohol‐induced cognitive dysfunction were sacrificed, the hippocampal tissues were taken and part of them was prepared into HE staining sections and Nissl staining sections. The liver of mice with long‐term alcohol‐induced liver injury. was collected and sliced by HE staining. Microscopic images were taken to assess damage to the hippocampus and liver.

Determination of serum biochemical indexes

According to the manufacturer's instructions, the contents of e alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TC), triacylglycerol (TG), high‐density lipoprotein cholesterol (HDL‐C) and low‐density lipoprotein cholesterol (LDL‐C) in serum of mice with long‐term alcohol‐induced liver injury group were determined using a commercially available kit (Jiangcheng Institute of Bioengineering).

Measurement of antioxidant indexes in the hippocampus and liver samples

According to the manufacturer's instructions, the contents of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH‐Px) in the hippocampus and liver samples were determined using a commercially available kit (Jianceng Bioengineering Institute).

Quantitative polymerase chain reaction

Total RNA from hippocampal and liver tissues was extracted using TRIzol (Thermo Fisher Scientific). Reverse transcription, as well as amplification and relative quantification of mRNA (2^−ΔΔCT method), were performed in triplicate using a Q2000A Quantitative Real‐time PCR System (LongGene) and HiScript II One Step qRT‐PCR SYBR Green Kit (Vazyme) and GADPH was chosen as the internal reference. The primer sequences are listed in Table S1.

Intestinal microbiota analysis

The bacterial DNA was extracted from faecal samples with a QIAamp Fast DNA stool Mini Kit (Qiagen, Cat# 51604) and PCR amplification was conducted with barcoded specific bacterial primers targeting the variable region 3–4 (V3–V4) of the 16S rRNA gene: forward primer 338F: 5′‐ACTCCTACGGGAGGCAGCA‐3′ and reverse primer 806R: 5′‐GGACTACHVGGGTWTCTAAT‐3′. Construction of sequencing libraries and paired‐end sequencing was performed on an Illumina NovaSeq6000 platform at Biomarker Technologies Co, Ltd. according to standard protocols. Paired‐end reads were merged using FLASH v1.2.7, and tags with more than six mismatches were discarded. The merged tags with an average quality score < 20 in a 50 bp sliding window were determined using Trimmomatic and those shorter than 350 bps were removed. Possible chimeras were further removed and the denoised sequences were clustered into operational taxonomic units (OTUs) with 97% similarity using USEARCH (version 10.0). Taxonomy was assigned to all OTUs by searching against the Silva databases (Release128) using QIIME software. Raw sequences were deposited in the Sequence Read Archive database (http://www.ncbinlm.nih.gov/sra) and the GenBank accession number is as follows: PRJNA940923.

Statistical analysis

All experimental results data were evaluated by mean ± standard error of the mean. Statistical significance was then analysed using one‐way ANOVA and finally using GraphPad Prism software and unpaired t tests. If data were not normally distributed, then the nonparametric Kruskal–Wallis test was used, and pairwise comparison was done using Dunn's multiple comparison tests; p < 0.05.

RESULTS

Cellular toxicity of A. pasteurianus BP2201

The selection of probiotics is contingent upon the crucial safety criterion that the chosen strain exhibits no haemolytic activity. The presence of γ‐haemolysis on the blood agar plate confirms the absence of haemolysis activity in A. pasteurianus BP2201, thereby establishing its suitability for subsequent experimental investigations. Furthermore, following a 4‐h co‐culture of A. pasteurianus BP2201 with monolayer cells, the release rate of lactate dehydrogenase (LDH) in the culture medium showed no significant difference compared to monolayer cells cultured alone (p > 0.05). These findings provide compelling evidence to support the conclusion that A. pasteurianus BP2201 does not exert cytotoxic effects.

Acetobacter pasteurianus BP2201 can relieve short‐term alcohol‐induced learning and memory impairment

The Morris water maze experiment is widely utilised to assess the spatial learning and memory performance of animals (Tian et al., 2019). The evaluation of spatial learning and memory within each group was conducted based on multiple parameters including latency to the platform, swimming trajectory, swimming speed and percentage of time spent in the target quadrant. Latency to the platform, which refers to the duration required for an animal to locate a concealed platform during a water maze experiment, is commonly used as a metric to quantify the learning and memory ability of experimental animals. A decreased latency to the platform is generally associated with enhanced learning and memory abilities. According to the results, when compared with the ConS group, the latency to the platform of the AES group was significantly prolonged (**p < 0.01). When compared with the alcohol group, the platform latency of the AcS group was significantly shortened (*p < 0.05). There was no observed significant difference between the ConS group and AcS group in platform latency (Figure 1A). And the AES group mice exhibited a disorderly swimming track and a circular swimming pattern, whereas the AcS group mice demonstrated a relatively simple swimming route that was similar to the ConS group mice (Figure 1B). Furthermore, the speed of the three groups of mice differed significantly. When compared to the ConS group, the AES group mice had a markedly reduced speed (**p < 0.01), while the AcS group mice showed a slight increase in speed (*p < 0.05) when compared with the AES group mice. However, there was no significant difference in the speed between the ConS and AcS mice groups (Figure 1C). In addition, the AES group exhibited a significantly shorter target quadrant time than the AcS and ConS groups (*p < 0.05) (Figure 1D). Taken together, these findings suggest that A. pasteurianus BP2201 can effectively ameliorate alcohol‐induced cognitive dysfunction and improve learning ability in mice.

FIGURE 1.

Acetobacter pasteurianus BP2201 can relieve short‐term alcohol‐induced learning and memory impairments. (A) The latency to the platform time of mice in each group in the Morris water maze test. (B) A representation of the swimming route of each group of mice. (C) Swimming speed of each group of mice. (D) Percentage of residence time in the target quadrant of each group. The number of independent samples used for each group was: ConS group, n = 6; AES group, n = 6; and AcS group, n = 6. *p < 0.05, **p < 0.01 and ***p < 0.001.

Acetobacter pasteurianus BP2201 can improve short‐term alcohol‐induced hippocampal morphological changes

Changes in the structure and cellular morphology of the hippocampus can have a profound impact on cognitive function, as this brain region plays a crucial role in learning and memory processes (Triviño‐Paredes et al., 2016). This study examined whether hippocampal changes occur in response to impaired learning and memory in mice, using HE and Nissl staining to evaluate alterations in cellular morphology (Figure 2). The results of this study showed that the hippocampal DG and CA3 regions in the ConS mice group exhibited normal morphology with closely arranged, uniform and neat cell layers as well as uniform staining. However, significant changes in the morphology of DG and CA3 regions were observed in the AES mice group, characterised by a loose, sparse and disordered cell arrangement, noticeable cell degeneration, necrosis and shedding, as well as an unclear cell membrane. Furthermore, a large number of Nissl bodies were reduced in the AES mice group. On the other hand, in the AcS group, the neurons in the hippocampus appeared to be normal, exhibiting a consistent and closely arranged pyramidal cell structure. This observation differed significantly from the AES group. The findings of this study indicate that A. pasteurianus BP2201 effectively repaired hippocampal damage and improved learning and memory ability in treated mice.

FIGURE 2.

Acetobacter pasteurianus BP2201 can improve alcohol‐induced hippocampal morphological changes. (A) H&E staining of the hippocampus from mice. (B) The Nissl staining of the hippocampus from mice. The number of independent samples used for each group was: ConS group, n = 6; AES group, n = 6; and AcS group, n = 6.

Acetobacter pasteurianus BP2201 can significantly improve the antioxidant capacity of the hippocampus

To assess the impact of A. pasteurianus BP2201 on hippocampal antioxidant capacity, levels of CAT, SOD and GSH‐Px antioxidant enzymes were measured in the hippocampus (Figure 3). According to the analysis results, the AES group exhibited a higher level of oxidative stress in the hippocampus compared to the ConS group, as evidenced by a significant reduction in the levels of CAT, SOD and GSH‐Px. Following treatment with A. pasteurianus BP2201, the levels of CAT, SOD and GSH‐Px were significantly higher in the AcS group when compared with the AES group. This suggests that A. pasteurianus BP2201 can effectively alleviate oxidative stress and enhance the antioxidant capacity of the hippocampus.

FIGURE 3.

Effects of Acetobacter pasteurianus BP2201 on CAT (A), SOD (B) and GSH‐Px (C) levels in the hippocampus of mice with short‐term alcohol‐induced cognitive dysfunction. **p < 0.05, **p < 0.01 and ***p < 0.001. The number of independent samples used for each group was: ConS group, n = 6; AES group, n = 6; and AcS group, n = 6.

Acetobacter pasteurianus BP2201 modulates NLRP3 expression and genes involved in neuronal development and function in the hippocampus

This study employed real‐time quantitative PCR to detect the mRNA expression levels of NLRP3, ERK, CREB, BDNF and TrkB genes in the hippocampus of mice with short‐term alcohol‐induced cognitive dysfunction (Figure 4). The findings indicated that in comparison to the ConS group, mice in the (AES) group exhibited a marked rise in the expression of NLRP3 inflammasome in their hippocampus (**p < 0.01). Additionally, there was a significant reduction in the expression levels of ERK, BDNF, TrkB and CREB. Moreover, the AcS group of mice exhibited a noteworthy reduction (*p < 0.01) in the expression of NLRP3 inflammation in their hippocampus compared to the AES group. Furthermore, the administration of A. pasteurianus BP2201 resulted in a significant increase in the expression levels of ERK, CREB, BDNF and TrkB in the hippocampus, as evidenced by statistical differences. These findings suggest that the TrkB/ERK/CREB/BDNF signalling pathway may play a crucial role in hippocampal neural injury induced by short‐term high‐concentration alcohol administration. Furthermore, the results suggest that the treatment with A. pasteurianus BP2201 could ameliorate neural damage in the hippocampus and improve cognitive ability, through its ability to inhibit the over‐expression of NLRP3 in the hippocampus and promote the expression of the TrkB/ERK/CREB/BDNF signalling pathway.

FIGURE 4.

Relative expression levels of mRNA in the hippocampus of mice with short‐term alcohol‐induced cognitive dysfunction hippocampal of NLRP3 (A), ERK (B), CREB (C), BDNF (D) and TrkB (E) genes. (n = 5 per group). *p < 0.05, **p < 0.01 and ***p < 0.001.

Intestinal microbiota analysis of short‐term alcohol‐induced cognitive dysfunction mice

This study additionally aimed to investigate the potential impact of A. pasteurianus BP2201 on the intestinal microbiota of mice with short‐term alcohol‐induced cognitive dysfunction. A total of 1,437,981 paired‐end reads were generated from 18 samples, consisting of the ConS (n = 6), AES (n = 6) and AcS (n = 6) groups. After quality control and splicing, 1,428,740 clean reads were obtained, with a minimum of 77,060 clean reads produced in each sample and an average of 79,374 clean reads per sample. By employing 16S rRNA sequencing of mouse faecal samples, various indicators of α and β diversity of mouse microbiota were evaluated. The PD whole tree analysis, based on the phylogenetic tree, revealed superior species diversity in the AcS group when compared to that of the AES group (Figure 5A). Moreover, the PCoA map, using the binary Jaccard algorithm, demonstrated notable dissimilarities in the gut microbiota among the three groups (Figure 5B). These findings suggest that A. pasteurianus BP2201 may potentially serve as a beneficial probiotic in treating alcohol‐related cognitive dysfunction, and provide valuable insights into the role of probiotics in modulating gut microbiota.

FIGURE 5.

Intestinal microbiota analysis of short‐term alcohol‐induced cognitive dysfunction mice. (A) PD whole tree Index. (B) Based on binary PCoA diagram of jaccard's algorithm. (C) Composition of the microbiota of the three mice groups at the phylum level. (D) Composition of the microbiota of the three mice groups at the genus level. (E) LEfSe cladogram. The taxa with LDA scores ≥3.5 between all three mice groups. (F) Species with relative abundance greater than 0.01% and multiGroup (p) less than 0.05 in the AcS group at the genus level. (G) Correlation network constructed by AES group and AcS group. (H) Correlations between species and indicators associated with short‐term alcohol‐induced cognitive dysfunction at the genus level. (I) Changes in bacterial metabolic pathways predicted based on characteristic sequences and KEGG database. *p < 0.05, **p < 0.01 and ***p < 0.001.

At the phylum level, the composition of microbiota in three groups was significantly different (Figure 5C). The ConS group was predominantly composed of Firmicutes (59.69%), Bacteroidota (31.5%), Verrucomicrobiota (3.54%), Actinobacteriota (3.23%) and Deferribacterota (0.75%). In contrast, short‐term high‐concentration alcohol intake led to a substantial reduction in Firmicutes (36.24%) and Actinobacteria (2.24%) and a significant increase in Proteobacteria (5.19%) in the AES group. Treatment with A. pasteurianus BP2201 caused a significant increase in Firmicutes (53.79%) and Verrucomicrobiota (8.29%), while Proteobacteria (0.71%) significantly decreased. The analysis was also conducted at the genus level (Figure 5D). Compared to the AES group, treatment with A. pasteurianus BP2201 resulted in the partial restoration of the abundance of the Lactobacillus genus. Simultaneously, there was a reduction in the relative abundance of Mucispirillum, unclassified Oscillospiraceae and Escherichia Shigella.

After which, LEfSe analysis was utilised to compare the cecal microbiota of three groups, wherein all species with LDA scores ≥3.5 were depicted graphically in a cladogram (Figure 5E). Notably, the ConS group was found to be enriched with Bacilli, Firmicutes, Lactobacillales, Lactobacillaceae and unclassified Lactobacillus. Conversely, analysis of the AES and AcS groups revealed a substantial abundance of harmful bacteria, such as Escherichia Shigella and Mucispirillum in the former, along with unclassified Rikenellaceae RC9 gut group and unclassified Oscillospiraceae. Furthermore, the AcS group demonstrated enrichment of unclassified Nostocaceae and uncultured Clostridiales bacterium. These findings suggest that treatment with A. pasteurianus BP2201 may effectively inhibit the growth of potentially pathogenic microorganisms, including Escherichia Shigella and Mucispirillum, thus leading to the amelioration of short‐term intestinal microbiota imbalance resulting from high concentrations of alcohol consumption.

Subsequentially, the genus‐level differences between the three groups were analysed using ANOVA, wherein all species with an abundance greater than 0.01% and multiGroup (p) less than 0.05 in the AcS group were presented graphically (Figure 5F). The administration of A. pasteurianus BP2201 successfully restores the bacterial community structure to that of healthy individuals, such as that seen in the ConS group. Specifically, the treatment significantly increases the relative abundance of Coriobacteriaceae UCG 002, Enterorhabdus and Turicibacter, which are known to be reduced by short‐term high‐concentration alcohol administration. Moreover, it significantly decreases the relative abundance of Mucispirillum, unclassified Oscillospiraceae, Rikenellaceae RC9 gut group and Escherichia Shigella. These findings suggest that A. pasteurianus BP2201 treatment may serve as a promising strategy for mitigating the negative effects of high concentrations of alcohol on the intestinal microbiota, ultimately leading to a restoration of a healthy bacterial community structure.

Furthermore, Spearman rank correlation analysis was used to investigate the dependency level between various species. Correlation networks were constructed using data exhibiting a correlation greater than 0.1 and a p‐value less than 0.05, to establish the interaction between different species (Figure 5G). To facilitate a comprehensive comparison of the effect of A. pasteurianus BP2201 treatment on species relationship, AES and AcS data were combined. The findings revealed a positive correlation between Enterorhabdus, Turicibacter and Acetobacter. Conversely, Mucispirillum, unclassified Oscillospiraceae, Escherichia Shigella, Alistipes, Oscillibacter, Rikenellaceae RC9 gut group and Acetobacter displayed a negative correlation. This suggests that A. pasteurianus BP2201 has the potential to inhibit the growth of harmful bacteria while promoting the re‐emergence of beneficial species.

Following this, the relationship between all indicators of significance and species at the genus level in the microbiota before and after treatment was comprehensively considered and displayed graphically (Figure 5H). The study considered species that displayed a negative correlation with the inflammatory factor NLRP3 and a positive correlation with the neurotrophic factor TrkB/ERK/CREB/BDNF as having positive significance. The species that were found to have such characteristics included Enterorhabdus and Turicibacter. Conversely, species that were positively associated with NLRP3 and negatively associated with TrkB/ERK/CREB/BDNF were considered to have negative significance, including Mucispirillum, unclassified Oscillospiraceae, Escherichia Shigella, Alistipes, Oscillibacter and Rikenellaceae RC9 gut group. The analysis revealed that the species with positive significance were positively correlated with Acetobacter, while those with negative significance were predominantly enriched in the AES group and negatively correlated with Acetobacter. These findings indicate that treatment with A. pasteurianus BP2201 can effectively inhibit the growth of potentially harmful species and promote the growth of beneficial ones, ultimately restoring the gut microbiota to a balanced state for therapeutic purposes.

Finally, PICRUSt2 was utilised to predict the metabolic pathways of the AES and ACS mice groups by utilising characteristic sequences combined with KEGG pathway information of genes (Figure 5I). The results of this analysis showed that the glycolysis, gluconeogenesis and pyruvate metabolic pathways, which are related to alcohol metabolism, decreased in the AES group, but increased in the ACS group following short‐term administration of large amounts of high concentration alcohol. Treatment with A. pasteurianus BP2201 not only upregulated these pathways, but also promoted the metabolism of phenylacetic acid, aspartic acid, glutamic acid, as well as glycine, serine and threonine. The gut microbiota‐brain axis. is an interaction between the gastrointestinal tract and the central nervous system. Amino acid metabolism is closely related to the gut microbiota‐brain axis. Amino acids present in the gastrointestinal tract can enter the blood circulation through intestinal epithelial cells and neurons, and can then be transmitted to the central nervous system. Serine and threonine are converted into neurotransmitters such as serotonin and dopamine, which play a role in cognitive function. Furthermore, phenylalanine metabolites such as tyrosine and norepinephrine are important neurotransmitters involved in the regulation of cognitive function. Based on these findings, it can be concluded that A. pasteurianus BP2201 can increase the metabolism of corresponding amino acids, which can affect the function of the central nervous system through various ways as neurotransmitters. This can regulate the interaction between the gut microbiota‐brain axis and improve cognitive function.

Acetobacter pasteurianus BP2201 can alleviate long‐term alcohol‐induced liver injury in mice

A comparison between the AEL group and the AcL group in terms of body weight after a 14‐day modelling period in mice with long‐term alcohol‐induced liver injury was conducted. The results of this comparison indicated a decreasing trend in body weight for both the AEL and AcL groups, with the AEL group exhibiting a more substantial weight loss and a statistically significant difference when compared to the AcL group (Figure 6A).

FIGURE 6.

Acetobacter pasteurianus BP2201 can alleviate long‐term alcohol‐induced liver injury in mice. (A). Changes in relative body weight in AEL and AcL groups, animals excluded the next day after death. (B). Gross examination of mice livers and Histological examination of liver sections stained with H&E. (C)The level of the liver index. Serum levels of ALT (D) and AST (E). The number of independent samples used for each group was: ConL group, n = 6; AEL group, n = 4; and AcL group, n = 6. *p < 0.05, **p < 0.01 and ***p < 0.001.

In addition, histopathological observations and H&E staining was used to evaluate the histopathological changes in the liver of mice with long‐term alcohol‐induced liver injury (Figure 6B). The findings revealed significant differences between the AEL group and the ConL group, with the liver in the AEL group exhibiting significant whitening, enlargement and roughening. Additionally, HE slices of liver tissue from the AEL group of mice showed vacuolar degeneration of hepatocytes, marked hepatocyte swelling and a large number of inflammatory cell infiltration, when compared to the ConL group. However, treatment with A. pasteurianus BP2201 in the AcL group significantly mitigated these changes, resulting in no obvious steatosis and inflammation in the liver of mice in the AcL group, with only a slight accumulation of lipid droplets. Notably, no significant histopathological difference was observed between the AcL and ConL groups. Furthermore, this study also evaluated the pathological changes in the liver using the liver index metric (Figure 6C). The findings demonstrated a marked increase in the liver index in the AEL group relative to the ConL group (***p < 0.001), whereas the liver index in the AcL group was significantly lower than that in the AEL group (*p < 0.05). These results suggest that A. pasteurianus BP2201 may have a protective effect against long‐term alcohol‐induced liver injury by ameliorating histopathological changes within the liver.

The assessment of serum enzyme activities, such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST), has become a conventional method to gauge the presence of alcoholic‐related liver diseases (Kim et al., 2008). Evidently, alcohol consumption exhibited a notable elevation in serum ALT and AST activities when compared to the ConL group (Figure 6D,E). In contrast, treatment with A. pasteurianus BP2201 effectively counteracted the observed increments in these enzymes. Thus, the findings propose that A. pasteurianus BP2201 holds considerable potential in ameliorating hepatic damage associated with prolonged alcohol consumption, by ameliorating liver steatosis, enhancing liver function and mitigating inflammatory responses.

Acetobacter pasteurianus BP2201 reduces serum lipid accumulation in mice with long‐term alcohol‐induced liver injury

Serum lipid levels serve as crucial indicators for the diagnosis and treatment of alcoholic liver injury (Arain et al., 2018). This study examined the effect of A. pasteurianus BP2201 on serum total cholesterol (TC), triglycerides (TG), low‐density lipoprotein cholesterol (LDL‐C) and high‐density lipoprotein cholesterol (HDL‐C) levels in a mouse model of long‐term alcohol‐induced liver injury (Figure 7). The results demonstrate that when compared to the ConL group, the AEL group exhibited increased concentrations of TC, TG and LDL‐C, while HDL‐C levels significantly decreased (*p < 0.01). Furthermore, the AcL group demonstrated significantly decreased levels of TC, TG and LDL‐C in comparison to the AEL group, while HDL‐C levels significantly increased (**p < 0.01). It is widely acknowledged that excessive alcohol consumption can lead to liver damage and abnormal lipid metabolism, characterised by elevated TG and LDL‐C levels and decreased HDL‐C levels, which are typical manifestations of alcoholic‐induced liver injury. In conclusion, this study successfully established a mouse model of alcoholic liver injury and revealed that the treatment of A. pasteurianus BP2201 effectively attenuated the severity of the liver injury.

FIGURE 7.

Effects of Acetobacter pasteurianus BP2201 on TG (A), T‐CHO (B), LDLC (C) and HDLC (D) levels in the serum of mice with long‐term alcohol‐induced liver injury. The number of independent samples used for each group was: ConL group, n = 6; AEL group, n = 4; and AcL group, n = 6. *p < 0.05, **p < 0.01 and ***p < 0.001.

Acetobacter pasteurianus BP2201 can significantly improve the antioxidant capacity of the liver

In order to assess the impact of A. pasteurianus BP2201 on liver antioxidant capacity, the levels of SOD, CAT and GSH‐Px antioxidant enzymes were measured in the liver (Figure 8). The results indicate that the levels of CAT, SOD and GSH‐Px the AEL group were significantly reduced in comparison to the ConL group, indicating a higher level of oxidative stress in the hippocampus of the AEL group. Following treatment with A. pasteurianus BP2201, the levels of CAT, SOD and GSH‐Px were significantly higher in the AcL group than in the AEL group. This suggests that A. pasteurianus BP2201 can effectively enhance the antioxidant capacity of the liver.

FIGURE 8.

Effects of Acetobacter pasteurianus BP2201 on CAT (A), SOD (B) and GSH‐Px (C) levels in the hippocampus of mice with long‐term alcohol‐induced liver injury. The number of independent samples used for each group was: ConL group, n = 6; AEL group, n = 4; and AcL group, n = 6. *p < 0.05, **p < 0.01 and ***p < 0.001.

Acetobacter pasteurianus BP2201 modulates inflammatory response‐related gene expression in the liver

In this study, real‐time quantitative PCR was employed to measure mRNA expression levels of several genes in the liver tissue of mice with alcohol‐induced liver injury (Figure 9). It was found that compared to the ConL group, the expression levels of CYP2E1, TLR4, MyD88, NF‐κB, TNF‐α and IL‐1β were significantly increased in the liver tissue of mice in the AEL group. In contrast, when compared to the AEL group, the expression levels of CYP2E1, TLR4, MyD88, NF‐κB, TNF‐α and IL‐1β were significantly decreased in the liver tissue of mice in the AcL group. These results suggest that the TLR4/MyD88/NF‐κB signalling pathway may play a direct role in liver injury caused by long‐term alcohol intragastric administration. Furthermore, treatment with A. pasteurianus can inhibit the expression of TLR4/MyD88/NF‐κB signalling pathway proteins in mouse liver and repair long‐term alcohol‐induced liver injury.

FIGURE 9.

Relative expression levels of mRNA in the liver of mice with long‐term alcohol‐induced liver injury of CYP2E1 (A), TLR4 (B), MyD88 (C), NF‐κB (D), TNF‐α (E) and IL‐1β (F) genes. n = 5 per group. *p < 0.05, **p < 0.01 and ***p < 0.001.

Intestinal microbiota analysis of long‐term alcohol‐induced liver injury mice

This study investigated the impact of A. pasteurianus BP2201 on the intestinal microbiota of mice with long‐term alcohol‐induced liver injury. Sixteen samples (ConL = 6, AEL = 4, AcL = 6) were subjected to sequencing, yielding 1,280,849 pairs of Reads. After quality control and splice, 1,272,325 Clean Reads were obtained, with an average of 79,520 Clean Reads per sample and at least 79,329 Clean Reads per sample. 16S rRNA sequencing was utilised to determine the α and β diversity indices of the intestinal microbiota in the mice suffering from long‐term alcohol‐induced liver injury. The ACE and Chao1 indices indicated that the species richness of the AEL and AcL group was significantly lower than that of the ConL group due to chronic alcohol intragastriction. Notably, the species richness was significantly higher following treatment with A. pasteurianus BP2201 than in the untreated AEL group (Figure 10A,B). NMDS analysis based on the bray Curtis algorithm revealed significant differences among the microbiomes of the three mice groups (Figure 10C).

FIGURE 10.

Intestinal microbiota analysis of long‐term alcohol‐induced liver injury mice. (A) ACE Index. (B) Chao 1 Index. (C). NMDS1 diagram. (D) Composition of the microbiota of the three groups at the phylum level. (E) Composition of the microbiota of the three groups at the genus level. (F) LEfSe cladogram. The taxa with LDA scores ≥3.5 between all three groups. (G). Species with relative abundance greater than 0.01% and multiGroup (p) less than 0.05 in the AcL group at the genus level. (H) Correlation network constructed by the AEL group and AcL group. (I) Correlations between species and indicators associated with Long‐term alcohol‐induced liver injury mice at the genus level. (J) Changes in bacterial metabolic pathways predicted based on characteristic sequences and the KEGG database. *p < 0.05, **p < 0.01 and ***p < 0.001.

At the phylum level, the micro microbiota composition among the three groups exhibited noteworthy variations (Figure 10D). Firmicutes (58.80%), Bacteroidota (25.46%), Verrucomicrobiota (6.46%), Actinobacteriota (4.88%) and Desulfobacterota (3.76%) was found to be the main constituents of the ConS group, ordered in decreasing proportions. Conversely, the AEL group demonstrated a significant reduction in the abundance of Bacteroidota (20.30%) and Actinobacteriota (0.23%), and a significant increase in Proteobacteria (3.48%), as a consequence of prolonged alcohol intake. Treatment with A. pasteurianus BP2201 was found to significantly increase Bacteroidota (53.79%) and slightly decrease Proteobacteria (3.27%). The analysis was also conducted at the genus level (Figure 10E). When compared to the AEL group, the restoration of the Lachnospiraceae NK4A136 group in the intestine was evident after treatment with A. pasteurianus BP2201. Furthermore, the relative abundance of Unclassified Lachnospiraceae and the significantly improved unclassified Oscillospiraceae, unclassified Muribaculaceae and unclassified Eubacterium coprostanoligenes group were also observed. The relative abundance of Alloprevotella, Akkermansia and Ligilactobacillus was noted to decrease after treatment with A. pasteurianus BP2201.

In addition to the above analysis, LEfSe analysis was also utilised to compare the diversity of colon microbiota across the three groups of mice with long‐term alcoholic liver damage. The corresponding figure displayed the abundance of all species possessing LDA scores ≥3.5 (Figure 10F). Among these species, unclassified Muribaculaceae, Lactobacillales and unclassified Lactobacillus were notably enriched in the ConL group. Furthermore, separate analyses and comparisons of the AEL and AcL groups were conducted. The findings demonstrated that the AEL group exhibited an abundance of harmful bacteria, including Prevotellaceae, Proteobacteria, unclassified Prevotellaceae UCG 001 and Lactobacillaceae, as well as Ligilactobacillus and Alloprevotella. In contrast, the AcL group was found to be enriched with an abundance of Bacteroidaceae, gIleibacterium and Oscillospiraceae. Consequently, the results suggest that the application of A. pasteurianus BP2201 treatment can lead to favourable changes in the intestinal microbiota of mice suffering from long‐term alcohol‐induced liver injury. Despite a lower species richness when compared to healthy individuals, restoring the microbial balance and suppressing the growth of pathogenic microorganisms, including Proteobacteria, can improve the intestinal microbial dysbiosis that arises from prolonged alcohol intragastric administration.

Next, an analysis of ANOVA at the genus level to identify differences among the three groups was conducted. The study focused on species with abundance greater than 0.01% and a multiGroup (p) less than 0.05 in the AcL group, as well as several important species (Figure 10G). The findings indicate that treatment with A. pasteurianus BP2201led to a microbiota structure that was more similar to that of healthy individuals, specifically that of the ConL group. Notably, the recovery of Turicibacter, Oscillibacter and an uncultured Muribaculaceae bacterium, which were previously depleted due to long‐term alcohol intragastric administration, was observed. Furthermore, the relative abundance of the Lachnospiraceae NK4A136 group, unclassified Clostridia and Eubacterium nodatum group was significantly increased, whereas the relative abundance of Alistipes and Prevotellaceae UCG 001 was reduced.

In addition, a Spearman rank correlation analysis at the taxonomic level was undertaken in order to construct a correlation network comprising data with correlation coefficients greater than 0.1 and p‐values less than 0.05, aiming to determine the interaction relationships between different species (Figure 10H). To comprehensively compare the changes in species interactions resulting from treatment with A. pasteurianus BP2201, the study pooled data from the AEL and AcL groups and identified a positive correlation between Lachnospiraceae NK4A136 group, unclassified Clostridia, [Eubacterium] nodatum group, Escherichia Shigella and Acetobacter, while Helicobacter, ASF356, Prevotellaceae UCG 001, Alistipes, unclassified Lachnospiraceae all displayed a negative correlation with Acetobacter. Thus, A. pasteurianus BP2201 can curb the growth of deleterious bacteria and facilitate the resurgence of beneficial microbial species. Then, a comprehensive evaluation of the relationship between various indicators of significance and species at the genus level in the microbiota, both before and after treatment was conducted (Figure 10I). The species that exhibited negative correlation with the detection indicators of alcoholic liver injury in this experiment, such as Lachnospiraceae NK4A136 group, Eubacterium nodatum group, unclassified Clostridia and Escherichia Shigella, were defined as positive. On the other hand, the species that were related to the detection index with negative significance, including ASF356, Alistipes, Helicobacter, Prevotellaceae UCG 001 and unclassified Lachnospiraceae, were defined as negative. The analysis revealed that the majority of species with positive significance were positively correlated with Acetobacter, while those with negative significance were primarily enriched in the AEL group and negatively correlated with Acetobacter. These findings suggest that both short‐term and long‐term alcohol‐induced treatment of A. pasteurianus BP2201 effectively suppressed many potentially threatening species, while also restoring the levels of beneficial bacteria and achieving a balanced gut microbiota for therapeutic purposes.

Finally, the study utilised PICRUSt2 to predict the AEL and AcL group's pathways based on gene characteristic sequences in combination with KEGG pathway information (Figure 10J). Following long‐term alcohol modelling, similar trends in the AEL group to the AES group were seen, with decreases in glycolysis and gluconeogenesis pathways and pyruvate metabolic pathways associated with alcohol metabolism. Conversely, these pathways increased in the ACL group. Treatment with A. pasteurianus BP2201 not only up‐regulated these pathways but also promoted microbial metabolism in diverse environments, including purine and pyrimidine metabolism and amino and nucleotide sugar metabolism. The gut microbiome is known to significantly impact the metabolism of purines and pyrimidines from the diet, which are basic building blocks of nucleic acids. Bacterial metabolic byproducts of these nucleotide precursors may enter the circulatory system and subsequently undergo liver processing. Changes in the gut microbiome composition can result in alterations in the levels of these metabolites, affecting the liver metabolism of purines and pyrimidines, thus influencing the gut microbiota‐liver axis. Similarly, microbial metabolism of amino and nucleotide sugars can impact the gut microbiota‐liver axis. In summary, microbial metabolism in various environments can influence the gut microbiota‐liver axis through multiple mechanisms such as metabolite production, regulation of intestinal barrier function and modulation of immune responses. A. pasteurianus BP2201 can promote the metabolism of nucleotide base components such as adenine and pyrimidine, generating related metabolites that potentially interact with the liver by influencing gut microorganisms. The liver can further metabolise these metabolites, potentially influencing the function of the gut microbiota‐liver axis.

DISCUSSION

Acetic acid bacteria are prevalent probiotics utilised in the preparation of food and beverages (Gomes et al., 2018). Nevertheless, hitherto, no investigations have been conducted to assess the therapeutic potential of these acetic acid bacteria against alcohol‐induced hepatic pathologies and neuropathies via modulation of the gut microbiota‐brain/liver axes. This study aimed to examine the potential of A. pasteurianus BP2201 in mitigating the deleterious effects of alcohol‐induced cognitive dysfunction and liver injury through modulation of the gut microbiota‐brain/liver axes. The study's findings suggest that A. pasteurianus BP2201 treatment in mice can lead to enhanced cognitive functions and improved liver health. These results emphasize the promising therapeutic potential of this intervention method, which is both safe and cost‐effective. This investigation presents compelling evidence in demonstrating the feasibility of mitigating alcohol‐induced pathologies through the administration of probiotics to modulate the gut microbiota‐brain/liver axes.

The above study effectively established two mice models, one exhibiting transient cognitive dysfunction induced by long‐term large doses of high‐concentration alcohol intake, and the other evincing chronic hepatic injury resulting from long‐term alcohol consumption. Numerous studies have documented that acute alcohol consumption leads to hippocampal neuronal damage and inflammatory response, ultimately resulting in cognitive dysfunctions (Mira et al., 2020; Quintanilla et al., 2020). In this study, utilising a mice model of short‐term alcohol‐induced cognitive dysfunction, it was observed that considerable pathological alterations in the hippocampus of mice within the AES group were present. Encouragingly, treatment with A. pasteurianus BP2201 was found to significantly mitigate these abnormal morphological changes observed within the hippocampus. The levels of SOD, CAT and GSH‐Px in the hippocampus were also assessed. As a crucial memory centre in the brain, the hippocampus' antioxidant potential is integral to normal cognitive functions (Lin et al., 2022). The AES group exhibited significantly decreased antioxidant potential in the hippocampus, accompanied by an elevation in hippocampal oxidative stress, potentially leading to cognitive dysfunction. Conversely, treatment with A. pasteurianus BP2201 demonstrated an increased antioxidant capacity in mice, providing protection to the hippocampus against oxidative stress and free radical damage, and possibly resulting in an enhancement of cognitive functions. Thus, in order to evaluate cognitive performance, a water maze test was conducted on the mice. The results unequivocally demonstrated that short‐term, high‐dose alcohol intake had a marked impact on cognitive function, which was notably ameliorated by A. pasteurianus BP2201 dietary intervention. These findings suggest that A. pasteurianus BP2201 possesses the capacity to mitigate alcohol‐induced cognitive dysfunction.

The ERK/CREB/BDNF/TrkB signalling pathway exerts a pivotal role in the hippocampus. CREB functions as a transcription factor contributing to neuronal plasticity, potentiating the activation of the BDNF and TrkB signalling pathways and fostering neuronal survival and development (Amidfar et al., 2020; Wang et al., 2018). The BDNF and TrkB signalling pathways are implicated in regulating neuronal growth and plasticity, while the ERK signalling pathway can activate CREB, ultimately fostering the survival and development of neurons (Ding et al., 2022; Wang et al., 2022). Normal operation of these signalling pathways is fundamental to learning and memory within the hippocampus. Impairment of these signalling pathways may lead to cognitive dysfunction (Zheng et al., 2020). Short‐term heavy alcohol consumption triggers the activation of the NLRP3 inflammasome in the hippocampus. Activation of the NLRP3 inflammasome results in the overstimulation of calpain, an enzyme associated with learning and memory processes in the nervous system (Lowe et al., 2018; Madhu et al., 2021; Xu et al., 2019; Yao et al., 2023). In cases of overactivation, calpain disrupts the normal functioning of the ERK/CREB/BDNF/TrkB signalling pathway in the hippocampus (Qiu et al., 2020). The findings of this study demonstrate a significant increase in NLRP3 expression, as well as decreased expression levels of the ERK/CREB/BDNF/TrkB pathway in the hippocampus following short‐term excessive alcohol consumption. Treatment with A. pasteurianus BP2201 resulted in reduced NLRP3 expression and a return to normal levels of ERK/CREB/BDNF/TrkB pathway expression. These results suggest that A. pasteurianus BP2201 may ameliorate alcohol‐induced neuroinflammatory responses and promote the survival and development of hippocampal neurons via the NLRP3‐ERK/CREB/BDNF/TrkB signalling pathway, thus improving cognitive function.

Chronic alcohol consumption has been found to be associated with alcoholic liver injury, which is closely linked to alterations in the gut microbiota‐liver axis and systemic inflammatory response (Shukla et al., 2021). ALT and AST are key liver transaminases widely used in medicine to assess liver damage and the extent of hepatocyte damage (Suciu et al., 2020). Inflammation resulting from alcohol metabolism may induce alterations in liver cell membrane permeability, causing a rise in serum ALT and AST levels (Ke et al., 2022). The results of this study indicate that the AcL group showed a significant reduction in ALT and AST levels in the mouse model of chronic alcohol‐induced liver injury. These findings suggest that A. pasteurianus BP2201 may provide strong protection against long‐term alcohol‐induced liver injury in mice. And the present study also investigated the therapeutic effect of A. pasteurianus BP2201 on chronic alcohol‐induced liver injury in mice. The histopathological analysis indicated that the treatment could ameliorate liver cell necrosis and inflammatory cell infiltration in the mice. The study also examined the impact of alcohol on lipid metabolism, as evidenced by altered levels of serum lipids, including TC, TG, HDL‐C and LDL‐C. The results showed that alcohol consumption led to a decrease in serum HDL‐C levels and an increase in serum TC, TG and LDL‐C levels, indicative of lipid metabolism disorders. However, treatment with A. pasteurianus BP2201 restored the serum lipid levels towards normal.

Alcohol consumption is known to induce oxidative stress, which is a critical mechanism leading to alcohol‐induced liver injury (Wu & Cederbaum, 2003). The intake of alcohol reduces the levels of key antioxidant enzymes, such as CAT, SOD and GSH‐Px, in the liver, leading to increased oxidative stress within the liver. In this study, it was observed that there was a significant reduction in the activities of SOD, CAT and GSH‐Px in the group with alcohol‐induced liver injury, indicating the presence of oxidative stress. However, the administration of A. pasteurianus BP2201 alleviated the alcohol‐induced oxidative stress in the liver by enhancing the antioxidant capacity, which may be one of the protective mechanisms of A. pasteurianus BP2201 against alcohol‐induced liver injury.

In addition to oxidative stress, chronic alcohol consumption induces inflammation, which is a critical factor in the pathogenesis of alcohol‐induced liver injury (Wu & Cederbaum, 2003). Alcohol and its metabolites trigger the release of pro‐inflammatory cytokines, which exacerbates liver inflammation and hepatocyte apoptosis. The TLR4/MyD88/NF‐κB signalling pathway is a classical inflammatory signalling pathway (Li, Ai, et al., 2020; Li, Niu, et al., 2020). Toll‐like receptors (TLRs) constitute a class of transmembrane proteins that exert a pivotal role within the realm of innate immunity (Brennan & Gilmore, 2018). To date, multiple TLRs have been identified in the human system, encompassing TLR2, TLR3, TLR4, among others, each endowed with distinct ligand specificity and signalling pathways (Takeda & Akira, 2004). Their activation serves as a crucial mechanism for the recognition of diverse pathogens, thereby initiating an appropriate immune response to combat infection (El‐Zayat et al., 2019). However, TLR4 is primarily activated by bacterial components, culminating in an immune response hallmarked by the generation of pro‐inflammatory cytokines, chemokines and other immunomodulatory factors (Vaure & Liu, 2014). Based on our hypothesis, we propose that TLR4 receptors within hepatic cells undergo activation subsequent to exposure to alcohol, and that A. pasteurianus BP2201 may harbour discrete molecular constituents capable of engaging with hepatic‐expressed TLR4. These interactions hold the potential to instigate TLR4 signalling, thereby instigating downstream pathways integral to immune responsiveness. The engagement of TLR4 signalling pathways typically triggers the recruitment of MyD88 adaptor molecules, thereby instigating the NF‐κB pathway. NF‐κB, a transcription factor, assumes a critical role in the regulation of immune and inflammatory responses. Nevertheless, A. pasteurianus BP2201 may exert an inhibitory influence on NF‐κB activity through regulatory means. Consequently, the suppression of NF‐κB by A. pasteurianus BP2201 culminates in the diminished production of pro‐inflammatory cytokines, exemplified by TNF‐α and IL‐1β, which both recognised contributors to hepatic damage and inflammation (Liu, Guo, et al., 2022; Liu, Vigorito, et al., 2022; Liu, Wu, et al., 2022). Moreover, alcohol promotes CYP2E1 activity in hepatocytes, leading to an increase in oxidative stress and further activation of NF‐κB (Albano, 2006). Therefore, inhibiting the TLR4/MyD88/NF‐κB pathway is an effective strategy for treating alcohol‐induced liver injury. It was found that A. pasteurianus BP2201 treatment could reduce CYP2E1 overexpression induced by alcohol, inhibit the TLR4/MyD88/NF‐κB pathway, and reduce the secretion of TNF‐α and IL‐1β, thereby alleviating alcohol‐induced liver injury.

The above study has demonstrated that the intestinal microbiota and their metabolites could potentially modulate the hepatic and neurological toxicity of alcohol through diverse mechanisms. These findings emphasize the significance of investigating the alcohol‐induced shifts in the gut microbiome and characterising the genomic and metabolic profile of the pertinent bacteria. Such an investigation would provide critical insights into the gut microbiota's contribution towards mitigating and treating alcohol‐induced disorders of the liver and nervous system. In this investigation, utilising both acute and chronic alcohol exposure models alongside assessing mRNA expression levels and changes in bacterial structure, the potential of A. pasteurianus BP2201 to hinder the growth of several bacteria that can potentially generate LPS, and dampen the expression of inflammatory factors via multiple pathways has been compellingly demonstrated. Moreover, the administration of A. pasteurianus BP2201 prompted the establishment of a healthy gut microbiota in individuals with normal bowel function. A. pasteurianus BP2201 demonstrated direct antagonistic effects against unfavourable bacterial species, significantly facilitated the growth of favourable species, and restored the levels of multiple core intestinal microbiota while broadly suppressing the abundance of potentially pathogenic bacteria.

However, a noteworthy observation from this investigation was the reduction in Escherichia Shigella abundance following short‐term alcohol exposure, while Escherichia Shigella exhibited detrimental effects in the corresponding heat map. Conversely, in the long‐term alcohol exposure model, treatment with A. pasteurianus BP2201 resulted in a significant enrichment of Escherichia Shigella and revealed its beneficial effects in the heat map. This anomalous phenomenon warrants further investigation. One plausible explanation is that A. pasteurianus BP2201 fosters the growth of beneficial bacteria, thus impeding the proliferation of pathogenic bacteria like Escherichia Shigella in the short‐term alcohol model. However, the chronic consumption of alcohol may bring about alterations in the gut environment, such as increased intestinal permeability and inflammatory responses, that stimulate the growth of both favourable and unfavourable bacteria, ultimately influencing the growth and enrichment of Escherichia Shigella. These observations indicate that the effects of alcohol on Escherichia Shigella are multifaceted and necessitate further research to elucidate the underlying mechanisms and outcomes.

Of utmost significance, our KEGG pathway analysis revealed that treatment with A. pasteurianus BP2201 leads to the upregulation of signalling pathways associated with the gut microbiota‐brain/liver axes, thereby ameliorating the deleterious effects of alcohol on the nervous system and liver. Specifically, in the short‐term alcohol‐induced cognitive dysfunction mice, A. pasteurianus BP2201 exhibited a favourable impact on cognitive function by augmenting the intestinal microecological milieu and enhancing the synthesis and secretion of neurotransmitters, such as phenylbenzene, aspartic acid and glutamate, through the gut microbiota‐brain axis, ultimately promoting cognitive function. Meanwhile, in the long‐term alcohol‐induced liver injury mice, A. pasteurianus BP2201 produced relevant metabolites by promoting the metabolism of nucleotide base components by intestinal microorganisms, and interacted with the liver via these metabolites, concurrently modulating intestinal barrier function and anti‐inflammatory effects, thereby affecting the gut microbiota‐liver axis.

The above study employed female C57BL/6J mice as the experimental model. Historically, investigations into alcohol‐related ailments have predominantly employed male mice, with female counterparts rarely being examined (Hartmann et al., 2018; Liu, Guo, et al., 2022; Liu, Vigorito, et al., 2022; Liu, Wu, et al., 2022; Zhu et al., 2022). Nevertheless, subsequent research revealed that mice exhibit reduced levels of alcohol dehydrogenase in the gastric region, and oestrogen‐mediated liver Kupffer cells manifest greater sensitivity to lipopolysaccharides and pro‐inflammatory agents (Maddur & Shah, 2020). Thus, the potential of mice as a subject for scrutinising alcohol‐related diseases has only recently been recognised. The adoption of mice in this study's experimental design offers a novel perspective into the impact of alcohol on the physiology of both sexes and the influence of sex hormones, thereby promoting an improved understanding of the disease aetiology.

CONCLUSIONS

This study demonstrates the potential therapeutic efficacy of A. pasteurianus BP2201 in ameliorating cognitive dysfunction and liver damage induced by alcohol consumption in mice. The findings indicate that treatment with A. pasteurianus BP2201 results in significant improvements in cognitive function, liver health and intestinal microbial community structure. These outcomes suggest that A. pasteurianus BP2201 may regulate the intestinal microbiome, thereby reducing alcohol‐induced damage, and may serve as a potential treatment for alcohol‐related diseases. However, additional research is required to fully elucidate the mechanisms underlying the therapeutic effects of A. pasteurianus BP2201 and to establish optimal dosages and administration protocols for probiotics. In general, this study provides a promising avenue for developing interventions aimed at preventing or treating alcohol‐induced brain and liver injuries by modulating the gut microbiota‐organ axes.

AUTHOR CONTRIBUTIONS

Xin Wen: Data curation (equal); formal analysis (equal); investigation (equal); methodology (equal). Zheng Wang: Data curation (equal); formal analysis (equal); investigation (equal); methodology (equal). Qi Liu: Data curation (equal); formal analysis (equal); investigation (equal); methodology (equal).

FUNDING INFORMATION

No funding information provided.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

The procedures involving the animals and their care were conducted in conformity with the national and international laws and policies. The animal experiments were approved by the Laboratory Animal Care and Use Committee of China Pharmaceutical University.

CONSENT FOR PUBLICATION

All authors gave their consent for publication.

Supporting information

Table S1.

Wen, X. , Wang, Z. , Liu, Q. , Lessing, D.J. & Chu, W. (2023) Acetobacter pasteurianus BP2201 alleviates alcohol‐induced hepatic and neuro‐toxicity and modulate gut microbiota in mice. Microbial Biotechnology, 16, 1834–1857. Available from: 10.1111/1751-7915.14303

DATA AVAILABILITY STATEMENT

The raw data for the 16S rRNA gene sequence have been deposited in the NCBI BioProject database (https://www.ncbi.nlm.nih.gov/bioproject/) under accession number: PRJNA940923. The other data of this study are available on request from W.C.