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. 2024 Oct 21;17(10):e70016. doi: 10.1111/1751-7915.70016

Lactobacillus helveticus attenuates alcoholic liver injury via regulation of gut microecology in mice

Jiawen Lv 1,2, Guanjing Lang 1, Qiangqiang Wang 1, Wenlong Zhao 3, Ding Shi 1, Ziyuan Zhou 1,2, Yangfan Shen 1,2, He Xia 1,2, Shengyi Han 1,2, Lanjuan Li 1,✉
PMCID: PMC11492535  PMID: 39431804

Abstract

Previous reports have demonstrated that alcohol consumption significantly reduces the abundance of Lactobacillus in the gut. In this study, we selected five species of the genus Lactobacillus, commonly found in fermented foods, and acknowledged them as safe, edible, and effective in preventing or treating certain diseases, to evaluate their effects on alcoholic liver disease (ALD). By comparing the liver damage indices in each group, we found that the type strain of Lactobacillus helveticus (LH, ATCC 15009) had the most marked alleviating effect on ALD‐induced liver injury. Furthermore, experiments combining microbiomics and metabolomics were conducted to explore the mechanisms underlying the hepatoprotective effects of LH. Finally, we discovered that LH mitigated ethanol‐induced liver steatosis and inflammation in ALD mice by altering the structure and function of the gut microbiome, increasing intestinal levels of short‐chain fatty acids (SCFAs), and enhancing gut barrier integrity. These findings suggest a potential strategy for the clinical management of patients with ALD.

We identified the hepatoprotective ability of five potential probiotics in genus Lactobacillus in ALD mice, the most effective species of which is Lactobacillus helveticus. To further explore the mechanism of it, we conducted inflammation‐relative factors testing in serum and liver, gut barrier function evaluating, 16S rRNA sequencing, and targeted metabolomics. In brief, we found that L.helveticus attenuated alcoholic liver injury by restoring the gut microbiome disorder, increasing the intestinal levels of beneficial metabolites, and enhancing the gut barrier integrity, which provided a potential strategy for the clinical management of ALD patients.

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INTRODUCTION

Excessive or chronic alcohol dependence is a serious global issue that has been identified as one of the leading risk factors for attributable disability‐adjusted life‐years from 2010 to 2019 (GBD 2019 Risk Factors Collaborators, 2020; Chen, 2022), with the majority of deaths attributed to alcoholic liver disease (ALD) or alcohol‐related liver disease (Marroni et al., 2018). ALD involves the liver manifestations of alcohol overconsumption. It usually begins with reversible fatty infiltration (alcoholic fatty liver disease [AFLD]), which may deteriorate into alcoholic hepatitis (AH) at any stage, and can eventually lead to cirrhosis or even hepatocellular carcinoma (HCC) with complications. (Louvet & Mathurin, 2015; Marroni et al., 2018; Wu et al., 2023) Accumulating evidence suggests that alcohol disease is a systemic disease that is not only limited to the liver. (Le Daré et al., 2019; Rocco et al., 2014; Thomes et al., 2021) Apart from the liver damage brought on by alcohol metabolites, gut microbiota is a direct target for alcohol intake as well. Long‐term alcohol abuse alters the richness and composition of the gut microbiota, reducing the abundance of beneficial bacteria (including Lactobacillus) and the overgrowth of pathogenic bacteria, contributing to an imbalance in the gut–liver axis. (Bishehsari et al., 2017; Gurwara et al., 2020; Pa et al., 2015) Alcohol and its metabolites disrupt intestinal microecology and impair the gut barrier function either directly or indirectly, resulting in increased intestinal permeability, causing the bacterial byproducts (pathogen‐associated molecular patterns [PAMPs], such as lipopolysaccharides [LPS]) to transfer from the gut to the liver via portal circulation. This induces the production of a large number of pro‐inflammatory cytokines, triggering inflammatory responses and exacerbating ALD. (Fan et al., 2018; Wu et al., 2023; Yan et al., 2011; Yang et al., 2019) Briefly, the translocation of PAMPs from the gut to the liver is necessary for alcohol‐induced liver injury and is thought to play a crucial role in the progression of ALD.

Currently, there is a lack of safe and effective medications for ALD. The clinical management of alcohol use disorder (AUD) involves the use of drugs that affect the central nervous system, such as baclofen and benzodiazepines. Alcohol‐induced liver injury is controlled with symptomatic supportive treatment, whereas corticosteroids suppress severe AH. (Singal et al., 2018) Nevertheless, alcohol addiction is a chronic relapsing disorder associated with compulsive alcohol drinking, which makes the treatment more challenging. In addition to the adverse reactions and economic burden associated with long‐term drug use, there is an urgent need to develop new ALD therapies.

In this study, we evaluated the efficacy of supplementation with the genus Lactobacillus, an intestinal commensal bacterium, mainly inhibited by alcohol. We selected five species commonly found in fermented foods to perform intervention experiments in ALD mice: including Lactobacillus helveticus (LH), Lactobacillus casei (LC), Lactobacillus paracasei (LP), Lactobacillus sakei (LS), and Lactobacillus delbrueckii (LD), which were approved as safe and edible by the Ministry of Health of the People's Republic of China (http://www.nhc.gov.cn/sps/s7892/202208/1d6c229d6f744b35827e98161c146afb.shtml). A few of them have been proven to be efficient in some diseases; for instance, studies have indicated that LH can ease depression through the gut microbiota–brain axis by promoting the level of 5‐hydroxytryptamine (Gao et al., 2022), and ameliorate autoimmune encephalomyelitis in mice (Yamashita et al., 2017). When mixed with Bifidobacterium longum and/or Lactiplantibacillus plantarum, LH can remit stress‐related disorders in humans (Edebol Carlman et al., 2022; Romijn et al., 2017); when combined with Lactobacillus gasseri, it can reduce colonisation of Candida in the murine alimentary‐canal and consequent inflammation (Authier et al., 2021). LC alleviates inflammatory bowel disease (IBD) in vitro (Liu et al., 2021) and in vivo (Dou et al., 2021). It can also control childhood diarrhoea (Lai et al., 2019) and the progression of kidney diseases (Zhu et al., 2021) through gut microbiome management. Moreover, LC has been demonstrated to relieve gut microbiota disorders and alcoholic liver injury in humans (Li, Liu, et al., 2021); however, its specific mechanism of action remains unknown. Furthermore, a few studies have assessed the role of LP in diseases such as hyperuricemia (Cao, Liu, et al., 2022) and dextran sulfate sodium‐induced IBD (Huang et al., 2021) in mice. Reportedly, LS attenuates nonalcoholic fatty liver disease (NAFLD) in mice (Nguyen et al., 2022), and ameliorates experimental atopic dermatitis by altering gut microbes (Kwon et al., 2018). LD can ease ethanol‐induced deterioration of DSS‐induced ulcerative colitis by upregulating IL‐22 (Cannon et al., 2022), and can protect the respiratory tract against allergic asthma through immunoregulation (Montuori‐Andrade et al., 2022) in mice. However, the specific effects of ALD remain unclear. Therefore, in this study, we applied LH, LC, LP, LS, and LD gavage on ALD mice to explore the effects of ethanol‐induced liver injury and the underlying mechanism. Our findings will provide a novel and secure method for the prevention and treatment of ALD.

EXPERIMENTAL PROCEDURES

Working bacterial fluid preparation

To prepare the treatment solution for daily intragastric gavage (IG), type strains of Lactobacillus helveticus (LH, ATCC 15009), Lactobacillus casei (LC, ATCC 393), Lactobacillus paracasei (LP, ATCC 25302), Lactobacillus sakei (LS, ATCC 15521), and Lactobacillus delbrueckii (LD, ATCC 9649) were cultured in De Man‐Rogosa‐Sharpe (MRS) broth under anaerobic conditions. After 48 h, all the bacterial solutions were centrifuged and resuspended in sterile phosphate‐buffered saline solution (PBS) twice, to remove the culture medium. Before IG, the final concentration of the treatment solution was adjusted to ~5 × 109 colony‐forming units (CFUs)/ml. The solutions were cultured and diluted daily to ensure probiotic activity.

ALD mice models establishment and experimental design

The National Institute on Alcohol Abuse and Alcoholism (NIAAA) model was selected for its resemblance to the chronic plus‐binge pattern in general patients with ALD. (Bertola et al., 2013) The procedures involving mice were approved by the Animal Care and Use Committee of the First Affiliated Hospital, School of Medicine, Zhejiang University (permit no. 2023–1349). Six‐ to eight‐week‐old female C57BL/6 mice, weighing approximately 20 g, were purchased from Hangzhou Medical College. After random grouping based on their weight, they were housed in cages under stable and specific pathogen‐free (SPF) conditions (temperature: 23°C ± 1°C; humidity: 50% ± 10%, in a 12 h light/dark cycle) with ad libitum feeding and watering. One week (adaption period) later, all mice were fed the Lieber‐DeCarli Regular Control Diet (Dyets, 710,027) for 5 days to accommodate the liquid diet. Then, five treatment groups (LH, LC, LP, LS, and LD groups) and the positive control (PC) group were changed diets to Lieber‐DeCarli Ethanol Rat Diet (Dyets, 710,260) for 14 days, which started at 1% ethanol and then increased 1% per day, allowing mice to adapt to the alcoholic food; 5% ethanol was maintained from day 17 to day 27 (see Figure 1A). Simultaneously, the treatment groups were administered IG with the prepared bacterial liquid once daily, and the PC group was orally administered an equivalent amount of PBS. The negative control (NC) group was fed the control diet and administered the same oral gavage as the PC group. During the experiment, the weight of each subject and the total liquid feed intake per cage were recorded every three or 5 days. On the last day, the PC group and five treatment groups received a gastric gavage of 31.5% (vol/vol) ethanol solution per gram weight at 20 μL, while the mice in the NC group were orally administered isocaloric 45% (vol/vol) maltodextrin solution equally. After 9 h, all mice were sacrificed, and the requisite tissues were collected and stored at −80°C until further analysis.

FIGURE 1.

FIGURE 1

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Effects of the selected Lactobacillus treatment on ethanol‐induced liver injury. (A) Experimental ALD model establishment methods. (B) Serum levels of liver transaminase and TBA. (C) Hepatic triglycerides quantification result. (D) The mRNA expression levels of inflammatory chemokines in liver. p < 0.05 was identified as the statistical significance (*p < 0.05, **p < 0.01, and ***p < 0.001).

Serum parameters detection

Serum indices of hepatic disease, including alanine transaminase (ALT), aspartate aminotransferase (AST), and total bile acid (TBA), were measured using an automatic biochemical analyser (BS‐200). Endotoxin levels were determined directly by serum LPS quantification using the LAL Chromogenic Endpoint assay (Hycult Biotech, HIT302). Serum cytokine levels were evaluated using the Bio‐Plex Pro Mouse Cytokine 23‐plex Assay kit (Bio‐Rad, M60009RDPD).

Histopathological examination

The liver and colon tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and then sliced into sections. The samples were stained with haematoxylin and eosin (H&E) staining. To display the mucus layer of the colon intuitively, an Alcian Blue Periodic acid Schiff (AB‐PAS) Stain Kit (Solarbio, G1285) was used, following the manufacturer's instructions. The contents of the colon tissues used for AB‐PAS staining were preserved to protect against damage to the mucus layer integrity.

Hepatic triglycerides quantification

Frozen hepatic sections stained with Oil Red O were used to microscopically assess lipid accumulation in the liver. The specific concentration of hepatic triglycerides (TG) was quantified using a Triglyceride Quantification Colorimetric/Fluorometric Kit (Sigma‐Aldrich, MAK266‐1KT), which was operated according to the manufacturer's protocols.

Immunohistochemical staining

To determine the degree of neutrophil infiltration into the liver, we performed immunohistochemical staining of the paraffin sections using BOND Polymer Refine Detection Kit (Leica, DS9800‐CN). First, a dewaxing solution and a gradient of ethanol were sequentially deparaffinised and rehydrated. After epitope retrieval and blockade, mouse myeloperoxidase monoclonal antibodies (Servicebio, 1:1000 dilution) were applied to the sections, followed by a post‐primary mouse linker. Finally, the figures were scanned and captured using a Pannoramic 250 FLASH (3DHISTECH).

RNA extraction and quantitative real‐time PCR (qPCR) analysis

The RNeasy Plus Mini Kit (Qiagen, 74,136) was used to purify total RNA from liver and colon tissues with gDNA eliminator columns or plates according to the manufacturer's protocol. The extracted RNA was then reverse‐translated into cDNA using PrimeScript RT Master Mix (TAKARA, RR036B) as soon as possible. mRNA transcription was measured by quantitative real‐time PCR (Applied Biosystems ViiA 7 Real‐Time PCR System) using SYBR Premix Ex Taq (TAKARA, RR420B). The gene sequences used in qPCR analysis are listed in Table S3.

Metabolism analysis

Using gas chromatography–mass spectrometry (GC–MS), we determined the concentration of short‐chain fatty acids (SCFA) in the colon contents, including acetic acid (AA), propionic acid (PA), isobutyric acid (IA), butyric acid (BA), 2‐methyl butyric acid (MBA), and valeric acid (VA). We also assessed the levels of tryptophan and its derivatives, including l‐tryptophan (Trp; HMDB0000929), indole‐3‐acetic acid (IAA; HMDB0000197), indole‐3‐propionic acid (IPA; HMDB0002302), indole‐3‐aldehyde (IAld; HMDB0029737) and indole‐3‐lactic acid (ILA; HMDB0000671) in cecum contents.

16S rRNA amplicon sequencing

A DNeasy PowerSoil Kit (Qiagen, 47,014) was used to extract total genomic DNA from the samples. DNA concentration and purity were monitored by NanoDrop Microvolume Spectrophotometers (Thermo Scientific) and diluted to 1 ng/μL with sterile water. Specific barcode sequences and linker primers were chosen to amplify distinct regions (16S V3‐V4) of the 16S rRNA genes, with diluted DNA as a template. After that, the PCR products were purified by Qiagen Gel Extraction Kit and established sequencing libraries with NEBNext® Ultra™ IIDNA Library Prep Kit. A Qubit@ 2.0 Fluorometer (Thermo Scientific) and an Agilent Bioanalyzer 2100 system were used to qualify the library. The obtained DNA library was sequenced on a NovaSeq PE250 sequencer, generating 250 bp paired‐end reads. The raw data were spliced and filtered to the clean data. The latter were denoised using DADA2, and sequences with abundances <5 were filtered out to produce final Amplicon Sequence Variants (ASVs).

Statistical analysis

Statistical analyses and figures were generated using the R software (version 4.1.2). The whole data were presented as means ± SEM. The Mann–Whitney U‐test was used to determine statistical significance between the two groups. p < 0.05 was identified as the statistical significance (*p < 0.05, **p < 0.01, and ***p < 0.001). Chao1, Shannon, and Simpson indices were calculated using Qiime2 (qiime2‐2020.6).

RESULTS

Effects of lactobacillus intervention on the biochemical and pathological indices of liver injury in ALD mice

We observed that the 15 days ingestion of alcohol inhibited weight gain in mice, with approximately equal feed intake (Figure 1A, Tables S1 and S2). By testing the most widely used clinical indicators for assessing hepatocyte damage, namely, ALT and AST, distinctions among the Lactobacillus treated groups and the PC group showed that liver transaminase levels in the serum were significantly suppressed after probiotic intervention, except in the LP group. Unlike the other four species, the diminishing part in the LP group was not statistically significant. Likewise, an analogous situation occurred in the serum level of TBA (p = 0.053 for group PC vs. NC; a trend existed), which is another sensitive indicator of liver injury (Figure 1B).

According to the H&E and Oil Red O staining results, prominent fatty degeneration of hepatocytes was observed in the alcohol intake group samples, mainly in the periphery of the central lobular vein. However, compared to the PC group, the treatment visibly alleviated steatosis with much smaller fat vacuoles, especially in the LH group (Figure 2). Moreover, the hepatic TG quantification results further confirmed the hepatoprotective effect of relieving fat deposits in the LH group; however, the performance of the other intervention species was not statistically significant (Figure 1C).

FIGURE 2.

FIGURE 2

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Representative images of histopathological examination results of livers (H&E staining, oil red O staining, and MPO immunohistochemical staining). Scale bars, 50 μm. MPO‐positive cells were stained dark brown, as indicated by the arrows in the image of the PC group. (x 200).

Myeloperoxidase (MPO) is a lysosomal enzyme primarily expressed in neutrophils and monocytes and is a specific marker of activated neutrophils involved in tissue damage and subsequent inflammation (Aratani, 2018). Here, liver MPO immunohistochemical staining exhibited neutrophil infiltration in group PC, but there were fewer MPO‐positive cells in intervention groups, especially group LH (Figure 2). Additionally, by assessing the mRNA expression levels of inflammatory chemokines in the liver, we found that ethanol intake significantly upregulated Cxcl2 and Cxcl5, which was partially suppressed by LH (p = 0.041 and p = 0.065, respectively) (Figure 1D), indicating that LH had a mitigating effect on immune infiltration in alcohol‐damaged livers.

From the evaluation results elucidated above, we found that out of the five species, the type strain LH was the most effective at attenuating ethanol‐induced liver damage, whereas the type strain LP was the least effective. Subsequently, mechanistic studies were conducted.

LH attenuated ethanol‐induced steatohepatitis by suppressing serum levels of endotoxin and inflammatory factors

Intestinal endotoxemia plays a crucial role in systemic and hepatic inflammatory responses in patients with ALD. (Gao & Bataller, 2011; Szabo, 2015) As expected, this experimental ALD mice model exhibited significantly higher serum LPS levels than control group but was strongly suppressed by the supplement of LH (Figure 3A). Moreover, the mRNA expression levels of Tlr4 and myeloid differential protein‐2 (Md2) in the liver were lower in the pair‐fed group than in the ethanol‐fed groups, whereas LH reversed this alteration (Figure 3B). Next, we assessed the protein expression levels of inflammatory cytokines and chemokines in serum, including TNF‐a, IL‐1b, IL‐6, granulocyte colony‐stimulating factor (G‐CSF), C‐X‐C motif chemokine ligand 1 (CXCL1), CC chemokine ligand (CCL) 4, and CCL11. We found that alcohol intake significantly increased the levels of these inflammatory factors in mice, which could be inhibited by oral gavage of LH (Figure 3C).

FIGURE 3.

FIGURE 3

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Administration of LH mitigated inflammatory response in ALD mice. (A) The level of serum lipopolysaccharide. (B) The mRNA expression level of Tlr4 and Md2 in liver. (C) The serum levels of inflammatory cytokines and chemokines. p < 0.05 was identified as the statistical significance (*p < 0.05, **p < 0.01, and ***p < 0.001).

LH strengthened the gut barrier integrity in ALD mice to reduce leakage of PAMPs

Excessive alcohol intake is associated with increased intestinal permeability, and the destruction of the colon barrier by alcohol may be a precursor to the onset of ALD. (Albillos et al., 2020; Chopyk & Grakoui, 2020) H&E staining was used to directly assess the variations in the colon barrier of mice under light microscopy. The colons of mice in the NC group exhibited typical histological characteristics and intact villus structures, whereas those in the PC and LP groups displayed severe colon damage, intestinal crypt atrophy, and a significant reduction in goblet cells. However, the oral administration of LH mitigated this pathological alteration (Figure 4A).

FIGURE 4.

FIGURE 4

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Effects of LH and LP supplementation on abnormalities to the gut barrier in ALD mice. (A) Representative histopathological pictures of H&E and AB‐PAS‐stained colons in each group (x 1000. Scale bars, 100 μm). (B) The mRNA expression levels of Claudin1 and Occludin. (C) Mucins mRNA relative expression levels. p < 0.05 was identified as the statistical significance (*p < 0.05, **p < 0.01, and ***p < 0.001).

Intestinal tight junction proteins are essential for maintaining the intestinal barrier function, and their absence or mutation can result in increased intestinal permeability and disruption of intestinal homeostasis. In this study, the vital components of the intestinal tight junction proteins, Claudin1 and Occludin, were assessed for their relative mRNA expression levels. The results showed that the ethanol‐fed group demonstrated a considerable reduction in both, but that LH effectively increased them, whereas LP did not (Figure 4B).

In addition to the aforementioned physical barriers, the mucus barrier is an essential component of the chemical barrier and is primarily composed of mucins released by goblet cells that promote intestinal homeostasis and tolerance. (Pelaseyed et al., 2014) Under light microscopy, AB‐PAS staining portrayed that the mucus barrier of the alcohol‐fed groups was thinner and lost its integrity compared with the NC group, yet LH rectified this change (Figure 4A). Furthermore, the mRNA expression levels of mucins were tested, suggesting that LH treatment reversed the decline in Muc3 and Muc4 expression caused by alcohol consumption. However, the trend in the level of Muc2 mRNA did not reach statistical significance compared with that in the PC group (Figure 4C).

LH alleviated alcohol‐induced intestinal microecology dysbiosis in mice

The ASVs obtained after noise reduction by the DADA2 method were analysed using the QIIME2 classify‐sklearn algorithm, and each ASV was annotated and sorted using a pre‐trained Naive Bayes classifier. The microbiome abundance at the kingdom, phylum, class, order, family, genus, and species levels were also determined. To evaluate the alpha diversity of the gut microbiome within the community, the Chao1, Shannon, and Simpson indices were calculated, which showed that the 15‐day alcohol intake contributed to a significant reduction in gut microbiota richness but had little impact on the diversity of intestinal microbes within the group. However, LH markedly reversed the decrease in the Chao1 index induced by the ethanol diet; however, the elevation of the Shannon and Simpson indices was not statistically significant, suggesting that LH recovered the richness of the gut microbiome yet had less influence on intra‐community diversity. However, the gavage of LP seemed to have no discernible impact on the gut microbiome richness or diversity of mice with ALD (Figure 5A). Beta diversity analysis revealed significant variations in gut microbial communities between the NC and PC groups. Moreover, the gut microbial structure of group LH was more similar to that of group NC, whereas that of group LP was closer to that of group PC (Figure S2A).

FIGURE 5.

FIGURE 5

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Effects of LH and LP gavage on Intestinal microbes' diversity in ALD mice. (A) The α diversity indices. (B) Stacked histogram of relative abundance intra groups at phylum and genus level. p < 0.05 was identified as the statistical significance (*p < 0.05, **p < 0.01, and ***p < 0.001).

Subsequently, the constituent alterations in the gut microbiota of the mice were analysed. At the phylum level, alcohol intake reduced the relative abundances of Verrucomicrobia, Proteobacteria, and Fusobacteriota and increased the relative abundances of Firmicutes and Bacteroidota (Bacteroidetes) in the murine gut. However, supplementation with LH reversed all of the alterations mentioned above, whereas LP failed to restore the richness of Proteobacteria and Fusobacteriota. At the genus level, ethanol consumption decreased the relative richness of Akkermansia, Blautia, Muribaculaceae, Fusobacterium, Lactobacillus and Alistipes in the mouse gut but increased the relative proportions of Bacteroides, Parabacteroides, Eubacterium coprostanoligenes, Faecalibaculum, Romboutsia, Monoglobus and Clostridia UCG 014., In contrast, administration of LH restored the abundance of Fusobacterium and Lactobacillus and decreased the abundance of Faecalibaculum, whereas LP did not (Figure 5B). The particular strains responsible for these intergroup distinctions were further explored, and detailed information is presented in Figure S3. Furthermore, based on 16S rRNA sequencing data and known microbial genome information, Picrust2 (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States 2) was used to predict the functional composition and metabolic potential of the microorganisms (Figure S4 for details).

LH enhanced the yield of gut microbial metabolites in ALD mice

Alcohol consumption alters the levels of intestinal metabolites, thereby affecting multiple systems throughout the body, including the liver (Albillos et al., 2020; Ohtani & Hara, 2021). As a member of the gut–liver axis, it has been proved that intestinal SCFAs are involved in the occurrence and development of ALD (Cassard & Ciocan, 2018), and can attenuate ethanol‐induced liver injury by regulating SCFA levels (Jiang et al., 2020; Wang et al., 2020). Therefore, the quantity of intestinal SCFAs in the colon content of each group was measured, including AA, PA, IA, BA, MBA, and VA. We discovered that alcohol intake led to substantial degradation in the levels of multiple SCFAs in the intestines of mice. However, LH intervention significantly corrected all the declines tested above, whereas LP only affected AA and BA (Figure 6). Tryptophanase, existing in Lactobacillus, can digest tryptophan from diets into a range of indole derivatives, which exert influence on the corresponding processes in the body (Agus et al., 2018). Thereby, the concentrations of five tryptophan derivatives in the cecal contents of each group of mice were assessed. We observed that alcohol gavage mainly altered the amount of IAA in the cecal contents of mice, whereas supplementation with LH tended to partially reverse this change, and the level of IAld was dramatically increased by the application of LH. However, the levels of L‐Trp, IAA, and IAld in the gut of mice in the LP group were considerably diminished (Figure S5).

FIGURE 6.

FIGURE 6

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Effects of LH and LP on gut microbial derivatives of ALD mice. The yields of six types of SCFAs in the colon contents of mice. p < 0.05 was identified as the statistical significance (*p < 0.05, **p < 0.01, and ***p < 0.001).

Correlation analysis of representative microbial genera, metabolites, gut barrier, and liver injury‐related indices

To further analyse the correlation among the metabolites, gut microbiome, gut barrier integrity, and liver injury, based on the Spearman correlation coefficient ρ (rho) as a measurement, the Spearman correlation analysis was performed. Heat map analysis revealed a robust positive correlation among serum inflammatory cytokines and chemokines, specifically IL‐1β, IL‐6, G‐CSF, CCL4, and CXCL1, as well as a pronounced positive association with LPS and hepatic injury markers, ALT and AST. Conversely, indices indicative of intestinal barrier integrity (Claudin1 and Occludin), exhibited a negative correlation with both ALT&AST and the inflammatory cytokines IL‐1β, IL‐6, and G‐CSF. The translocation of LPS into the blood induced systemic inflammatory responses, with reciprocal amplification of inflammatory mediators culminating in hepatic injury; however, the enhancement of gut barrier function mitigated systemic inflammation and liver injury. Furthermore, Faecalibaculum, which was significantly enriched in groups PC and LP, was markedly positively correlated with liver injury markers and inflammatory cytokines IL‐1b and CCL4 but negatively correlated with the gut barrier function‐related indices Claudin1 and Occludin. In contrast, Fusobacterium genus, which was more abundant in the NC and LH groups, was significantly negatively correlated with liver injury indicators. Additionally, the gut microbial metabolites PA and VA were negatively correlated with the chemokine CCL4, further implicating a complex interplay between the gut microbiota, intestinal barrier integrity, and liver injury. Hence, the correlation analysis demonstrated that regulating the gut microbiome and its derivatives could strengthen gut barrier integrity and relieve inflammation in the circulatory system and liver (Figure 7).

FIGURE 7.

FIGURE 7

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Intestinal metabolites, gut microbiota, gut barrier function, and liver injury association analysis heat map. The symbol * denotes that the difference between the two is statistically significant (p < 0.05).

DISCUSSION

The interaction between the gut microbiota and ALD has been extensively studied, with a focus on the genus Lactobacillus. Lactobacillus rhamnosus, Lactobacillus plantarum, and Lactobacillus acidophilus exert hepatoprotective effects by alleviating intestinal barrier dysfunction and gut microbiome disturbance, and in turn, reduce the translocation of intestinal bacteria and their derivatives (Ding et al., 2022; Hong et al., 2015; Li, Cheng, et al., 2022; Li, Wang, et al., 2022; Zhu et al., 2022). Lactobacillus casei modulates gut flora disorder and has a beneficial impact on the lipid metabolism of the liver in ALD patients (Li, Liu, et al., 2021). A randomised, double‐blind clinical trial discovered that daily supplementation with prescribed doses of Lactobacillus and Bifidobacteria (comprising Lactobacillus gasseri CBT LGA1, Lactobacillus casei CBT LC5, Bifidobacterium lactis CBT BL3, and Bifidobacterium breve CBT BR3) prior drinking can improve the capacity of the body to metabolise alcohol and relieve acute alcoholic liver impairment (Jung et al., 2021). A distinguishing feature of gut microbiome imbalance in ALD patients is severe symbiotic‐bacteria inhibition, of which Lactobacillus is one of the taxa strongly suppressed by alcohol intake (Yan et al., 2011). In this study, mice were fed alcoholic diets with supplementation of five culturable and edible type‐strains belonging to the genus Lactobacillus. Here, 16 s rRNA sequencing of gut microbes revealed that IG species failed to colonise massively to become the dominant strain, which may be due to the strong bactericidal ability of gastric acid and the short‐term intervention (15 days). Nonetheless, uncolonised Lactobacillus still demonstrated considerable hepatoprotective activity in ALD mice, particularly in the LH‐type strain, and the underlying mechanism was further explored.

Concerning the specific effects of the intervention species on the gut flora, alcohol intake decreased the richness of the gut microbiome but had less effect on its diversity, whereas LH markedly boosted the richness of intestinal microbes in ALD mice. Furthermore, alcohol intake resulted in G. Faecalibaculum (Fae) and Clostridia UCG‐014 being the primary discriminating taxa in the intestines of mice, which is not consistent with previous studies, suggesting that Fae and hepatic transaminases are negatively correlated (Yi et al., 2020). Fae is a member of F. Erysipelotrichaceae mainly existed in mice gut, whose human congener is Holdemanella biformis. (Chang et al., 2015; Cox et al., 2017; Zagato et al., 2020) Fae has not received enough attention yet, and previous researchers hold different views. A recent study found that a high‐sugar diet encourages Fae overgrowth in the mouse gut, which replaces certain microbiota that induces specific Th17 cells, resulting in the dysfunction of IL‐17‐relied intestinal epithelial cell lipid absorption, causing obesity and metabolic syndrome (Kawano et al., 2022). Also, Fae is involved in the metabolic disorder of the progression of diabetic kidney disease (Zhang et al., 2022). However, other studies reported that Fae inhibits retinoic acid in the proximal small intestine to reduce the proportion of eosinophils, accelerating intestinal epithelial proliferation and turnover, thereby maintaining epithelium stability while damaged; nevertheless, this report points out that Fae exacerbates anti‐CD3 induced injury, which may enhance susceptibility to the intestinal epithelium (Cao, Bae, et al., 2022). Besides, applying Fae and its metabolites to intestinal tumour mice models could inhibit malignant growth. (Zagato et al., 2020) Despite these encouraging findings, further studies are needed to fully understand the specific role of Faecalibaculum in ALD. Another dominant bacterium in the PC group, Clostridia UCG‐014, was correlated with impaired gut barrier function (Leibovitzh et al., 2022). Notably, ethanol feeding has been reported to enhance Escherichia coli population, but no significant increase in Escherichia coli was observed at the phylum or genus levels in our study. Escherichia coli belongs to the genus Escherichia, which belongs to the phylum Proteobacteria. Additionally, 16 s sequencing did not show Escherichia among the top 12 taxa at the genus level, which may be related to the depth of data mining. However, at the phylum level, Proteobacteria decreased in the PC group. However, Proteobacteria phylum has a wide variety of genera; therefore, it could not be concluded that E. coli decreased in the PC group. Consequently, we further consulted the relevant literature and found that there were studies indicating a significant reduction in Proteobacteria in the gut at the beginning of the alcoholic diet (~2 weeks) compared to the pair‐food group (Bull‐Otterson et al., 2013; Ferrere et al., 2017). Although the species chosen in this study belong to the genus Lactobacillus, the gut microbiome composition of each group differs following the intervention. After the supplementation of LH, G. Romboutsia (Rom), one of the dominant bacteria in the gut after supplementation with LH, is poorly studied. In studies on alcoholic liver injury in mice, the administration of ganoderic acids considerably improved alcohol‐induced hepatic oxidative stress and increased Rom levels in the gut; (Cao, Huang, et al., 2022) yet, in another study with fucoidan treatment, a decrease both in the expression of hepatic lipid metabolism‐related factors and the amount of Rom were observed (Xue et al., 2021). Furthermore, a drug treatment experiment targeting T2D rats demonstrates that Rom is negatively correlated with ALT, TC, TG, and LDL‐C but positively correlated with HDL‐C (Li, Jia, et al., 2021). This agrees with our data, which showed that the mice in group LH with Rom as one of the dominant gut flora had considerably lower ALT and TG levels. Currently, there are few reports on the relationship between Paludicola, the primary discriminating taxa in the LP group, and illness. In our study, LP had no significant effect on ALD liver damage. Another dominant taxon in group LP is Muribaculaceae, which has been identified as a producer of SCFAs and has demonstrated protective properties in numerous investigations on the effects of chemical treatment on ALD (Du et al., 2022; Lin et al., 2021; Lv et al., 2022; Xu, Li, et al., 2022). In our study, the gut microbiota in the LP group showed SCFA‐producing ability for AA and BA but was weaker than that in the LH group.

Our follow‐up studies revealed that alcohol altered not only the structure of the gut microbiome in mice but also its function. We conducted deeper data mining using Picrust2, performed functional prediction analysis on the top 35 taxa of each group, and found that alcohol significantly enhanced 80% of the pathways on biosynthesis, including deoxynucleotides, carbohydrates, branched‐chain amino acids (BCAA), and aromatic amino acids (AAA), which were markedly reversed by the administration of LH. Correspondingly, studies on NAFLD have shown that adolescents with this disease have higher plasma levels of BCAA and AAA, which are positively correlated with liver TG levels and insulin resistance (Goffredo et al., 2017; Lischka et al., 2021). However, another report suggested that long‐term alcohol intake is a cause of protein malnutrition and circulatory BCAA drop in human beings (Tedesco et al., 2018), and BCAA supplementation can alleviate the prognosis of patients with advanced liver cirrhosis (Charlton, 2006; Hiraoka et al., 2017; Mahpour et al., 2020). Hence, we hypothesised that the changes in BCAA metabolism of gut microbes in the alcohol‐fed group may be related to an early increase in compensatory behaviour.

SCFAs can suppress liver inflammation via T‐cell regulation and macrophage polarisation (Cassard & Ciocan, 2018; Ohtani & Hara, 2021; Wang et al., 2020). Furthermore, it also has a protective effect against ethanol‐induced gastrointestinal injury (Liu et al., 2017; Xu, Zhang, et al., 2022). Tryptophan derivatives improve alcohol‐induced liver injury by inducing aryl hydrocarbon receptor activation (Wrzosek et al., 2021). Correspondingly, lower production of indoles promotes inflammation, contributes to hepatocyte death, and results in a fibrotic response (Mendes & Schnabl, 2020). Consistent with the findings of our study, alcohol intake significantly decreases the levels of SCFAs in the gut tract of mice. However, the administration of LH demonstrated a potent reversal of this inhibitory effect, thereby maintaining elevated SCFA levels, which is positively correlated with the extent of liver damage in ALD mice. In contrast, the effects of alcohol and LH on the gut levels of tryptophan and its derivatives were comparatively less pronounced; however, they collectively manifested a trend analogous to that observed for SCFAs.

From the results of ALT, AST, and TBA, the chosen Lactobacillus strains had a certain degree of alleviating impact on alcoholic liver damage, except for LP. Images of liver pathology combined with Oil Red O staining demonstrated that the lipid droplets of hepatocytes in all intervention groups decreased, but only the LH and LC groups exhibited a statistically significant decrease in liver TG content by quantitative detection. It is reasonable to speculate that LH and LC not only shrink the volume of lipid droplets but also reduce their quantity, whereas the LS and LD groups only diminished in size, which may relieve liver injury through other means, such as inhibiting the inflammatory response, as confirmed by follow‐up MPO immunohistochemistry results and mRNA expression levels of chemokines Cxcl2 and Cxcl5. TLRs can be used to identify intestine‐derived PAMPs that play key roles in the incidence and progression of inflammation in ALD (Du et al., 2022; Li, Shi, et al., 2021; Yang et al., 2019). Long‐term alcohol abuse leads to weakened intestinal barrier function and gut microecology disorder, thereby raising the level of circulatory LPS, stimulating the TLRs in the liver and peripheral blood tissues, followed by inflammatory cell chemotaxis and cascade inflammatory responses (Szabo, 2015). Tlr4‐deficient mice fed with alcoholic diets own significantly lower serum hepatic transaminase content and liver inflammation levels than wild‐type mice (Uesugi et al., 2001), and another report further proves that Tlr4 ablation of mice hepatocytes or myeloid cells can prevent elevated serum hepatic transaminase caused by alcoholic liver damage (Jia et al., 2018, 4). Correspondingly, in this study, the mRNA expression levels of Tlr4 and its helper Md2 in the livers of mice in the LH group were markedly suppressed, and the levels of liver transaminases and liver neutrophil infiltration were significantly mitigated.

The mucus layer is one of the first protective lines of the gastrointestinal tract, and its structural integrity is highly dependent on Mucin‐2 (MUC‐2), which is regarded as a crucial component of the intestinal barrier that prevents the migration of gut bacteria and helps maintain intestinal homeostasis (Paone & Cani, 2020). For instance, barrier dysfunction of the mucus layer plays an essential role in the occurrence and persistence of IBD (including Crohn's disease and ulcerative colitis), which is provoked by gut microbes invading the colon lacking MUC2, inducing mucosal and submucosal inflammatory responses but can be alleviated by mucus barrier repairt (Al‐Shaibi et al., 2021; Yao et al., 2021). Additionally, the intestinal mucus layer, part of the gut–liver axis, is closely related to the incidence and development of NAFLD (Zhang et al., 2023). Nevertheless, studies that evaluate functions of the intestinal mucus layer in ALD are currently scarce, and most of them suggest that alcohol intake reduces MUC‐2 levels in the colon, leading to intestinal barrier dysfunction and inducing liver injury (Jiang et al., 2020; Kirpich et al., 2013; Lamas‐Paz et al., 2020), which is consistent with our research. However, reports claim that alcohol intake thickens the intestinal mucus layer (Wang et al., 2023), and in a study of Muc2 (−/−) ALD mice, it was reported that the deficiency of Muc2 showed lower plasma LPS levels and less alcohol‐induced liver injury (Hartmann et al., 2013). Notably, intestinal Lactobacillus was significantly higher in Muc2 (−/−) ALD mice than in alcohol‐fed WT mice. This has been proven to be associated with the reactive activation of the innate immune system of the mucosa and augmentation of the expression of related antimicrobial molecules (such as Reg3b and Reg3g), which reduce the bacterial load in the gut. This study further emphasises that the process described above is not a general response in Muc‐2‐deficient mice encountering intestinal damage or inflammation, but a specific reaction to alcohol. Furthermore, in our study, supplementation with LH markedly increased Muc3 and Muc4 mRNA expression; however, the increase in Muc2 expression was not statistically significant. This phenomenon may be related to LH traversing the thinner mucus layer (caused by an alcohol‐induced decrease in secretory mucins, mainly Muc‐2), eliciting an interaction with membrane‐bound mucins (Muc‐3 and Muc‐4) (van Tassell & Miller, 2011), thereby regulating the epithelial immune system, reducing the leakage of LPS through the gut barrier, and resulting in less liver injury (assessed by serum ALT and AST levels).

However, this study has some limitations. First, our study demonstrated the efficacy of particular strains against ALD in the experimental animals; however, their impact on humans requires further evaluation. Additionally, owing to the distinctive variations in the baseline gut microbiome of each host, it is necessary to employ a more accurate assessment on an individual basis.

In conclusion, this study pioneered the experimental treatment of five type strains of species from the genus Lactobacillus which are safe and culturable but rarely studied as single‐bacterial interventions for ALD, combining microbiomics, metabolomics, and machine learning. First, we found that LH, LC, LS, and LD alleviated liver injury in ALD mice, but LP did not. Then, we demonstrated that in group LH, this hepatoprotective effect was mainly achieved by regulating the structure and function of gut microbes, promoting the production of intestinal SCFAs, thereby enhancing the intestinal barrier function, suppressing the circulatory inflammation, and reducing hepatic lipid deposition. Finally, this study provides a new method for the prevention and treatment of ALD.

AUTHOR CONTRIBUTIONS

Jiawen Lv: Conceptualization; data curation; methodology; project administration; writing – original draft; writing – review and editing. Guanjing Lang: Validation; writing – review and editing. Qiangqiang Wang: Formal analysis; software; visualization. Wenlong Zhao: Formal analysis; software; visualization. Ding Shi: Funding acquisition; resources; supervision. Ziyuan Zhou: Methodology. Yangfan Shen: Methodology. He Xia: Methodology. Shengyi Han: Methodology. Lanjuan Li: Funding acquisition; resources; supervision.

CONFLICT OF INTEREST STATEMENT

Authors declare no conflict of interest.

ETHICS STATEMENT

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Supporting information

Data S1.

MBT2-17-e70016-s001.docx (1.3MB, docx)

ACKNOWLEDGEMENTS

The authors would like to thank the laboratory animal center of the First Affiliated Hospital of Zhejiang University for providing an SPF environment. This study was funded by the Fundamental Research Funds for the Central Universities (grant number 2022ZFJH003); the Shandong Provincial Laboratory Project (grant number SYS202202); and the Research Project of Jinan Microecological Biomedicine Shandong Laboratory (grant number JNL‐2022001A).

Lv, J. , Lang, G. , Wang, Q. , Zhao, W. , Shi, D. , Zhou, Z. et al. (2024) Lactobacillus helveticus attenuates alcoholic liver injury via regulation of gut microecology in mice. Microbial Biotechnology, 17, e70016. Available from: 10.1111/1751-7915.70016

Jiawen Lv and Guanjing Lang contributed equally to this work.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available in [Sequence Read Archive (SRA)] at [https://www.ncbi.nlm.nih.gov/sra/PRJNA1018882], reference number [PRJNA1018882].

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Data S1.

MBT2-17-e70016-s001.docx (1.3MB, docx)

Data Availability Statement

The data that support the findings of this study are available in [Sequence Read Archive (SRA)] at [https://www.ncbi.nlm.nih.gov/sra/PRJNA1018882], reference number [PRJNA1018882].

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