Liver cirrhosis and complications from the perspective of dysbiosis

The gut-liver axis refers to the intimate relationship and rigorous interaction between the gut and the liver. The intestinal barrier’s integrity is critical for maintaining liver homeostasis. The liver operates as a second firewall in this interaction, limiting the movement of potentially dangerous compounds from the gut and, as a result, contributing in barrier management. An increasing amount of evidence shows that increased intestinal permeability and subsequent bacterial translocation play a role in liver damage development. The major pathogenic causes in cirrhotic individuals include poor intestinal permeability, nutrition, and intestinal flora dysbiosis. Portal hypertension promotes intestinal permeability and bacterial translocation in advanced liver disease, increasing liver damage. Bacterial dysbiosis is closely related to the development of cirrhosis and its related complications. This article describes the potential mechanisms of dysbiosis in liver cirrhosis and related complications, such as spontaneous bacterial peritonitis, hepatorenal syndrome, portal vein thrombosis, hepatic encephalopathy, and hepatocellular carcinoma, using dysbiosis of the intestinal flora as an entry point.


Introduction
Liver cirrhosis is a primary cause of morbidity and mortality worldwide.The mortality rate of patients with compensated liver cirrhosis increased five-fold and increased 10-fold in patients with decompensated liver cirrhosis compared with the general population (1).Liver cirrhosis is an advanced chronic liver disease caused by various etiologies, including alcohol abuse, obesity, and hepatitis virus infection, and prevalence of cirrhosis is increasing worldwide (2).Although there are many etiologies of liver cirrhosis, alcoholic liver disease (ALD), nonalcoholic fatty liver disease (NAFLD), and viral hepatitis are the prevalent etiologies of chronic liver disease globally (3,4).Liver cirrhosis is the end-stage pathological manifestation of chronic liver disease charactered by chronic inflammation, hepatic lobe reconstruction, and the formation of pseudolobule and tubercle (5).The bidirectional interactions of the liver and the intestinal microbiota provide a new perspective for the occurrence and development of liver cirrhosis and its complications (6).
Microbes inhabit specific locations of the body, such as the skin, mouth, respiratory tract, gastrointestinal tract, genitourinary system, etc., and the highest density residing in the gastrointestinal tract (7,8).The exact time of gut microbial colonization is not known, but intestinal microbial colonization does not occur before birth (9).A variety of internal and external factors will affect the intestinal flora after birth, such as dietary, disease and sleep, etc. (10)(11)(12)(13).Metagenomics, metatranscriptomic, and metaproteomic of the human intestinal microbiota show that the intestinal microbiota is an important constituent of the organism and plays a crucial role in human health and disease (14).The portal vein is an important channel for the interaction between the liver and the intestinal microbiota.The portal vein can transport intestinal nutrients, bacteria, and microbial products to the liver (15).Under normal physiological conditions, the intestinal microbiota is essential for liver metabolic functions, maturation and maintenance of immune homeostasis (16), and in turn, the liver regulates the intestinal microbiota and metabolic functions mainly through secreting bile acids (BA) (17).
We review clinical studies on cirrhosis and dysbiosis, and then turn to the topic of cirrhosis-related complications and dysbiosis.Dysbiosis may provide new perspectives on cirrhosis and complications.

Liver cirrhosis and dysbiosis
Liver cirrhosis affects the intestinal microbiota mainly through two aspects: on the one hand, liver cirrhosis can reduce the synthesis of BA, and resulting in the composition and function of the intestinal microbiota (18,19).On the other hand, liver cirrhosis can cause portal hypertension, gastrointestinal blood stasis, impair intestinal barrier, and gut dysbiosis.Bacteria and metabolites can enter the portal vein and systemic circulation through translocation of the damaged intestinal barrier, causing an inflammatory state in the body and endotoxemia (20), which have different effects on both cirrhosis and cirrhosis-related complications (21).
Bile acids are the major functional components of bile and are generated by a classical and alternative pathway in the liver (22).BA primarily promote the emulsification of fats and aid the absorption of lipid and fat-soluble vitamins (23,24).BA and the intestinal microbiota can interact with each other, and BA play an important role in regulating the diversity of intestinal flora and small intestine bacterial overgrowth (25).BA can not only directly affect the integrity of the intestinal barrier via regulating the composition of intestinal flora but promoting the synthesis and antimicrobial peptides secretion of the intestinal epithelial cells (26).Moreover, BA regulate metabolic function, energy consumption, and inflammation through interaction with its receptors, such as farnesoid-X-receptor (FXR) and the vitamin D receptor (VDR) as well as the Takeda G-protein coupled BA receptor (TGR5) (27).The activation of FXR can protect the integrity of the intestinal barrier, reduce bacterial translocation and intestinal inflammation (28,29).
The intestinal microbiota plays a unique role in BA metabolism and homeostasis in the host (19).The metabolites of the intestinal microbiota with a variety of biological functions, also known as postbiotics, such as short-chain fatty acids, secondary BA, choline metabolites, indole derivatives, vitamins, polyamines, lipids, neurotransmitters, neuroactive compounds, and thalamic-pituitaryadrenal axis hormones play an important role in the body (30).ALD, NAFLD, and viral hepatitis are the leading associated causes of liver cirrhosis worldwide, we have mainly summarized the clinical studies related to dysbiosis in these diseases.
In general, the 16S rRNA gene sequencing analysis showed a significant decrease in the levels of Lachnospiraceae and Ruminococcaceae in patients with liver cirrhosis, while the wholemetagenome shotgun sequencing analysis showed that the level of Faecalibacterium prausnitzii from the Ruminococcaceae and of Coprococcus spp.from the Lachnospiraceae are significantly reduced (31,32).Dysbiosis of intestinal flora further aggravates liver injury.Studies have shown that the severity of liver injury is closely related to the severity of intestinal flora dysbiosis (33).Changes in the fecal bacterial flora are manifested by changes in the composition of the major Bacteroidetes and Sclerotiniaceae, which produce short-chain fatty acids (SCFA) that are a source of energy for intestinal epithelial cells, and also regulate BA metabolism and induce modulation of the immune function of the intestinal barrier (34).Dysbiosis of the intestinal flora affects changes in intestinal permeability and intestinal metabolites that may be involved in the progression of cirrhosis and its associated complications (35).

ALD and NAFLD
Liver cirrhosis is the common pathologic of the advanced stage of ALD and NAFLD (36).Alcohol and its metabolites can affect the tight junction between intestinal epithelial cells, impair intestinal barrier function, induce bacterial translocation and endotoxemia (37, 38).Gut dysbiosis is closely related to the occurrence and development of NAFLD and ALD, but the mechanism is not clear (39,40).The intestinal microbiota can promote the development of human NAFLD and ALD into end-stage liver disease (41,42), indicating that gut dysbiosis may be the common changes of these diseases.A higher abundance of Enterobacteriaceae and Halomonadaceae, and lower Lachnospiraceae, Ruminococcaceae, and Clostridialies XIV in alcoholicrelated liver cirrhosis than non-alcoholic cirrhotics, whereas non-alcoholic steatohepatitis-related liver cirrhosis (NASH) had a higher level of Porphyromonadaceae, Bacterioidaceae, and lower Veillonellaceae compared to those without NASH etiology (43).The number of Escherichia coli, anaerobes, Lactobacillus, and streptococci in intestinal microflora of patients with NAFLD is higher than that of healthy controls (44).Compared to healthy controls, the proportion of Bacteroidetes was significantly reduced, whereas Proteobacteria and Fusobacteria were highly enriched in the ARLC patients with different etiologies (45).The median abundance of Bacteroidetes was lower and the median abundance of Proteobacteria was higher in the intestines of patients with ALD, and these changes appeared to be associated with higher serum endotoxin levels in some of the samples (46).Studies have shown that alcohol causes a significant increase in Veillonellaceae and a decreasing trend in Bacteroidaceae and Porphyromonadaceae (47).16S rRNA gene sequencing revealed that Peptostreptococcacae, Proteobacteria, Pasteurellaceae and Bacillales were significantly increased, while Lachnospiraceae, Ruminococcaeae, Clostridiales cluster XIV, Prevotellaceae and Bacteroidaceae significantly decreased in ARLC patients (48).Metagenomic sequencing revealed that Bifidobacterium, Streptococcus and Lactobacillusspecies were significantly increased, while Akkermansia, Coprococcus, Unclassified and Clostridiales significantly decreased in ARLC patients (49).
For NASH patients, there was a significant increase in Clostridium coccoides, Porphyromonadaceae and Actioidaceae and a decrease in

PVT
PVT is a common complication observed in liver cirrhosis and occurs in intrahepatic branches of the portal vein, with or without superior mesenteric vein and splenic vein thrombosis (67).The prevalence of PVT is approximately 1%-26% (68).The pathogenesis of liver cirrhosis with PVT is unclear.Slow portal vein blood flow caused by liver cirrhosis is the important factor for PVT (69).The decrease of symbiotic anaerobes and the increase of pathogen abundance after gut dysbiosis, especially the increase of Gram-negative Enterobacteriaceae, are closely related to the occurrence of PVT (70).Bacterial LPS is the glycolipid on the outer membrane of Gram-negative bacteria and is one of the key factors in the hypercoagulable state of liver cirrhosis (71,72).The translocation of bacteria and pathogen-associated molecular patterns (PAMPs), especially LPS, will cause systemic inflammation, endotoxemia, and platelet activation, at the same time, systemic inflammation and elevated levels of systemic inflammatory factors exacerbate the risk of PVT formation (73,74).Liver cirrhosis patients usually have higher endotoxemia and systemic inflammation due to gut dysbiosis.Endotoxin can increase thrombosis through the production of tissue factor (TF) (75).With the increase of intestinal permeability and bacterial translocation, the level of LPS in the blood of patients with liver cirrhosis increased significantly.Due to the immune function of the liver, the level of LPS in the portal system was significantly higher than that in the systemic circulation (76,77).
After entering the circulation system, LPS interacts to toll-like receptors (TLRs) and initiating a series of pathophysiological changes linked to the formation of PVT.On the one hand, LPS binds to TLRs expressed on hepatocytes and immune cells.Activation of these cells will release a huge amount of inflammatory cytokines, chemokines, vasoactive factors, adhesion molecules, and reactive oxygen species (ROS) (78-80), resulting in systemic inflammation, the proliferation of hepatic stellate cells, and the development of liver cirrhosis (81).On the other hand, LPS binds to TLRs receptors expressed on vascular endothelial cells, platelet, and neutrophils, promoting blood hypercoagulability and PVT in the context of liver cirrhosis (70) (Figure 2).

LPS and endothelial cells
LPS binds to TLRs receptors on vascular endothelial cells and activates endothelial cells to release von Willebrand factor (vWF) and factor VII (43,82).Animal experiments showed that LPS could also bind to toll-like receptor 2 (TLR2) on hepatic endothelial cells to promote the synthesis of the vWF precursor and promote platelet integrin-dependent thrombus growth, while the synthesis of vWF in hepatic endothelial cells decreased and the level of plasma vWF decreased significantly in TLR2 knockout mice (83).In vitro studies have shown that LPS can stimulate the formation and secretion of Weibel-Palade bodies in endothelial cells through toll-like receptor 4 (TLR4), and promote the release of VIII and vWf into the blood resulting in a blood hypercoagulable state.TLR receptor blockers can significantly inhibit the release of VIII and vWf, indicating that LPS in the intestinal microbiota increases the level of systemic factor VIII by stimulating the release of endothelial cells (43).The level of Plasminogen activator inhibitor-1 (PAI-1) and tissue plasminogen activator (TPA), key determinants in thrombosis, were shown to be related to the gut microbiota.In vivo and in vitro studies have shown that bacterial lipoprotein TLR2 agonists can increase vascular endothelial cell permeability, increase plasma PAI-1 and decrease TPA (84).

LPS and platelets
Platelet activation in patients with liver cirrhosis may be associated with increased levels of circulating LPS (85).TLR2 and TLR4 are widely distributed on the surface of platelets.The activation of the TLR2 in platelets causes the activation of platelets, which can cause platelets to release their own stores of α-particles  Dysbiosis and liver cirrhosis and complications.and dense particles to interact with vascular endothelial cells and promote platelet-dependent thrombosis (86,87).In vitro studies have shown that platelets in patients with liver cirrhosis are more reactive to TLRs agonists than healthy people and have nothing to do with the number of platelets.The reactivity is significantly weakened in the presence of TLR4 blockers, indicating that LPS may activate platelets through TLR4 and may lead to liver cirrhosisrelated thrombotic complications (70).Activated platelets release P-selectin to stimulate monocytes and macrophages to release chemokines, promote platelet-monocyte aggregation, and release inflammatory molecules to change the chemotaxis and adhesion of endothelial cells (88).

LPS and neutrophils
Activation of neutrophils can induce thrombosis through the neutrophil extracellular bactericidal networks (NETs) (89,90).In vitro animal models have shown that the elimination of NETs can prevent thrombosis (91).NETs are formed by neutrophils releasing nuclear contents (DNA-histone complex, double-stranded DNA, and neutrophil elastase) into extracellular space (92).Histone-DNA complex can activate coagulation factor XIIa activated by coagulation factor XII, and then activate coagulation factor XI and downstream endogenous coagulation pathway (93).Histone of NETs can promote thrombin production, endothelial cell activation, and thrombus formation through the platelet-dependent mechanism mediated by TLR2 and TLR4 (94).

Trimethylamine-N-oxide
Trimethylamine (TMA) lyase from intestinal microorganisms metabolizes phosphatidylcholine, choline and carnitine to TMA, which is further processed to trimethylamine-N-oxide (TMAO) by flavin monooxygenase (FMO) in the liver (95).TMAO not only aggravates cardiovascular events but also is closely related to the formation of thrombosis (96).TMAO activates platelets by increasing the release of Ca 2+ stored platelets intracellular, while platelets with hyperreactivity enhance the thrombosis risk (97).The animal experiment showed that a high choline diet can enhance platelet hyperresponsiveness, but this will not occur with the intervention of antibiotics or a high choline diet in germ-free mice (98).Therefore, TMAO acts as a medium to closely connect intestinal microbiota with thrombosis.
Liver cirrhosis is usually accompanying by acquired factors of thrombophilia, such as hyperhomocysteinemia, secondary to vitamin B and folate deficiencies, and antiphospholipid antibody syndrome (67).Moreover, the levels of albumin and protein C were lower in patients with liver cirrhosis compared to healthy controls, and low levels of albumin and protein C were associated with the increased risk of PVT formation (99,100).Abnormal metabolic state, hemodynamic changes, and PAI-1 in patients with NAFLD may contribute to prethrombotic state and hypercoagulable state (101,102).In a cross-sectional study, NASH-related cirrhosis was the strongest independent risk factor for the independent diagnosis of PVT in patients undergoing liver transplantation (103).

SBP
SBP is a common and serious complication in cirrhotic patients (104).It refers to primary peritonitis that occurs in patients with cirrhosis and ascites without abdominal infection lesions (105).Animal experiments showed that the bacteria strain isolated from intestinal mucosa lymphoid tissue and ascites was similar (106).These studies provide strong evidence for the role of bacterial translocation in SBP.The bacterial translocation and the increase of intestinal permeability is the main mechanism of SBP in the setting of liver cirrhosis, at the same time, the decrease of host immune clearance ability is also closely related to the occurrence of SBP (43).Gram-negative bacteria, such as Escherichia coli and Klebsiella, and Gram-positive bacteria are common causes of SBP (107,108).The patients with liver cirrhosis were accompanied by decreased diversity of the gut microbiota, which was characterized by a significant reduction in autochthonous taxa and a significant increase in pathogenic taxa (109).Clinical studies have shown that Streptococcus spp., Klebsiella, Escherichia, and Citrobacter spp.were mainly infectious organisms on routine culture in liver cirrhosis patients with SBP (43).
The gut-liver-immune axis plays a key role in SBP.The changes in intestinal motility, mucosal immunity, and drug usage in patients with liver cirrhosis will lead to significant changes in the composition of gastrointestinal microorganisms and aggravate the translocation of the intestinal microbiota (110,111).Moreover, interaction between BA and its receptor farnesoid X receptor (FXR) helps to maintain intestinal barrier function and reduce bacterial translocation (112).Animal experiments showed that FXR agonists could reduce intestinal permeability and bacterial translocation through FXR (29, 113).In addition to FXR agonists, selective intestinal decontamination is an important measure for the treatment of SBP.Intestinal decontamination with rifaximin and norfloxacin significantly decreased the incidence of SBP in cirrhotic patients with ascites (114, 115), but rifaximin was more effective than norfloxacin in the secondary prevention of SBP (116).Moreover, non-selective β-blockers (NSBB) can reduce intestinal permeability, markers of bacterial translocation (IL-6/LPS binding protein), and SBP by improving intestinal motility and reducing intestinal bacterial overgrowth (117,118).

HE
HE is a severe complication of advanced liver cirrhosis and is closely linked to the gut-liver-brain axis (119).The impaired hepatic clearance ability with the progression of liver cirrhosis is reconsider phrasing for clarity, neurotoxic substances, and false neurotransmitters produced by the intestinal microbiota (120).These substances have an important effect on HE.Moreover, the formation of portal shunts further facilitates the entrance of the microbial metabolites into the blood (121).
A series of inflammatory cytokines and endotoxemia caused by gut dysbiosis can impair blood-brain barriers, neuroinflammation, and affect cognition (122).Compared with healthy people, the specific bacteria (Alcaligenes, Porphyromonas, and Enterobacteriaceae) in fecal microbiota were significantly increased in liver cirrhosis patients complicated with HE and were strongly related to the cognition and inflammation of HE (123).A study analyzed the relationship between cognition, Magnetic resonance imaging parameters, and intestinal microflora and found that patients with HE had a significantly lower cognitive ability, systemic inflammation, gut dysbiosis, and hyperammonemia than controls and cirrhotic patients without HE (119).Specific microbial families (autochthonous taxa negatively and Enterobacteriaceae positively) correlated with changes in astrocytes associated with magnetic resonance spectroscopy and hyperammonemia (119).Treatment methods such as fecal microbial transplantation, intestinal decontamination, and diet regulation for the intestinal microbiota can improve the cognitive level of patients with HE (124-126).Therefore, Gut dysbiosis is associated with the development of HE (43).
Emerging evidence also shows that brain cholesterol accumulation contributes to the progression of HE through BA-mediated effects on the FXR (127).Moreover, serotonin and tryptophan metabolism via the gut microbiota is a key factor for the occurrence of central nervous system diseases (128).Recent studies have found that bacteria (Stenotrophomonas pavanii, Methylobacterium extorquens) and metabolites (methanol, threonine) in the blood and feces of patients with liver cirrhosis are positively correlated with HE, while fecal Enterobacteriaceae and TMA were positively correlated with blood proinflammatory cytokines (129).Therefore, bacteria or their metabolites in the blood are correlated with systemic inflammation and HE in patients with liver cirrhosis.Those studies provide new perspectives and treatment strategies for the pathogenesis of HE.

PH
Liver structural disorder and nodular regeneration in liver cirrhosis will cause compression of hepatic sinusoids and blood vessels, and increase intrahepatic resistance, resulting in (PH) (130).PH has an important effect on intestinal permeability, gut microbiota, and bacterial translocation.PH reduces intestinal mucosal blood flow, causes neoangiogenesis, ischemia, and edema of the intestine, destroying intestinal barrier function (131).Therefore, the gut dysbiosis in liver cirrhosis causes the translocation of a large number of bacteria and products, especially LPS, into the blood, leading to activate the immune system and causes systemic inflammation via interacting with TLRs (43, 132), and activation of the liver immune system and systemic inflammation promote the progression of liver cirrhosis and aggravate PH (133).In the animal model, intraperitoneal injection of LPS activates the expression of TLR4 and increases inflammatory mediators, leading to escalating PH (134).The release of systemic inflammatory factors (nitric oxide, NO), can also reduce systemic vascular resistance and induce hyperdynamic circulation, thus affecting PH (135,136).Therefore, there is an important pathophysiological relationship between PH and the gut-liver axis.
FXR not only plays an important role in intestinal microflora homeostasis, BA metabolism, and intestinal barrier function, but also in anti-fibrosis and reducing PH.In the experimental liver cirrhosis, non-steroidal FXR agonist PX20606 improves PH via reducing intestinal bacterial migration, liver fibrosis, vascular remodeling, and hepatic sinusoid dysfunction (137).Studies in preclinical models of cirrhosis shows that intestinal decontamination can improve the portal vein pressure and hyperdynamic circulation in liver cirrhosis (138).These studies provide insight into molecular mechanisms and novel therapeutic targets in PH.

HRS
HRS refers to functional acute renal failure in patients with severe liver disease (139).HRS is a severe complication of advanced liver cirrhosis with a prevalence between 11% and 20% (140).The mechanism of HRS is not clear, but it is mainly related to the following two aspects: on the one hand, the production of ascites increases, and the circulating blood volume decreases after decompensation of liver cirrhosis, resulting in prerenal renal failure (141); on the other hand,

HPS
HPS is a pulmonary complication in liver cirrhosis patients and charactered by pulmonary microvascular dilatation and hypoxemia (149).The pathogenesis of HPS is unknown.The bacterial translocation, intestinal endotoxemia, and pulmonary microvascular dilatation may closely relate to the pathogenesis of HPS (150-152).Studies on animal models have shown that bacterial translocation, intestinal endotoxemia, and related inflammatory factors are closely related to the occurrence of HPS (153).Moreover, bacterial translocation can increase the incidence and severity of HPS in cirrhotic rats, and prophylactic norfloxacin usage can reduce the incidence and severity of HPS (154).However, antibiotic norfloxacin usage in clinical patients does not improve the gas exchange of HPS (155).Therefore, there need to be further studies on the effect of antibiotics in patients with HPS and the role of the specific intestinal microbiota in the pathogenesis of HPS.

HCC
HCC is the most common type of liver cancer as well as the common cause of death in patients with advanced cirrhosis (156, 157).Moreover, different etiologies may affect the composition of the microbiota in HCC patients (158).As aforementioned, systemic inflammation caused by the translocation of bacteria, LPS, bacterial DNA, and peptidoglycans by activating the TLRs is crucial for the development of HCC (42,159).LPS activates TLR4 signal to promote the production of interleukin-6 (IL-6) and TNF-α, mediating the differentiation of hepatic progenitor cells into myofibroblasts, and promoting the proliferation and malignant transformation of hepatic progenitor cells (160).Therefore, LPS and TLR4 are closely related to the development of cirrhotic patients to HCC (161).Compared to NAFLD cirrhotic patients without HCC, the NAFLD cirrhotic patients with HCC have a higher abundance of Bacteroidetes at the phylum level, Bacteroidaceae, Streptococcaceae, Enterococcaceae, and Gemellaceae at the family level, and Phascolarctobacterium, Enterococcus, Streptococcus, Gemella, and Bilophila at the genus level (162).In another study, intestinal microbiota constitution in cirrhotic patients with HCC is distinguished from those without HCC.Compared patients without HCC, Haemophilus, Eggerthella, Bifidobacterium, Butyricimonas, Christensella, Odoribacter, an unknown genus phylum Tenericutes, and an unknown genus, phylum Firmicutes, family Erysipelotrichaceae were all elevated in cirrhotic patients with HCC, while Fusobacterium, Prevotella, Streptococcus, S24-7 (Phylum Bacteroidetes) and an unknown genus were all decreased.Thus, gut dysbiosis is a crucial factor in cirrhotic patients with HCC (163).

Conclusion
The intestinal microbiota plays a significant role in human health and disease.Gut dysbiosis is associated with the onset and progression of liver cirrhotic and its complications.The correlation between specific intestinal microbiota and pathogenesis of liver cirrhosis related complications needs further study.The gut microbiota can be used as a potential diagnosis biomarker and treatment target for liver cirrhosis and its complications.It has been reported that bacteriophage therapy (164), microRNA therapy (165), and carbon nanoparticles (166) that based on targeting the intestinal microbiota in liver cirrhosis.It is important to assess the role of the gut microbiota in the pathogenesis of liver cirrhosis and its complications.With the rise of personalized medicine and nanomedical technology, treatment options that targeting specific intestinal microbiota composition may be the most promising treatment for liver cirrhosis and complications in the future.

TABLE 1
The changed gut microbiota in alcohol and NASH-related liver disease.

TABLE 2
The changed gut microbiota in viral hepatitis-related liver disease.