Ethanol exposure frequently induces intestinal and liver injury, dysbiosis of the gut microbiota and vitamin C (VC) deficiency. Gut microbiota-targeted therapy is emerging as an important adjuvant method for protecting the body against ethanol-induced injury, particularly probiotics containing
Excessive alcohol consumption has become one of the major causes of diverse diseases (
The mammalian gastrointestinal tract is colonized by trillions of microorganisms that coevolved with their hosts (
It is well recognized that temperance is the best way to prevent the damage of alcohol to human body (
Therefore, the aim of the present study was to determine whether symbiotic supplementation of LA plus VC is able to reduce ethanol-induced intestine and liver injury by modulating gut microbiota dysbiosis and restoring intestinal barrier function in mice.
The animal experiments of the present study were approved by the Ethics and Clinical Research Committee of Tianjin Medical University (Tianjin, China). Forty male C57BL/6J mice (age, 7-8 weeks; weight, 21-23 g) were obtained from Huafukang Biological Technology Co., Ltd. and maintained in a specific pathogen-free environment at 23˚C and 40-60% humidity with a 12-h light/12-h dark cycle. The ethanol feeding mouse model was constructed based on the Lieber-DeCarli diet (cat. no. TP 4030C/TP 4030A; TROPHIC Animal Feed High-Tech Co. Ltd.) (
To determine intestinal permeability, FITC-dextran (cat. no. 68059; Sigma-Aldrich; Merck KGaA) was orally administered to mice (600 mg/kg body weight) at 4 h prior to sacrifice. These blood samples were collected from the isoflurane-anesthetised mice and blood samples were centrifuged (2,000 x g, 4˚C) for 10 min to obtain serum (200 µl). The fluorescence of these serum samples was immediately recorded by a spectrophotometer (Tecan) at an excitation wavelength of 485 nm and emission wavelength of 528 nm.
For all mice, the middle part of colon tissues was collected and the colon lamina propria lymphocytes (LPMCs) were prepared as described previously (
Colon samples (100 mg) were homogenized with a mini-bead beater (cat. no. KZ-II) and glass beads (cat. no. G0101-200G; both from Wuhan Servicebio Technology Co., Ltd.) in 1 ml 1X PBS buffer and 700 µl supernatant was transferred to a new centrifuge tube after centrifugation. The superoxide dismutase (SOD) kit (cat. no. BC0170; Solarbio Life Sciences), myeloperoxidase (MPO) kit (cat. no. ab105136; Abcam) and glutathione peroxidase (GSH-PX) kit (cat. no. BC1190; Solarbio Life Sciences) were used to determine the SOD activity, MPO activity and GSH-PX activity according to the manufacturer's protocols. Serum aspartate transaminase (AST) and alanine transaminase (ALT) were determined by a blood biochemical analyser (Fujifilm DRI-CHEM 3500s; Fujifilm) according to the manufacturer's protocol. Mouse ELISA Kits were used to determine the serum levels of LPS (cat. no. JL20691-96T; Jiang Lai Biological), TNF-α (cat. no. ml002095; Enzyme Link Biotechnology Co., Ltd.), IL-1β (cat. no. ml301814; Enzyme Link Biotechnology Co., Ltd.) and IL-6 (cat. no. M*ml002301; Enzyme Link Biotechnology Co., Ltd.), and spectrophotometric methods were used to measure hepatic triglyceride (cat. no. JL46662-96T; Jiang Lai Biological) and hepatic malonaldehyde (MDA; cat. no. JL13329-96T; Jiang Lai Biological) via a spectrophotometer (Infinite F50; Tecan Group, Ltd.). All the assays were performed in triplicate and all the experiments were performed according to the manufacturer's protocol.
For the histological analysis, the liver and colonic tissues were stained with hematoxylin and eosin (H&E). In brief, the tissues were fixed in 10% formalin for 48 h at room temperature, and paraffin-embedded sections (5 µM) were stained with H&E. For the evaluation of mucins (Muc), Alcian Blue-Periodic acid-Schiff (AB-PAS) Stain Kit (cat. no. G1285; Beijing Solarbio Science & Technology Co., Ltd.) was used to stain the paraffin-embedded intestinal tissue sections according to the manufacturer's protocol. H&E- and AB-PAS-stained colonic sections were observed under an optical microscope (IX73; Olympus Corporation) at x100 magnification.
Total RNA was isolated using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and the RNA concentration was quantified using the NanoPhotometer® N50 (Implen) and RT was performed with the PrimeScript RT reagent kit with gDNA Eraser (cat. no. RR047Q; Takara Biotechnology Co. Ltd.) according to the manufacturer's protocol. RT-qPCR was performed on a LightCycler 96 System (Roche) using TB Green Premix Ex Taq II (Tli RNaseH Plus; cat. no. RR820A; Takara Biotechnology Co. Ltd.) and a cycling program of initial denaturation for 10 min at 95˚C, then 40 cycles of 10 sec at 95˚C, 10 sec at 62˚C and 10 sec at 72˚C, followed by 95˚C for 60 sec and a dissociation curve analysis. The primer sequences are listed in
Faecal genomic DNA was collected from 150-200 mg of fecal samples by the QIAamp PowerFecal DNA Kit (cat. no. 51804; Qiagen GmbH). The hypervariable V3-V4 region (341F and 805R) was amplified and purified. Sequencing was performed on the paired-end Illumina MiSeq PE300 (2x300 bp) platform (Illumina, Inc.) at Novogene Corp. according to the manufacturer's protocol. These raw sequences were processed using the QIIME (v1.9.1) pipeline (
All experimental results were obtained from at least three independent experiments. Values are expressed as the mean ± standard deviation. Statistical comparisons were performed by one-way ANOVA and Tukey's post-hoc test. GraphPad Prism 8.0 (GraphPad Software, Inc.) was used for statistical analysis and R (version 3.6.3) was used for plotting the graphs. P<0.05 was considered to indicate a statistically significant difference.
The effects of LA plus VC to reduce ethanol-induced injury were explored using the NIAAA model (mouse model of chronic and binge ethanol feeding) as described previously (
Chronic ethanol consumption is a major cause of gut microbiota dysbiosis, which may support the pathophysiology of ethanol-related morbidity (
Subsequently, the taxonomic changes in the bacterial community were explored. At the phylum level, Firmicutes and Bacteroidetes were dominant in the faecal microbiota of the control group, whereas Firmicutes, Proteobacteria and Bacteroidetes were dominant in the EH group (
Ethanol and ethanol-associated gut microbiota dysbiosis directly influence the physiological status of the intestine (
Since the immune imbalance and intestinal inflammation are essential for the onset and progression of ethanol-associated intestinal injury (
To evaluate the effects of LA plus VC on oxidative stress, SOD activity and GSH-Px activity in the colon were determined. The results indicated that ethanol exposure significantly reduced the activity of SOD from 20.80±1.29 to 17.57±1.14 U/mg protein (P<0.05;
To determine whether LA+VC protects the liver against ethanol-induced damage, HE staining of liver sections was performed. The results indicated distinct pathological alterations upon ethanol exposure, including neutrophil infiltration and steatosis, whereas LA+VC led to improvement of these pathological alterations (
Furthermore, the mRNA expression of genes related to steatosis was determined. The RT-qPCR results revealed that ethanol exposure markedly increased the expression of genes encoding peroxisome proliferator activated receptor-γ (PPAR-γ) and transporter CD36 for fatty acids (P<0.01;
Ethanol exposure markedly increased the gut permeability and caused LPS translocation into the bloodstream, which contributed to ethanol-associated liver inflammation (
Mounting evidence revealed that gut microbiota dysbiosis has a crucial role in ethanol-associated organ injury and gut microbiota-targeted therapy is emerging as an important adjuvant therapy for protecting the body against ethanol-induced damage (
Ethanol exposure leads to significant gut microbiota dysbiosis and reduces the abundance of
Previous studies have revealed that alcohol consumption results in depletion of GSH levels and declined antioxidant activity (
Increased LPS in the circulatory system triggers the innate immune response and leads to inflammatory response via the TLR4 pathway (
The primary mechanisms by which LA+VC significantly attenuated alcohol-induced intestinal injury involved restoring the gut microbiota, reinstating the immune balance, inhibiting pro-inflammatory cytokines, reducing oxidative stress and maintaining gut barrier function. Based on all of these results, it may be concluded that LA+VC attenuated intestinal inflammatory responses and oxidative stress, and restored the intestinal tight junction and mucus excretion, which markedly alleviated the translocation of gut-derived LPS. In addition, the decrease of LPS in serum contributed to the relief of inflammatory cytokine expression in the Myd88-dependent TLR4 pathway, which was responsible for the amelioration of liver function in ethanol-challenged mice. These results provide mechanisms by which LA+VC attenuated ethanol-induced intestinal and liver injury, and hence, guide the further exploration of synbiotics based on
Not applicable.
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Raw sequencing reads of 16S rRNA sequencing have been deposited in the NCBI Sequence Read Archive (accession nos. PRJNA732292 and SRX10973300;
FW designed the experiments; XL performed experimental experiments and data analysis under the supervision of FW. XL and FW wrote the manuscript, and confirmed the authenticity of the raw data. Both authors read and approved the final manuscript.
The animal experiments of the present study were approved by the Ethics and Clinical Research Committee of Tianjin Medical University (Tianjin, China).
Not applicable.
The authors declare that they have no competing interests.
Effects of LA plus VC on an ethanol-fed mouse model. (A) Treatment schedule: The mice were divided into 5 groups: Ctrl, EH, LA, VC and LA+VC. LA (1x108 colony-forming units/mouse) and VC (100 mg/kg body weight) were administered to mice by gavage daily. (B) Body weight gain (initial, from day -5 to day 0). (C) Liver/body weight ratio (%). (D) IL-6 levels in serum. Values are expressed as the mean ± standard deviation of at least three independent experiments. *P<0.05; **P<0.01. VC, vitamin C; LA,
LA plus VC improve ethanol-induced gut microbiota dysbiosis. (A) Effects of LA plus VC on the α-diversity (Chao1, Shannon and Simpson index) of the fecal microbiota. LA+VC treatment increased the Chao1 and Shannon index and reduced the Simpson index in comparison to the EH group. (B) Effects of LA plus VC on the β-diversity of the fecal microbiota as assessed by PCoA. (C) Effects of LA plus VC on the microbiota composition at the phylum level. (D) Relative abundance of Firmicutes and Proteobacteria. LA+VC treatment decreased the abundance of proteobacteria in comparison to the EH group. (E) Relative abundance of Lachnospiraceae and Enterobacteriaceae at the family level. The markedly increased abundance of Enterbacteriaceae induced by ethanol was significantly suppressed by the LA+VC treatment. Values are expressed as the mean ± standard deviation of at least three independent experiments. *P<0.05; **P<0.01. VC, vitamin C; LA,
LA plus VC improves ethanol-induced intestinal barrier dysfunction. (A) Serum concentration of FITC-dextran. (B) LPS levels in serum. (C) Representative histology images of colon sections (magnification, x100; scale bar, 100 mm; H&E). (D) Quantified crypt depth in colon tissues. (E) Relative mRNA expression of Muc2, ZO-1 and occludin in colon tissues. (F) Representative colon sections with AB-PAS staining (magnification, x100; scale bar, 100 mm). (G) Quantified goblet cells per crypt. (H) Relative mRNA expression of mucus secretion-associated genes and Klf4 gene in colon tissues. The markedly decreased expression of Muc2/3/4 and Klf4 in the EH mice was significantly improved by the LA+VC treatment. Values are expressed as the mean ± standard deviation of at least three independent experiments. *P<0.05; **P<0.01. ns, no significance; VC, vitamin C; LA,
LA plus VC alleviates ethanol-induced inflammatory responses. (A) Flow cytometry plots and quantification of Treg cells (CD4+CD45+Foxp3+) in the colon lamina propria. (B) Quantified percentage of Treg cells in the colon lamina propria in the different groups. (C) Relative mRNA expression of pro-inflammatory and anti-inflammatory genes. (D) Liver IL-10 levels in colon tissues. (E) Liver IL-17A levels in colon tissues. (F) MPO activity (U/g protein) in colon tissues. (G) SOD activity (U/mg protein) in colon tissues. (H) GSH-Px activity (U/mg protein) in colon tissues. Values are expressed as the mean ± standard deviation of at least three independent experiments. *P<0.05; **P<0.01. ns, no significance; VC, vitamin C; LA,
LA plus VC restores ethanol-induced liver function disorders. (A) Representative histological sections of the liver (H&E; magnification, x100; scale bar, 100 µm; red arrows indicate areas of steatosis and the black arrows indicate the sites of neutrophil infiltration in liver tissues). (B) Serum ALT levels. (C) Serum AST levels. (D) Hepatic triglyceride levels. (E) Hepatic MDA levels. Relative mRNA expression of (F) PPAR-γ, (G) CD36, (H) Fas, (I) Scd1 and (J) Srebp-1c in liver tissues. Values are expressed as the mean ± standard deviation of at least three independent experiments. *P<0.05; **P<0.01. VC, vitamin C; LA,
LA plus VC alleviates ethanol-induced liver inflammation. (A) TNF-α levels in liver tissues. (B) IL-1β levels in liver tissues. (C) Relative mRNA expression of TLR4, Myd88, IRAK4 and TRAF6 in liver tissues. (D) Relative mRNA expression of the proinflammatory cytokines TNF-α, NF-κB and IL-1β and the chemokine MCP-1 in liver tissues. Values are expressed as the mean ± standard deviation of at least three independent experiments. *P<0.05; **P<0.01. ns, no significance; MCP, monocyte chemoattractant protein; TLR4, Toll-like receptor 4; IRAK4, IL-1 receptor associated kinase 4; TRAF6, TNF receptor associated factor 6; Myd88, myeloid differentiation primary response 88; VC, vitamin C; LA,