Lactobacillus protects against lead-induced hepatotoxicity by preserving the gut barrier and microbiota remodeling

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This study explored the protective effects of lactobacilli against Pb(II)-induced hepatotoxicity in mice. Three strains of lactobacilli— Lacticaseibacillus paracasei GMNL-32, Limosilactobacillus fermentum GMNL-93, and Lacticaseibacillus casei GMNL-277—were evaluated for Pb adsorption and cytoprotective properties. The results indicated that probiotic treatment reduced the liver-to-body weight ratio, aspartate transaminase and alanine transaminase levels, and liver damage without increasing Pb excretion. It also upregulated the expression of gut tight junction proteins, reduced the levels of inflammatory cytokines (tumor necrosis factor-α and interleukin-6), and modulated the diversity and composition of the gut microbiota. Strong correlations were observed between probiotics, microbial abundance, metabolic pathways, and reduced liver inflammation. Overall, this study suggests that GMNL-32, GMNL-93, and GMNL-277 can mitigate Pb-induced hepatotoxicity by modulating the gut microbiota and regulating metabolism. Thus, these probiotics hold promise as protective agents against Pb-induced hepatotoxicity. lead toxicity Lacticaseibacillus paracasei Limosilactobacillus fermentum Lacticaseibacillus casei hepatotoxicity gut microbiota intestinal barrier Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Heavy metals are naturally occurring elements with high atomic weights and densities at least five times greater than that of water. Some heavy metals, such as iron and zinc, serve as essential nutrients at low concentrations. Other heavy metals, such as lead (Pb), mercury, and arsenic, have no known biological function and can be toxic even in trace amounts [ 1 ]. Heavy metal toxicity occurs when these metals accumulate in the body to levels that impair physiological function and cause harm. Since 2017, public health and ecological concerns regarding heavy metal toxicity have increased as a result of the global increase in environmental pollution and industrial activity [ 2 ]. Pb, one of the most dangerous heavy metals, poses major risks to human and animal health and the environment [ 3 ]. Once absorbed, Pb has a half-life of approximately 30 days in the blood. Thereafter, it diffuses into soft tissues, such as the kidneys, brain, and liver, and is subsequently distributed to the bones, teeth, and hair in the form of Pb(II) phosphate [ 4 ]. This pathway indicates that the toxic effects of Pb are multifaceted and can affect multiple organ systems. Pb is well known for its neurotoxic effect on both the central and peripheral nervous systems [ 5 ]. Studies have indicated that Pb-induced brain damage can result in various neurological disorders, such as cognitive impairment, motor dysfunction, and encephalopathy, particularly in children [ 5 – 7 ]. In adults, chronic Pb exposure has been linked to health problems such as reduced fertility [ 8 ], cataracts [ 9 ], neuromuscular disorders [ 10 ], and memory and concentration deficits [ 11 ]. Multiple studies have highlighted other health risks associated with Pb exposure. For example, hypertension, cardiovascular disease [ 12 ], renal disease, liver damage, and hematologic diseases [ 13 ] are currently recognized as potential consequences of Pb exposure. These findings underscore the importance of monitoring and mitigating Pb exposure to safeguard public health. Although the use of Pb has been restricted in many countries, Pb exposure remains a concern, particularly in developing countries, necessitating effective management of Pb accumulation in these regions [ 14 ]. Evidence suggests that chelation therapy with edetate calcium disodium and meso-2,3-dimercaptosuccinic acid can protect against Pb poisoning by promoting the excretion of this heavy metal [ 15 , 16 ], but this therapy is reserved only for preclinical use. In addition to chelators, diets supplemented with essential metals, vitamin C, and phytochemicals have been reported to reduce the absorption of Pb in the body [ 16 ]. Probiotics are live microorganisms that confer health benefits when administered in adequate amounts. Recent research has assessed certain probiotics for their potential to prevent Pb absorption or alleviate Pb poisoning [ 17 , 18 ]. Some probiotic strains, particularly lactic acid bacteria, can bind to Pb ions in the gastrointestinal tract and reduce their absorption into the body [ 19 ]. This binding effect may be attributable to the cell wall components of the probiotic bacteria. Evidence also suggests that probiotics can modulate the composition and function of the gut microbiota, which may be disrupted by the administration of Pb, and thus regulate the metabolism and excretion of Pb [ 20 , 21 ]. In addition, probiotics can strengthen the intestinal barrier, whose integrity is impaired by Pb, thereby hindering the translocation of Pb or bacteria from the gut lumen into the bloodstream [ 22 , 23 ]. Lactic acid bacterial strains can bind to Pb and reduce its absorption, thus mitigating Pb poisoning. However, limited in vivo and clinical evidence is available supporting the effectiveness of these strains in preventing Pb-induced organ damage. In this study, we identified specific lactic acid bacterial strains capable of adsorbing Pb or mitigating its toxicity. Furthermore, we investigated whether these probiotic bacteria can protect organs from Pb-induced damage and explored the mechanisms underlying the potential protective effects. Materials and methods Bacterial strains and culture conditions Eight Lactobacillus strains were obtained from GenMont Biotech (Tainan, Taiwan) and screened for their Pb(II) adsorption, Pb(II) tolerance, and antioxidative properties (Table 1 ). These strains were cultured in De Man–Rogosa–Sharpe broth (BD Difco, Franklin Lakes, NJ, USA) at 37°C for 18 h under anaerobic conditions. They were then subcultured twice before using in in vitro experiments. Lactobacillus lysates were prepared by thoroughly homogenizing the bacteria by using a bacteriolytic device (FastPrep-24; MP Biomedicals, Irvine, CA, USA) while ensuring complete cell disruption to generate whole-cell lysates. These lysates were subjected to high-speed centrifugation at 20,000 × g for 5 min at 4°C to spin down unbroken bacterial cell debris. For details on this experimental procedure, please refer to the study by [ 24 ]. Cell culture Human colon carcinoma (HT-29; ATCC® HTB-38™) and hepatocellular carcinoma (HepG2; ATCC® HB-8065™) cell lines were obtained. HT-29 cells were cultured in Roswell Park Memorial Institute 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. HepG2 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Pb(II) chemicals Pb chloride (PbCl 2 ) and Pb sulfide (PbS) were procured from Thermo Fisher Scientific (Waltham, MA, USA) and dissolved in sterile distilled water for experimental use. Pb(II) adsorption assay A mixture of 1 mL of PbCl 2 solution (Pb(II) concentration: 30 mg/L) and 1 mL of 4 × 10 8 CFU/mL Lactobacillus in HEPES buffer (100 mM; pH 6.8) was incubated at 37°C for 1 h. This mixture was then centrifuged at 10,000 x g or 5 min, and the supernatant and bacterial pellets were separately subjected to aqua regia digestion. The concentration of Pb(II) was quantified through flame atomic absorption spectrophotometry (SensAA; GBC Scientific Equipment, Keysborough, Australia). All assays were performed in triplicate, and Pb(II) adsorption capacity was calculated as follows: $$\:\%\:Pb\:adsorption\:=\:\frac{{C}_{pellet}}{{C}_{supernatant}\:+{C}_{pellet}}\:\times\:100$$ Pb(II) tolerance assay A minimum inhibitory concentration assay was performed to determine the Pb(II) tolerance of Lactobacillus . Each Lactobacillus strain was cultured (density: 1 × 10 6 CFU/well) with varying concentrations of PbCl 2 solution (Pb(II) concentration: 10–1000 mg/L) on a 96-well plate at 37°C for 24 h. The minimum inhibitory concentration was identified as the lowest Pb(II) concentration that completely inhibited bacterial growth. Antioxidant assay After probiotic samples were centrifuged, they were washed, resuspended in methanol (10 mg/mL), and filter-sterilized. Ascorbic acid was used as a positive control (10 µg/mL). Each sample or control solution (100 µL) was mixed with 100 µL of 2,2-diphenyl-1-picrylhydrazyl (DPPH) solution (0.1 mM) in a 96-well microplate and incubated in the dark for 30 min. Finally, absorbance was measured at 517 nm to determine DPPH-scavenging activity. Cell viability assay HepG2 or HT-29 cells were seeded at a density of 2 × 10 4 cells/well on 96-well plates and incubated overnight. These cells were then treated with PbCl 2 solution alone or in combination with Lactobacillus lysates for 48 h. Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Animal experiment Male C57BL/6 mice (age: 8 weeks) were obtained from the Animal Center of National Cheng Kung University (Tainan, Taiwan) and housed under controlled environmental conditions (temperature: 25°C; humidity: 58%; photoperiod: 12-h light/dark) with ad libitum access to food and water. The animal study protocol was approved by the Institutional Animal Care and Use Committee of National Cheng Kung University (approval number: 110289). The mice were randomly divided into five groups, each comprising five mice: naïve control (untreated), Pb(II) + H 2 O (Pb alone), Pb(II) + GMNL-32, Pb(II) + GMNL-93, and Pb(II) + GMNL-277. Lyophilized live probiotic powders were orally administered at a dosage of 1.64 × 10 7 CFU/mouse in 0.2 mL of sterile water once daily for 5 days a week for a total of 8 weeks. The mice received either normal drinking water or water containing 1000 mg/mL Pb(II), which was replaced every week. The body weight and water consumption of each mouse were recorded on a weekly basis. At the end of week 8, all mice were euthanized using CO 2 . Pb contents in tissues and feces Liver, kidney, and fecal samples were homogenized in aqua regia by using a FastPrep-24 instrument (MP Biomedicals). These samples were centrifuged at 13,000 x g for 3 min. The supernatant was analyzed to determine the concentration of Pb through atomic absorption spectrophotometry. Pb content was expressed in milligrams/liter per gram of tissue or feces wet weight. Blood biochemistry and hematology Serum levels of alanine transaminase (ALT), aspartate transaminase (AST), and creatinine were measured using a DRI-CHEM 4000i chemistry analyzer (Fujifilm, Tokyo, Japan). Hematological parameters were assessed in ethylenediaminetetraacetate–whole blood by using an Scil Vet Focus 5 analyzer (Seneca Scientific, Denver, CO, USA). Expression levels of tight junction proteins and proinflammatory cytokines in the gut Total RNA was extracted from mouse jejunum tissues and converted into complementary DNA by using a reverse transcription kit (Thermo Fisher Scientific). The expression levels of tight junction proteins and proinflammatory cytokines were determined through quantitative real-time polymerase chain reaction (PCR) with specific primers (Supplementary Table S1 ) and a SYBR Green PCR Kit (Qiagen, Hilden, Germany). The expression of target genes was normalized to that of mouse Gapdh and calculated using the 2 −ΔΔCt method. Histology Liver samples were dissected and fixed in neutral buffered formalin for 24 h, routinely processed, and embedded in paraffin wax. Subsequently, 5-µm sections were dewaxed, rehydrated, and stained using standard hematoxylin and eosin. Then, these sections were examined for liver pathology. The number of oval cells in portal areas was counted in 10 high-power fields under a microscope. 16S ribosomal DNA sequencing After each mouse’s ileum was homogenized, the supernatant containing intestinal microorganisms was collected. DNA extraction was performed using a Qiagen DNA kit, following the manufacturer’s protocol. The extracted DNA was analyzed; the 260/280 optical density ratio ranged from 1.8 to 2.0. Subsequently, 16S ribosomal DNA was amplified using metagenomic DNA (template) and bacteria-specific primers (S17 and A21). The size of the amplified DNA was verified using a fragment analyzer (Agilent 5300 Fragment Analyzer; Agilent Technologies, Santa Clara, CA, USA). Then, it was sequenced on an Illumina MiSeq platform (Illumina, San Diego, CA, USA). DNA libraries were constructed using a Nextera XT Index Kit v2, mixed using a 600-cycle MiSeq Reagent Kit v3, and sequenced in a 2 × 300-bp paired-end run. The resulting sequences were filtered for quality and merged, and low-quality or chimera sequences were removed. Operational taxonomic units were clustered at 97% similarity by using the Greengenes database (v.13.8). Further analysis was performed using a Qiagen CLC Microbial Genomics Module (v.10.1.1). Processing and analysis of metataxonomic data The alpha diversity of taxonomic composition was assessed using the Shannon diversity index, which accounts for both species richness and distribution evenness within each group. Beta diversity, reflecting differences in microbial composition across groups, was measured through a UniFrac analysis weighted by a principal coordinate analysis. Linear discriminant analysis effect size (LEfSe) was performed to identify microbial markers by using the Galaxy/Hutlab webtool. To analyze LEfSe data, pairwise comparisons were performed using Wilcoxon’s rank-sum exact test and permutational multivariate analysis of variance. Functional abundance was predicted on the basis of marker gene sequences by using the PICRUSt2 tool (v.2.3.0b0). Intergroup comparisons were performed using two-tailed Student’s t tests. Statistical significance was set at p < 0.05. Prism (v.8; GraphPad Software, San Diego, CA, USA) was used to determine taxonomic differences and generate relative abundance plots. Statistical analysis Comparisons involving more than two groups were evaluated by one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test to assess pairwise differences. For comparisons between two independent groups, an unpaired Student’s t -test was applied. A p -value < 0.05 was considered statistically significant. Results Pb(II) adsorption capacity varies across Lactobacillus strains Multiple studies have demonstrated the biosorption of heavy metals by lactic acid bacteria to reduce the concentrations of Pb and cadmium in milk [ 25 , 26 ]. In the present study, to select lactic acid bacterial strains suitable for our experiments, we measured the Pb(II)Cl 2 adsorption capacities of eight single Lactobacillus strains (Table 1 ). Our results indicated that five of the tested strains absorbed at least 61% of the total Pb(II): Lacticaseibacillus paracasei GMNL-32 (61.4% ± 0.4%), Limosilactobacillus fermentum GMNL-93 (97.6% ± 1.4%), Limosilactobacillus fermentum BCRC 910720 (74.6% ± 6.7%), Limosilactobacillus reuteri BCRC 910340 (61.1% ± 2.0%), and Lacticaseibacillus casei GMNL-277 (67.9% ± 7.2%). Among these strains, GMNL-93 exhibited a Pb(II) adsorption rate of > 97% and a tolerance level of > 1000 mg/L, which highlighted its potential efficacy in Pb(II) removal. Regarding DPPH clearance, GMNL-32, GMNL-93, and GMNL-277 exhibited clearance rates of > 50%, indicating strong free radical–scavenging capacity. This antioxidant activity is particularly noteworthy because it enables the probiotics to mitigate oxidative stress induced by heavy metal exposure—a key factor in Pb-induced cellular damage. Thus, GMNL-32, GMNL-93, and GMNL-277 were used in subsequent experiments. Table 1 Pb absorption, tolerance, and antioxidant activity of selected Lactobacillus strains. Bacteria species * Strain Pb absorption (%) Pb tolerance (mg/L) # DPPH clearance rate (%) 1 L. acidophilus BCRC 910774 44.7 ± 1.9 > 1000 2 L. casei BCRC 910585 (GMNL-277) 67.9 ± 7.2 > 1000 55.10 ± 1.14 3 L. paracasei BCRC 910220 (GMNL-32) 61.4 ± 0.4 > 1000 50.53 ± 2.15 4 L. fermentum BCRC 910259 (GMNL-93) 97.6 ± 1.4 > 1000 84.46 ± 0.45 5 L. fermentum BCRC 910720 74.6 ± 6.7 > 1000 6 L. plantarum BCRC 911066 44.7 ± 0.7 > 1000 7 L. plantarum BCRC 910776 44.3 ± 0.1 > 1000 8 L. reuteri BCRC 910340 61.1 ± 2.0 > 1000 Lead (Pb) absorption capacity, Pb tolerance, and antioxidant activity (DPPH clearance rate) of different Lactobacillus and Lactobacillus reuteri strains. Pb absorption values are expressed as mean ± SD, Pb tolerance as the maximum concentration tolerated, and antioxidant activity was determined using the DPPH assay. * Bioresource Collection and Research Center (BCRC), Taiwan. #2,2-Diphenyl-1-picrylhydrazyl (DPPH)-scavenging assay. Scanning electron microscopy visually confirmed the adsorption of Pb(II) into bacterial cell surfaces (Fig. 1 A and B). To determine whether the S-layers of probiotics are essential for Pb(II) adsorption, we removed the S-layers from GMNL-93 and GMNL-277. This removal markedly reduced the rate of Pb(II) adsorption by GMNL-93 from 97% to 44% (Fig. 1 C); only a minor reduction of approximately 10% was noted for GMNL-277 (Fig. 1 D). Taken together, these results indicate that although some Lactobacillus strains adsorb Pb(II) in an S-layer-dependent manner, others do not rely on S-layers for adsorption (Fig. 1 ). Protective effects of Lactobacillus against Pb(II)-induced cytotoxicity To assess the cytoprotective effects of GMNL-93 and GMNL-277, human HepG2 hepatoma cells and HT-29 intestinal cells were exposed to Pb(II)Cl 2 either alone or in combination with Lactobacillus probiotic lysates. Cell viability was measured after each treatment. In HepG2 cells, cell viability decreased with increasing Pb(II) concentrations, with a half-maximal inhibitory concentration (IC 50 ) of 98 mg/L (Fig. 2 A). Depending on the IC 50 value for Pb-induced cytotoxicity, cell death caused by 100 mg/L Pb(II) was significantly reduced when HepG2 cells were treated with GMNL-93 lysates (Fig. 2 B). Consistent with the results in HepG2 cells, in vitro assays determining the protective effects of probiotics against Pb(II) poisoning in HT-29 cells revealed marked reductions in Pb(II)-induced cytotoxicity after treatment with GMNL-277 lysates (Fig. 2 C and D). Collectively, these in vitro results suggest that Lactobacillus can protect hepatic and intestinal cells against Pb(II)-induced damage. Protective effects of Lactobacillus against Pb(II)-induced liver injury To understand the role of Lactobacillus strains in chronic Pb(II)-induced toxicity, we treated mice with Pb(II)Cl 2 at a concentration of 1000 mg/L either alone or in combination with probiotics (GMNL-32, GMNL-93, and GMNL-277) for 8 weeks. Figure 3 A presents the timeline of each treatment and the tissue samples collected. During this experiment, no significant difference was observed in body weight between mice drinking Pb(II)-containing water with or without probiotics (GMNL-32, GMNL-93, and GMNL-277) and the control group (Fig. 3 B). Supplementary Figure S1 depicts the body weight of each mouse. No significant between-group difference was observed in the amount of water consumed (Supplementary Fig. S1 ). Moreover, no significant between-group differences were observed in white blood cell count, but a slight reduction in neutrophil count was observed in mice treated with GMNL-93 plus Pb(II) (Supplementary Table S2). Notably, all mice exposed to Pb(II) exhibited a slight reduction in mean corpuscular volume, suggesting Pb(II)-induced toxicity of red blood cells (Supplementary Table S3). Mice exposed to Pb(II) alone developed moderate hepatomegaly, and a significant increase was observed in the liver-to-body weight ratio (Fig. 3 C). Treatment with probiotics led to various degrees of reduction in this ratio, with GMNL-277 being the most effective probiotic (Fig. 3 C). Mice treated with Pb(II) exhibited significantly elevated levels of AST (mean: 436.8 U/L) and ALT (mean: 227.8 U/L), indicating liver damage (Fig. 3 D and E). Furthermore, mice treated with probiotics exhibited varying levels of AST, with GMNL-93 significantly reducing the level of AST (Fig. 3 D). Similarly, treatment with probiotics, particularly with GMNL-93 and GMNL-277, significantly reduced the levels of ALT (Fig. 3 E). A histological comparison of mice exposed to Pb(II) and mice treated with probiotics revealed a marked reduction in liver damage in probiotic-treated mice, with fewer oval cells and a more intact liver architecture (Fig. 3 F; Supplementary Fig. S2). Taken together, these results suggest that Lactobacillus strains can protect against Pb(II)-induced liver damage. Hepatoprotective effects of Lactobacillus are not mediated through enhanced Pb(II) excretion To determine whether the ability of Lactobacillus to adsorb Pb directly contributes to its protective effects against Pb(II)-induced hepatotoxicity, we measured the concentrations of Pb(II) in the livers, kidneys, and feces of mice after treatment with different probiotic strains. In addition, we measured corresponding Pb concentrations in the control group. However, our results indicated that hepatic Pb concentration was significantly higher in mice treated with Pb(II) alone or in combination with GMNL-32, GMNL-93, and GMNL-277 than in control mice (Supplementary Fig. S3A). Similarly, renal Pb concentration was significantly elevated in all the Pb-treated groups relative to the control (Supplementary Fig. S3B). During the 8-week experiment period, the concentrations of Pb in the feces did not significantly differ between the groups (Supplementary Fig. S3C). Collectively, these results suggest that Lactobacillus strains do not enhance the excretion of Pb(II) through feces and, therefore, do not reduce the accumulation of Pb in the kidneys or liver. Lactobacillus protects mucosal barriers from Pb(II)-induced intestinal damage Lactobacillus strains strengthen the gut mucosal barrier, which is essential for maintaining gut health and preventing inflammation [ 27 ]. Therefore, liver health is closely associated with gastrointestinal integrity, and a compromised intestinal barrier may precipitate hepatic pathology [ 28 ]. To confirm whether the probiotic strains used in this study promoted gut epithelial integrity, we analyzed the mRNA expression levels of various tight junction proteins, such as Claudin 3 (Cldn3), Claudin 5 (Cldn5), and Mucin-5 ( Muc5 ), in an in vivo experiment. Our results indicated that the expression of Cldn3 , Cldn5 , and Muc5 was significantly upregulated in response to Pb(II) exposure with GMNL-277 treatment (Fig. 4 A–C). Furthermore, treatment with GMNL-32 and GMNL-93 significantly upregulated the expression of Cldn5 (Fig. 4 B). Regarding inflammatory cytokines, none of the probiotics modulated the expression of Tnf with Pb(II) exposure (Fig. 4 D). However, all probiotics exhibited a tendency to reduce Pb(II)-induced interleukin (IL)-6 upregulation (Fig. 4 E). Collectively, these results indicate that Lactobacillus can protect intestinal cells from Pb(II)-induced damage and can exert anti-inflammatory effects. Lactobacillus modulates gut microbiome composition in response to Pb(II) exposure To determine how intestinal microbiota respond to Pb(II) exposure and lactobacilli, we analyzed the gut microbiome composition of each mouse. For this, 16S ribosomal DNA sequencing was performed for each sample to profile each mouse’s gut microbiome. In addition, LEfSe was performed to identify differentially abundant bacterial taxa in the Pb(II) alone group and the Pb(II) + probiotic groups. The Chao1 and Shannon index values indicated that mice treated with GMNL-32 and GMNL-277 exhibited higher alpha diversity values than did those exposed to Pb(II) alone. However, mice treated with GMNL-93 exhibited significantly lower alpha diversity values than did those exposed to Pb(II) alone, as indicated by the Shannon index (Fig. 5 A). Principal coordinate analysis revealed that the GMNL-32 and GMNL-277 groups had similar microbiome compositions, whereas the GMNL-93 group had a different composition (Fig. 5 B). Moreover, significant differences were observed between the microbiome compositions of the GMNL-32 and GMNL-93 groups and that of the Pb(II) alone group (Fig. 5 B). We further evaluated the relative abundance of gut microbes in each mouse (Fig. 5 C and D) and noted patterns of microbial shifts. Exposure to Pb(II) led to dysbiosis, characterized by reduced abundances of Bacteroides spp., Parabacteroides spp., and Prevotella spp. and an increase abundance of Turicibacter spp. Treatment with GMNL-32 and GMNL-277 restored the gut microbiome composition (Supplementary Fig. S4). Compared with the Pb(II) alone group, the GMNL-32 group exhibited significantly increased abundances of Bacteroides spp., Parabacteroides spp., and Prevotella spp. (Fig. 6 A), whereas the GMNL-32 and GMNL-277 groups exhibited increased abundances of Anaerostipes spp., Alistipes spp., and Streptococcus spp. (Fig. 6 B). Furthermore, the relative abundances of the dominant microbiota exhibited distinct patterns in the Pb(II) alone and Pb(II) + GMNL-93 groups, differing from those observed in the GMNL-32 and GMNL-277 groups, which exhibited the abundances of Bacillus , Firmicutes, and Bifidobacterium pseudolongum in the gut (Fig. 6 C). Lactobacillus may exert its hepatoprotective effects by modulating gut microbiome and metabolism In this study, we observed a distinct bacterial profile in each mouse group. This distinction suggests that Lactobacillus -induced variations in gut microbiome diversity lead to unique health outcomes and metabolic shifts. To test this hypothesis, we investigated the correlation between bacterial abundance and body weight gain. The presence of Bacteroides caccae , which was significantly abundant in the GMNL-32 ( p = 0.037 vs. Pb(II) alone) and GMNL-277 groups, was strongly and positively correlated with weight gain ( r = 0.409; p = 0.047) in mice (Fig. 7 A and B). Similarly, the presence of Prevotella copri , which was also significantly abundant in the GMNL-32 ( p = 0.007 vs. Pb(II) alone) and GMNL-277 groups, was positively correlated with weight gain ( r = 0.405; p = 0.049) in mice, confirming the role of P. copri in maintaining healthy weight in the presence of Pb(II) (Fig. 7 C and D). A PICRUSt2 analysis was performed to identify active metabolic pathways across different mouse groups (Supplementary Fig. S5). The results revealed that the thiamine metabolism pathway was particularly active in the GMNL-277 group, and this pathway exhibited a significant negative correlation with ALT ( r = − 0.562; p = 0.004) and AST ( r = − 0.522; p = 0.009) levels (Fig. 7 E and F). Additionally, the pantothenate and coenzyme A (CoA) biosynthesis pathways were particularly active in the GMNL-32 and GMNL-277 groups ( p < 0.001 and p = 0.023, respectively; Fig. 7 G). In these groups, key bacterial species, such as Bacteroides uniformis , B. caccae , Bacteroides fragilis , Gemmiger formicilis , P. copri , and Faecalibacterium prausnitzii , were positively associated with the pantothenate and CoA biosynthesis pathways (Fig. 7 H). A significant reduction was observed in the abundance of Bacteroides plebeius in the GMNL-93 + Pb(II) group ( p = 0.003, Fig. 8 A). A positive correlation was observed between the abundance of this bacterium and the severity of liver pathology, measured in terms of the liver-to-body weight ratio ( r = 0.511; p = 0.011; Fig. 8 B). Treatment with GMNL-93 significantly reduced the abundance of Burkholderia spp. ( p = 0.016). The abundance of Burkholderia spp. was strongly and positively correlated with elevated levels of ALT ( r = 0.686; p < 0.001; Fig. 8 C) and AST ( r = 0.599; p = 0.002; Fig. 8 D). Pb(II) exposure plus GMNL-93 treatment substantially enhanced the metabolism of D-glutamate (Glu) and D-glutamine (Gln) ( p = 0.016; Fig. 8 E). This enhancement was significantly and negatively correlated with ALT ( r = − 0.606; p = 0.002) and AST ( r = − 0.437; p = 0.033) levels (Fig. 8 F). Both the pentose phosphate pathway (PPP) and the secondary bile acid synthesis pathway were significantly upregulated in the GMNL-93 + Pb(II) group (Fig. 8 G and I). These metabolic changes were strongly and negatively correlated with ALT level (PPP pathway: r = − 0.616 [ p = 0.001]; secondary bile acid synthesis pathway: r = − 0.558 [ p = 0.005]; Fig. 8 H) and AST level (PPP pathway: r = − 0.446 [ p = 0.029]; secondary bile acid synthesis pathway: r = − 0.385 [ p = 0.063]; Fig. 8 J). Taken together, these findings suggest that GMNL-93 mitigates liver damage by modulating key metabolic pathways in response to Pb(II) exposure. Discussion Our study indicated that the Lactobacillus strains GMNL-32, GMNL-93, and GMNL-277 exhibited varying levels of Pb(II) adsorption, with GMNL-93 exhibiting the highest rate of adsorption. These findings are consistent with those of studies indicating the ability of lactic acid bacteria to reduce the concentrations of Pb and cadmium in milk through biosorption [ 25 , 26 ]. According to the literature, the cell wall of Lactobacillus , which is rich in negatively charged molecules such as peptidoglycans and teichoic acids, may bind positively charged Pb(II); this binding leads to the sequestration of Pb and reduces its bioavailability [ 29 ]. The adsorption process may prevent Pb from participating in harmful biochemical reactions, particularly those generating reactive oxygen species (ROS), which cause oxidative stress and cell damage [ 30 ]. In the present study, we discovered that S-layers played a key role in the biosorption of Pb(II) by Lactobacillus . This finding is consistent with those of [ 31 ], who stated that Pb(II) binds to bacterial cell wall components such as fatty acids, polysaccharides, S-layer proteins, and teichoic acids. [ 31 ] also reported that the heavy-metal-binding capacity of a certain Lactobacillus bulgaricus strain mitigated Pb(II)-induced toxicity in mice by reducing systemic absorption. Our in vivo experiments revealed that various probiotic strains exerted protective effects against Pb(II)-induced liver damage, as evidenced by reductions in the liver-to-body weight ratio and AST and ALT levels. These findings are consistent with those of studies highlighting the hepatoprotective effects of probiotics against various toxins and stressors [ 27 , 28 ]. As shown in Supplementary Figure S3, the hepatoprotective effects of Lactobacillus were not attributable to enhanced Pb(II) excretion or to postbiotics, given that the concentrations of Pb(II) in the liver, kidneys, and feces did not vary significantly across the treatment groups. Therefore, these protective effects may be mediated by indirect mechanisms that involve interactions with the gut microbiota or the regulation of gut function, as evidenced by the fact that Lactobacillus protected the gut mucosal barrier against Pb(II)-induced damage and upregulated the expression of tight junction proteins while maintaining normal IL-6 levels (Fig. 4 E). These results are consistent with those of studies indicating that probiotics strengthen the gut mucosal barrier, a key player in gut health and inflammation prevention [ 22 , 23 ]. Chronic Pb exposure induced dysbiosis, characterized by a reduction in the abundance of beneficial bacteria belonging to phylum Firmicutes, which play vital roles in maintaining gut health and metabolic balance [ 32 ]. This phylum includes key genera such as Lactobacillus , Bacillus , Enterococcus , and Ruminococcus [ 33 ]. Our results indicated that treatment with lactic acid bacteria, particularly GMNL-32 and GMNL-277, reversed the process of Pb(II)-induced dysbiosis by enriching Firmicutes species, such as Bacillus and Ruminococcus spp., as well as beneficial Bacteroides spp. and P. copri (Fig. 6 A and B). As shown in Fig. 6 C, Pb(II)-induced dysbiosis promoted the growth of pathogens, particularly those belonging to phylum Proteobacteria. Treatment with GMNL-93 significantly reduced the abundance of Proteobacteria while increasing that of Firmicutes (Fig. 6 C). Collectively, these results underscore the potential protective effect of GMNL-93 against gut dysbiosis. Our study indicated that certain probiotic strains substantially influenced the composition and metabolic activity of the gut microbiome in response to Pb(II) exposure. Gut dysbiosis contributes to the onset and progression of liver diseases such as nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, cirrhosis, and hepatocellular carcinoma [ 34 ]. In the present study, treatment with GMNL-32 and GMNL-277 substantially increased biodiversity and resulted in microbiome profiles similar to those of untreated mice, characterized by a high abundance of Bacteroides (Fig. 7 A and C). Studies have highlighted the roles of Bacteroides in maintaining gut health [ 35 ] and regulating inflammation through organ-to-organ communication [ 36 ]. Research has also indicated that B. caccae enhances mucus degradation, which in turn mitigates intestinal inflammation by minimizing bacterial interaction with the intestinal epithelium [ 37 ]. In the present study, the abundance of B. caccae was strongly correlated with the improvement of liver function in Pb(II)-treated mice; the abundance of other Bacteroides species, such as B. fragilis and B. uniformis , was significantly correlated with the biosynthesis of pantothenate and CoA (Fig. 7 H). These processes protect the liver from fat accumulation and fibrosis [ 38 ]. Together, these findings confirm that Bacteroides mitigates liver disease by restoring the metabolic and microbial environment of the gut–liver axis [ 39 ]. In the present study, treatment with GMNL-32 and GMNL-277 increased the abundances of B. caccae and P. copri in Pb(II)-exposed mice (Fig. 7 A and C). According to the literature, B. caccae enhances mucus degradation, which in turn reduces intestinal inflammation by minimizing bacterial interaction with the intestinal epithelium [ 37 ]. Notably, P. copri is one of the most prevalent species in the human gut microbiome [ 40 ]. This species has been extensively studied for its protective effects against insulin insensitivity and liver fibrosis, potentially achieved through the modulation of gut microbial activity [ 40 – 43 ]. In this study, the abundances of B. caccae and P. copri were strongly correlated with the amelioration of Pb(II)-induced toxicity. This finding is consistent with those of studies indicating that Bacteroides and Prevotella alleviate hepatic disease by restoring the metabolic and microbial environment of the gut–liver axis [ 39 , 44 ]. Our results indicated that GMNL-277 exerted significant effects on the thiamine metabolism pathway (Fig. 7 E), which was negatively correlated with AST and ALT levels in Pb(II)-exposed mice treated with GMNL-277 (Fig. 7 F). The thiamine metabolism pathway may play a role in probiotic-mediated liver protection. Thiamine (vitamin B1) is a crucial component of several key metabolic processes in the liver, particularly those involving the metabolism of carbohydrates and amino acids [ 45 , 46 ]. Adequate thiamine levels are essential for ensuring smooth functionality of these processes, which prevents the accumulation of toxic substances that may damage the liver. Our findings are consistent with those of Wang et al. (2007a), who reported that vitamin C and B1 supplementation can mitigate Pb-induced liver damage (Fig. 7 E and F). In the present study, GMNL-32 and GMNL-277 significantly regulated the pantothenate and CoA biosynthesis pathways in Pb(II)-exposed mice (Fig. 7 G). Pantothenate, a precursor of CoA, is a key component of cellular metabolism, particularly in the tricarboxylic acid cycle; its deficiency has been associated with various metabolic diseases [ 47 ]. Because the liver plays a central role in tricarboxylic-acid-related processes such as gluconeogenesis, lipogenesis, and ureagenesis, maintaining appropriate CoA levels is crucial for optimal liver function [ 48 ]. In this study, the abundances of various Bacteroides species, such as B. caccae , B. fragilis , and B. uniformis , were strongly associated with pantothenate and CoA biosynthesis (Fig. 7 H). These pathways were particularly active in mice treated with GMNL-32 and GMNL-277, indicating the crucial role of these probiotics in supporting the metabolic function of the liver. Compared with GMNL-32 and GMNL-277, GMNL-93 led to a distinct gut microbiota profile with fewer potentially harmful bacteria in Pb(II)-exposed mice (Fig. 6 B). The GMNL-93 group exhibited reduced abundances of B. plebeius and Burkholderia spp., which were significantly associated with improved liver health after Pb(II) exposure (Fig. 8 A–D). Very few studies have directly associated B. plebeius with liver injury. Under certain conditions, some characteristics of Bacteroides may exacerbate liver injury, likely by increasing gut permeability and reducing anti-inflammatory responses [ 49 ]. Burkholderia spp. have been implicated in some cases of liver or spleen abscess [ 50 ]. The majority of Burkholderia infections occur as complications in patients with other underlying conditions, such as liver cirrhosis [ 51 ] and cystic fibrosis [ 52 ], highlighting these bacteria as opportunistic pathogens. Taken together, these findings suggest that Burkholderia spp. exacerbate Pb(II)-induced intestinal and hepatic injury, and reducing the abundance of these bacteria through GMNL-93 administration may help mitigate Pb(II)-induced liver damage. Compared with GMNL-32 and GMNL-277, GMNL-93 resulted in a distinct gut microbiota profile with an elevated abundance of B. pseudolongum (Fig. 6 B) and reduced abundances of B. plebeius and Burkholderia spp. (Fig. 8 A and C). The literature suggests that B. pseudolongum increases the integrity of the gut barrier to prevent leakage and colitis [ 53 ], thereby contributing to liver health. Pretreatment with B. pseudolongum considerably mitigates lipopolysaccharide-induced acute liver injury in mice, as indicated by reduced serum levels of ALT and AST [ 54 ]. Treatment with B. pseudolongum also alleviates liver inflammation by reducing the concentrations of proinflammatory cytokines such as tumor necrosis factor-α, IL-1β, and IL-6 and mitigates oxidative stress by enhancing the activity of antioxidative enzymes, such as superoxide dismutase, catalase, and glutathione peroxidase [ 54 ]. Regarding gut microbiota composition, our intervention increased the relative abundances of Alistipes and Bifidobacterium spp. and reduced those of Bacteroides , Muribaculum , and Parasutterella and species belonging to the Ruminococcaceae family [ 54 ]. These findings are consistent with those of studies investigating the role of B. pseudolongum , which underscore the importance of maintaining a balanced gut microbiota for overall liver health and inflammation prevention. Overall, the metabolic pathways influenced by Pb(II) exposure plus GMNL-93 treatment differ from those influenced by GMNL-32 and GMNL-277 treatment alone. Specifically, the pathways modulated by GMNL-93 are associated with detoxification and antioxidative activities, which may confer liver protection (Fig. 8 ). For example, the PPP generates nicotinamide adenine dinucleotide phosphate (NADPH), a key reducing agent in the synthesis of fatty acids and cholesterol, which are essential for liver function [ 55 ]. NADPH plays a vital role in the regeneration of reduced glutathione (GSH), which protects hepatocytes from oxidative stress and damage [ 56 ]. GSH and cysteine depletion, coupled with reduced NADPH levels, has been associated with ferroptosis in mice with hepatic ischemia/reperfusion injury [ 56 ]. Furthermore, the PPP supports hepatic detoxification; NADPH generated by the pathway is used in various detoxification processes, including the reduction of oxidized cytochrome P450 enzymes, which are essential for metabolizing and detoxifying drugs and xenobiotics [ 57 ]. As shown in Fig. 8 I,J, treatment with GMNL-93 induced the biosynthesis of secondary bile acids, which may protect the liver from Pb(II)-induced toxicity. By exerting antimicrobial effects, secondary bile acids regulate the gut microbiota and prevent pathogen overgrowth, thereby mitigating the risk of liver infection [ 58 ]. In intestinal diseases, disruption of the gut microbiota leads to an imbalance in the homeostasis of bile acids [ 59 ]. The metabolism of D-Glu and D-Gln is crucial for the conversion of amino acids into corresponding D-forms that can be utilized by the host [ 60 ]. GSH—synthesized from Glu—neutralizes ROS, protecting hepatocytes from oxidative damage [ 61 ]. This process is particularly essential during Pb(II) exposure, which generates ROS and induces thus oxidative stress, thereby causing liver damage [ 60 , 62 ]. Gln serves as a precursor of glutamate and supports the synthesis of glutathione [ 60 ]. In addition, Gln protects the gut barrier from atrophy and injury, reducing the translocation of harmful substances, such as Pb(II), from the gut to the liver [ 63 ]. Consistent with the literature, this study highlights multiple roles of three Lactobacillus strains in mitigating Pb poisoning. It suggests that probiotics play crucial roles in protecting liver function, strengthening gut barrier integrity, and maintaining gut microbiota balance in response to Pb exposure. Our findings indicate that lactobacilli can mitigate Pb(II)-induced hepatotoxicity while positively modulating gut microbiota composition and related metabolic pathways. These findings highlight the therapeutic potential of probiotics as a complementary approach to conventional methods for mitigating and preventing Pb-induced liver damage. Moreover, this study underscores a link between gut health and liver function in the context of heavy metal toxicity. Further research is required to elucidate the mechanisms underlying the interactions between probiotics, gut microbiota, and metabolic pathways in response to Pb exposure. Understanding these mechanisms may facilitate the development of probiotic-based strategies aimed at heavy metal detoxification and overall health improvement. Declarations Conflicts of Interest YLH, WHT, and YTF are employed by GenMont Biotech. YTF is an academic consultant at GenMont Biotech. YCC, CCH, YTY, and SWH declare that they have no conflicts of interest. Author Contribution YTY analyzed the gut microbiomes and prepared the figures. YCC, CCH, and YLH performed the experiments. YLH, WHT, and YTF analyzed the data and prepared the figures. SWH conceptualized the study, drafted the manuscript, and revised the final manuscript. All authors have approved the final manuscript for journal submission. Acknowledgement The authors gratefully acknowledge Wallace Academic Editing for their professional assistance in manuscript editing. Data Availability The data supporting the findings of this study are available from SWH upon reasonable request. References Tchounwou PB, Yedjou CG, Patlolla AK, Sutton DJ. Heavy metal toxicity and the environment. Exp Suppl. 2012;101:133–64. 10.1007/978-3-7643-8340-4_6 . Vareda JP, Valente AJM, Duraes L. 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14:07:53","extension":"html","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":196503,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8195284/v1/c2266669cb89b4f1f3e499fe.html"},{"id":96784378,"identity":"8767502f-4310-4b4d-858e-4d3e59d2881f","added_by":"auto","created_at":"2025-11-26 05:33:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1373891,"visible":true,"origin":"","legend":"\u003cp\u003ePb(II) adsorption by \u003cem\u003eLactobacillus\u003c/em\u003e strains.\u003cstrong\u003e \u003c/strong\u003eThe absorption of Pb(II) by GMNL-93 and GMNL-277 was analyzed. (A) Results of scanning electron microscopy combined with energy-dispersive X-ray spectroscopy. (B) Results of X-ray diffraction performed to identify crystalline phases in Pb(II)-treated samples. Levels of Pb(II) adsorption by (C) GMNL-93 and (D) GMNL-277 (D), along with corresponding S-layer protein deletion patterns. Scanning electron microscopy images in back-scattered electron mode provided enhanced contrast. The diffractograms of Pb(II) + GMNL-93 (blue) and Pb(II) + GMNL-277 (red) were compared with those of untreated GMNL-93 (black) and GMNL-277 (gray), with the peaks highlighting PbS (*) and Pb(OH)\u003csub\u003e2\u003c/sub\u003e (▲). Data are presented as mean ± standard deviation (SD) values (\u003cem\u003en\u003c/em\u003e = 3). Statistical significance is indicated by \u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8195284/v1/1ffd135604096aae8bd3fe39.png"},{"id":96784380,"identity":"60469fd8-7732-40c5-8f71-5f5bb889cdd0","added_by":"auto","created_at":"2025-11-26 05:33:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":476296,"visible":true,"origin":"","legend":"\u003cp\u003eCytoprotective effects of\u003cem\u003e Lactobacillus\u003c/em\u003e strains against Pb(II)-induced damage.\u003cstrong\u003e \u003c/strong\u003eViability of (A and B) HepG2 cells and (C and D) HT-29 cells after exposure to varying concentrations of Pb(II). The IC\u003csub\u003e50\u003c/sub\u003e value of Pb(II) was 98 mg/L for HepG2 cells and 330 mg/L for HT-29 cells. Cell viability was further evaluated after treating HepG2 cells with 100 mg/L Pb(II) and HT-29 cells with 350 mg/L Pb(II) in the presence or absence of \u003cem\u003eLactobacillus\u003c/em\u003e lysates (10 μg/mL for HepG2; 25 μg/mL for HT-29). Data are presented as mean ± SD (\u003cem\u003en\u003c/em\u003e = 3). Differences in cell viability among groups were evaluated by one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test. Statistical significance was defined as \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 and \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01. \u003cstrong\u003eAbbreviations:\u003c/strong\u003e 32, GMNL-32; 93, GMNL-93; 277, GMNL-277; Naïve Ctrl, untreated control group.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8195284/v1/c50024e4b78cd73b401f0284.png"},{"id":96784388,"identity":"08d48117-0342-4cd9-9a8e-f245a1ed3565","added_by":"auto","created_at":"2025-11-26 05:33:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":730870,"visible":true,"origin":"","legend":"\u003cp\u003eHepatoprotective effects of \u003cem\u003eLactobacillus\u003c/em\u003e strains in mice exposed to Pb(II).\u003cstrong\u003e \u003c/strong\u003e(A) Schematic depicting the \u003cem\u003ein vivo\u003c/em\u003e experimental design of this study. (B) Body weight measurements. (C) Liver-to-body weight ratios. (D and E) Serum levels of AST and ALT. Liver sections were subjected to histological analysis. (F) Levels of Pb(II)-induced hepatotoxicity, determined focusing on the proliferation of oval cells. Data are presented as mean ± SD, with four to five mice per group. Statistical significance was evaluated using one-way ANOVA, followed by Tukey’s post hoc test (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, and \u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). \u003cstrong\u003eAbbreviations:\u003c/strong\u003e AST, aspartate transaminase; ALT, alanine transaminase; 32, GMNL-32; 93, GMNL-93; 277, GMNL-277; Naïve Ctrl, untreated control group.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8195284/v1/578a89b044c07f158007b02b.png"},{"id":96784385,"identity":"0f895035-34a5-433d-88c4-d5414b62d444","added_by":"auto","created_at":"2025-11-26 05:33:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":372739,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of \u003cem\u003eLactobacillus\u003c/em\u003e strains on gut epithelial integrity and inflammation in Pb(II)-exposed mice. Expression levels (2\u003csup\u003e−∆∆Ct\u003c/sup\u003e) of tight junction genes: (A) \u003cem\u003eCldn3\u003c/em\u003e, (B) \u003cem\u003eCldn5\u003c/em\u003e, and (C) \u003cem\u003eMuc5\u003c/em\u003e. Expression levels (2\u003csup\u003e−∆∆Ct\u003c/sup\u003e) of proinflammatory cytokines: (D) \u003cem\u003eTnf\u003c/em\u003e and (E) \u003cem\u003eIl-6\u003c/em\u003e. Data are presented as mean ± SD (\u003cem\u003en\u003c/em\u003e = 4 to 5 per group). Statistical significance was evaluated using using one-way ANOVA, followed by Tukey’s post hoc test (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, and \u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). \u003cstrong\u003eAbbreviations:\u003c/strong\u003e \u003cem\u003eCldn3\u003c/em\u003e, Claudin 3; \u003cem\u003eCldn5\u003c/em\u003e, Claudin 5; \u003cem\u003eMuc5\u003c/em\u003e, Mucin-5; \u003cem\u003eTnf\u003c/em\u003e, tumor necrosis factor-α; \u003cem\u003eIl-6\u003c/em\u003e, interleukin-6; 32, GMNL-32; 93, GMNL-93; 277, GMNL-277; Naïve Ctrl, untreated control group.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8195284/v1/266947933811561075639ff4.png"},{"id":96784399,"identity":"adf0e9de-8cea-40a7-8d79-6cc50183fa0c","added_by":"auto","created_at":"2025-11-26 05:33:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1107856,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of \u003cem\u003eLactobacillus\u003c/em\u003e strains on gut microbiome composition in Pb(II)-exposed mice. (A) Alpha diversity indices (Chao1 and Shannon indices). (B) Results of principal coordinate analysis. (C) Relative abundances of the (C) top 7 phyla and (D) top 10 genera in each group. Abbreviations: 32, GMNL-32; 93, GMNL-93; 277, GMNL-277; Naïve Ctrl, untreated control group.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8195284/v1/c42b66c5198cda604aa268fa.png"},{"id":96915579,"identity":"049daae8-f7df-4f83-ab3b-447941a3b894","added_by":"auto","created_at":"2025-11-27 14:07:24","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1379926,"visible":true,"origin":"","legend":"\u003cp\u003eDifferential abundance of bacterial taxa in mice treated with Pb(II) alone or in combination with probiotics. Linear discriminant analysis effect size was performed to analyze the differential abundance of bacterial taxa in mice treated with Pb(II) alone and those treated with Pb(II) plus different \u003cem\u003eLactobacillus\u003c/em\u003e strains. Comparison of bacterial taxa between (A) mice treated with Pb(II) (red) and those treated with Pb(II) + GMNL-32 (Pb+32; green), (B) mice treated with Pb(II) (red) and those treated with Pb(II) + GMNL-93 (Pb+93; green), and (C) mice treated with Pb(II) (red) and those treated with Pb(II) + GMNL-277 (Pb+277; green).\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-8195284/v1/456b0d9e4222b3da36180d88.png"},{"id":96784381,"identity":"f4335c4c-15e5-4239-bfcb-55f40cd96f82","added_by":"auto","created_at":"2025-11-26 05:33:17","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":967844,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelations between liver function changes, microbial abundance, and predicted metabolic pathways in mice treated with Pb(II) and GMNL-32 or GMNL-277. (A,B) \u003cem\u003eBacteroides caccae\u003c/em\u003e abundance and correlation with weight gain. (C,D) \u003cem\u003ePrevotella copri\u003c/em\u003e abundance and correlation with weight gain. (E,F) Thiamine metabolism pathway proportions and correlation with AST and ALT. (G,H) Pantothenate and CoA biosynthesis proportions and correlation specific gut microbial taxa (\u003cem\u003eBacteroides uniformis\u003c/em\u003e, \u003cem\u003eGemmiger formicilis\u003c/em\u003e, \u003cem\u003eB. caccae\u003c/em\u003e, \u003cem\u003eP. copri\u003c/em\u003e, \u003cem\u003eBacteroides fragilis\u003c/em\u003e, and \u003cem\u003eFaecalibacterium prausnitzii\u003c/em\u003e). Data are mean ± SD (n = 4–5/group). One-way ANOVA with Tukey’s test; *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001. Abbreviations: AST, aspartate transaminase; ALT, alanine transaminase; 32/93/277, GMNL-32/-93/-277; Naïve Ctrl, control.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-8195284/v1/a0c193cb44d36c055c0dbe22.png"},{"id":96784404,"identity":"6915f76f-fe90-436e-bf00-fd4d6355a1ec","added_by":"auto","created_at":"2025-11-26 05:33:18","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1120110,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelations between liver function changes, microbial abundance, and predicted metabolic pathways in mice treated with Pb(II) and GMNL-93.\u003cstrong\u003e \u003c/strong\u003e(A,B) \u003cem\u003eBacteroides plebeius\u003c/em\u003e abundance and correlation with liver/body weight ratio. (C,D) \u003cem\u003eBurkholderia\u003c/em\u003e spp. abundance and correlation with AST and ALT. (E,F) D-Gln/D-Glu metabolism proportions and correlation with AST and ALT. (G,H) PPP proportions and correlation with AST and ALT. (I,J) Secondary bile acid biosynthesis proportions and correlation with AST and ALT. Data are mean ± SD (n = 4–5/group). One-way ANOVA with Tukey’s test; *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001. Abbreviations: AST, aspartate transaminase; ALT, alanine transaminase; 32/93/277, GMNL-32/-93/-277; Naïve Ctrl, control; D-Gln, D-glutamine; D-Glu, D-glutamate; PPP, pentose phosphate pathway.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-8195284/v1/d041e7ad9fd361d6e2112fee.png"},{"id":105224481,"identity":"9a38ac1a-664a-4fca-87f6-3b32668d5d4b","added_by":"auto","created_at":"2026-03-23 16:14:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8715871,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8195284/v1/f9ad18f8-fca1-491d-87a2-20333f8a8686.pdf"},{"id":96784383,"identity":"bb330938-0f43-4c3f-bc36-32f2cd7517ba","added_by":"auto","created_at":"2025-11-26 05:33:17","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":3713066,"visible":true,"origin":"","legend":"","description":"","filename":"Suplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-8195284/v1/77ff213acdf0cdbc56718108.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Lactobacillus protects against lead-induced hepatotoxicity by preserving the gut barrier and microbiota remodeling","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHeavy metals are naturally occurring elements with high atomic weights and densities at least five times greater than that of water. Some heavy metals, such as iron and zinc, serve as essential nutrients at low concentrations. Other heavy metals, such as lead (Pb), mercury, and arsenic, have no known biological function and can be toxic even in trace amounts [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Heavy metal toxicity occurs when these metals accumulate in the body to levels that impair physiological function and cause harm. Since 2017, public health and ecological concerns regarding heavy metal toxicity have increased as a result of the global increase in environmental pollution and industrial activity [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePb, one of the most dangerous heavy metals, poses major risks to human and animal health and the environment [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Once absorbed, Pb has a half-life of approximately 30 days in the blood. Thereafter, it diffuses into soft tissues, such as the kidneys, brain, and liver, and is subsequently distributed to the bones, teeth, and hair in the form of Pb(II) phosphate [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. This pathway indicates that the toxic effects of Pb are multifaceted and can affect multiple organ systems.\u003c/p\u003e\u003cp\u003ePb is well known for its neurotoxic effect on both the central and peripheral nervous systems [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Studies have indicated that Pb-induced brain damage can result in various neurological disorders, such as cognitive impairment, motor dysfunction, and encephalopathy, particularly in children [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In adults, chronic Pb exposure has been linked to health problems such as reduced fertility [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], cataracts [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], neuromuscular disorders [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], and memory and concentration deficits [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Multiple studies have highlighted other health risks associated with Pb exposure. For example, hypertension, cardiovascular disease [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], renal disease, liver damage, and hematologic diseases [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] are currently recognized as potential consequences of Pb exposure. These findings underscore the importance of monitoring and mitigating Pb exposure to safeguard public health.\u003c/p\u003e\u003cp\u003eAlthough the use of Pb has been restricted in many countries, Pb exposure remains a concern, particularly in developing countries, necessitating effective management of Pb accumulation in these regions [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Evidence suggests that chelation therapy with edetate calcium disodium and meso-2,3-dimercaptosuccinic acid can protect against Pb poisoning by promoting the excretion of this heavy metal [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], but this therapy is reserved only for preclinical use. In addition to chelators, diets supplemented with essential metals, vitamin C, and phytochemicals have been reported to reduce the absorption of Pb in the body [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eProbiotics are live microorganisms that confer health benefits when administered in adequate amounts. Recent research has assessed certain probiotics for their potential to prevent Pb absorption or alleviate Pb poisoning [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Some probiotic strains, particularly lactic acid bacteria, can bind to Pb ions in the gastrointestinal tract and reduce their absorption into the body [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. This binding effect may be attributable to the cell wall components of the probiotic bacteria. Evidence also suggests that probiotics can modulate the composition and function of the gut microbiota, which may be disrupted by the administration of Pb, and thus regulate the metabolism and excretion of Pb [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In addition, probiotics can strengthen the intestinal barrier, whose integrity is impaired by Pb, thereby hindering the translocation of Pb or bacteria from the gut lumen into the bloodstream [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eLactic acid bacterial strains can bind to Pb and reduce its absorption, thus mitigating Pb poisoning. However, limited \u003cem\u003ein vivo\u003c/em\u003e and clinical evidence is available supporting the effectiveness of these strains in preventing Pb-induced organ damage. In this study, we identified specific lactic acid bacterial strains capable of adsorbing Pb or mitigating its toxicity. Furthermore, we investigated whether these probiotic bacteria can protect organs from Pb-induced damage and explored the mechanisms underlying the potential protective effects.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eBacterial strains and culture conditions\u003c/h2\u003e\u003cp\u003eEight \u003cem\u003eLactobacillus\u003c/em\u003e strains were obtained from GenMont Biotech (Tainan, Taiwan) and screened for their Pb(II) adsorption, Pb(II) tolerance, and antioxidative properties (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These strains were cultured in De Man\u0026ndash;Rogosa\u0026ndash;Sharpe broth (BD Difco, Franklin Lakes, NJ, USA) at 37\u0026deg;C for 18 h under anaerobic conditions. They were then subcultured twice before using in \u003cem\u003ein vitro\u003c/em\u003e experiments. \u003cem\u003eLactobacillus\u003c/em\u003e lysates were prepared by thoroughly homogenizing the bacteria by using a bacteriolytic device (FastPrep-24; MP Biomedicals, Irvine, CA, USA) while ensuring complete cell disruption to generate whole-cell lysates. These lysates were subjected to high-speed centrifugation at 20,000 \u0026times;\u003cem\u003eg\u003c/em\u003e for 5 min at 4\u0026deg;C to spin down unbroken bacterial cell debris. For details on this experimental procedure, please refer to the study by [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCell culture\u003c/h3\u003e\n\u003cp\u003eHuman colon carcinoma (HT-29; ATCC\u0026reg; HTB-38\u0026trade;) and hepatocellular carcinoma (HepG2; ATCC\u0026reg; HB-8065\u0026trade;) cell lines were obtained. HT-29 cells were cultured in Roswell Park Memorial Institute 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. HepG2 cells were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin.\u003c/p\u003e\n\u003ch3\u003ePb(II) chemicals\u003c/h3\u003e\n\u003cp\u003ePb chloride (PbCl\u003csub\u003e2\u003c/sub\u003e) and Pb sulfide (PbS) were procured from Thermo Fisher Scientific (Waltham, MA, USA) and dissolved in sterile distilled water for experimental use.\u003c/p\u003e\n\u003ch3\u003ePb(II) adsorption assay\u003c/h3\u003e\n\u003cp\u003eA mixture of 1 mL of PbCl\u003csub\u003e2\u003c/sub\u003e solution (Pb(II) concentration: 30 mg/L) and 1 mL of 4 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e CFU/mL \u003cem\u003eLactobacillus\u003c/em\u003e in HEPES buffer (100 mM; pH 6.8) was incubated at 37\u0026deg;C for 1 h. This mixture was then centrifuged at 10,000 x g or 5 min, and the supernatant and bacterial pellets were separately subjected to aqua regia digestion. The concentration of Pb(II) was quantified through flame atomic absorption spectrophotometry (SensAA; GBC Scientific Equipment, Keysborough, Australia). All assays were performed in triplicate, and Pb(II) adsorption capacity was calculated as follows:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\%\\:Pb\\:adsorption\\:=\\:\\frac{{C}_{pellet}}{{C}_{supernatant}\\:+{C}_{pellet}}\\:\\times\\:100$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003ePb(II) tolerance assay\u003c/h3\u003e\n\u003cp\u003eA minimum inhibitory concentration assay was performed to determine the Pb(II) tolerance of \u003cem\u003eLactobacillus\u003c/em\u003e. Each \u003cem\u003eLactobacillus\u003c/em\u003e strain was cultured (density: 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e CFU/well) with varying concentrations of PbCl\u003csub\u003e2\u003c/sub\u003e solution (Pb(II) concentration: 10\u0026ndash;1000 mg/L) on a 96-well plate at 37\u0026deg;C for 24 h. The minimum inhibitory concentration was identified as the lowest Pb(II) concentration that completely inhibited bacterial growth.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eAntioxidant assay\u003c/h2\u003e\u003cp\u003eAfter probiotic samples were centrifuged, they were washed, resuspended in methanol (10 mg/mL), and filter-sterilized. Ascorbic acid was used as a positive control (10 \u0026micro;g/mL). Each sample or control solution (100 \u0026micro;L) was mixed with 100 \u0026micro;L of 2,2-diphenyl-1-picrylhydrazyl (DPPH) solution (0.1 mM) in a 96-well microplate and incubated in the dark for 30 min. Finally, absorbance was measured at 517 nm to determine DPPH-scavenging activity.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCell viability assay\u003c/h3\u003e\n\u003cp\u003eHepG2 or HT-29 cells were seeded at a density of 2 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well on 96-well plates and incubated overnight. These cells were then treated with PbCl\u003csub\u003e2\u003c/sub\u003e solution alone or in combination with \u003cem\u003eLactobacillus\u003c/em\u003e lysates for 48 h. Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.\u003c/p\u003e\n\u003ch3\u003eAnimal experiment\u003c/h3\u003e\n\u003cp\u003eMale C57BL/6 mice (age: 8 weeks) were obtained from the Animal Center of National Cheng Kung University (Tainan, Taiwan) and housed under controlled environmental conditions (temperature: 25\u0026deg;C; humidity: 58%; photoperiod: 12-h light/dark) with \u003cem\u003ead libitum\u003c/em\u003e access to food and water. The animal study protocol was approved by the Institutional Animal Care and Use Committee of National Cheng Kung University (approval number: 110289). The mice were randomly divided into five groups, each comprising five mice: na\u0026iuml;ve control (untreated), Pb(II)\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO (Pb alone), Pb(II)\u0026thinsp;+\u0026thinsp;GMNL-32, Pb(II)\u0026thinsp;+\u0026thinsp;GMNL-93, and Pb(II)\u0026thinsp;+\u0026thinsp;GMNL-277. Lyophilized live probiotic powders were orally administered at a dosage of 1.64 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e CFU/mouse in 0.2 mL of sterile water once daily for 5 days a week for a total of 8 weeks. The mice received either normal drinking water or water containing 1000 mg/mL Pb(II), which was replaced every week. The body weight and water consumption of each mouse were recorded on a weekly basis. At the end of week 8, all mice were euthanized using CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003ePb contents in tissues and feces\u003c/h2\u003e\u003cp\u003eLiver, kidney, and fecal samples were homogenized in aqua regia by using a FastPrep-24 instrument (MP Biomedicals). These samples were centrifuged at 13,000 x g for 3 min. The supernatant was analyzed to determine the concentration of Pb through atomic absorption spectrophotometry. Pb content was expressed in milligrams/liter per gram of tissue or feces wet weight.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eBlood biochemistry and hematology\u003c/h2\u003e\u003cp\u003eSerum levels of alanine transaminase (ALT), aspartate transaminase (AST), and creatinine were measured using a DRI-CHEM 4000i chemistry analyzer (Fujifilm, Tokyo, Japan). Hematological parameters were assessed in ethylenediaminetetraacetate\u0026ndash;whole blood by using an Scil Vet Focus 5 analyzer (Seneca Scientific, Denver, CO, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eExpression levels of tight junction proteins and proinflammatory cytokines in the gut\u003c/h2\u003e\u003cp\u003eTotal RNA was extracted from mouse jejunum tissues and converted into complementary DNA by using a reverse transcription kit (Thermo Fisher Scientific). The expression levels of tight junction proteins and proinflammatory cytokines were determined through quantitative real-time polymerase chain reaction (PCR) with specific primers (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) and a SYBR Green PCR Kit (Qiagen, Hilden, Germany). The expression of target genes was normalized to that of mouse \u003cem\u003eGapdh\u003c/em\u003e and calculated using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eHistology\u003c/h2\u003e\u003cp\u003eLiver samples were dissected and fixed in neutral buffered formalin for 24 h, routinely processed, and embedded in paraffin wax. Subsequently, 5-\u0026micro;m sections were dewaxed, rehydrated, and stained using standard hematoxylin and eosin. Then, these sections were examined for liver pathology. The number of oval cells in portal areas was counted in 10 high-power fields under a microscope.\u003c/p\u003e\u003cp\u003e\u003cb\u003e16S ribosomal DNA sequencing\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAfter each mouse\u0026rsquo;s ileum was homogenized, the supernatant containing intestinal microorganisms was collected. DNA extraction was performed using a Qiagen DNA kit, following the manufacturer\u0026rsquo;s protocol. The extracted DNA was analyzed; the 260/280 optical density ratio ranged from 1.8 to 2.0. Subsequently, 16S ribosomal DNA was amplified using metagenomic DNA (template) and bacteria-specific primers (S17 and A21). The size of the amplified DNA was verified using a fragment analyzer (Agilent 5300 Fragment Analyzer; Agilent Technologies, Santa Clara, CA, USA). Then, it was sequenced on an Illumina MiSeq platform (Illumina, San Diego, CA, USA). DNA libraries were constructed using a Nextera XT Index Kit v2, mixed using a 600-cycle MiSeq Reagent Kit v3, and sequenced in a 2 \u0026times; 300-bp paired-end run. The resulting sequences were filtered for quality and merged, and low-quality or chimera sequences were removed. Operational taxonomic units were clustered at 97% similarity by using the Greengenes database (v.13.8). Further analysis was performed using a Qiagen CLC Microbial Genomics Module (v.10.1.1).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eProcessing and analysis of metataxonomic data\u003c/h2\u003e\u003cp\u003eThe alpha diversity of taxonomic composition was assessed using the Shannon diversity index, which accounts for both species richness and distribution evenness within each group. Beta diversity, reflecting differences in microbial composition across groups, was measured through a UniFrac analysis weighted by a principal coordinate analysis. Linear discriminant analysis effect size (LEfSe) was performed to identify microbial markers by using the Galaxy/Hutlab webtool. To analyze LEfSe data, pairwise comparisons were performed using Wilcoxon\u0026rsquo;s rank-sum exact test and permutational multivariate analysis of variance. Functional abundance was predicted on the basis of marker gene sequences by using the PICRUSt2 tool (v.2.3.0b0). Intergroup comparisons were performed using two-tailed Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e tests. Statistical significance was set at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Prism (v.8; GraphPad Software, San Diego, CA, USA) was used to determine taxonomic differences and generate relative abundance plots.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eComparisons involving more than two groups were evaluated by one-way analysis of variance (ANOVA), followed by Tukey\u0026rsquo;s post hoc test to assess pairwise differences. For comparisons between two independent groups, an unpaired Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test was applied. A \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003ePb(II) adsorption capacity varies across\u003c/b\u003e \u003cb\u003eLactobacillus\u003c/b\u003e \u003cb\u003estrains\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMultiple studies have demonstrated the biosorption of heavy metals by lactic acid bacteria to reduce the concentrations of Pb and cadmium in milk [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In the present study, to select lactic acid bacterial strains suitable for our experiments, we measured the Pb(II)Cl\u003csub\u003e2\u003c/sub\u003e adsorption capacities of eight single \u003cem\u003eLactobacillus\u003c/em\u003e strains (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Our results indicated that five of the tested strains absorbed at least 61% of the total Pb(II): \u003cem\u003eLacticaseibacillus paracasei\u003c/em\u003e GMNL-32 (61.4% \u0026plusmn; 0.4%), \u003cem\u003eLimosilactobacillus fermentum\u003c/em\u003e GMNL-93 (97.6% \u0026plusmn; 1.4%), \u003cem\u003eLimosilactobacillus fermentum\u003c/em\u003e BCRC 910720 (74.6% \u0026plusmn; 6.7%), \u003cem\u003eLimosilactobacillus reuteri\u003c/em\u003e BCRC 910340 (61.1% \u0026plusmn; 2.0%), and \u003cem\u003eLacticaseibacillus casei\u003c/em\u003e GMNL-277 (67.9% \u0026plusmn; 7.2%). Among these strains, GMNL-93 exhibited a Pb(II) adsorption rate of \u0026gt;\u0026thinsp;97% and a tolerance level of \u0026gt;\u0026thinsp;1000 mg/L, which highlighted its potential efficacy in Pb(II) removal. Regarding DPPH clearance, GMNL-32, GMNL-93, and GMNL-277 exhibited clearance rates of \u0026gt;\u0026thinsp;50%, indicating strong free radical\u0026ndash;scavenging capacity. This antioxidant activity is particularly noteworthy because it enables the probiotics to mitigate oxidative stress induced by heavy metal exposure\u0026mdash;a key factor in Pb-induced cellular damage. Thus, GMNL-32, GMNL-93, and GMNL-277 were used in subsequent experiments.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePb absorption, tolerance, and antioxidant activity of selected Lactobacillus strains.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBacteria species\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003csup\u003e*\u003c/sup\u003eStrain\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePb absorption (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePb tolerance (mg/L)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003csup\u003e#\u003c/sup\u003eDPPH clearance rate (%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eL. acidophilus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBCRC 910774\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e44.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;1000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eL. casei\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBCRC 910585 (GMNL-277)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e67.9\u0026thinsp;\u0026plusmn;\u0026thinsp;7.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;1000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e55.10\u0026thinsp;\u0026plusmn;\u0026thinsp;1.14\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eL. paracasei\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBCRC 910220 (GMNL-32)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e61.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;1000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e50.53\u0026thinsp;\u0026plusmn;\u0026thinsp;2.15\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eL. fermentum\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBCRC 910259 (GMNL-93)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e97.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;1000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e84.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eL. fermentum\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBCRC 910720\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e74.6\u0026thinsp;\u0026plusmn;\u0026thinsp;6.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;1000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eL. plantarum\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBCRC 911066\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e44.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;1000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eL. plantarum\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBCRC 910776\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e44.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;1000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eL. reuteri\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBCRC 910340\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e61.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;1000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e\u003cp\u003eLead (Pb) absorption capacity, Pb tolerance, and antioxidant activity (DPPH clearance rate) of different \u003cem\u003eLactobacillus\u003c/em\u003e and \u003cem\u003eLactobacillus reuteri\u003c/em\u003e strains. Pb absorption values are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, Pb tolerance as the maximum concentration tolerated, and antioxidant activity was determined using the DPPH assay.\u003c/p\u003e\u003cp\u003e\u003csup\u003e*\u003c/sup\u003eBioresource Collection and Research Center (BCRC), Taiwan.\u003c/p\u003e\u003cp\u003e#2,2-Diphenyl-1-picrylhydrazyl (DPPH)-scavenging assay.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eScanning electron microscopy visually confirmed the adsorption of Pb(II) into bacterial cell surfaces (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and B). To determine whether the S-layers of probiotics are essential for Pb(II) adsorption, we removed the S-layers from GMNL-93 and GMNL-277. This removal markedly reduced the rate of Pb(II) adsorption by GMNL-93 from 97% to 44% (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC); only a minor reduction of approximately 10% was noted for GMNL-277 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Taken together, these results indicate that although some \u003cem\u003eLactobacillus\u003c/em\u003e strains adsorb Pb(II) in an S-layer-dependent manner, others do not rely on S-layers for adsorption (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eProtective effects of\u003c/b\u003e \u003cb\u003eLactobacillus\u003c/b\u003e \u003cb\u003eagainst Pb(II)-induced cytotoxicity\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo assess the cytoprotective effects of GMNL-93 and GMNL-277, human HepG2 hepatoma cells and HT-29 intestinal cells were exposed to Pb(II)Cl\u003csub\u003e2\u003c/sub\u003e either alone or in combination with \u003cem\u003eLactobacillus\u003c/em\u003e probiotic lysates. Cell viability was measured after each treatment. In HepG2 cells, cell viability decreased with increasing Pb(II) concentrations, with a half-maximal inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) of 98 mg/L (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Depending on the IC\u003csub\u003e50\u003c/sub\u003e value for Pb-induced cytotoxicity, cell death caused by 100 mg/L Pb(II) was significantly reduced when HepG2 cells were treated with GMNL-93 lysates (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Consistent with the results in HepG2 cells, \u003cem\u003ein vitro\u003c/em\u003e assays determining the protective effects of probiotics against Pb(II) poisoning in HT-29 cells revealed marked reductions in Pb(II)-induced cytotoxicity after treatment with GMNL-277 lysates (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and D). Collectively, these \u003cem\u003ein vitro\u003c/em\u003e results suggest that \u003cem\u003eLactobacillus\u003c/em\u003e can protect hepatic and intestinal cells against Pb(II)-induced damage.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eProtective effects of\u003c/b\u003e \u003cb\u003eLactobacillus\u003c/b\u003e \u003cb\u003eagainst Pb(II)-induced liver injury\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo understand the role of \u003cem\u003eLactobacillus\u003c/em\u003e strains in chronic Pb(II)-induced toxicity, we treated mice with Pb(II)Cl\u003csub\u003e2\u003c/sub\u003e at a concentration of 1000 mg/L either alone or in combination with probiotics (GMNL-32, GMNL-93, and GMNL-277) for 8 weeks. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA presents the timeline of each treatment and the tissue samples collected. During this experiment, no significant difference was observed in body weight between mice drinking Pb(II)-containing water with or without probiotics (GMNL-32, GMNL-93, and GMNL-277) and the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e depicts the body weight of each mouse. No significant between-group difference was observed in the amount of water consumed (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Moreover, no significant between-group differences were observed in white blood cell count, but a slight reduction in neutrophil count was observed in mice treated with GMNL-93 plus Pb(II) (Supplementary Table S2). Notably, all mice exposed to Pb(II) exhibited a slight reduction in mean corpuscular volume, suggesting Pb(II)-induced toxicity of red blood cells (Supplementary Table S3). Mice exposed to Pb(II) alone developed moderate hepatomegaly, and a significant increase was observed in the liver-to-body weight ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Treatment with probiotics led to various degrees of reduction in this ratio, with GMNL-277 being the most effective probiotic (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Mice treated with Pb(II) exhibited significantly elevated levels of AST (mean: 436.8 U/L) and ALT (mean: 227.8 U/L), indicating liver damage (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD and E). Furthermore, mice treated with probiotics exhibited varying levels of AST, with GMNL-93 significantly reducing the level of AST (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Similarly, treatment with probiotics, particularly with GMNL-93 and GMNL-277, significantly reduced the levels of ALT (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). A histological comparison of mice exposed to Pb(II) and mice treated with probiotics revealed a marked reduction in liver damage in probiotic-treated mice, with fewer oval cells and a more intact liver architecture (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF; Supplementary Fig. S2). Taken together, these results suggest that \u003cem\u003eLactobacillus\u003c/em\u003e strains can protect against Pb(II)-induced liver damage.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eHepatoprotective effects of Lactobacillus are not mediated through enhanced Pb(II) excretion\u003c/h2\u003e\u003cp\u003eTo determine whether the ability of \u003cem\u003eLactobacillus\u003c/em\u003e to adsorb Pb directly contributes to its protective effects against Pb(II)-induced hepatotoxicity, we measured the concentrations of Pb(II) in the livers, kidneys, and feces of mice after treatment with different probiotic strains. In addition, we measured corresponding Pb concentrations in the control group. However, our results indicated that hepatic Pb concentration was significantly higher in mice treated with Pb(II) alone or in combination with GMNL-32, GMNL-93, and GMNL-277 than in control mice (Supplementary Fig. S3A). Similarly, renal Pb concentration was significantly elevated in all the Pb-treated groups relative to the control (Supplementary Fig. S3B). During the 8-week experiment period, the concentrations of Pb in the feces did not significantly differ between the groups (Supplementary Fig. S3C). Collectively, these results suggest that \u003cem\u003eLactobacillus\u003c/em\u003e strains do not enhance the excretion of Pb(II) through feces and, therefore, do not reduce the accumulation of Pb in the kidneys or liver.\u003c/p\u003e\u003cp\u003e\u003cb\u003eLactobacillus\u003c/b\u003e \u003cb\u003eprotects mucosal barriers from Pb(II)-induced intestinal damage\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eLactobacillus\u003c/em\u003e strains strengthen the gut mucosal barrier, which is essential for maintaining gut health and preventing inflammation [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Therefore, liver health is closely associated with gastrointestinal integrity, and a compromised intestinal barrier may precipitate hepatic pathology [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. To confirm whether the probiotic strains used in this study promoted gut epithelial integrity, we analyzed the mRNA expression levels of various tight junction proteins, such as Claudin 3 (Cldn3), Claudin 5 (Cldn5), and Mucin-5 (\u003cem\u003eMuc5\u003c/em\u003e), in an \u003cem\u003ein vivo\u003c/em\u003e experiment. Our results indicated that the expression of \u003cem\u003eCldn3\u003c/em\u003e, \u003cem\u003eCldn5\u003c/em\u003e, and \u003cem\u003eMuc5\u003c/em\u003e was significantly upregulated in response to Pb(II) exposure with GMNL-277 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u0026ndash;C). Furthermore, treatment with GMNL-32 and GMNL-93 significantly upregulated the expression of \u003cem\u003eCldn5\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Regarding inflammatory cytokines, none of the probiotics modulated the expression of \u003cem\u003eTnf\u003c/em\u003e with Pb(II) exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). However, all probiotics exhibited a tendency to reduce Pb(II)-induced interleukin (IL)-6 upregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Collectively, these results indicate that \u003cem\u003eLactobacillus\u003c/em\u003e can protect intestinal cells from Pb(II)-induced damage and can exert anti-inflammatory effects.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eLactobacillus\u003c/b\u003e \u003cb\u003emodulates gut microbiome composition in response to Pb(II) exposure\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo determine how intestinal microbiota respond to Pb(II) exposure and lactobacilli, we analyzed the gut microbiome composition of each mouse. For this, 16S ribosomal DNA sequencing was performed for each sample to profile each mouse\u0026rsquo;s gut microbiome. In addition, LEfSe was performed to identify differentially abundant bacterial taxa in the Pb(II) alone group and the Pb(II)\u0026thinsp;+\u0026thinsp;probiotic groups. The Chao1 and Shannon index values indicated that mice treated with GMNL-32 and GMNL-277 exhibited higher alpha diversity values than did those exposed to Pb(II) alone. However, mice treated with GMNL-93 exhibited significantly lower alpha diversity values than did those exposed to Pb(II) alone, as indicated by the Shannon index (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Principal coordinate analysis revealed that the GMNL-32 and GMNL-277 groups had similar microbiome compositions, whereas the GMNL-93 group had a different composition (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Moreover, significant differences were observed between the microbiome compositions of the GMNL-32 and GMNL-93 groups and that of the Pb(II) alone group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). We further evaluated the relative abundance of gut microbes in each mouse (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and D) and noted patterns of microbial shifts. Exposure to Pb(II) led to dysbiosis, characterized by reduced abundances of \u003cem\u003eBacteroides\u003c/em\u003e spp., \u003cem\u003eParabacteroides\u003c/em\u003e spp., and \u003cem\u003ePrevotella\u003c/em\u003e spp. and an increase abundance of \u003cem\u003eTuricibacter\u003c/em\u003e spp. Treatment with GMNL-32 and GMNL-277 restored the gut microbiome composition (Supplementary Fig. S4). Compared with the Pb(II) alone group, the GMNL-32 group exhibited significantly increased abundances of \u003cem\u003eBacteroides\u003c/em\u003e spp., \u003cem\u003eParabacteroides\u003c/em\u003e spp., and \u003cem\u003ePrevotella\u003c/em\u003e spp. (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), whereas the GMNL-32 and GMNL-277 groups exhibited increased abundances of \u003cem\u003eAnaerostipes\u003c/em\u003e spp., \u003cem\u003eAlistipes\u003c/em\u003e spp., and \u003cem\u003eStreptococcus\u003c/em\u003e spp. (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Furthermore, the relative abundances of the dominant microbiota exhibited distinct patterns in the Pb(II) alone and Pb(II)\u0026thinsp;+\u0026thinsp;GMNL-93 groups, differing from those observed in the GMNL-32 and GMNL-277 groups, which exhibited the abundances of \u003cem\u003eBacillus\u003c/em\u003e, Firmicutes, and \u003cem\u003eBifidobacterium pseudolongum\u003c/em\u003e in the gut (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eLactobacillus\u003c/b\u003e \u003cb\u003emay exert its hepatoprotective effects by modulating gut microbiome and metabolism\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn this study, we observed a distinct bacterial profile in each mouse group. This distinction suggests that \u003cem\u003eLactobacillus\u003c/em\u003e-induced variations in gut microbiome diversity lead to unique health outcomes and metabolic shifts. To test this hypothesis, we investigated the correlation between bacterial abundance and body weight gain. The presence of \u003cem\u003eBacteroides caccae\u003c/em\u003e, which was significantly abundant in the GMNL-32 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.037 vs. Pb(II) alone) and GMNL-277 groups, was strongly and positively correlated with weight gain (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.409; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.047) in mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA and B). Similarly, the presence of \u003cem\u003ePrevotella copri\u003c/em\u003e, which was also significantly abundant in the GMNL-32 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.007 vs. Pb(II) alone) and GMNL-277 groups, was positively correlated with weight gain (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.405; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.049) in mice, confirming the role of \u003cem\u003eP. copri\u003c/em\u003e in maintaining healthy weight in the presence of Pb(II) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC and D).\u003c/p\u003e\u003cp\u003eA PICRUSt2 analysis was performed to identify active metabolic pathways across different mouse groups (Supplementary Fig. S5). The results revealed that the thiamine metabolism pathway was particularly active in the GMNL-277 group, and this pathway exhibited a significant negative correlation with ALT (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;0.562; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.004) and AST (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;0.522; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.009) levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE and F). Additionally, the pantothenate and coenzyme A (CoA) biosynthesis pathways were particularly active in the GMNL-32 and GMNL-277 groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.023, respectively; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG). In these groups, key bacterial species, such as \u003cem\u003eBacteroides uniformis\u003c/em\u003e, \u003cem\u003eB. caccae\u003c/em\u003e, \u003cem\u003eBacteroides fragilis\u003c/em\u003e, \u003cem\u003eGemmiger formicilis\u003c/em\u003e, \u003cem\u003eP. copri\u003c/em\u003e, and \u003cem\u003eFaecalibacterium prausnitzii\u003c/em\u003e, were positively associated with the pantothenate and CoA biosynthesis pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eA significant reduction was observed in the abundance of \u003cem\u003eBacteroides plebeius\u003c/em\u003e in the GMNL-93\u0026thinsp;+\u0026thinsp;Pb(II) group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003, Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). A positive correlation was observed between the abundance of this bacterium and the severity of liver pathology, measured in terms of the liver-to-body weight ratio (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.511; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.011; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). Treatment with GMNL-93 significantly reduced the abundance of \u003cem\u003eBurkholderia\u003c/em\u003e spp. (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.016). The abundance of \u003cem\u003eBurkholderia\u003c/em\u003e spp. was strongly and positively correlated with elevated levels of ALT (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.686; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC) and AST (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.599; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003ePb(II) exposure plus GMNL-93 treatment substantially enhanced the metabolism of D-glutamate (Glu) and D-glutamine (Gln) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.016; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE). This enhancement was significantly and negatively correlated with ALT (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;0.606; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002) and AST (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;0.437; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.033) levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF). Both the pentose phosphate pathway (PPP) and the secondary bile acid synthesis pathway were significantly upregulated in the GMNL-93\u0026thinsp;+\u0026thinsp;Pb(II) group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eG and I). These metabolic changes were strongly and negatively correlated with ALT level (PPP pathway: \u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;0.616 [\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001]; secondary bile acid synthesis pathway: \u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;0.558 [\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.005]; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eH) and AST level (PPP pathway: \u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;0.446 [\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.029]; secondary bile acid synthesis pathway: \u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;0.385 [\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.063]; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eJ). Taken together, these findings suggest that GMNL-93 mitigates liver damage by modulating key metabolic pathways in response to Pb(II) exposure.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur study indicated that the \u003cem\u003eLactobacillus\u003c/em\u003e strains GMNL-32, GMNL-93, and GMNL-277 exhibited varying levels of Pb(II) adsorption, with GMNL-93 exhibiting the highest rate of adsorption. These findings are consistent with those of studies indicating the ability of lactic acid bacteria to reduce the concentrations of Pb and cadmium in milk through biosorption [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. According to the literature, the cell wall of \u003cem\u003eLactobacillus\u003c/em\u003e, which is rich in negatively charged molecules such as peptidoglycans and teichoic acids, may bind positively charged Pb(II); this binding leads to the sequestration of Pb and reduces its bioavailability [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The adsorption process may prevent Pb from participating in harmful biochemical reactions, particularly those generating reactive oxygen species (ROS), which cause oxidative stress and cell damage [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In the present study, we discovered that S-layers played a key role in the biosorption of Pb(II) by \u003cem\u003eLactobacillus\u003c/em\u003e. This finding is consistent with those of [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], who stated that Pb(II) binds to bacterial cell wall components such as fatty acids, polysaccharides, S-layer proteins, and teichoic acids. [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] also reported that the heavy-metal-binding capacity of a certain \u003cem\u003eLactobacillus bulgaricus\u003c/em\u003e strain mitigated Pb(II)-induced toxicity in mice by reducing systemic absorption.\u003c/p\u003e\u003cp\u003eOur \u003cem\u003ein vivo\u003c/em\u003e experiments revealed that various probiotic strains exerted protective effects against Pb(II)-induced liver damage, as evidenced by reductions in the liver-to-body weight ratio and AST and ALT levels. These findings are consistent with those of studies highlighting the hepatoprotective effects of probiotics against various toxins and stressors [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. As shown in Supplementary Figure S3, the hepatoprotective effects of \u003cem\u003eLactobacillus\u003c/em\u003e were not attributable to enhanced Pb(II) excretion or to postbiotics, given that the concentrations of Pb(II) in the liver, kidneys, and feces did not vary significantly across the treatment groups. Therefore, these protective effects may be mediated by indirect mechanisms that involve interactions with the gut microbiota or the regulation of gut function, as evidenced by the fact that \u003cem\u003eLactobacillus\u003c/em\u003e protected the gut mucosal barrier against Pb(II)-induced damage and upregulated the expression of tight junction proteins while maintaining normal IL-6 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). These results are consistent with those of studies indicating that probiotics strengthen the gut mucosal barrier, a key player in gut health and inflammation prevention [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eChronic Pb exposure induced dysbiosis, characterized by a reduction in the abundance of beneficial bacteria belonging to phylum Firmicutes, which play vital roles in maintaining gut health and metabolic balance [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. This phylum includes key genera such as \u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003eEnterococcus\u003c/em\u003e, and \u003cem\u003eRuminococcus\u003c/em\u003e [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Our results indicated that treatment with lactic acid bacteria, particularly GMNL-32 and GMNL-277, reversed the process of Pb(II)-induced dysbiosis by enriching Firmicutes species, such as \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003eRuminococcus\u003c/em\u003e spp., as well as beneficial \u003cem\u003eBacteroides\u003c/em\u003e spp. and \u003cem\u003eP. copri\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and B). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, Pb(II)-induced dysbiosis promoted the growth of pathogens, particularly those belonging to phylum Proteobacteria. Treatment with GMNL-93 significantly reduced the abundance of Proteobacteria while increasing that of Firmicutes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Collectively, these results underscore the potential protective effect of GMNL-93 against gut dysbiosis.\u003c/p\u003e\u003cp\u003eOur study indicated that certain probiotic strains substantially influenced the composition and metabolic activity of the gut microbiome in response to Pb(II) exposure. Gut dysbiosis contributes to the onset and progression of liver diseases such as nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, cirrhosis, and hepatocellular carcinoma [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In the present study, treatment with GMNL-32 and GMNL-277 substantially increased biodiversity and resulted in microbiome profiles similar to those of untreated mice, characterized by a high abundance of \u003cem\u003eBacteroides\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA and C). Studies have highlighted the roles of \u003cem\u003eBacteroides\u003c/em\u003e in maintaining gut health [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] and regulating inflammation through organ-to-organ communication [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Research has also indicated that \u003cem\u003eB. caccae\u003c/em\u003e enhances mucus degradation, which in turn mitigates intestinal inflammation by minimizing bacterial interaction with the intestinal epithelium [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In the present study, the abundance of \u003cem\u003eB. caccae\u003c/em\u003e was strongly correlated with the improvement of liver function in Pb(II)-treated mice; the abundance of other \u003cem\u003eBacteroides\u003c/em\u003e species, such as \u003cem\u003eB. fragilis\u003c/em\u003e and \u003cem\u003eB. uniformis\u003c/em\u003e, was significantly correlated with the biosynthesis of pantothenate and CoA (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH). These processes protect the liver from fat accumulation and fibrosis [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Together, these findings confirm that \u003cem\u003eBacteroides\u003c/em\u003e mitigates liver disease by restoring the metabolic and microbial environment of the gut\u0026ndash;liver axis [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn the present study, treatment with GMNL-32 and GMNL-277 increased the abundances of \u003cem\u003eB. caccae\u003c/em\u003e and \u003cem\u003eP. copri\u003c/em\u003e in Pb(II)-exposed mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA and C). According to the literature, \u003cem\u003eB. caccae\u003c/em\u003e enhances mucus degradation, which in turn reduces intestinal inflammation by minimizing bacterial interaction with the intestinal epithelium [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Notably, \u003cem\u003eP. copri\u003c/em\u003e is one of the most prevalent species in the human gut microbiome [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. This species has been extensively studied for its protective effects against insulin insensitivity and liver fibrosis, potentially achieved through the modulation of gut microbial activity [\u003cspan additionalcitationids=\"CR41 CR42\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. In this study, the abundances of \u003cem\u003eB. caccae\u003c/em\u003e and \u003cem\u003eP. copri\u003c/em\u003e were strongly correlated with the amelioration of Pb(II)-induced toxicity. This finding is consistent with those of studies indicating that \u003cem\u003eBacteroides\u003c/em\u003e and \u003cem\u003ePrevotella\u003c/em\u003e alleviate hepatic disease by restoring the metabolic and microbial environment of the gut\u0026ndash;liver axis [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOur results indicated that GMNL-277 exerted significant effects on the thiamine metabolism pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE), which was negatively correlated with AST and ALT levels in Pb(II)-exposed mice treated with GMNL-277 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF). The thiamine metabolism pathway may play a role in probiotic-mediated liver protection. Thiamine (vitamin B1) is a crucial component of several key metabolic processes in the liver, particularly those involving the metabolism of carbohydrates and amino acids [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Adequate thiamine levels are essential for ensuring smooth functionality of these processes, which prevents the accumulation of toxic substances that may damage the liver. Our findings are consistent with those of Wang et al. (2007a), who reported that vitamin C and B1 supplementation can mitigate Pb-induced liver damage (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE and F). In the present study, GMNL-32 and GMNL-277 significantly regulated the pantothenate and CoA biosynthesis pathways in Pb(II)-exposed mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG). Pantothenate, a precursor of CoA, is a key component of cellular metabolism, particularly in the tricarboxylic acid cycle; its deficiency has been associated with various metabolic diseases [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Because the liver plays a central role in tricarboxylic-acid-related processes such as gluconeogenesis, lipogenesis, and ureagenesis, maintaining appropriate CoA levels is crucial for optimal liver function [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. In this study, the abundances of various \u003cem\u003eBacteroides\u003c/em\u003e species, such as \u003cem\u003eB. caccae\u003c/em\u003e, \u003cem\u003eB. fragilis\u003c/em\u003e, and \u003cem\u003eB. uniformis\u003c/em\u003e, were strongly associated with pantothenate and CoA biosynthesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH). These pathways were particularly active in mice treated with GMNL-32 and GMNL-277, indicating the crucial role of these probiotics in supporting the metabolic function of the liver.\u003c/p\u003e\u003cp\u003eCompared with GMNL-32 and GMNL-277, GMNL-93 led to a distinct gut microbiota profile with fewer potentially harmful bacteria in Pb(II)-exposed mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). The GMNL-93 group exhibited reduced abundances of \u003cem\u003eB. plebeius\u003c/em\u003e and \u003cem\u003eBurkholderia\u003c/em\u003e spp., which were significantly associated with improved liver health after Pb(II) exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA\u0026ndash;D). Very few studies have directly associated \u003cem\u003eB. plebeius\u003c/em\u003e with liver injury. Under certain conditions, some characteristics of \u003cem\u003eBacteroides\u003c/em\u003e may exacerbate liver injury, likely by increasing gut permeability and reducing anti-inflammatory responses [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. \u003cem\u003eBurkholderia\u003c/em\u003e spp. have been implicated in some cases of liver or spleen abscess [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. The majority of \u003cem\u003eBurkholderia\u003c/em\u003e infections occur as complications in patients with other underlying conditions, such as liver cirrhosis [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e] and cystic fibrosis [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], highlighting these bacteria as opportunistic pathogens. Taken together, these findings suggest that \u003cem\u003eBurkholderia\u003c/em\u003e spp. exacerbate Pb(II)-induced intestinal and hepatic injury, and reducing the abundance of these bacteria through GMNL-93 administration may help mitigate Pb(II)-induced liver damage.\u003c/p\u003e\u003cp\u003eCompared with GMNL-32 and GMNL-277, GMNL-93 resulted in a distinct gut microbiota profile with an elevated abundance of \u003cem\u003eB. pseudolongum\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) and reduced abundances of \u003cem\u003eB. plebeius\u003c/em\u003e and \u003cem\u003eBurkholderia\u003c/em\u003e spp. (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA and C). The literature suggests that \u003cem\u003eB. pseudolongum\u003c/em\u003e increases the integrity of the gut barrier to prevent leakage and colitis [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], thereby contributing to liver health. Pretreatment with \u003cem\u003eB. pseudolongum\u003c/em\u003e considerably mitigates lipopolysaccharide-induced acute liver injury in mice, as indicated by reduced serum levels of ALT and AST [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Treatment with \u003cem\u003eB. pseudolongum\u003c/em\u003e also alleviates liver inflammation by reducing the concentrations of proinflammatory cytokines such as tumor necrosis factor-α, IL-1β, and IL-6 and mitigates oxidative stress by enhancing the activity of antioxidative enzymes, such as superoxide dismutase, catalase, and glutathione peroxidase [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Regarding gut microbiota composition, our intervention increased the relative abundances of \u003cem\u003eAlistipes\u003c/em\u003e and \u003cem\u003eBifidobacterium\u003c/em\u003e spp. and reduced those of \u003cem\u003eBacteroides\u003c/em\u003e, \u003cem\u003eMuribaculum\u003c/em\u003e, and \u003cem\u003eParasutterella\u003c/em\u003e and species belonging to the \u003cem\u003eRuminococcaceae\u003c/em\u003e family [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. These findings are consistent with those of studies investigating the role of \u003cem\u003eB. pseudolongum\u003c/em\u003e, which underscore the importance of maintaining a balanced gut microbiota for overall liver health and inflammation prevention.\u003c/p\u003e\u003cp\u003eOverall, the metabolic pathways influenced by Pb(II) exposure plus GMNL-93 treatment differ from those influenced by GMNL-32 and GMNL-277 treatment alone. Specifically, the pathways modulated by GMNL-93 are associated with detoxification and antioxidative activities, which may confer liver protection (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). For example, the PPP generates nicotinamide adenine dinucleotide phosphate (NADPH), a key reducing agent in the synthesis of fatty acids and cholesterol, which are essential for liver function [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. NADPH plays a vital role in the regeneration of reduced glutathione (GSH), which protects hepatocytes from oxidative stress and damage [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. GSH and cysteine depletion, coupled with reduced NADPH levels, has been associated with ferroptosis in mice with hepatic ischemia/reperfusion injury [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Furthermore, the PPP supports hepatic detoxification; NADPH generated by the pathway is used in various detoxification processes, including the reduction of oxidized cytochrome P450 enzymes, which are essential for metabolizing and detoxifying drugs and xenobiotics [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eI,J, treatment with GMNL-93 induced the biosynthesis of secondary bile acids, which may protect the liver from Pb(II)-induced toxicity. By exerting antimicrobial effects, secondary bile acids regulate the gut microbiota and prevent pathogen overgrowth, thereby mitigating the risk of liver infection [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. In intestinal diseases, disruption of the gut microbiota leads to an imbalance in the homeostasis of bile acids [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. The metabolism of D-Glu and D-Gln is crucial for the conversion of amino acids into corresponding D-forms that can be utilized by the host [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. GSH\u0026mdash;synthesized from Glu\u0026mdash;neutralizes ROS, protecting hepatocytes from oxidative damage [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. This process is particularly essential during Pb(II) exposure, which generates ROS and induces thus oxidative stress, thereby causing liver damage [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Gln serves as a precursor of glutamate and supports the synthesis of glutathione [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. In addition, Gln protects the gut barrier from atrophy and injury, reducing the translocation of harmful substances, such as Pb(II), from the gut to the liver [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eConsistent with the literature, this study highlights multiple roles of three \u003cem\u003eLactobacillus\u003c/em\u003e strains in mitigating Pb poisoning. It suggests that probiotics play crucial roles in protecting liver function, strengthening gut barrier integrity, and maintaining gut microbiota balance in response to Pb exposure. Our findings indicate that lactobacilli can mitigate Pb(II)-induced hepatotoxicity while positively modulating gut microbiota composition and related metabolic pathways. These findings highlight the therapeutic potential of probiotics as a complementary approach to conventional methods for mitigating and preventing Pb-induced liver damage. Moreover, this study underscores a link between gut health and liver function in the context of heavy metal toxicity. Further research is required to elucidate the mechanisms underlying the interactions between probiotics, gut microbiota, and metabolic pathways in response to Pb exposure. Understanding these mechanisms may facilitate the development of probiotic-based strategies aimed at heavy metal detoxification and overall health improvement.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConflicts of Interest\u003c/h2\u003e\u003cp\u003eYLH, WHT, and YTF are employed by GenMont Biotech. YTF is an academic consultant at GenMont Biotech. YCC, CCH, YTY, and SWH declare that they have no conflicts of interest.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eYTY analyzed the gut microbiomes and prepared the figures. YCC, CCH, and YLH performed the experiments. YLH, WHT, and YTF analyzed the data and prepared the figures. SWH conceptualized the study, drafted the manuscript, and revised the final manuscript. All authors have approved the final manuscript for journal submission.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors gratefully acknowledge Wallace Academic Editing for their professional assistance in manuscript editing.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data supporting the findings of this study are available from SWH upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTchounwou PB, Yedjou CG, Patlolla AK, Sutton DJ. 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Amino Acids. 2015;47(10):2143\u0026ndash;54. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00726-014-1773-4\u003c/span\u003e\u003cspan address=\"10.1007/s00726-014-1773-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"lead toxicity, Lacticaseibacillus paracasei, Limosilactobacillus fermentum, Lacticaseibacillus casei, hepatotoxicity, gut microbiota, intestinal barrier","lastPublishedDoi":"10.21203/rs.3.rs-8195284/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8195284/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLead (Pb) toxicity is a global health concern that primarily affects the liver. This study explored the protective effects of lactobacilli against Pb(II)-induced hepatotoxicity in mice. Three strains of lactobacilli\u0026mdash;\u003cem\u003eLacticaseibacillus paracasei\u003c/em\u003e GMNL-32, \u003cem\u003eLimosilactobacillus fermentum\u003c/em\u003e GMNL-93, and \u003cem\u003eLacticaseibacillus casei\u003c/em\u003e GMNL-277\u0026mdash;were evaluated for Pb adsorption and cytoprotective properties. The results indicated that probiotic treatment reduced the liver-to-body weight ratio, aspartate transaminase and alanine transaminase levels, and liver damage without increasing Pb excretion. It also upregulated the expression of gut tight junction proteins, reduced the levels of inflammatory cytokines (tumor necrosis factor-α and interleukin-6), and modulated the diversity and composition of the gut microbiota. Strong correlations were observed between probiotics, microbial abundance, metabolic pathways, and reduced liver inflammation. Overall, this study suggests that GMNL-32, GMNL-93, and GMNL-277 can mitigate Pb-induced hepatotoxicity by modulating the gut microbiota and regulating metabolism. Thus, these probiotics hold promise as protective agents against Pb-induced hepatotoxicity.\u003c/p\u003e","manuscriptTitle":"Lactobacillus protects against lead-induced hepatotoxicity by preserving the gut barrier and microbiota remodeling","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-26 05:33:12","doi":"10.21203/rs.3.rs-8195284/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-02T08:08:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-29T09:25:10+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-29T09:23:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Microbiology","date":"2025-11-24T15:42:02+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"85a0a736-e3ab-4ae7-aad2-bd4cceb551e5","owner":[],"postedDate":"November 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-23T16:10:58+00:00","versionOfRecord":{"articleIdentity":"rs-8195284","link":"https://doi.org/10.1186/s12866-026-04956-2","journal":{"identity":"bmc-microbiology","isVorOnly":false,"title":"BMC Microbiology"},"publishedOn":"2026-03-17 15:58:25","publishedOnDateReadable":"March 17th, 2026"},"versionCreatedAt":"2025-11-26 05:33:12","video":"","vorDoi":"10.1186/s12866-026-04956-2","vorDoiUrl":"https://doi.org/10.1186/s12866-026-04956-2","workflowStages":[]},"version":"v1","identity":"rs-8195284","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8195284","identity":"rs-8195284","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}