Gut–Liver Axis Is the Bridge between Gut Microbes and Liver

Over the last decade, the term “gut–liver axis” has emerged from a collection of evidence that
expanding the influence of gut microbiota-generated components and metabolites, intestinal barrier
permeability, and bacterial translocation on liver diseases. Venous blood carries nutrients absorbed
from food, factors derived from intestinal microbiota, and immune response products, which enters
the hepatic tissue through the portal vein. Meanwhile, bile acids synthesized in hepatocytes are
conjugated with glycine or taurine, forming bile salts in the liver and being stored in gallbladder and
then passing into the small intestine. This close connection between gut and liver determines the
crucial regulatory effect of the gut microbiota on liver health. Long-term consumption of diet high in
calories and saturated fat may lead to dysbiosis in gut microbiota; this in turn evokes imbalanced bile
acid pool and intestinal barrier dysfunction, followed by an increase in bacterial translocation and
pro-inflammatory components and metabolites from bacteria entering into the liver. Ultimately, it induces
an acceleration in occurrence and development of NAFLD.


Gut Microbiome Profiles in NAFLD Patients

Bacteroidetes and Firmicutes account for the majority (above 90%) of human gut microbiota, while
other phylums of bacteria exist in small amount, including Proteobacteria, Verrucomicrobia, Actinobacteria,
Fusobacteria and Cyanobacteria. Three major classes (Bacilli, Clostridia, and Mollicutes) encompassing
over 250 genera are included in Firmicutes, almost all of which are gram-positive. In contrast,
all gut bacteria belonging to the phylum of Bacteroidetes are gram-negative, composed mainly of the
genera of Bacteroides, Prevotella, Alistipes, and Parabacteroides. Accumulating clinical and animal
studies have indicated that NAFLD is intimately associated with disruption of the balance between
Firmicutes and Bacteroidetes. Although available studies have pointed out the association between
the increased abundance of gut Firmicutes and NAFLD, other findings confirm that overgrowth of
Bacteroides plays a key role in the development of NAFLD. In addition, the severity of NAFLD
is associated with an increase in fecal abundance of Bacteroides, accompanied by a decrease in the level
of Prevotella. Other evidence suggests the fecal abundance of Anaerosporobacter and Faecalibacterium
are lower, whereas Allisonella and Parabacteroides are higher in non-alcoholic steatohepatitis (NASH)
patients. Metagenome sequencing revealed Bacteroides vulgatus and Eubacterium rectale present
the highest abundance in the feces from mild and moderate NAFLD, while Bacteroides vulgatus and
Escherichia colireach the most abundant in liver fibrosis. To sum up, the above evidence supports the
theory that the development and progression of NAFLD is closely linked to gut microbiota dysbiosis.


Gut Microbiota-Derived Components and Metabolites That Accelerating NAFLD

A disturbance of the intestinal microbiota in response to unbalanced diet (e.g., diet high in saturated
fat and fructose) may elicit an increase in intestinal permeability, leading to chronic inflammatory
condition. Remarkably, a continuous low-grade inflammatory state may result in an acceleration
in the progression from hepatic simple steatosis to NASH. Serving as a key constituent of
the outer membrane of cell wall in most gram-negative bacteria, lipopolysaccharides (LPS) (also
known as endotoxins) has well been identified as a major factor in activation of innate immunity
and are therefore suggested as a vital pathogenesis for NAFLD . Indeed, patients with hepatic
steatosis and inflammation present an elevation in the serum LPS. This may be associated with a
rise in the abundance of specific gram-negative bacterial genus, including Bacteroides, Enterobacteria,
Escherichia, and Proteus, as observed in NAFLD patients. Recently, study on germfree mice
have uncovered that colonization of one of the three nonvirulent LPS-producing strains identified
from obese human gut induces NAFLD in combination with high-fat diet feeding. These results
were further corroborated by either interference in the synthetic pathway of bacterial LPS or deletion
of the LPS receptor toll-like receptor 4 (TLR4). As a main TLR4 ligand governing the release of
pro-inflammatory cytokines, LPS can potentiate the susceptibility of NAFLD through the augmentation
of inflammatory responses. In addition to LPS, other pro-inflammatory bacterial components
such as peptidoglycans, lipoteichoic acids, bacterial DNA, and extracellular vesicles have also been
noted in recent years. However, the underlying mechanism by which the role of these components
produced by specific gut bacterial strains in NAFLD requires more extensive and explicit studies.
Considerable attention has been also paid to the role of gut microbiota-derived metabolites in the
pathological process of NAFLD. A clinical research with 330 subjects by Barrea et al. reveals that the
circulating levels of trimethylamine-N-oxide (TMAO) is a novel indicator of metabolic syndrome and
NAFLD. Although TMAO is synthesized in the liver, trimethylamine (TMA), the precursor of
TMAO, is generated from gut bacteria. L-carnitine, choline, or betaine have been the major substrates
for TMA synthesis by gut bacterial strains (e.g., Clostridium asparagiforme, Clostridium sporogenes,
Clostridium hathewayi, Escherichia fergusonii, Anaerococcus hydrogenalis, and Proteus penneri). TMAO
is formed by the oxidation of TMA following the catalysis of flavin-containing monooxygenase
(FMO) enzymes in the liver. The precise mechanisms involving the relationship between
TMAO and the initiation and progression of NAFLD remains to be clarified. However, it has been
verified that TMAO exacerbates hepatic steatosis by blocking the farnesoid X receptor (FXR) signaling
activated by bile acid. By contrast, the activation of FXR signaling have shown a protective
effect against NAFLD including liver steatosis and inflammation. It should be mentioned that
N,N,N-trimethyl-5-aminovaleric acid (TMAVA), a newly identified gut microbiota metabolite, appears
as a key molecule exacerbating high-fat diet-induced liver steatosis through the inhibition of carnitine
synthesis accompanied by a reduction in mitochondrial fatty acid β-oxidation in hepatic tissue.
This metabolite is yielded by Enterococcus faecalis and Pseudomonas aeruginosa using trimethyllysine.
In future study, more metabolites are required to be identified and examined from particular gut strains
to provide more extensive knowledge concerning the interaction of gut microbes with NAFLD.

Impaired Intestinal Barrier Function is an Important Reason for the Development of NAFLD

Intestinal barrier performs an effective defensive action against the translocation of harmful
substances including bacteria, bowel luminal antigens and inflammatory factors, and is usually
assessed by intestinal permeability. Gut microflora dysbiosis has been closely followed by
increased intestinal permeability and subsequent bacterial translocation to the liver, leading to the
release of inflammatory cytokines and free radicals from activated Kupffer cells. Ample evidence
reveals that NAFLD individuals develop an imbalanced gut microbiome and compromised intestinal
permeability. Inflammatory bowel disease (IBD) patients have a high incidence (up to 33.6%)
of NAFLD even independent of metabolic risk, which has been closely associated with impaired
intestinal barrier function. An impairment of the integrity of intestinal barrier initiated by disordered
gut microbiome has been a prerequisite for NASH. Consequently, interventions preserving the
integrity of the intestinal barrier may contribute to attenuating or preventing the progression of NAFLD.
It should be noted that zonulin serves as a key protein regulating intestinal permeability reversibly
by adjusting the size of tight junctions between epithelial cells. Serum circulation levels of
zonulin present a significant positive correlation with pathological indicators in NAFLD, especially in
NASH. Of note, it has been shown that the endogenous ethanol content of NAFLD patients is
substantially higher than that of healthy individuals. The impaired intestinal integrity is partially
associated with the endogenous production of ethanol. Intriguingly, recent evidence found that specific
gut strains (Klebsiella pneumoniae) from NAFLD patients possess the ability to produce high level
of endogenous ethanol, thus exacerbating the compromised intestinal barrier function and hepatic
steatosis.


Gut–Liver Crosstalk Mediated by Bile Acids

The circulation of bile acids between liver and intestine is quite active. Bile acids are known as
amphipathic hydroxylated steroids synthesized from catabolism of cholesterol in the liver and are
released into the small intestine in the form of bile salts from gallbladder. They function not only
to facilitate the emulsification, transport, and absorption of lipids and fat-soluble vitamins but also to
regulate the balance of glucolipid metabolism and immune responses. The synthesis of primary bile
acids cholic acid (CA) from cholesterol is initiated by rate-limiting enzyme cholesterol 7a-hydroxylase
(CYP7A1) in hepatocytes. Alternatively, sterol-27-hydroxylase (CYP27A1) catalyzes the production
of chenodeoxycholic acid (CDCA) or muricholic acids (MCAs, only in mice) from cholesterol. The
formation of secondary bile acids (deoxycholic acid (DCA), lithocholic acid (LCA), and ursodeoxycholic
acid (UDCA)) is achieved via the modification of bile salts (primary bile acids conjugated with
glycine or taurine) by gut microbiota. In mice, other secondary bile acids including Omega-MCA
(ωMCA), hyocholic acid (HCA), murideoxycholic acid (MDCA), and hyodeoxycholic acid (HDCA)
are formed from MCAs by microbial epimerization and dehydroxylation. About 95% of the bile
acids are reabsorbed into the terminal ileum and are transferred to the liver via the portal vein.
Importantly, bile acids regulate bile acid receptors including FXR and Takeda G-protein receptor 5
(TGR5) which are known to modulate insulin sensitivity, glucolipid metabolism, energy homoeostasis,
inflammatory responses, and intestinal barrier function. FXR and TGR5, in turn, play a crucial
role in the feedback regulation of bile acid homeostasis. Imbalance of intestinal flora may lead
to abnormal size and composition of bile acids and dysregulation of FXR and TGR5 signaling.
Specific bile acid conjugates esteem differential feedback of regulatory influences in FXR signaling.
Since the FXR is a primary controlling actor for bile acid synthesis and bile flow, this would imply
that patients with NAFLD manifest altered bile acid kinetics. For instance, a metabolomic study
revealed that NASH patients presented higher plasma concentration of glycine or taurine-conjugated
CA, and glycine-conjugated CDCA, compared with healthy subjects. FXR and TGR5 have become
important intervention targets for NAFLD. Activation of FXR ameliorates NASH through multiple
mechanisms, including the suppression of monocyte and neutrophil infiltration and the attenuation of
NF-κB-mediated inflammation signaling. TGR5 activation by agonist also has been proved to
improve liver steatosis in a mouse model of NAFLD.


Targeting the Gut–Liver Axis as a Nutritional Treatment for NAFLD

Disturbances in intestinal flora composition and gut–liver axis following unbalanced diets has a
profound influence on the progression of NAFLD. However, a large number of dietary supplements
supporting the homeostasis of intestinal bacteria have been reported, including probiotics, functional
oligosaccharides, dietary fibers, ω-3 polyunsaturated fatty acids (ω-3 PUFAs), functional amino acids
(L-tryptophan and L-glutamine), carotenoids, and polyphenols. These nutritional interventions have
been turned out to protect and improve NAFLD through targeting the gut–liver axis, including

1. balance of gut microbiome;
2. preservation of bile acid homeostasis;
3. reinforcement of intestinal barrier function;
4. reduction in bacterial translocation;
5. decrease in the unwholesome components and metabolites from gut bacteria; and
6. supply of gut bacteria-derived beneficial metabolites (e.g.,short chain fatty acids (SCFAs), indoles, and urolithins).

Probiotics for liver health

The use of probiotics has become an enticing and promising approach for the prevention and
therapy of NAFLD. Probiotics refer to the live microorganism strains with sufficient quantity which
provide the host with a health benefit or improved pathological conditions. These strains
induce a competitive exclusion of pathogenic bacteria to ensure a healthy and balanced intestinal
microflora ecosystem favoring epithelial barrier and host immune function. Plentiful studies
have shown the extensive salubrious effects of probiotics (e.g., Lactobacillus and Bifidobacterium) on
liver disease. As shown in high-fructose or high-fat diet-induced experimental NAFLD models,
MAF-Liver ameliorated NAFLD by

1. control of gut microbiome;
2. repair of intestinal barrier; and
3. suppression of hepatic steatosis, inflammation, and lipid accumulation.

Bifidobacterium serves to protect against secretion of pro-inflammatory cytokines and dysfunction in
intestinal barrier both in vitro and in vivo. Results from a randomized clinical trial indicates
that patients with NAFLD receiving a complex-probiotic of MAF-Liver presented a reduction in
fat accumulation in liver and aminotransferase activity, and pro-inflammatory factor levels including
tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6) in serum. 

In conclusion, the gut–liver axis has been a key component for the onset and progression of
NAFLD. The exact mechanisms that connect gut microbiota with liver are complex and deserved
additional thorough exploration. Intestinal flora perturbation and its concomitant pro-inflammatory
initiators and disrupted bile acid homeostasis as well as gut barrier integrity have emerged as key
regulators for metabolic dysfunction, leading to an acceleration of NAFLD progress. The targeting
of gut–liver axis has been in the spotlight of metabolic diseases and may become imperative for
the prevention and therapy of NAFLD in the future. The gut bacterial species serving to impact
the gut–liver axis have been summarized. Nutritional supplements are involved in the
attenuation of NAFLD, since they facilitate the maintenance of homeostasis in gut microbiome, thereby
improving the intestinal barrier function and bile acid profiles as well as reducing the migration
of bacteria and harmful factors into liver.