Intestinal microbiomics and metabolomics insights into the hepatoprotective effects of Lactobacillus paracasei CCFM1222 against the acute liver injury in mice

doi:10.21203/rs.3.rs-1868070/v1

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Intestinal microbiomics and metabolomics insights into the hepatoprotective effects of Lactobacillus paracasei CCFM1222 against the acute liver injury in mice

https://doi.org/10.21203/rs.3.rs-1868070/v1

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In recent years, acute liver injury (ALI) has received wide-range attention in the world due to its relatively high morbidity and mortality. This study aimed to explore the hepatoprotective effect of Lactobacillus paracasei CCFM1222 against lipopolysaccharide (LPS)-induced ALI mice, and further elaborate its mechanism of action from the perspective of intestinal microbiomics and metabolomics. The results displayed that L. paracasei CCFM1222 pretreatment significantly decreased the serum ALT, and AST levels, inhibited the releases of hepatic TNF-α, IL-1β, and IL-6 levels, and activated the SOD, CAT, and GSH-Px activities in LPS-treated mice. The cecal short-chain fatty acid (SCFAs) levels were increased in LPS-treated mice with L. paracasei CCFM1222 pretreatment. In addition, L. paracasei CCFM1222 pretreatment remarkably shifted the intestinal microbiota composition, including the higher abundance of Faecalibaculum, Bifidobacterium, and lower abundance of Prevotellaceae NK3B31 group, which is positively associated with the cecal propionic, butyric, valeric, isobutyric, and isovaleric acids. The metabolomics based on UPLC-QTOF/MS revealed that L. paracasei CCFM1222 pretreatment significantly regulated the composition of feces metabolites in LPS-treated mice, especially the potential biomarkers-related butanoate metabolism, vitamin B6 metabolism, D-glutamine and D-glutamate metabolism, tryptophan metabolism, caffeine metabolism, arginine biosynthesis, arginine, and proline metabolism. Moreover, L. paracasei CCFM1222 pretreatment remarkably regulated the expression of genes-associated ALI (including Tlr4, Myd88, Nf-kβ, iNOS, Cox2, Iκ-Bα, Nrf2, and Sirt-1). In conclusion, these results suggest the possibility that L. paracasei CCFM1222 supplementation has beneficial effects on preventing the occurrence and development of ALI by inhibiting the inflammatory responses, altering intestinal microbiota composition and their metabolites, and mediating the Tlr4/Nf-kβ pathway.

Lactobacillus paracasei

acute liver injury

metagenomic

metabolomics

Tlr4/Nf-κB pathway

Due to unhealthy dietary habits and lifestyle, hyperglycemia, atherosclerosis acute liver injury (ALI) has gradually become serious problems that threatening human health worldwide [1]. Among them, ALI has attracted more and more attention due to its relatively high morbidity and mortality [2]. According to the investigation by the European Association, the mortality rates of ALI patients reached 30% in 28 days [3]. ALI is characterized mainly by the higher activities of serum alanine transaminase (ALT) and aspartate amino transferase (AST), massive apoptosis of hepatocytes, abnormal inflammation responses, and excessive oxidative stress [4–6]. Among those, the abnormal inflammatory response is considered to be a common detrimental factor that is strongly associated with the occurrence and development of various clinical disorders. Lipopolysaccharide (LPS) or carbon tetrachloride (CCl₄) promotes the secretion of inflammatory cytokines, additional cytokines, and chemokines from the hepatic cell, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 [4, 6]. Our previous study suggested the lower activities of antioxidant enzymes and higher concentrations of inflammatory cytokines in ALI mice induced by LPS [7]. Consequently, the intraperitoneal injection of LPS is widely applied to establishing the ALI model in animals, which is beneficial for further exploring the pathogenesis of ALI and the process of pharmacological interventions [4]. Although various drugs have been widely used to treat ALI, most of the therapeutic drugs have various side effects [8]. Thus, searching for new functional food supplements with the hepatoprotective effects is a prospective strategy to attenuate inflammation and symptom-related ALI.

Probiotics are living microorganisms that confer health benefits to the host when supplemented in sufficient amounts, have drawn great attention from both local and international research communities. Among commensal bacteria, probiotic Lactobacillus and Bifidobacterium are the most numerous probiotics in the mammalian gut and are widely applied in the field of medicine and food [9]. Especially, Lactobacillus has been reported to ameliorate intestinal microbiota composition, improve the inflammatory response, reduce serum cholesterol and elevate antioxidant stress [10]. For example, L. paracasei has been proved to suppress the inflammatory cytokines secretion (including TNF-α, IL-6, IFN-γ), and elevate the antioxidant enzymes activities [including catalase (CAT) and superoxide dismutase (SOD)] [11]. L. paracasei S16 intervention also ameliorated the aberrant inflammation response and reduced the percentage of Th1 and Th2 cells and Th17/Treg ratio [12]. Furthermore, a previous study elaborated that Long-term intervention of L. paracasei remarkably elevated the proportion of beneficial bacteria and remarkably decreased the proportion of harmful bacteria [13]. L. plantarum ZY08 attenuates the long-time alcohol consumption-induced hepatic steatosis and liver damage by regulate the intestinal microbiota composition [14]. In addition, recently report also suggested that the “gut-liver” axis takes a vital role in the pathological process of some liver diseases [15]. Metabolites from intestinal flora are regarded as the vital medium in the “gut-liver” axis, such as short-chain fatty acids (SCFAs), amino acids, and bile acids. Among them, SCFAs are commonly considered anti-inflammatory substances, which stem from the fermentation of dietary carbohydrates by intestinal microbiota [16]. For example, butyrate promotes the production of IL-22 by activating the expression of aryl hydrocarbon receptor (AhR) and hypoxia-inducible factor 1α (HIF-1α) [17]. Nevertheless, the functional mechanism by which L. paracasei prevents liver injury induced by endotoxin is less explored. Recently years, with the development of untargeted metabolomics based on UPLC-QTOF/MS, many novel microbial metabolites were detected and identified in the faeces [18]. For example, recently report have exhibited that Lactobacillus modulates the host’s immune system by producing inosine [19]. Our previous study also exhibited that inosine pretreatment remarkably reduced the serum TNF-α, IL-1β, and IL-6 levels [7]. Although the hepatoprotective efficacy of L. paracasei have been explored in vivo, there are few reports on the potential mechanism of L. paracasei intervention on ALI by UPLC-QTOF/MS-based metabolomics.

L. paracasei CCFM1222, a newly identified probiotic, was isolated from the feces of an infant and stored in our laboratory. The aim of this study was to explore the underlying mechanism of L. paracasei CCFM1222 against ALI in mice with LPS treatment through high-throughput sequencing and untargeted metabolomics based on UPLC-QTOF/MS. Moreover, the association between the key intestinal flora and key liver indicators-related ALI was also uncovered by correlation analysis, which supplies a strong theoretical basis for developing L. paracasei CCFM1222 to improve ALI.

2.1. Materials and chemical reagents

L. paracasei CCFM1222 was isolated from the feces of an infant and stored in Culture Collection of Food Microorganisms in Jiangnan University (Wuxi, China). LPS was supplied from Sigma-Aldrich (Shanghai, China). Cholesterol (TC), triacylglycerols (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), alanine transaminase (ALT), aspartate aminotransferase (AST), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) kits were purchased from Jiancheng Bioengineering Institute (Nanjing, China). TNF-α, IL-1β, IL-6, and LPS kits were purchased from SenBeiJia Biological Technology Co., Ltd. (Nanjing, China). SCFAs were obtained from Solarbio Co., Ltd. (Beijing, China).

2.2 Probiotic strain and culture conditions

The original strain of L. paracasei CCFM1222 was stored in -80 ℃ environment. During the animal experiment, L. paracasei CCFM1222 has activated in MRS broth (at 37 ℃ for 18 h) every four days. After four generations, strains were washed three with 0.9% NaCl, and then re-suspended in sterile saline (fine concentrations: 5.0 × 10⁹ CFU/mL) for the animal experiment.

2.3 Animal protocols

Twenty-four SPF male C57BL/6J mice (seven-week-old, 21 ± 1 g) were purchased from Animal Research Center (Hangzhou, China) and maintained in an appropriate environment (temperature 22.5 ± 2°C, relative humidity 55 ± 10%, and 12 h light/dark cycles) with free to intake food and water. The whole experimental procedures were approved by the Animal Ethics Committee of Jiangnan University (JN.No20211130S0360525[491]).

After a 7-day adaptation period, mice were stochastically distributed to 3 groups (n = 8), namely: Control, Model, and CCFM1222 groups. During the whole experiments, mice in the Control and Model groups were fed with 0.2 mL of 0.9% NaCl, while mice in the CCFM1222 group were fed with 0.2 mL of L. paracasei CCFM1222 (5.0 × 10⁹ CFU/mL) every day. At the end of the animal experiment, mice in the Control group were intraperitone ally injected with 0.2 mL of 0.9% NaCl after 6-hour starvation, while other mice were intraperitoneally injected with 0.2 mL of LPS (5 mg/kg). All mice were euthanized at 4 h after LPS intraperitoneally injection. During the whole experiments, the body weight, food intake, and water intake were measured every two days. After 14 days of intervention, fecal samples were obtained and stored in the -80℃ refrigerator.

2.4 Serum and hepatic biochemical assays

The Blood was obtained and placed at room temperature for 90 min, and then centrifugation (5000 rpm, 25℃, 10 min). The serum was obtained and placed at -80℃ environment. The levels of serum TC, TG, LDL-C, HDL-C, AST, and ALT were analyzed using the commercial kits (Jiancheng, Nanjing, China).

Partial liver tissue was homogenated using RIPA Lysis Buffer (1:9, g:mL), and then the supernatant was obtained by centrifugation (10000 rpm, 4℃, 15 min). The levels of hepatic TNF-α, IL-1β, IL-6, IL-10, and LPS were analyzed. In addition, some liver tissue was homogenated using 0.9% NaCl (1:9, g:mL), and the supernatant was obtained by centrifugation (10000 rpm, 4℃, 15 min). The concentrations of hepatic MDA, MPO, SOD, CAT, and GSH-Px were the commercial kits.

2.5 Histological analysis

Histopathological examination was conducted using liver sections fixed in 4% paraformaldehyde for 24 h and dehydrated using ethanol solutions. Following embedding in paraffin, the sections were cut into 5 μm thick sections and the photos were taken with 3Dhistech Pannoramic Scan (Budapest, Hungary) after the sections were stained with hematoxylin eosin. At least 3 different random fields per liver tissue were measured in the three groups.

2.6 Short-chain fatty acids (SCFAs)

Cecal contents were collected and freeze-drying. 0.5 ml of saturated NaCl solution was added to 0.05 g samples. Samples were placed at 25℃ for 30 min, and then homogenated (65 MHz, 15s) five times. Ten microliters of sulphuric acid (10%, v/v) and the samples were mixed, and then centrifugated (12,000 rpm, 10 min, 4 °C). The supernatant and 0.8 ml of C₂H₅OC₂H₅ were mixed and centrifugated (12,000 rpm, 10 min, 4 °C). 0.25 g of anhydrous Na₂SO₄was added to the supernatant and placed at 25℃ for 30 min. The solution was centrifugated (12,000 rpm, 10 min, 4 °C), and the supernatant was obtained. SCFAs of supernatants were measured using gas chromatography. The oven temperature process was 60 ℃ maintained for 2 min, and the rose to180°C at a rate of 10 °C/min, followed by 180 °C maintained for 2 min.

2.7 Intestinal flora analysis

Bacterial genomic DNAs of faeces were extracted using fecal genomic DNA extraction kit (MoBio, USA), and then used as a template to amplify the V3-V4 hypervariable region of the 16S rRNA gene with the forward primer 341 F and the reverse primer 806 R. Validated libraries were used for sequencing on the Illumina MiSeq platform at Shanghai Majorbio Co., Ltd. (Shanghai, China). The quality control and clustering results were analyzed by QIIME 2.0 software (version 2019.7). Optimized sequences were clustered at a similarity of 97% and blasted with the GreenGenes database (Ver. 13.8) to identify microbial phylotypes at the genus level. Principal coordinate analysis (PCA) and hierarchical clustering analysis (HCA) of intestinal microbiota were implemented by SIMCA software (14.1). The differences in intestinal microbiota among three groups by an online website (http://huttenhower.sph.harvard.edu/galaxy/). The correlations between the key parameters-related ALI and the significant changes of intestinal microbiota were computed and visualized using R software (4.1.3) and Cytoscape (3.9.0).

2.8 Untargeted feces metabolomics analysis

Feces (25 mg) were handled using 0.8 mL 80% methanol, and vortexed for 60 s. After centrifugation (14000 rpm, 20 min, 4 °C), the liquid was transferred into the EP tube and dried at 25 °C under a vacuum environment. All samples were resuspended in 0.2 mL of 80% methanol, and centrifugated at 14000 rpm for 20 min at 4 °C. The samples was obtained and passed by a 0.2 μm aqueous membrane. Ultimately, all samples were injected into UPLC-QTOF/MS system for metabolomics analysis. Raw data were converted and analyzed by Compound Discoverer software (v 3.3.0). PCA, partial least squares discriminant analysis (PLS-DA) of fecal metabolites was performed using MetaboAnalyst 5.0. Fecal metabolites of remarkable discrepancy between the model and CCFM1222 groups are determined using orthogonal partial least-squares discrimination analysis (OPLS-DA) and S-plot (VIP value > 1.0, p value < 0.05). Pathway analysis of fecal metabolites with remark differences was carried out using MetaboAnalyst 5.0.

2.9 Real-time quantitative polymerase chain reaction (RT-qPCR)

Hepatic RNA was extracted by commercial kits (Sangon, Shanghai, China). Reverse transcription of RNA and qPCR was carried out using PrimeScript™ RT reagent Kit with gDNA Eraser (Takala, Beijing, China) and One Step TB Green® PrimeScript™ RT-PCR Kit II (Takala, Beijing, China), respectively. The qRT-PCR process contained the initial denaturation step at 95 °C for 30 s, followed by 40 cycles of 95 °C for 10 s, 60 °C for 15 s, and 75 °C for 30 s. The expressions of mRNA were calculated according to the expression of Gapdh and 2^−ΔΔCt method, and the primer sequences are presented in Table S1.

2.10 Immunohistochemical analysis

Immunohistochemical analysis was implemented at Wuhan Saiweier Co., Ltd. (Wuhan, China). Liver tissues were soaked in a primary antibody to p65, Iκ-Bα, and Nrf2 for 45 min, and then transferred to a secondary antibody and streptavidin-biotin peroxidase for 45 min. Finally, the brown color was analyzed under 3,3′-diaminobenzidine tetrahydrochloride by a light microscope. Sections were observed using a panoramic scanner (Pannoramic MIDI, 3D HISTECH, Hungary).

2.11 Statistical analysis

To calculate the significance of differences between groups, the data were analyzed using one-way ANOVA with GraphPad Prism (Ver. 7.0) software followed by Duncanʼs multiple range test. ^*p < 0.05, and ^**p < 0.01 compared with the Model group, n.s. represents no significance.

3.1 Influences of L. paracasei CCFM1222 pretreatment on body weight and serum biometric indexes in mice

As displayed in Figure 1, there was no remarkable discrepancy in the body weight of mice among the three groups at the beginning of animal experiment. After 14 days of treatment, the body weight of mice in CCFM1222 group was higher than that in the Control and Model groups, but no remarkable discrepancy among the three groups (p > 0.05). Furthermore, there was no remarkable difference in food intake and water intake of mice among the Control, Model, and CCFM1222 groups (p > 0.05).

Notably, L. paracasei CCFM1222 pretreatment did not remarkably altered the serum TC, TC, HDL-C, LDL-C levels relative to the Control and Model groups (p > 0.05), suggesting L. paracasei CCFM1222 did not increase the risk of hyperlipidemia and atherosclerosis (Figure 1). However, the serum AST, and ALT activities were obviously increased in the Model group relative to that in mice without LPS treatment (p < 0.05), but L. paracasei CCFM1222 preintervention obviously inhibited the serum AST, and ALT activities in LPS-treated mice (p < 0.05). This phenomenon suggesting L. paracasei CCFM1222 is beneficial for protecting the liver tissue against LPS.

3.2 L. paracasei CCFM1222 pretreatment attenuated the inflammatory response and oxidative stress in LPS-treated mice

The hepatic IL-6, IL-1β, TNF-α, and LPS levels were detected after the 4 h of LPS treatment (Figure 2A). As relative to the Control group, intraperitoneal injection of LPS remarkably elevated the hepatic IL-6, IL-1β, TNF-α, and LPS levels (p < 0.05), implying that ALI model was successfully established. Interestingly, pretreatment with L. paracasei CCFM1222 significantly reduced the IL-1β, TNF-α, and LPS levels in LPS-treat mice (p < 0.05), and slightly reduced the hepatic IL-6 levels (p > 0.05). The hepatic IL-10 levels were remarkably decreased in the Model group, relative to the Control group (p < 0.05). L. paracasei CCFM1222 pretreatment significantly recovered the hepatic IL-10 levels in LPS-treated mice (p < 0.05), which is closed to the Control group. Furthermore, the results of H&E staining exhibited that intraperitoneal injection of LPS caused tissue architecture disorder, inflammatory cell infiltration, and hemorrhage compared with mice without LPS treatment (Figure 2B). Nevertheless, these symptoms were significantly ameliorated in LPS-treated mice with L. paracasei CCFM1222 pretreatment for 14 days. These appearances indicate that the ALI-related inflammation induced by LPS was significantly improved by L. paracasei CCFM1222 pretreatment.

The influence of L. paracasei CCFM1222 pretreatment on oxidative stress in LPS-treated mice was analyzed (Figure 3). Relative to the Control group, LPS treatment results in an increase in hepatic MDA levels and MPO activities (p < 0.05). However, pretreatment with L. paracasei CCFM1222 remarkably decreased the hepatic MPO activities in LPS-treated mice (p < 0.05), while no remarkably changed the MDA levels (p > 0.05). Moreover, the hepatic T-AOC, SOD, and CAT activities of mice in the Model group were obviously higher than that in the Control group (p < 0.05). Interestingly, pretreatment with L. paracasei CCFM1222 significantly restored the activities of hepatic T-AOC, SOD, and CAT (p < 0.05). Moreover, pretreatment with L. paracasei CCFM1222 had a limited effect on hepatic GSH-Px activity (p > 0.05). Therefore, pretreatment with L. paracasei CCFM1222 might alleviate these LPS-induced the inflammatory response and oxidative stress in mice

3.3 L. paracasei CCFM1222 pretreatment elevated the levels of cecal SCFAs and colonic inosine in LPS-treated mice

SCFAs take an essential role in reducing the pH of the gut, inhibiting pathogens, and regulating host immunity. Therefore, the influence of L. paracasei CCFM1222 pretreatment on cecal SCFAs concentrations in ALI mice was analyzed (Figure 4). There was no remarkably changed in the acetic, propionic, isobutyric, butyric, valeric, and isovaleric acids between the Control and Model groups (p > 0.05). Notably, after the 14 days of L. paracasei CCFM1222 intervention, the propionic, isobutyric, butyric, and valeric acids were remarkably increased in LPS-treated mice (p < 0.05).

3.4 L. paracasei CCFM1222 pretreatment altered the intestinal flora composition

High-throughput sequencing was implemented to analysis the structural alters of intestinal flora from the feces samples of different groups (control, model, and CCFM1222 groups). PCA and HCA based on the intestinal flora of operational taxonomic units (OTUs) directly exhibited distinct clustering of microbe communities for three groups. The first, two, and three main components of PCA accounted for 36.3%, 16.6%, and 10.8% of the total data change, respectively (Figure 5A and Figure S1). There was an obvious discrepancy in the intestinal flora between the control and model groups. However, L. paracasei CCFM1222 pretreatment obviously altered the intestinal flora composition relative to that in the model group. These results were further confirmed by PLS-DA, and HCA (Figure 5B).

To deeply explore the alters in intestinal flora induced by L. paracasei CCFM1222 treatment, LEfSe taxonomy cladogram, and linear discriminant analysis (LDA, more than 2.0) score was carried out. As can be derived from Figure 5C and 5D, f_Erysipelotrichaceae, o_Erysipelotrichales, c_ Erysipelotrichia, g_Faecalibaculum, f_Bifidobacteriaceae, g_Bifidobacterium, c_Actinobacteria, o_Bifidobacteriales, c_Mollicutes, and p_Tenericutes were enriched in the CCFM1222 group. g_ Prevotellaceae NK3B31 group and g_Ruminiclostridium 5 were enriched in the model and control groups, respectively.

3.5 Associations analysis of the key intestinal flora and biochemical parameters

The association between the abundance of mainly intestinal flora and the important parameters-related inflammation and oxidative stress was explored by Spearman correlation analysis (Figure 6A and 6B). The results displayed that the relative abundance of f_Erysipelotrichaceae, o_Erysipelotrichales, c_ Erysipelotrichia, g_Faecalibaculum, f_Bifidobacteriaceae, g_Bifidobacterium, c_Actinobacteria, o_Bifidobacteriales, c_Mollicutes, p_Tenericutes, and g_Ruminiclostridium 5 were negatively correlated with liver oxidative stress parameters (MDA, and MPO), and inflammation parameters (IL-6, IL-1β, and TNF-α), but positively correlated with liver anti-oxidative enzymes (SOD, and T-AOC), and cecal SCFAs (acetic, propionic, butyric, valeric, isobutyric acid, and isovaleric acids). g_ Prevotellaceae NK3B31 group was negatively correlated with cecal SCFAs levels, and positively associated with inflammatory cytokines (IL-6, IL-1β, and TNF-α), and oxidative stress parameters (MDA, and MPO). These results indicated that the increases in these beneficial bacteria caused by L. paracasei CCFM1222 could help to the restrain of the excessive oxidative stress and inflammatory cytokines proliferation.

3.6 L. paracasei CCFM1222 pretreatment altered the fecal metabolomic profiling

Untargeted metabolomics based on UPLC-QTOF/MS is used to identify the changes in fecal metabolites in ALI mice caused by L. paracasei CCFM1222 intervention. As can be derived from Figure 7A, there was not remarkable discrepancy in fecal metabolites between the Control and Model groups in PCA score plots. However, there was an obvious discrepancy in the fecal metabolic profile between the Model and CCFM1222 groups, which is further confirmed by LPS-DA score plots (Figure 7B). The OLPS-DA and S-plots displayed the discrepancy in fecal metabolites between the Model and CCFM1222 groups (Figure 7C and 7D).

To further explore the therapeutic effect of L. paracasei CCFM1222, 51 potential biomarkers (VIP > 1.0 and p < 0.05) were successfully identified (Figure 7E), of which 22 potential biomarkers (deoxycytidine, lysergicacid, 2'-deoxyinosine, 5-hydroxyindole, bis(2 -ethylhexyl)phthalate, (R)-3-hydroxybutanoate, catechol, D-Ala-D-Ala, hexanoylglycine, succinic acid, palmitic acid, lmidazoleaceticacid, methylglyoxal, D-(+)-proline, L-(+)-citrulline, L-methionine sulfoxide, thymidine, 2-deoxystreptamine, L-lysine, L-glutamic acid, and sphingosine (d18:1)) were remarkably reduced, and of which 29 potential biomarkers (alpha-tocotrienol, D-tryptophan, valylproline, (+/-)-6-hydroxy-3-oxo-alpha-ionol, pyridoxamine, L-kynurenine, 1,5-lsoquinolinediol, D-glutamate, benzaldehyde, 1-methylxanthine, 2,4-quinolinediol, 4-nitroacetophenone, N-acetyl-DL-tryptophan, 4-(hydroxymethyl)benzoic acid, D-(+)-pipecolinic acid, 3-indolepropionicacid, D-O-phosphoserine, glycitein, (s)-4-hydroxymandelonitrile, butein, N-carbamoylputrescine, L-histidinol, 3,4-dihydroxybenzoate, 3,5-dihydroxy-phenylglycine, D-saccharicacid, beta-glycerophosphoricacid, pyridoxine, indole-3-acetic acid, and tropine) were remarkably increased. These potential biomarkers were connected with multiple metabolic pathways, such as butanoate metabolism, vitamin B6 metabolism, synthesis and degradation of ketone bodies, alanine, aspartate and glutamate metabolism, D-glutamine and D-glutamate metabolism, tryptophan metabolism, caffeine metabolism, arginine biosynthesis, and arginine and proline metabolism (Figure 7F).

3.7 L. paracasei CCFM1222 pretreatment regulated the expression of genes related to ALI in LPS-treated mice

The liver is an essential organ involved in the regulation of host immunity. To reveal the potential mechanism of the hepatoprotective effects of L. paracasei CCFM1222 treatment, the mRNA levels of genes associated with the inflammatory response and oxidative stress in the liver were measured. As can be derived from Figure 8A, the translocations of hepatic Tlr4, Myd88, Nf-kβ, iNOS, and Cox2 were remarkably increased and the translocations of hepatic Iκ-Bα, Nrf2, and Sirt-1were remarkably reduced in ALI mice induced by LPS, relative to mice without LPS treatment (p < 0.05). Interestingly, pretreatment with L. paracasei CCFM1222 remarkably suppressed the hepatic Tlr4, Myd88, Nf-kβ, and iNOS translocations, and remarkably promoted Iκ-Bα, Nrf2, and Sirt-1 translocations (p < 0.05), compared with ALI mice induced by LPS. In immunohistochemistry experiments, L. paracasei CCFM1222 could up-regulate the expressions of hepatic Iκ-Bα, Nrf2, and down-regulate the expression of p65 in ALI mice induced by LPS (p < 0.05) (Figure 8B and Figure 8C).

To establish drug hepatitis-caused liver function injury, animals were intraperitoneally injected with LPS, which is a widely recognized model for the induction of ALI and inflammation response because of its symptoms being similar to those in patients with ALI [6, 20]. Therefore, ALI model induced by LPS is frequently used for developing natural active substances, probiotics, and novel drugs that possess hepatoprotective, anti-inflammation, and anti-oxidative stress properties. Consequently, the aim of this study was to explore the hepatoprotective and anti-inflammatory effects of L. paracasei CCFM1222 on ALI mice induced by LPS. Our results displayed that L. paracasei CCFM1222 could remarkably suppress the inflammatory responses, inhibit oxidative stress, improved the intestinal flora composition, shift the fecal metabolites, and regulate the gene expression-related liver injury in ALI mice induced by LPS, indicating that L. paracasei CCFM1222 is a potential probiotic that can relieve/treat ALI and inflammation.

Liver injury is frequently accompanied by the higher activities of serum ALT and AST which are widely regarded as the most credible biochemical markers for assessing liver health. Clinic evidence also confirmed that the injury of liver tissue results in the hepatic ALT and AST being transferred to the circulation [21]. The activities of ALT reflect the injury of the cell membrane to a certain degree, while the activities of AST major reflect the injury of mitochondria to a certain degree [22]. The result of this research exhibited that intraperitoneally injected with LPS elevated the serum ALT and AST activities, indicating that ALI model was successfully established, which is in agreement with the previous report [5]. Our study suggested that L. paracasei CCFM1222 pretreatment obviously reduced the serum ALT and AST activities, and slightly increased body weight in LPS-induced ALI mice. The increase in body weight is considered an important index of lipid metabolism disorder. However, whether L. paracasei CCFM1222 can destroy host lipid metabolism need to be further explored. Accumulating evidence has elaborated that lipid metabolism disorder is strongly related to the accumulation of serum lipid profile, such as the higher levels of serum TC, TG, and LDL-C. It has been reported that the increases in serum TC and TG levels promote the development of cardiovascular disease [23]. Therefore, serum TC and TG are usually applied for estimating the state of host energy metabolism. Interestingly, there were no remarkable discrepancy in serum TC, TG, and LDL-C levels among the three groups.

Abnormal inflammation cytokines (TNF-α, IL-1β, and IL-6) are is a trait of the ALI symptom and accelerate the process of comorbidities. Our results displayed that LPS treatment elevates the hepatic TNF-α, IL-1β, and IL-6 levels relative to mice without LPS treatment. TNF-α serves as a vital regulator of immunity and inflammation, which causes cytotoxic effects and induces hepatocyte necrosis [24–25]. However, the secretion of TNF-α is monitored by the levels of IL-1β to a certain extent. IL-1β is a proinflammatory cytokine that mainly stems from activated macrophages, which are involved in multiply inflammatory and autoimmune diseases, such as hepatitis, osteomyelitis, and alcoholic liver injury [26]. Moreover, IL-6 is traditionally regarded a modulator of acute-phase responses, and its concentrations are positively related to glucolipid metabolism disorder and liver functional decline [27]. Contrarily, IL-10 is an anti-inflammatory factor that takes a beneficial role in limiting the development of inflammation. A previous report displayed that IL-10 could suppress the secretion of TNF-α, and then relieve the inflammatory responses [24]. Interestingly, we found that L. paracasei CCFM1222 pretreatment effectively suppressed LPS-aroused the higher levels of hepatic IL-6, IL-1β, TNF-α, and the lower levels of IL-10, indicating that L. paracasei CCFM1222 prevents the development of ALI partly by regulating the secretion of proinflammatory cytokines. In addition, it is widely accepted that overload oxidative stress destroys liver function. MDA is a secondary metabolite that stems from the end product of lipid peroxidation, which is regarded as a vital index for assessing the host oxidative stress. High concentrations of MDA directly resulted in cell cytotoxicity, and even cause damage to organ function. LPS treatment is reported to increase the hepatic MDA concentration, leading to destroy to the cell membrane and the DNA structure, which is agreement with this study [28]. Moreover, lipid peroxidation induces the production of peroxides and reactive oxygen species. The function of SOD is the depressor of reactive oxygen species and promoting the formation of hydrogen peroxide in vivo, which was further decomposed to the non-toxic substance (oxygen and water) by up-regulating GSH-Px activity [15]. Our study displayed that L. paracasei CCFM1222 pretreatment obviously suppressed the MDA concentration, and up-regulated the SOD and GSH-Px activities in LPS-treated mice. In addition, the hepatic CAT activity was remarkably increased in LPS-treated mice with L. paracasei CCFM1222 pretreatment. CAT is a vital endogenous enzyme that relieves the symptoms of inflammation by eliminating the reactive oxygen species in the body [29].

It is reported that Lactobacillus produces the SCFAs by fermenting the undigested carbohydrates. SCFAs play multiple roles in human health, including it serve as energy substrates and signaling molecules. The appropriate concentration of cecal SCFAs is help to keep the stable intestinal function, thus preventing the injury of liver function [30]. For example, acetic acid controls the host glucose metabolism by affecting the sensitivity of insulin [15]. Butyric acid not only supplied energy to intestinal epithelial cells, and promoted the mitosis of the immune cell, which is help to increase protection for the host [31]. In the present study, we also found that L. paracasei CCFM1222 pretreatment effectively elevated the cecal SCFAs levels in LPS-induced ALI mice.

Recently years, the “gut-liver” axis is received wide-range attention in the world. Gut-liver axis is composed of intestinal flora, microbial metabolites, intestinal barrier, and liver function. Our study showed that L. paracasei CCFM1222 treatment significantly altered the intestinal flora composition, including the enrichment in f_Erysipelotrichaceae, g_Faecalibaculum, and g_Bifidobacterium. A previous study exhibited that the enrichment in Erysipelotrichaceae was observed in high-fat diet-induced obese animals and individuals because it promotes the energy extraction from the diet [32]. Our result suggested that L. paracasei CCFM1222 treatment significantly increased the body weight of mice, which may associate with the abundance of Erysipelotrichaceae. However, it has also been reported that the relative abundances of Erysipelotrichaceae (butyrate-producing bacteria) were negatively related to the expression of inflammatory cytokines [33]. Among Erysipelotrichaceae family, Faecalibaculum genus promotes SCFAs production, carbohydrate metabolism, and lactate metabolism [34]. A higher concentration of butyric acid improves the inflammation and oxidative stress of ALI by stimulating the secretion of IL-22 from intestinal epithelial cells [35]. In addition, Bifidobacterium is one of the most important probiotics that have beneficial effects on the repair of liver function. A previous study exhibited that Bifidobacterium could suppress the inflammation responses by inducing a morphologic, phenotypic, and functional transitional change [36]. Our results exhibited that the relative abundance of f_Erysipelotrichaceae, g_Faecalibaculum, and g_Bifidobacterium were negatively associated with the liver inflammatory cytokines.

More and more evidence suggested that microbiome-related intestinal metabolites may have a vital role in the “gut-liver” axis [13, 37]. Oral supplementary of L. paracasei CCFM1222 remarkably reduced fecal levels of 2'-deoxyinosine, (R)-3-hydroxybutanoate, succinic acid, and methylglyoxal. 2'-deoxyinosine is one kind of purine derivatives that is absorbed into the systemic blood circulation. Higher concentrations of 2'-deoxyinosine were frequently discovered in the inflammatory mouse model [38]. (R)-3-hydroxybutanoate is the intermediate products of the incomplete oxidative degradation of fatty acids, and it could stimulate the abnormal inflammation response and excessive oxidative stress [39]. Succinic acid and palmitic acid serve as the intermediate products of energy metabolism, which play an anti-inflammatory role in the improvement of liver injury. However, succinic acid promotes the development of autoimmune uveitis by elevating the production of IFN-γ/IL-17A and neutrophil extracellular trap generation [40]. In addition, methylglyoxal is a reactive dicarbonyl byproduct of glucose and other reducing sugars, which accumulate in mitochondria during diabetes [41]. However, the concentrations of alpha-tocotrienol, pyridoxamine, tryptophan, pipecolinic acid, 3-indole propionic acid, indole-3-acetic acid, and glycitein in the CCFM1222 group were higher than that in the Model group. Alpha-tocotrienol belongs to vitamin E, have antioxidant and anti-inflammatory effects, have been shown to be protective to liver function [42]. Pyridoxamine is a structural analog of vitamin B6 that could attenuate liver fibrosis by inhibiting the inflammatory cascades and combating oxidative stress [43]. Tryptophan and pipecolinic acid, the essential amino acid, has been reported that possesses the capacity to regulate host susceptibility to acute liver failure by regulating the activity of aryl hydrocarbon receptor [44]. A previous study suggested that Lactobacillus casei intervention ameliorates the gut healthy by promoting the production of pipecolinic acid [45]. Gut microbiome-derived 3-indole propionic acid and indole-3-acetic acid act as the microbiota-specific AhR ligand, and their concentrations are negatively associated with the relative abundance of Bifidobacterium [46–47]. Among these, indole-3-acetic acid alleviates LPS-stimulated inflammatory response and free radical generation by regulating the expression of HO-1 and eliminating the free radicals [48]. The increases of fecal glycitein were remarkably increased in AOM/DSS-treated mice after Lactiplantibacillus plantarum-12 intervention, which is agreement with this study [49].

To reveal the latent mechanism whereby L. paracasei CCFM1222 pretreatment relieves LPS-induced ALI and inflammation in mice, the translocation of genes-related ALI and inflammation were measured in the liver, such as Tlr4, Myd88, Nf-kβ, iNOS, Cox2, Iκ-Bα, Nrf2, and Sirt-1. Tlr4 is the first identified Toll protein that takes a radical character in sensing invading causative agents or endogenous damage signals, especially LPS [50]. Elevating the expression of Tlr4 promotes the nuclear translocation of Nf-kβ by increasing the recruitment of Myd88, and further intervenes the secretion of inflammatory cytokines. Deficiency Myd88 is beneficial for hampering the development of inflammation [51]. In addition, Nf-kβ expression is monitored by the concentrations of Iκ-Bα and Sirt-1 in the liver. Overexpression of Iκ-Bα has been beneficial in blocking the expression of Nf-kβ. The function of Sirt-1 is similar to Iκ-Bα, and take participates in cell survival, stress resistance, and other physiological activations [52]. Therefore, up-regulation of Iκ-Bα, Sirt-1 expression helps to hamper the occurrence and development of ALI. We found that pretreatment with L. paracasei CCFM1222 down-regulated the hepatic Tlr4, Myd88, Nf-kβ translocation, and up-regulated the hepatic Iκ-Bα, Sirt-1 translocation in LPS-treated mice. Moreover, the higher expression of hepatic iNOS and Cox2 was significantly inhibited in ALI mice with L. paracasei CCFM1222 pretreatment. Overexpression of Nf-kβ accelerated the expression of iNOS and Cox2 in liver. iNOS and Cox2 serve as the proinflammatory enzymes that involved in the pathogenesis of various biological and inflammatory disorders by controlling the production of nitric oxide [53]. According to these results, we speculate preliminarily that pretreatment with L. paracasei CCFM1222 relieves ALI by controlling the Tlr4/Nf-kβ pathway.

This research supplied the evidence that L. paracasei CCFM1222 pretreatment has the potential capacity to alleviate ALI-associated inflammation in LPS-treated mice. The hepatoprotective effects of L. paracasei CCFM1222 on ALI mice were achieved by altering the intestinal flora composition and its metabolites, and mediating the Tlr4/Nf-κB pathway. In future research, we need to explore the metabolite in the gut that alleviates/treats the occurrence and development of ALI using liver transcriptomics and proteomics.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgements

The authors wish to thank the staff at the Animal Ethics Committee of Jiangnan University for their technical assistance during this study.

Author’s Contribution

W.G., B.M. and S.C. performed the experiments and wrote the manuscript. X.T. and Q.Z. checked the logicality and language of this manuscript. J.Z. and H.Z. conceived and designed the study. B.M. was responsible for overall study coordination of this manuscript. All authors read and approved the final manuscript.

Data Availability

The datasets generated and/or analyzed in the current study are available from the corresponding author on reasonable request.

Funding

This work was supported by National Natural Science Foundation of China (Grant No. 31972086, 32172173, 32072197), Yongjiang Talent Introduction Programme (2021C-003-T) and Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province.

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Intestinal microbiomics and metabolomics insights into the hepatoprotective effects of Lactobacillus paracasei CCFM1222 against the acute liver injury in mice

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