The role of gut microbiota in liver disease development and treatment

Abstract

Liver cancer is the sixth most common cancer worldwide, and the third most common cause of cancer-related death. Hepatocellular carcinoma (HCC), which accounts for more than 90% of primary liver cancers, is an important public health problem. In addition to cirrhosis caused by hepatitis B viral (HBV) or hepatitis C viral (HCV) infection, non-alcoholic fatty liver disease (NAFLD) is becoming a major risk factor for liver cancer because of the prevalence of obesity. Non-alcoholic steatohepatitis (NASH) will likely become the leading indication for liver transplantation in the future. It is well recognized that gut microbiota is a key environmental factor in the pathogenesis of liver disease and cancer. The interplay between gut microbiota and liver disease has been investigated in animal and clinical studies. In this article, we summarize the roles of gut microbiota in the development of liver disease as well as gut microbiota-targeted therapies.

1. Introduction

There are about 100 trillion (10¹⁴) microorganisms and approximately 2000 different bacterial species in the human digestive tract.¹ The gut microbiota colonizes immediately after birth and plays an essential role in keeping the host healthy by assisting digestion, producing vitamins, generating bile acids, and modulating local and systemic immunity.^2–5 Many factors, including diet, age, medication, illness, stress, and lifestyle, influence the gut microbiota community structure, which has an impact on disease development.⁶ It is important to note that genetic factors only contribute to 5–15% of most cancers. About 80% of cancers are caused by the environment or lifestyle.⁷ Emerging evidence reveals that the gut microbiota is a major environmental and etiological factor for liver disease development.^8–12 In this review, we summarize publications on the topics of gut microbiota in liver disease development, as well as treatment, focusing on non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma (HCC).

We performed a literature search in PubMed for papers published within the past 10 years, using the keywords: microorganism, microbiota, bacteria, liver, liver disease, HCC, hepatocellular carcinogenesis, NAFLD, NASH, cirrhosis, probiotics, prebiotics, synbiotics, and their combinations.

2. Role of gut microbiota in liver diseases

2.1. Gut microbiota and NAFLD as well as NASH

NAFLD is a global public health problem because of the prevalence of obesity.¹³ NAFLD is a spectrum of chronic liver diseases, including simple steatosis, NASH, advanced fibrosis, cirrhosis, and HCC.⁶ Dysbiosis refers to unfavorable alteration of the microbiota. It is commonly characterized with a decreased ratio of autochthonous to nonautochthonous taxa. Increasing evidence indicates that gut dysbiosis has an important role in the development of NASH via regulation of inflammation, insulin resistance, bile acids, and choline metabolism.^4,14–16

Data generated from human studies have established the relationship between gut microbiota and NAFLD (Table 1).^14,17–24 In, 2013, Mouzaki et al.¹⁷ reported that NASH patients have significantly lower levels of Bacteroidetes compared to healthy individuals. Shen et al.¹⁸ showed similar findings. However, Wang et al.¹⁹ found that non-obese patients with NAFLD have significantly higher levels of Bacteroidetes and lower abundance of Firmicutes in addition to reduced diversity. In these non-obese patients with NAFLD, the depletion of Firmicutes included Lachnospiraceae, Ruminococcaceae, and Lactobacillaceae, which generated short-chain fatty acids (SCFAs).¹⁹ Additionally, a different Bacteroidetes abundance pattern in adolescents was revealed in a study conducted by Stanislawski et al.²⁰ The abundance of Bacteroides showed a U-shaped pattern based on hepatic fat; both low and high abundances were associated with elevated hepatic fat, while a moderate level was associated with reduced hepatic fat.²⁰ In addition, Bacteroides is associated with a high-fat diet (HFD).²⁵ However, certain species of Bacteroides have protective roles in obesity.²⁶

Table 1.

Gut microbiota alteration in NAFLD and NASH patients.

Authors	Population	N	Comparison	Implicated microbiota			Methodology
				Phylum	Family	Genus
Boursier et al.14	F0/F1 fibrosis without NASH F0/F1 fibrosis with NASH	20 10	NASH vs no NASH	Bacteroidetes Bacteroidetes	Bacteroidaceae↑ Prevotellaceae ↓	Bacteroides↑ Prevotella↓	16S rRNA gene sequencing (Stool sample)
	F ≥ 2 fibrosis without NASH F ≥ 2 fibrosis with NASH	2 25	F ≥ 2 fibrosis vs F0/1 fibrosis	Bacteroidetes Bacteroidetes Firmicutes Firmicutes	Bacteroidaceae↑ Prevotellaceae↓ Ruminococcaceae Erysipelotrichaceae↓	Bacteroides↑ Prevotella ↓ Ruminococcus↑ N/A
Mouzaki et al.17	Steatosis patients NASH patients Healthy controls	11 22 17	NASH vs Healthy NASH vs Steatosis	Bacteroidetes ↓ Bacteroidetes ↓ Firmicutes	N/A N/A Lachnospiraceae	N/A N/A Clostridium coccoides↑	Quantitative real-time PCR (Stool sample)
Shen et al.18	NAFLD patients Healthy controls	25 22	NAFLD vs Healthy	Proteobacteria ↑ Fusobacteria ↑ Firmicutes Firmicutes Firmicutes Bacteroidetes↓	Enterobacteriaceae↑ N/A Lachnospiraceae ↑ Erysipelotrichaceae ↑ Streptococcaceae↑ Prevotellaceae ↓	Escherichia_Shigella ↑ N/A Lachnospiraceae_Incenae_Sedis ↑, Blautia↑ Clostridium_XVIII ↑ Streptococcus↑ Prevotella ↓	16S rDNAamplicon sequencing (Stool sample)
	NAFLD patients with NASH NAFLD patients with fibrosis(F≥2)	6 4	NASH vs no NASH F ≥ 2 fibrosis vs F0/F1 fibrosis	Firmicutes Protenbacteria	Lachnospiraceae ↑ Enterobacteriaceae ↑	Blautia ↑ Escherichia_Shigella ↑
Wang et al.19	NAFLD patients Healthy controls	43 83	NAFLD vs Healthy	Bacteroidetes ↑ Firmicutes ↓ Proteobacteria (Gramnegative bacteria↑)	N/A Lachnospiraceae ↑ Enterobacteriales	N/A N/A Escherichia_Shigella ↑	454 pyrosequencing of thel6S rRNA V3 region (Stool sample)
Stanislawski et al.20	Adolescents exposure to gestational diabetes mellitus during singleton pregnancies	107	HFF vs non HFF	Proteobacteria Bacteroidetes Proteobacteria Bacteroidetes Firmicutes	Desulfovibrionaceae Prevotellaceae RF32 Bacteroidaceae Ruminococcaceae	Bilophila↑ Paraprevotella ↑ Suturella ↑ RF32↑ Bacteroides (U-shaped pattern; ↑ or ↓) oscillospira ↓	16S rRNA gene sequencing (Stool sample)
Del Chierico et al.21	NAFLD patients	61	NAFLD vs Healthy	Proteobacteria	Bradyrhizobiaceae	Bradyrhizobium ↑	454 pyrosequencing of the16S rRNA V1-V3 region (Stool sample)
	Healthy controls	54		Firmicutes Firmicutes Actinobacteria Firmicutes Firmicutes Firmicutes Bacteroidetes	unassigned unassigned Propionibacteriaceae Lachnospiraceae Ruminococcaceae Ruminococcaceae Rikenellaceae	Anaerococcust ↑ Peptoniphilus↑ Propionibacterium acnes↑ Dorea ↑ Ruminococcus↑ oscillospira↑ Rikenellaceae ↓
Zhu et al.22	NASH patients	22	NASH or Obese vs Healthy	Actinobacteria ↓	Bifidobacteriaceae ↓	Bifidobacterium ↓	16S rRNA pyrosequencing (Stool sample)
	Obese patients	25		Bacteroidetes↑	Bactemidaceae (−)	Bacteroides (−)
	Healthy controls	16		Bacteroidetes↑ Bacteroidetes↑ Bacteroidetes ↑ Bacteroidetes↑ Firmicutes ↓ Firmicutes ↓ Firmicutes ↓ Firmicutes ↓ Firmicutes ↓ Firmicutes ↓ Firmicutes ↓ Firmicutes ↓ Firmicutes↓ Firmicutes↓ Firmicutes ↓ Firmicutes ↓ Firmicutes ↓ Firmicutes↓ Firmicutes ↓ Proteobacteria ↓ Proteobacteria ↓ Proteobacteria ↓	Porphyromonadaceae (−) Porphyromonadaceae Prevotellaceae ↑ Rikenellaceae ↑ Clostridiales family XI (−) Peptoniphilaceae Peptoniphilaceae Lachnospiraceae ↓ Clostridiaceae Lachnospiraceae Eubacteriaceae Lachnospiraceae Ruminococcaceae Ruminococcaceae ↓ Clostridiaceae Ruminococcaceae Veillonellaceae (-} Veillonellaceae (−) Veillonellaceae (−) Alcaligenaceae↓ Campylobacteraceae (−) Enterobacteriaceae↑	Parabacteroides (−) Porphyromonas (−) Prevotella ↑ Alistipes↑ Anaerococcus (−) Finegoldia (−) Peptoniphilus ↑ Blautia ↓ Closm’dlum (−) Coprococcus↓ Eubacteriutnm↓ Roseburia↓ Ruminococcus (−) Faecalibacterium (−1) Oscillospira ↓ Ruminococcus ↓ Acidaminococcus (−) Dialister (−) Megamonas (−) N/A Campylobacter (−) Escherichia ↑
Koniffkoff et al.23	Mild/moderate NAFLD (Stage 0–2 fibrosis)	72	Stage 0–2 fibrosis vs Stage 3 or 4 fibrosis	Proteobacteria ↑	N/A	N/A	Whole genome shotgun sequencing of DNA (Stool sample)
	Advanced fibrosis (Stage 3 or 4 fibrosis)	14		Firmicutes ↓	Eubacteriaceae	Eubacteriutn rectale ↓
				Firmicutes ↓	Ruminococcaceae	Ruminococcus obeum CAG:39↓, Ruminococcus obeum ↓
Roma et al.24	NAFLD	30	NAFLD vs Healthy	Proteobacteria	Kiloniellaceae ↑	N/A	16S rRNA gene pyrosequencing (Stool sampie)
	Healthy controls	30		Proteobacteria Firmicutes Firmicutes	Pasteurellaceae↑ Lactobacillaceae ↑ Lachnospiraceae ↑	N/A Lactobacillus↑ Robinsoniella ↑,Roseburia↑, Dorea ↑
				Firmicutes Firmicutes Bacteroidetes	Ruminococcaceae ↓ Veillonellaceae ↑ Porphyromonadaceae ↓	Oscillibacter↓ N/A N/A

Comparison of condition A vs condition B:

^↑signifies an increase in condition A relative to condition B.

^↓signifies a decrease in condition A relative to condition B.

(−) signifies no changes in condition A relative to condition B.

Abbreviations: NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; HFF, hepatic fat fraction; N/A, not applicable.

Stanislawski et al.²⁰ also found that Bilophila, Paraprevotella, Suturella, and RF32 have a positive relationship with hepatic fat, while Oscillospira and Varibaculum correlate negatively. The positive association between hepatic fat and Bilophila is accompanied by reduced Oscillospira. These results suggest that Bilophila might contribute to fatty liver, while Oscillospira might counteract its effects.²¹ The abundance of Bilophila wadworthia increases in response to a western diet or HFD.^27,28 Bilophila wadworthia is also associated with T helper 1 (Thl)-mediated intestinal inflammation. Oscillospira is reduced in pediatric NAFLD and NASH.^21,22 Reduced Oscillospira accompanied by increased 2-butanone has been identified as a gut microbiota signature of NAFLD onset. Increases in Ruminococcus and Dorea have been identified as gut microbiota signatures of NAFLD and NASH progression.²¹ Oscillospira is generally linked to leanness and health.²³ Bilophila, Oscillospira, and Bacteroides are associated with diets high in animal products.^29–31 In addition, increased levels of Lactobacillus and selected members of the Firmicutes (Lachnospiraceae; genera, Dorea, Robinsoniella, and Roseburia) have been observed in NAFLD patients.²⁴ NAFLD patients and healthy subjects have a distinct intestinal microbiota community structure.

Further evidence shows that gut dysbiosis and altered metabolic function are linked with the severity of NAFLD. A study by Boursier et al.¹⁴ demonstrated that Bacteroides and Ruminococcus are associated with NASH and the severity of fibrosis. Patients with NASH and fibrosis severity F ≥ 2 have higher abundance of Bacteroides and lower abundance of Prevotella compared to those without NASH. Patients with F ≥ 2 fibrosis have higher abundance of Bacteroides and Ruminococcus and lower abundance of Prevotella compared with those with F0/F1 fibrosis.¹⁴ Patients with mild/moderate NAFLD have a higher abundance of Firmicutes, while patients with advanced fibrosis NAFLD have a higher abundance of Proteobacteria. Patients with advanced fibrosis have lower abundance of Ruminococcus obeum CAG: 39, Ruminococcus obeum, and Eubacterium rectale compared to those with mild/moderate NAFLD.³²

Small intestinal bacterial overgrowth (SIBO) is defined as bacterial culture >10⁵ CFU/ml in upper jejunal aspirate.^33,34 SIBO has a direct relationship with the severity of liver disease. Many patients with chronic liver disease have dysbiosis with SIBO.^3,35 SIBO in patients with NAFLD/NASH has an estimated prevalence of 39–85%.^36–41 As a consequence of reduced intestinal motility and decreased bile acid production, SIBO has a role in NAFLD progression.⁴² Miele et al.³⁷ have reported that SIBO is implicated in increased intestinal permeability and development of fatty liver. SIBO increases lipopolysaccharide (LPS) secretion and inflammation. Hepatic expression of Toll-like receptor 4 (TLR4), together with release of interleukin-8 (IL-8) induced by SIBO, promotes inflammation.⁴ SIBO increases endogenous ethanol and intestinal permeability, favoring LPS production and increased inflammation via TLR4 signaling.^43,44 SIBO is considered as an independent risk factor for the severity of NAFLD and is essential for NAFLD to progress into NASH, followed by development of cirrhosis.^{15,19,38,45,46}

Enteric dysbiosis or intestinal inflammation induced by HFD and dextran sulfate sodium significantly promotes liver fibrosis in mice with NASH.⁴⁷ The inflammasome-mediated dysbiosis, including increased Prevotellaceae and Porphyromonadaceae families as well as the TM7 taxa, promote NAFLD progression in mouse models.⁶ Apart from providing bacterial byproducts and increasing intestinal permeability, the gut microbiota might also inhibit small intestinal secretion of fasting-induced adipocyte factor, resulting in increased hepatic triglyceride deposition.⁴⁸ Antibiotic treatment or surgical removal of the bypassed section of the intestine can reverse SIBO and steatohepatitis.^36,42 SIBO might be an important target for using antibiotics in treating NAFLD as well as NASH.^49,50

Patients with liver cirrhosis and liver or colon cancer have reduced bile acid receptor farnesoid X receptor (FXR).^51–53 Wan’s group has shown that the sex of an animal can affect the gut microbiota, which is implicated in the dissimilar development of steatosis in both western-diet-fed mice and FXR knockout (KO) mouse models according to sex.⁵⁴ Decreased S24–7, in parallel with increased Bacteroidaceae, Rikeneilaceae, Lactobacillaceae, and Verrucomicrobiaceae, has been observed in wild-type female mice compared to their male counterparts. However, these sex differences are abolished in FXR KO mice, indicating that sex difference in steatosis is FXR dependent.⁵⁴ Western-diet-fed male FXR KO mice develop advanced NASH with massive hepatic lymphocyte infiltration, and have decreased Firmicutes and increased Proteo-bacteria.⁵⁵ Broad-spectrum as well as a Gram-negative coverage antibiotics are useful in treating NASH in male FXR KO mice, but are relatively ineffective when FXR K0 male mice are on a western diet.⁵⁵ In the Proteobacteria, the relative abundance of Heli-cobacteraceae and Desulfovibrionaceae substantially increases because of FXR inactivation. Consistently, antibiotic-reduced hepatic inflammation is accompanied by their reduction. In contrast, Lactococcus, Lactobacillus, and Coprococcus have a protective effect in hepatic inflammation.⁵⁵ The basic mechanisms of dysbiosis affecting liver disease are summarized in Fig. 1.

Fig. 1. The mechanisms by which gut microbiota affects liver health and diseases.

Under healthy condition, intestinal barrier and integrity prevent the entry of bacterial products, such as endotoxin, from the gut into the portal circulation. Liver immune cells rapidly clear the microbial products and bacteria passing though the gut barrier, thereby establishing immune tolerance without inflammation, Gut microbiota contributes to improving insulin sensitivity, reducing inflammation, and hepatic lipid accumulation via modulating the productions of bile acids, short-chain fatty acids, glucagon-like peptide 1, etc. Factors such as antibiotics, injury, infection, and high-fat diet can cause dysbiosis. Dysbiosis increases endogenous ethanol, endotoxin, and intestinal permeability, thereby leading the translocations of bacteria and bacterial metabolites from the intestine to the liver. Bacteria and their metabolites can activate the innate immune system via toll-like receptors and cause inflammation and subsequent liver damage. Moreover, dysbiosis-associated bile acid dysregulation increases insulin resistance, hepatic lipid accumulation, and inflammatory signaling. Furthermore, dysbiosis converts choling to trimethylamine, Which leads to choline deficiency. All these metabolites and factors contribute to liver diseases. Abbreviations: NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; TLRs, Toll-like receptors.

2.2. Gut microbiota and liver cirrhosis as well as cirrhosis-associated complications

2.2.1. Gut microbiota and liver cirrhosis

Liver cirrhosis is the end stage of chronic liver diseases and is characterized by fibrosis, abnormal hepatic architecture, and portal hypertension. Liver cirrhosis may lead to progressive hepatic failure and cancer. It has been shown that dysbiosis can affect clinical outcomes, including 90-day-hospitalization, organ failure, and death.^56–58

Cirrhosis-associated gut dysbiosis is accompanied by reduced Bacteroidetes, increased Proteobacteria at the phylum level, and reduced Lachnospiraceae as well as increased Enterobacteriaceae and Veillonelaceae at the family level⁵. Potentially pathogenic overgrowth of Enterobacteriaceae is linked to the severity of cirrhosis and its complications, such as hepatic encephalopathy.⁵ Chen et al.⁵⁹ demonstrated an increase in Proteobacteria and Fusobacteria, along with a decrease in Bacteroidetes and change in Firmicutes at the phylum level in fecal samples from cirrhotic patients. In addition, cirrhotic patients have increased fecal Entero-bacteriaceae, Veillonelaceae, and Streptococcaceae, and reduced Lachnospiraceae.⁵⁹ Moreover, several commensal genera, such as, Coprococcus, Pseudobutyrivibrio, and Roseburia in the Lachnospiraceae family, are beneficial to the host via production of SCFAs.⁵⁹

In 2014, Bajaj et al.⁵⁶ compared fecal microbiota analysis in cirrhotic patients and healthy controls. They reported that the reduction of autochthonous taxa, including Lachnospiraceae, Ruminococcaceae, and Clostridiales XIV, and increase of non-autochthonous taxa including Staphylococcaceae, Enter-ococcaceae, and Enterobacteriaceae, are linked to liver failure and plasma LPS levels in cirrhosis patients. In addition, Enterobacteriaceae and endotoxemia are enriched in patients with alcoholic compared with non-alcoholic cirrhosis.⁵⁶ Enterobacteriaceae are also frequently found in spontaneous bacterial peritonitis; an infection in decompensated cirrhosis.⁶⁰ Enterobacteriaceae are more abundant in patients with decompensated cirrhosis compared to patients with compensated cirrhosis and healthy controls.⁵¹ The mucosal microbiota in the duodenum also differs markedly between cirrhotic patients and healthy controls.³⁴ Based on the predicted metagenomes analyzed, pathways related to nutrient absorption are enriched in the duodenal microbiota of patients with cirrhosis, while bacterial proliferation and colonization, including bacterial motility proteins and secretory systems, are over-represented in control subjects.³⁴

Bile acid pool size and composition are major regulators of microbiome structure.^61,62 Increased primary bile acid, cholic acid (CA) can cause dysbiosis with a dramatic shift toward the Firmicutes, particularly Clostridium cluster XlVa and can increase production of deoxycholic acid (DCA).^61,62 Cirrhosis-associated dysbiosis increases inflammation via metabolism, LPS, and translocation. Inflammation can suppress synthesis of bile acids in the liver.^61,62 Secondary bile acids, which are generated by the Clostridiales cluster, are reduced in cirrhotic patients.^63,64 Bile acids have an important role in the pathogenesis of cirrhosis,^54,55,65 Reduced bile acid secretion facilitates oral microbiota migration to the distal gut and boosts SIBO. In contrast, activation of FXR stimulates bile acid excretion and induces production of antimicrobial peptides.^66,67 The interaction between bile acids and microbiota plays an important role in cirrhosis.^61,62 The data related to alteration of gut microbiota in cirrhotic patients are summarized in Table 2.^{34,56,57,59,64}

Table 2.

Gut microbiota alteration in cirrhotic patients.

Authors	Population	N	Comparison	Implicated microbiota			Methodology
				Phylum	Family	Genus
Chen et al.34	Cirrhotic patients with HBV	24	Cirrhosis vs Healthy	Actinobacteria Firmicutes	Coriobacteriaceae Veillonellaceae	Atopobium↑ Dialister↑, Veillonella↑ and Megasphera↑	16S rRNA gene pyrosequencing (Mucosa of the distal duodenum sample)
	Cirrhotic patients with PBC	6		Proteobacteria	Pasteurellaceae	Hemophilus↓, Neisseria↓ and SR 1 genera incertae sedis↓
	Healthy controls	28
Bajaj et al.56	Patients with liver cirrhosis	219	Cirrhosis vs Healthy	Firmicutes	Lachnospiraceae↓, Ruminococcaceae↓ and Clostridiales XIV ↓,	N/A	Multi-tagged pyrosequencing (Stool sample)
	Healthy controls	25		Firmicutes	Staphylococcaceae ↑, Enterococcaceae ↑	N/A
				Proteobacteria Firmicutes Bacteroidetes	Enterobacteriaceae↑ Veillonellaceae ↓, Porphyromonadaceae ↓	N/A N/A
Bajaj et al.57	Patients with liver cirrhosis	278 out of 335	Hospitalized vs non Hospitalized	Bacteroidetes	Bacteroidaceae ↓	N/A	16S rRNA pyrosequencing (Stool sample)
	Non hospitalized patients with liver cirrhosis within 90 days	162		Firmicutes	Clostridiales XIV↓, Lachnospiraceae↓, Ruminococcacae↓	N/A
				Firmicutes Proteobacteria	Enterococcaceae↓ Enterobacteriaceae ↓	N/A N/A
	Hospitalized patients with liver cirrhosis	94		Bacteroidetes ↓	BacterDidetes_Bacteroidaceae ↓,	N/A
				Bacteroidetes↓	Bacteroidetes_Porphyromonadaceae ↓	N/A
				Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Proteobacteria Proteobacteria	Firmicutes_Lactobacillaceae ↑ Firmicutes_Enterococcaceae ↑ Firmicutes_Clostridiales XIV↓ Firmicutes_Lachnospiraceae ↓ Firmicutes_Ruminococcaceae ↓ Proteobacteria-Enterobacteriaceae↑ Proteobacteria_Pasteurellaceae ↑	N/A N/A N/A N/A N/A N/A N/A N/A
	Non DM	191	DM vs non DM	Bacteroidetes	Bacteroidetes_Bacteroidaceae ↓	N/A
	DM	87		Proteobacteria Firmicutes Firmicutes Firmicutes Actinobacteria	Firmicutes_Eubacteriaceae ↓ Firmicutes_Ruminococcaceae ↑ Firmicutes_VeilIonellaceae↓ Firmicutes_Streptococcaceae↓ Actinobacteria_Streptomycetae ↓	N/A N/A N/A N/A N/A	Mucosal sample
				Firmicutes Bacteroidetes Fusobacteria	Firmicutes_Clostridiacaeae ↓ Bacteroidetes_Prevotellaceae ↑ Fusobacteria_Fusobacteriaceae ↑	N/A N/A N/A
Chen et al.59	Patients with liver cirrhosis Healthy controls	36 24	Cirrhosis vs Healthy	Bacteroidetes↓ Proteobacteria ↓ Fusobacteria↓ Firmicutes Firraicutes Firmicutes	Enterobacteriaceae ↑ N/A N/A Veillonellaceae↑ Streptococcaceae↑ Lachnospiraceae↓	N/A N/A N/A N/A N/A Coprococcus ↓, Pseudobutyrivibrio ↓ Roseburia ↓	The 16S rRNA V3 region pyrosequencing; Real-time PCR (Stool sample)
Kakiyama et al.64	Early cirrhotics	23	Cirrhosis vs Healthy	Firmicutes	Lachonospiraceae↓, Ruminococcaceae ↓	N/A	Culture-independent multitagged-pyrosequencing (Stool sample)
				Firmicutes	Lachnospiraceae	Blautia ↓
	Advanced cirrhotics	24		Bacteroidetes	Rikenellaceae↓	N/A
	Healthy controls	14		Proteobacteria	Enterobacteriaceae ↑	N/A

Comparison of condition A vs condition B:

^↑signifies an increase in condition A relative to condition B.

^↓signifies a decrease in condition A relative to condition B.

Abbreviations: HBV, hepatitis B virus; PBC, primary biliary cirrhosis; DM, diabetes mellitus; N/A, not applicable.

2.2.2. Gut microbiota and complications associated with liver cirrhosis

Bacterial translocation (BT) plays a crucial role in the development of complications associated with hepatic cirrhosis.⁶⁸ By inoculating an equal amount of Escherichia coli (E. coli) into small and large intestines, it was found that BT predominantly occurs in the small intestine.⁶⁹ Consistently, the small intestine is a preferred site for BT in cirrhotic patients.⁷⁰ In addition, BT is closely associated with SIBO as well as intestinal barrier injury in cirrhotic rats.⁷¹

Spontaneous bacterial peritonitis is a common complication of liver cirrhosis because bacterial infections occur in cirrhotic patients with ascites.^72–74 Most of the bacteria in patients with spontaneous bacterial peritonitis are E. coli, Klebsiella pneumoniae, coagulase-negative Staphylococcus, and Enterococcus.⁷⁴ E. coli is the predominant pathogen of spontaneous bacterial peritonitis. ^72–74

Hepatic encephalopathy is a common complication of liver cirrhosis and a result of liver failure.^75,76 Hepatic encephalopathy affects brain astrocytes, microglia, and neurons.^75,76 A decrease in autochthonous bacteria and increase in Gram-negative bacteria are observed in cirrhotic patients with hepatic encephalopathy. It has been shown that elevated serum ammonia levels are linked to astrocytic impairment⁷⁷ Moreover, ammonia-associated brain magnetic resonance imaging changes are associated with autochthonous taxa and Enterobacteriaceae, while white matter inflammatory changes are associated with oral taxa such as Porphyromonadaceae.⁷⁷ Only mucosal and not fecal microbiota is altered significantly in patients with hepatic encephalopathy. The Firmicutes phylum, including Veillonella, Megasphaera, Bifidobacterium,.and Enterococcus, is highly enriched in hepatic encephalopathy, whereas Roseburia is more abundant in the non-hepatic encephalopathy group.⁷³

2.2.3. Gut microbiota and liver transplantation

Liver transplantation is one option used to treat cirrhosis or cirrhosis-associated complications,^78,79. Liver transplantation affects the recipient’s microbiota (Table 3).^79–81 Gut microbiota diversity is increased after liver transplantation, but does not reach the levels in healthy controls.⁸⁰ Alteration of Proteobacteria and Firmicutes links with improved cognitive level of patients with liver transplantation.⁸⁰ In 2016, the Wan laboratory established the relationship between intestinal microbiota and expression of hepatic genes in regenerating the liver, using partial hepatectomy mouse models.⁸² Removal of two-thirds of mouse liver led to rapid changes in gut microbiota, with increased Bacteroidetes S24–7 and Rikenellaceae as well as decreased Firmicutes Clostridiales, Lach-nospiraceae, and Ruminococcaceae.⁸² The abundance of Rumino coccacea, Lachnospiraceae, and S24–7 was closely linked with liver metabolism and immune functions.⁸² Hepatic secondary bile acids are positively correlated with Firmicutes and negatively with Bacteroidetes, while tauro-conjugated bile acids show positive correlations with Bacteroidetes and negative correlations with Firmicutes.⁸² Priming mice with all-trans retinoic acid lowers the ratio of Firmicutes to Bacteroidetes and increases hydrophilic bile acids, which is linked with facilitated metabolism and enhanced cell proliferation in regenerating mouse livers.⁸³

Table 3.

Gut microbiota alteration and liver transplantation.

Authors	Population	N	Comparison	Implicated microbiota			Methodology
				Phylum	Family	Genus
Sun et al.79	Post-LT patients Healthy controls	9 15	Post-LT vs Pre-LT	Proteobacteria Proteobacteria Actinobacteria Proteobacteria Proteobacteria Verrucomicrobia	Pasteurellaceae Enterobacteriaceae Micromonosporaceae Desulfobacteraceae Eubacteriaceae Akkermansiaceae	Actinobacillus ↓ Escherichia↓ and Shigella↓ Micromonosporaceae ↑ Desulfobacterales ↑ the Sarcina genus of Eubacteriaceae ↑ Akkermansia ↑	MiSeq-PE250 sequencing of the V4 region of 16S rRNA (Stool sample)
Bajaj et al.80	Outpatient patients with cirrhosis on the LT list Healthy controls	45 45	Improved cognition post-LT vs Pre-LT	Proteobacteria ↓ and Firmicutes ↑	N/A	N/A	Multitagged sequencing; 16s rRNA (V1-V2) sequencing (Stool sample)
			Not improved cognition after LT vs Healthy	Proteobacteria ↑ and Firmicutes ↓	N/A	N/A
			post-LT vs Pre-LT	Firmicutes (−)	Ruminococcaceae ↑ and Lachnospiraceae ↑	N/A
				Bacteroidetes (−)	N/A	N/A
				Proteobacteria (−)	Enterobacteriaceae	Escherichia ↓, Salmonella↓ and Shigella ↓
			Pre-LT patients vs Healthy Post-LT patients vs Healthy	Proteobacteria (−) Firmicutes (−) Actinobacteria Bacteroidetes	Enterobacteriaceae Ruminococcaceae ↑ and Lachnospiraceae ↑ Bifidobacteriaceae ↓ Bacteroidaceae↑	Escherichia, ↑ Shigella, ↑ Salmonella ↑ N/A N/A N/A
Bajaj et al.81	Patients with cirrhosis	40	Post-LT vs Pre-LT	Proteobacteria Proteobacteria Proteobacteria Actinobacteria Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Bacteroidetes	Enterobacteriaceae Sutterellaceae Desulfovibrionales Bifidobacteriaceae Clostridiales Incertae Sedis XI Ruminococcaceae Clostridiales Incertae Sedis XIII Lachnospiraceae Streptococcaceae Clostridiaceae Rikenellaceae	Shigella↓, Escherichia ↓, and Salmone↓ Suterella ↑ Bilophila↑ Bifidobacterium ↓ Desulfatibacter ↑, and Sporanaerobacter↑ ClostridiumlV↑, Osdiiibocte↑, Anaerovorax ↑ Anaerostipes↑, Clostridium XIVb ↑, Blautia↑ Roseburia↑, and Dorea↑ Streptococcus↑ Butyricicoccus↑, CtostridiumXIVa ↑ Alistipes↑	Multitagged sequencing (Stool sample)

Comparison of condition A vs condition B:

^↑signifies an increase in condition A relative to condition B.

^↓signifies a decrease in condition A relative to condition B.

(−) signifies no changes in condition A relative to condition B.

Abbreviations: LT, liver transplantation; N/A, not applicable.

Bajaj et al.⁸¹ have reported the effect of liver transplantation on microbial composition and functionality in patients. Successful liver transplantation increases the microbial diversity accompanied by an increase in autochthonous and a decrease in potentially pathogenic taxa.⁸¹ The favorable changes in the gut microbiota also have the benefit of increasing fecal bile acids and urinary phenyl-acetylglutamine, accompanied with a reduction in serum ammonia and endotoxemia.⁸¹

2.2.4. Fungal dysbiosis and complications associated with liver cirrhosis

In addition to bacteria, microbiota includes archaea, protists, fungi, viruses, and bacteriophages.⁸⁴ A recent study showed fungal dysbiosis in cirrhotic patients. Bajaj et al.⁸⁵ have demonstrated a link between fungal and bacterial diversity in patients with liver cirrhosis, and Bacteroidetes/Ascomycota ratio can affect 90-day-hospitalization (Table 4). Moreover, Candida overgrowth and reduced intestinal fungal diversity are observed in patients with alcoholic cirrhosis (Table 4).⁸⁶

Table 4.

Fungal dysbiosis in complications associated with liver cirrhosis.

Authors	Population	N	Comparison	Implicated microbiota			Methodology
				Phylum	Family	Genus
Bajaj et al.85	Outpatients cirrhotics	77	Inpatients vs Outpatients	Basidiomycota↓ Ascomycota	Bacteroidetes/Ascomycota ratio ↑ Saccharomycetaceae	N/A Candida↑	Metagenomics (Stool sample)
	Inpatients cirrhotics	66	After antibiotics vs before antibiotics	Ascomycota	Saccharomycetaceae	Candida↑
	Controls	26	Inpatients vs Outpatients	Ascomycota	Saccharomycetaceae	Candida↑
				Proteobacteria	Enterobacteriaceae↑	N/A
				Firmicutes	Enterococcaceae↑	N/A
			Outpatients vs Controls	Basidiomycota↓	N/A	N/A
				Ascomycota	Saccharomycetaceae	Candida↑
			Inpatients vs Controls	Proteobacteria	Enterobacteriaceae ↑	N/A
				Firmicutes	Enterococcaceae↑	N/A
			Outpatients on antibiotics	Ascomycota ↑	Saccharomycetaceae	Candida↑
				Proteobacteria	Pasteurellaceae↑	N/A
Yang et al.86	Healthy individuals	8	Patients vs Healthy individuals	Ascomycota	Saccharomycetaceae	Candida↑	lllumina MiSeq platform
	Alcohol-dependent	10					sequencing of the V4 region of 16S rRNA (Stool sample)
	patients (nonprogressive liver disease)
	Patients with alcoholic liver cirrhosis	4

Comparison of condition A vs condition B:

^↑signifies an increase in condition A relative to condition B.

^↓signifies a decrease in condition A relative to condition B.

Abbreviation: N/A, not applicable.

2.2.5. Oral microbiota and liver cirrhosis

Oral microbiota contributes to the progression of liver diseases. Elevated oral Streptococcus and Veillonella are found in cirrhotic patients.⁸⁷ Increased Enterobacteriaceae and Enterococcaceae, as well as reduced autochthonous bacteria, are found in patients with previous episodes of hepatic encephalopathy.⁸⁷ Oral microbiota has a significant impact on duodenal microbiota. At the genus level, the most distinctive taxa found in cirrhotic patients and controls include Veillonella, Prevotella, Neisseria, and Haemophilus, which are commonly found in the oral cavity.⁸⁸

Bajaj et al.⁸⁹ performed a direct comparison of the salivary microbiome between healthy controls and patients with cirrhosis. Relative abundance of potentially pathogenic taxa (Prevotella and Fusobacteriaceae) increased whereas autochthonous taxa (Lach-nospiraceae and Ruminococcaceae) decreased in oral microbiota of cirrhotic patients with previous hepatic encephalopathy.⁸³ Mi-crobes of oral origin can be present in the duodenum. Duodenal Prevotella and Fusobacterium are also increased significantly in, cirrhotic patients.³⁴ Proton pump inhibitors increase the microbiota of oral origin in patients with cirrhosis.⁹⁰ The removed pH barrier in the gastrointestinal tract allows the microbiota of oral origin to migrate along the gastrointestinal tract and even into feces.⁹⁰ Certain oral bacteria can produce high levels of hydrogen sulfide (H₂S) and methyl mercaptan (CH₃SH).⁹¹ Higher proportions of Neisseria, Porphyromonas, and SRI are linked to H₂S production that can damage deoxyribonucleic acid (DNA). Prevotella, Veillonella, Atopobium, Megasphaera, and Selenomonas are associated with production of CH₃SH, which contributes to development of hepatic encephalopathy.^91,92 Literature related to oral microbiota and liver disease is summarized in Table 5.^34,87,89,90

Table 5.

Oral microbiota alteration in patients with cirrhosis.

Authors	Population	N	Comparison	Impiicated microbiota			Methodology
				Phylum	Family	Genus
Chen et al.34	Cirrhotic patients	30	Patients vs Controls	Actinobacteria	Coriobacteriaceae	Atopobium↑	16S rRNA gene pyrosequencing
				Firmicutes	Veillonellaceae	Dialister↑, Veillonella↑, and Megasphera↑	(Mucosal from the distal duodenum sample)
	Healthy controls	28		Proteobacteria	Pasteurellaceae	Hemophilus ↓
				Proteobacteria	Neisseriaceae	Neisseria ↓
				undefined	undefined	SR 1 genera incertae sedis↓
Qin et al.87	Patients with cirrhosis	98	Patients vs Controls	Firmicutes	Streptococcaceae	Streptococcus↑	Quantitative metagenomics (Stool sample)
				Firmicutes	Veillonellaceae	Veillonella ↑
	Healthy controls	83		Firmicutes	Enterococcaceae ↑	N/A
				Proteobacteria	Enterobacteriaceae↑	N/A
Bajaj et al.89	Patients with cirrhosis without HE	59	Patients vs Controls	Bacteroidetes	Prevotellaceae	Prevotella ↑	Quantitative metagenomics (Stool or saliva sample)
				Fusobacteria	Fusobacteriaceae ↑	N/A
	Patients with cirrhosis with previous HE	43		Firmicutes	Lachnospiraceae ↓ and	N/A
					Ruminococcaceae ↓
	age-matched controls	32		Proteobacteria	Enterobacteriaceae↑	N/A
				Firmicutes	Enterococcaceae ↑	N/A
Bajaj et al.90	Cirrhotic outpatients on PPI	59	PPI users vs Patients without PPI and Controls	Firmicutes	Streptococcaceae↑	N/A	Multi-tagged sequencing (Stool sample)
				Firmicutes	Lachnospiraceae↓	N/A
	Cirrhotic outpatients not on PP1	78
	Healthy controls	45
	Cirrhotic outpatients not on PPI	15	After vs Before PPI initiation	Bacteroidetes Firmicutes	Porphyromonadaceae ↑ Streptococcaceae↑	N/A N/A
	Patients with decompensated cirrhosis on chronic PPI	15	PPI withdrawal vs Pre-PPI therapy	Bacteroidetes	Porphyromonadaceae ↓	N/A
				Firmicutes	Streptococcaceae ↓, and Veillonellaceae↓	N/A

Comparison of condition A vs condition B:

^↑signifies an increase in condition A relative to condition B.

^↓signifies a decrease in condition A relative to condition B.

Abbreviations: HE, hepatic encephalopathy; PP1, proton pump inhibitors; N/A, not applicable.

2.3. Gut microbiota and HCC

Gut microbes are implicated in liver carcinogenesis.^5,93,94 Helicobacter species are important pathogens that may be directly involved in the occurrence of liver cancer, and are found in human HCC specimens.⁹⁵ A human study has shown that Helicobacter is present in the liver of patients with primary liver carcinoma but not in controls without primary liver carcinoma.⁹⁶ However, Helicobacter hepaticus (H. hepaticus) is not present in HCC patients with chronic hepatitis B or C.⁹⁷

H. hepaticus infection promotes HCC in chemical and viral transgenic liver cancer models.⁹⁸ However, HCV transgene or H. hepaticus exposure alone is not sufficient to initiate liver cancer.⁹⁸ Moreover, increased risk of HCC is not dependent on translocation of H. hepaticus to the liver.⁹⁸ Gut H. hepaticus colonization induces nuclear factor κ-light-chain-enhancer of activated B cell signaling, which activates innate and Th1-type adaptive immunity.⁹⁸ Thus, H. hepaticus in the intestinal niche without translocation to the liver can change the immune signaling and play a synergistic role with chemical and viral carcinogenic factors.⁹⁸

Gut dysbiosis is found in patients with liver cirrhosis and HCC as well as animal models using streptozotocin-HFD, diethylnitrosamine (DEN), or carbon tetrachloride (CCI₄). Blooming of E. coli is found in cirrhotic patients who have HCC compared to those without HCC.⁹⁹ In the C57BL/6J mouse model of NASH and HCC induced by streptozotocin-HFD, a significant increase of Atopobium spp., Bacteroides spp., Bacteroides vulgatus, Bacteroides acidifaciens, Bacteroides uniformis, Clostridium cocleatum, Clostridium xylanolyticum, and Desulfovibrio spp. is associated with disease progression.¹⁰⁰ A significant reduction of Lactobacillus, Bifidobacterium, and Enterococcus species, along with increased E. coli and Atopobiumcluster, has been found in rat models of HCC induced by DEN.¹⁰¹ Moreover, Clostridium spp. are reduced in CCl₄-induced liver carcinogenesis models.¹⁰² When DEN is used in combination with CCl₄, gut sterilization or TLR4 deletion reduces tumor number and volume but does not affect tumor incidence, while continuous low-dose LPS administration increases tumor number and size.¹⁰³

Changes in the microbiota of the tongue coating have been noted in patients with HCC.¹⁰⁴ Moreover, enrichment of tongue Oribacterium and Fusobacterium could be microbial biomarkers of HCC.¹⁰⁴ Microbial genes in the categories related to nickel/iron transport, amino acid transport, energy-producing systems, and metabolism differ in abundance between HCC patients and healthy controls.¹⁰⁴

The steatohepatitis-inducing HFD (STHD-01) is a NASH-inducing HFD, which promotes HCC without chemical carcinogens.¹⁰⁵ A recent study revealed that gut bacteria associated with secondary bile acid production promote STHD-01-induced HCC development that can be prevented by antibiotics.^105,106 In addition, Prevotella and Oscilibacter, producers of anti-inflammatory metabolites, can inhibit carcinogenesis. This anti-cancer effect may result from increased regulatory T (Treg) cells and reduced, migration of Thl7 cells to the liver.⁹⁴ The gut microbiota plays a key role in HCC development and can potentially be used to treat HCC. Literature related to microbiota alteration in human HCC and animal model of HCC is summarized in Tables 6 and 7,^{95–97,99–102,104} respectively.

Table 6.

Gut microbiota alteration in human HCC.

Authors	Population	N	Comparison	Implicated microbiota			Methodology
				Phylum	Family	Genus
Nilsson et al. 95	Liver specimens of patients with cholangiocarcinoma HCC human specimens Controls (liver tissue from patients with resected métastasés from colorectal cancers)	14 16 20	HCC or cholangiocarcinoma specimens vs Controls	Proteobacteria	Helicobacteraceae	Helicobacter spp.↑	PCR and DNA sequencing (HCC human specimens)
Huang et al.96	HCC human specimens Controls without HCC	20 16	HCC specimens vs Controls	Proteobacteria	Helicobacteraceae	Helicobacter pylori↑	PCR, DNA sequencing, and immunostaining (Liver specimens)
Krüttgen et al.97	Patients with viral-induced HCC Control patients	14 11	Patients with viral-induced HCC vs Control patients	Proteobacteria	Helicobacteraceae	H. hepaticus (no exist)	PCR (Stool sample)
Grat et al.99	Patients with HCC Non HCC patients	15 15	HCC vs non HCC	Proteobacteria	Enterobacteriaceae	Escherichia coli ↑	Culturing on enriching and selective agar media (Stool sample)
Lu et al.104	Early liver carcinoma patients with cirrhosis Healthy controls	35 25	HCC vs Healthy	Firmicutes Fusobacteria	Lachnospiraceae Fusobacteriaceae	Oribacterium changes Fusobacterium changes	16S rRNA gene sequencing (Tongue coat sample)

Comparison of condition A vs condition B:

^↑signifies an increase in condition A relative to condition B.

Abbreviation: HCC, hepatocellular carcinoma.

Table 7.

Gut microbiota alteration in HCC animal models.

Authors	Model	Agent	Comparison	Implicated microbiota			Methodology
				Phylum	Family	Genus
Xie et al.100	NASH-HCC C57BL/6J mouse model	STZ-HFD	NASH-HCC vs Controls	Actinobacteria Bacteroidetes	Coriobacteriaceae Bacteroidaceae	Atopobium spp.↑ Bacteroides spp. ↑ Bacteroides vulgcitus,↑ Bacteroides acidifaciens↑ and Bacteroides uniformis ↑	16S rDNA gene pyrosequencing (Stool sample)
				Firmicutes	Clostridiaceae	Clostridium cocleatum, ↑ Clostridium and xylanolyticum ↑
				Proteobacteria	Desulfovibrionaceae	Desulfovibrio spp.↑
Zhang et al.101	Male Sprague-Dawley HCC rats	DEN	HCC rats vs Controls	Firmicutes Firmicutes Actinobacteria	Lactobacillaceae Enterococcaceae Bifidobacteriaceae	Lactobacillus ↓ Enterococcus ↓ Bifidobacterium ↓	16S rRNA based quantitative real-time PCR (Stool sample)
Gómez-Hurtado et al.102	Female Balb/c fibrosis mice	CCI4	Fibrosis mice vs Controls	Firmicutes Firmicutes Firmicutes	Clostridiaceae Clostridiaceae Clostridiaceae	Clostiidia spp.↓ Clostiidium coccoides ↓ Clostridium leptum ↓	Quantitative real-time PCR (Stool sample)

Comparison of condition A vs condition B:

^↑signifies an increase in condition A relative to condition B.

^↓signifies a decrease in condition A relative to condition B.

Abbreviations: STZ-HFD, streptozotocin-high fat diet; DEN, diethylnitrosamine; HCC, hepatocellular carcinoma; CCL₄ carbon tetrachloride.

3. Gut microbiota-targeted therapy

Dysbiosis contributes to the development of liver diseases. Thus, restructuring the gut microbiota community to establish eubiosis can be effective in preventing or treating liver diseases.

3.1. Probiotics

Probiotics are live microorganisms that provide health benefits for the host when consumed in adequate amounts.¹⁰⁷ In addition to the beneficial effects on gastrointestinal diseases, probiotics also exert a beneficial effect in liver diseases.^108–113

Li et al.⁸³ reported that using Prohep for feeding reduces the liver tumor size in xenograft mouse models. Prohep consists of Lactobacillus rhamnosus (L. rhamnosus) GG, E. coli Nissle 1917, and heat-inactivated VSL#3. Prohep feeding increases the abundance of Prevotella and Oscillibacter and generates anti-inflammatory metabolites, which lead to reduced Th17 polarization and increased differentiation of Treg/Trl cells in the gut. In addition, L. rhamnosus GG protects mice from high-fructose-induced NAFLD and reduces cholesterol in HFD-fed mice.^114,115 Lactobacillus casei shirota protects against NAFLD in multiple mouse NAFLD models via improved insulin sensitivity, reduced plasma LPS-binding protein, and inhibition of LPS/TLR4 signaling in the liver.^116–118 Other probiotics such as Lactobacillus plantarum MA2, Lactobacillus plantarum NCU116, Lactobacillus johnsonii BS15, Lactobacillus reuteri GMNL-263, and Lactobacillus gasseri BNR17 also have protective roles in improving dyslipidemiaand NAFLD.^119–122 Moreover, Bifidobacterium prevents fat accumulation and increases insulin sensitivity in HFD-fed rats.¹²³ Probiotics of Bifidobacterium are superior to Lactobacillus acidophilus in decreasing hepatic fat accumulation.¹²⁴

Probiotics of Clostridium butyricum MIYAIRI 588, a butyrate-producing bacterium, reduce hepatic lipid droplets and improve insulin sensitivity in rats with HFD-induced NAFLD.¹²⁵ This strain also decreases hepatic lipids and LPS in rats with NAFLD induced by choline-deficient/L-amino acid-defined diet.¹²⁶ Kumar et al.¹²⁷ have demonstrated that probiotic-fermented milk and chlorophyllin significantly reduce the incidence of aflatoxin B1-associated HCC.

Although the health effects of probiotics are mainly obtained from animal studies, some consistent results have been generated in clinical studies. Administration of L. rhamnosus GG and a mixture of Lactobacillus bulgaricus and Streptococcus thermophiles has, beneficial effects on obese children with NAFLD.^128,129 VSL#3 im, proves liver function and increases glucagon-like peptide 1 levels in obese children with NASH.¹³⁰ Moreover, L. rhamnosus GG alters gut microbiota in patients with cirrhosis.¹³¹ Compared with placebo, L. rhamnosus GG increases the beneficial autochthonous Clostridiales Incertae Sedis XIV and Lachnospiraceae and reduces the abundance of Enterobacteriaceae and Porphyromonadaceae in patients with stable cirrhosis and minimal hepatic encephalopathy.¹³¹ Combination of Bifidobacterium longum and fructooligosaccharides (FOSs, a mixture of fermentable dietary fibers) improved minimal and overt hepatic encephalopathy in clinical studies.^132,133 In addition, VSL#3 prevented hepatic encephalopathy in a randomized controlled clinical study.¹³⁴ Compared with baseline, 3 months treatment with VSL#3 increased psychometric hepatic encephalopathy scores and reduced the levels of arterial ammonia, SIBO, and orocecal transit time.¹³⁴ Over 6 months, VSL#3 treatment reduced the recurrence of hepatic encephalopathy in, cirrhotic patients compared with the placebo-treated controls.¹³⁵ VSL#3 also decreased the hepatic venous pressure gradient, cardiac index, and heart rate, and increases systemic vascular resistance and mean arterial pressure in patients with cirrhosis and ascites.¹³⁶ This indicates that VSL#3 improves the hepatic and systemic hemodynamics in patients with cirrhosis.¹³⁶ A probiotics combination of eight strains of Lactobacillus, Bifidobacterium, and Streptococcus, is also effective in preventing secondary hepatic encephalopathy in patients with cirrhosis.¹³⁷ However, in a study conducted by Solga et al.¹³⁸ 4 months supplementation with VSL#3 increased hepatic lipid content in four patients who already had steatosis. Another randomized double-blind study conducted by And reasen et al ¹³⁹ revealed that 4 weeks intake of L acidophilus NCFM improved insulin sensitivity but did not affect systemic inflammatory response. Additionally, 6 weeks supplementation with L acidophilus did not change serum lipids in volunteers who had elevated cholesterol.¹⁴⁰ More well-designed trials are needed to further study the effects of probiotics in preventing liver diseases.

3.2. Prebiotics

Prebiotics are food ingredients that selectively stimulate the growth or activity of beneficial microorganisms, such as bacteria and fungi.^141,142 They can alter the composition and/or activity of gut microbiota. Prebiotics are useful in preventing NAFLD in laboratory animals and clinical studies.^{109,143–145}

Prebiotics of FOSs prevent NAFLD via restoring the gut microbiota composition and intestinal epithelial barrier function, leading to reduced serum LPS, hepatic inflammation, and hepatic cholesterol content in NAFLD mice.^{146–148,157} FOS supplementation significantly reduces serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in NASH patients.¹⁴³ Lactulose increases the growth of lactic acid bacteria and Bifidobacterium.¹⁴⁹ It also decreases serum LPS and hepatic inflammation in rats with NASH.¹⁵⁰ Chitin-glucan, a prebiotic from a fungal source, reduces hepatic triglyceride, body weight gain, and glucose intolerance via restoring clostridial cluster XlVa in HFD-induced obese mice.¹⁵¹ Treatment with isomaltooligosaccharides plus lycopene increases adipose tissue fat mobilization, reduces body weight gain, and improves insulin sensitivity in HFD-induced NAFLD mice.¹⁵² Prebiotics have great potential for prevention of liver disease through improving metabolism and the intestinal barrier, as well as reducing endotoxemia.

3.3. Synbiotics

Synbiotics refer to the combination of probiotics and prebiotics in a form of synergism.¹⁵³ Synbiotics that consist of Lactobacillus paracasei B21060 plus arabinogalactan and FOSs reduce hepatic inflammation in diet-induced NAFLD.¹⁵⁴ Supplementation with seven probiotics consisting of L. casei, L. rhamnosus, S. thermophilus. Bifidobacterium breve, L. acidophilus, B. longum, and L bulgaricus plus FOSs improves fasting blood glucose, serum triglycerides, and inflammatory cytokines in both lean and obese NAFLD patients.^155,156 Compared to lifestyle intervention alone, synbiotics of B. longum plus FOSs have added benefits for NASH patients. This intervention reduces serum tumor necrosis factor α (TNFα), C-reactive protein, endotoxin, and AST.¹⁵⁷

Milk oligosaccharides (MOs) selectively increase the growth of Bifidobacterium infantis (B. infantis). Synbiotics of B. infantis and MOs prevent occurrence of cancer-prone NASH in western-diet-fed FXR KO male mice. B. infantis and MOs increase G protein-coupled bile acid receptor 1 (also known as Takeda G protein-coupled receptor, TGR5)-regulated signaling, thereby generating beneficial effects. B. infantis and/or MO treatment also improves ileal SCFA, signaling in western-diet-fed FXR KO mice. Furthermore, MOs alone and B. infantis plus MOs inhibit the growth of genus Bilophila and reduce the abundance of bacterial genes including dissimilatory sulfite reductase (dsrA) and methyl coenzyme M reductase A (mcrA), which are increased in mice with NASH.¹⁵⁹

3.4. Other approaches

3.4.1. Bacterial metabolite butyrate

Butyrate is generated by bacterial fermentation of non-digestible polysaccharides.^15,160 Sodium butyrate treatment reduces inflammation and fat accumulation in diet-induced NAFLD, potentially via enriching beneficial bacteria Christensenellaceae, Blautia, and Lactobacillus.¹⁶¹ Additionally, butyrate supplementation reverses NASH via reducing hepatic β-muricholic acid (β-MCA) as well as DCA, which are implicated in the development of NASH in western-diet-fed FXR-KO mice.^65,69 It has been shown that Lactobacillus and Bifidobacterium reduce adiposity and inflammation in NAFLD rats via butyrate production and butyrate receptor G-protein-coupled receptor 109A-regulated signaling.¹⁶⁰ Butyrate and its synthetic derivative, N-(l-carbamoyl-2-phenyl-ethyl) butyramide, reduce the intracellular lipid accumulation and oxidative stress in diet-induced insulin-resistant obese mice.¹⁶² Furthermore, sodium butyrate has a protective role in NAFLD pathogenesis via increased duodenal melatonin synthesis, as well as decreased hepatic inducible nitric oxide synthase in fructose-induced NAFLD mice.¹⁶³

3.4.2. Fecal microbiota transplantation

A randomized clinical trial in patients with cirrhosis and recurrent hepatic encephalopathy was conducted to compare the safety of fecal microbiota transplantation with no such intervention.¹⁶⁴ Fecal microbiota transplantation reduced hospitalization and improved cognition and dysbiosis in patients with cirrhosis with recurrent hepatic encephalopathy, when compared with standard of care (SOC).¹⁶⁴ Fecal microbiota transplantation has protective effects in rats with CCl₄-induced hepatic encephalopathy.¹⁶⁵ Fecal microbiota transplantation reduces intestinal permeability and improves the TLR response of the liver, leading to improved cognitive function and reduced liver function indexes.¹⁶⁵

3.4.3. Diet

Diet is a contributing factor to liver diseases. Fructose-enriched diet alters liver metabolism and gut barrier function, increases endotoxemia, decreases Bifidobacterium and Lactobacillus, and eventually leads to NAFLD.¹⁶⁶ Long-term fructose consumption increases lipogenic enzymes via activation of sterol regulatory element binding protein-lc (SRFBPlc) and carbohydrate responsive element binding protein (ChREBP).¹⁶⁷ It promotes lipogenesis, hypertriglyceridemia, hepatic insulin resistance, and hepatic steatosis.¹⁶⁷ A diet rich in fermented milk, vegetables, cereals, coffee, and tea contributes to a higher microbial diversity in patients with cirrhosis.¹⁶⁸ Microbial diversity is an independent factor that reduces the risk of 90-day hospitalization.¹⁶⁸

4. Conclusions and perspectives

Gut microbiota plays a pivotal role in the pathogenesis of metabolic liver diseases. Re-establishing eubiosis using probiotics, prebiotics, and synbiotics, as well as natural products, is a promising avenue to prevent and treat liver diseases and, potentially, liver cancer. Although bile acid and SCFA-regulated pathways can explain how diet through gut microbiota affects health and disease processes, other molecular links remain to be uncovered. With the advancement of sequencing technology as well as cultural techniques, specific bacterial species and microbial functions can be uncovered to establish a causal relationship. There is no doubt that metabolomics and epigenetic genomics are powerful tools to elucidate the underlying mechanism for disease processes, leading to innovative treatment strategies. The generated information should have an impact on personalized nutrition as well as precision medicine.