According to the “Global Status Report on Alcohol and Health 2014” released by the World Health Organization (WHO), the use of alcohol has reached a detrimental level, resulting in more than 3 million deaths every year and accounting for 5.9% of all deaths worldwide. As the metabolism of alcohol in the organism mainly depends on the liver, the long-term consumption and over-consumption of alcohol leads to liver damage and triggers alcoholic fatty liver disease (AFLD) [1
], thereafter causing health issues ranging from fatty liver to alcoholic hepatitis and even cirrhosis [2
]. The first two phases can be reversible through alcohol abstinence and lifestyle intervention. Thus, the detection of potential functional ingredients for AFLD prevention is significant.
Astaxanthin is one of the major xanthophyll carotenoids in marine organisms, and is found in shrimp, crabs, fish, algae, yeast, and feathers of birds [4
]. As astaxanthin cannot be synthesized in humans, its uptake fully depends on dietary sources. Astaxanthin-associated protection against aging as well as cardiovascular and cancerous diseases is attributed to its great antioxidative and anti-inflammatory activity [4
]. In particular, it has been suggested that astaxanthin is protective against various types of liver damage [6
], such as non-alcoholic fatty liver disease (NAFLD) [8
] and liver fibrosis [11
]; however, further investigation is required to determine the effect of astaxanthin on AFLD protection.
Previous studies have confirmed that gut microbiota imbalance is related to a variety of chronic diseases, such as obesity, cardiovascular disease, and cancer [12
]. The relationship between gut microbiota and AFLD has become a topic of increasing concern to researchers. In healthy humans, the intestinal microbiota remains in symbiotic balance, but alcohol intake induces the modification of its composition, thereby inhibiting dominant intestinal bacteria and promoting a small amount of pathogenic bacteria overgrowth [15
], resulting in the impairment of intestinal physiological function. The long-term intake of alcohol breaks the barrier function of the intestine and promotes the growth of intestinal pathogenic bacteria and harmful metabolites (such as lipopolysaccharides). These health-threatening components lead to the development of AFLD by invading into other tissues and organs through blood circulation [16
]. Akkermansia muciniphila
), which can degrade mucin and maintain intestinal barrier integrity, colonizes in the human gut mucus layer and is negatively associated with certain diseases like obesity, diabetes, inflammation, and metabolic disorders [18
]. Long-term dietary intervention can restore the gut microbiota and improve intestinal physiological function [19
]. The concentration of A. muciniphila
can be increased through the ingestion of Bifidobacteria
, fructo-oligosaccharides, short-chain carbohydrates, metformin, rhubarb extract, or specific antibiotics [18
]. Bacterial imbalance is an important factor in inducing disease in AFLD patients. An increase in probiotics species and the inhibition of pathogenic bacteria may improve AFLD status.
Astaxanthin has been reported to prevent liver damage [6
], but there has been little research on the effect of astaxanthin on AFLD. Studying intestinal microbiota regulation in AFLD may provide new insight into the pathogenesis and therapeutic target of AFLD. In the present study, we investigated whether astaxanthin can protect against alcohol-induced liver injury and its association with the intestinal microecology. We also determined the main influence of bacteria during the astaxanthin intervention.
2. Materials and Methods
2.1. Animal Experimentation
Male C57BL/6J mice (22 ± 2 g, six weeks old) were purchased from the Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and housed individually in cages with a 12-h light/dark cycle at 23 ± 2 °C with full access to chow diet and water. All experiment protocols were approved by the Institutional Animal Care and Use Committee at the Jilin Institute of Traditional Chinese Medicine (approval number: SYXK (JI) 2015-0009).
The alcoholic liver disease (AFLD) mouse model was established using the modified Lieber–DeCarli liquid diet [21
]. The 60 mice were randomly separated into five groups, each consisting of 12 mice. The first group was fed with a normal standard growth diet while the other four groups were fed high-fat liquid diets (35% fat, 18% protein, 47% carbohydrates, provided by TROPHIC Animal Feed High-Tech Co., Ltd., Nantong, China) for an acclimation period of two weeks acclimation. Then the four high-fat diet groups were fed with either only the high-fat liquid diet (Con), or combined with the astaxanthin (AST group, 50 mg/kg bw), ethanol-containing (Et group, 5% ethanol v
, accounted for 36% of the total caloric intake), or ethanol plus astaxanthin (EtAST group) treatments for 12 weeks. Compositions of the diets are shown in Supplementary Table S1
. The astaxanthin was purchased from Sigma-Aldrich (St. Louis, MO, USA; purity ≥97%, SML0982), isolated from Blakeslea trispora
and dissolved in corn oil for utilization in this experiment. The dose selection of astaxanthin was chosen in accordance with previous research [22
], while the amount of ethanol included increased over two weeks to reach a final concentration of 5% (v
). Body weight gain and food intake were assessed once a week. The pair-fed control group (Con) was included in this model.
After 12 weeks of treatment, the fasted mice were euthanized and blood samples were collected. The feces in the colon were also harvested into 2-mL sterile tubes to assess the gut microbiota. The tissues were immediately removed and weighed, and the liver coefficient (liver weight/body weight) was calculated. The left lobe of the liver was instantly fixed in 10% buffered formalin for histology analysis with the rest of the tissues frozen in liquid nitrogen and stored at −80 °C until further use.
2.2. Biochemical Analyses
The serum was obtained from the collected blood by centrifugation at 5000 rpm for 10 min at 4 °C and stored at −80 °C. Plasma triacylglycerol (TG), total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were measured by enzymatic colorimetric assays using commercial detection kits (Nanjing Jiancheng of Bioengineering Institute, Nanjing, China).
2.3. Hepatic Triglyceride Staining
The left lobe of the liver tissue was separated and rapidly fixed in 4% neutral buffered formalin solution for 24 h and then processed for paraffin embedding. Five-micrometer-thick paraffin sections were stained with hematoxylin and eosin (H&E) and oil-red solution. The liver steatosis status was examined under a light microscope (Olympus, Tokyo, Japan), and photographed at 200× magnification.
2.4. Quantification of Genes Expression in Liver Tissue
Total RNA was extracted and purified in the liver tissue using TRIzol reagent (TAKARA, Beijing, China. RNAiso Plus, Code No. 9108). The purity, concentration, and quality of RNA were measured. High-quality RNA was converted to cDNA using PrimeScript™ RT reagent Kit with gDNA Eraser (TAKARA, Beijing China. Code No. RR047A). The SYBR fluorescent dye method (TAKARA, Beijing China. Code No. RR420A) and Agilent Stratagene Mx3000P Real-Time PCR System (Santa Clara, CA, USA) were used to detect the gene expression. The primers used are shown in Supplementary Table S2
. The data were calculated using the 2−ΔΔCt
relative quantification method and normalized to β-actin.
2.5. Fecal DNA Extraction
Total bacterial genomic DNA was extracted from the fecal material in the colon using Fast DNA SPIN extraction kits (MP Biomedicals, Santa Ana, CA, USA), following the manufacturer’s instructions. The DNA yield was measured for quantity using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and the quality was analyzed by 0.8% agarose gel electrophoresis; the DNA was then stored at −20 °C until further analysis.
2.6. Amplification and Sequencing of the 16S rRNA Genes
The V3–V4 region of bacterial 16S rRNA genes was subjected to PCR amplification using the forward primer 5′-ACTCCTACGGGAGGCAGCA-3′ and the reverse primer 5′-GGACTACHVGGGTWTCTAAT-3′. The specific system was a 25-µL reaction including 5 µL of Q5 High-Fidelity DNA Polymerase (New England Biolabs (Beijing) Ltd., Beijing, China). PCR amplicons were purified with Agencourt AMPure Beads (Beckman Coulter, Indianapolis, IN, USA) and quantified using the PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). The amplicons were pooled and normalized, and then paired-end 2 × 300 bp sequencing was performed using the Illumina MiSeq platform with the MiSeq Reagent Kit v3 at Shanghai Personal Biotechnology Co., Ltd. (Shanghai, China).
Sequencing data for the 16S rRNA sequences have been deposited in the SRA database under GenBank accession NO. SRP148082.
2.7. Bioinformatics and Statistical Analysis
Sequence data were processed using Quantitative Insights into Microbial Ecology (QIIME, v1.8.0), as previously described [23
]. The low-quality sequences, which had lengths of <150 bp and average Phred scores of <20, and contained ambiguous bases and mononucleotide repeats of >8 bp, were filtered with the following criteria. Paired-end reads were assembled using FLASH. The remaining high-quality sequences were clustered into operational taxonomic units (OTUs) at 97% sequence identity by UCLUST (Edgar 2010) [24
]. OTU taxonomic classification was conducted by BLAST and the OTUs containing more than 99.999% of total sequences across all samples were reserved.
All results are presented as means ± standard deviation. Data were analyzed with SPSS 19.0 using one-way analysis of variance (ANOVA), and, when appropriate, using a two-tailed Student’s t-test between different groups. Differences among groups were evaluated for significance with the comparable variances, followed by Tukey’s and least significant difference (LSD) tests. p < 0.05 was considered statistically significant. GraphPad Prism 7.0 software (GraphPad Software, La Jolla, CA, USA) was used for graph-making.
Sequence data analysis was mainly performed using QIIME (v1.8.0, University of Colorado, Denver, CO, USA) and R packages (v3.2.0, Bell Labs Technology Showcase, Murray Hill, NJ, USA). The alpha diversity indices, Chao1 richness estimator, and the Shannon diversity index were calculated using the OTU table in QIIME. Beta diversity analysis was performed using UniFrac distance metrics and visualized by principle coordinate analysis (PCoA), and the unweighted pair-group method with arithmetic means (UPGMA) hierarchical clustering. The significance of microbiota structure differentiation among groups was assessed by PERMANOVA (permutational multivariate analysis of variance) and ANOSIM (analysis of similarities) using the R package “vegan”. Taxa abundances at different taxonomies were statistically compared among groups by Metastats and visualized as box plots. Microbial functions were predicted by PICRUSt (phylogenetic investigation of communities by reconstruction of unobserved states), based on high-quality sequences.
Increasing evidence indicates that the gut microbiota could significantly affect the emergence and development of AFLD [25
]. Astaxanthin can prevent liver injury by suppressing inflammation, fibrosis [27
], and fat accumulation [10
]. However, previously, there was little evidence suggesting an effect of astaxanthin on AFLD protection. Here, we demonstrated that astaxanthin intervention can mitigate ethanol-induced hepatic steatosis through the reconstruction of the gut microbiota structure and a subsequent increased abundance of Akkermansia
Organ dysfunction is mainly associated with prolonged excessive drinking, which leads to tissue injury-related conditions, such as AFLD, with initial symptoms like fatty liver, and may be responsible for the development of alcoholic hepatitis, liver fibrosis, and cirrhosis. Proper dietary habits can reverse the pathological state of fatty liver in the early stages [29
]. Astaxanthin has been proven to protect against liver injury in mice suffering from NAFLD [27
] and liver fibrosis in mice [7
] due to its anti-inflammation and antioxidant ability. In addition, Zheng et al. has demonstrated that astaxanthin can protect against maternal ethanol-induced embryonic developmental retardation in C57BL/6J mice [22
]. In the present study, we found that astaxanthin can protect against ethanol-induced liver injury in mice by alleviating lipid accumulation, inflammatory cell infiltration, and necrosis in the liver. Furthermore, we explored whether astaxanthin could reverse the AFLD development associated with inflammatory responses, and we detected inflammatory gene expression. Various inflammatory mediators and cytokines can be generated after the activation of inflammatory signaling, particularly the major inflammatory markers TNF-α and IL-6 [30
]. IL-1α and MIP-2 are important pro-inflammatory cytokines [31
] that are significantly induced by ethanol. Astaxanthin significantly decreased these mRNA expression level and partly protected the liver from inflammation and injury.
Increasing evidence indicates that the gut microbiota is responsible for the pathogenesis and development of liver disease. A large microbial ecosystem exists in the human gastrointestinal tract, which is associated closely with health and disease control [33
]. The gut–liver circulation pathway plays a critical role in alcohol metabolism and is strongly regulated by the gut microbiota. It has been proven that dietary perturbations have dominant effects on the gut microbiota [34
]. We further identified bacterial groups that are significantly affected by ethanol and astaxanthin supplementation. Phyla and genera imbalance are associated with health disorders. In this model of alcohol-induced liver injury, we found that several Bacteroidetes
, including the genera Bacteroides
, and Parabacteroides
, were significantly increased. Consistent with our results, Llopis et al. reported that after five weeks’ consumption of a diet including alcohol, Bacteroides
was significantly more represented in severe alcoholic hepatitis mice relative to the control group [35
]. Similarly, other studies have reported that the relative abundance of Bacteroidetes
increases in alcohol-fed mice [36
]. The phylum of Bacteroidetes
is composed of three major classes of Gram-negative bacteria, normally resident in the intestines, mouth, upper respiratory tract, and genital tracts of humans and animals, which have been described as having both beneficial and detrimental features. Bacteroidetes
can lead to endogenous infections due to a micro-ecological imbalance. In particular, Bacteroides fragilis
can produce polysaccharide A to relieve colitis in animals [39
]. However, it can also produce a toxin, which triggers a pro-carcinogenic effect, to induce colon tumorigenesis [40
]. In addition, Proteobacteria
, as pro-inflammatory intestinal microbes, can multiply in the gut in response to an imbalanced microbial composition and are associated with disease occurrence and development [41
]. During this experiment, astaxanthin intervention was found to significantly reverse the ethanol-induced increases in Bacteroidetes
, restoring their proportions to the levels of the ND group. This indicates that the protective effect of astaxanthin is likely associated with its anti-inflammatory activity.
Cyanobacteria was completely suppressed in mice after treatment with ethanol as compared to the ethanol-free treatment. Although the abundance of Cyanobacteria is extremely low in the microbiota, we focused on this phylum because Cyanobacteria is completely inhibited in the gut when the diet contains ethanol, either with or without astaxanthin. This phenomenon indicated that astaxanthin did not affect the action of alcohol.
Accumulating reports have focused on the beneficial effects of Akkermansia
on host metabolism. Akkermansia
is a dominant genus in Verrucomicrobia
and can degrade intestinal mucin [42
], increasing mucus thickness and enhancing gut barrier function, which correlates inversely with the incidence of inflammation [43
] and metabolic syndrome [18
]. Lack of Akkermansia
has been determined to be an early marker of alcohol-induced gut dysbiosis [37
]. Moreover, ethanol exposure reduces the abundance of Akkermansia
in both mice and humans, and the status of AFLD can be improved by oral supplementation of the genus, directly demonstrating the protective effect of this bacterium in AFLD [44
]. As a probiotic, the abundance of Akkermansia
can be affected by the administration of specific dietary components [45
]. In the current research, the abundance of Akkermansia
in ethanol-exposed AFLD mice dramatically reduced, and astaxanthin intervention obviously recovered its abundance, to even higher than that of the ND group. Our data suggest that mice with AFLD might benefit from astaxanthin supplementation increasing Akkermansia
. Studies have proved that Akkermansia
can play a protective role in ethanol-induced liver injury as a result of its function of improving the gut barrier [46
], and the present research requires the further investigation of astaxanthin barrier function in the future.
The different bacterial species in the same genus may reflect different responses by the same treatment. Thus, it is indispensable to identify changes in the microbiota at the species level. In the present study, among the 13 OTUs altered by ethanol and reversed by astaxanthin intervention, the proportions of Akkermansia muciniphila
(OTU36578) were significantly decreased in the Et group, while they were enriched in the EtAST and ND groups. Akkermansia muciniphila
is a species of the Akkermansia
genus, and extensive research has shown that it can improve the status of obesity, diabetes, and inflammation [47
]. On the other hand, the species from Butyricimonas
(OTU 61092, OTU22852, and OTU43831), S24-7 (OTU52622, OTU52789, OTU67103, OTU4618, and OTU11187), Oscillospira
(OTU60609 and OTU71112), and Bilophila
(OTU2261) were significantly increased by ethanol diet and markedly reversed by astaxanthin supplementation. It has been reported that the abundance of Oscillospira
increased in inflammatory response and is associated with the barrier injury of intestinal mucosa [48
]. Stanislawski et al. demonstrated that Bilophila
was positively correlated with the fat fraction of the liver [50
]. Our research found that astaxanthin treatment significantly decreased the levels of ALT, AST, TG, LDL, and the index of LW/BW, which were increased by ethanol diet. Among the 13 OTUs altered by ethanol and reversed by astaxanthin supplementation, Spearman’s correlation analysis suggested that Akkermansia muciniphila
, enriched in the EtAST group, was negatively associated with the AFLD phenotype. On the other hand, the 12 enriched OTUs in the Et group were positively associated with the AFLD phenotype. These results indicate that astaxanthin relieves the AFLD phenotype through its ability to enrich the bacteria responsible for intestinal integrity and anti-inflammation.
The typical characteristic of AFLD is excessive hepatic lipid accumulation. Therefore, lipid metabolic regulation plays an important role in the pathogenesis of metabolic disorders. In this study, the predicted metabolic function of the gut microbiota showed that the lipid metabolism pathway was increased after astaxanthin treatment, while further research proved that Akkermansia
acts as a probiotic to promote lipid metabolism and avoid lipid excessive accumulation [51
]. The results indicate that the inhibition by astaxanthin of excessive lipid accumulation in the liver may be associated with gut bacteria that promote lipid metabolism. In addition, astaxanthin increased the xenobiotic biodegradation and metabolism pathways. Xenobiotics are a kind of foreign chemical in living systems, which, after entering an organism, may induce adverse or even very serious consequences [52
]. The liver is the major organ of metabolism, and astaxanthin supplementation can increase the abundance of bacteria that promote hepatic xenobiotic biodegradation and metabolism, resulting in detoxification and liver injury protection. However, the current findings need to be verified in larger samples.
Interestingly, astaxanthin was more effective in ameliorating alcohol-induced liver injury rather than that related to HFD. There is evidence that astaxanthin can protect against liver damage conditions like NAFLD and AFLD through multiple mechanisms, including antioxidant and anti-inflammatory effects [10
]. In this research, mice in the Con group fed a high-fat diet did not show obvious inflammation, as measured by the liver index, histopathology, and liver gene expression. However, ethanol interference induced a marked inflammatory response which promoted astaxanthin to exert its anti-inflammatory effects to protect the liver from injury. This can also be explained from another aspect; specifically, astaxanthin will not disturb the metabolic balance under non-inflammatory conditions [54
], which is consistent with the result that most responses in the AST group were similar to those in the Con group in our research. Future research should determine the content of astaxanthin in the mouse (liver and serum) and its feces.