Research progress on the correlation between intestinal microflora disorder and liver disease

ZHAO Jing-fang, YOU Jing

1.Department of Infectious Diseases, The First Affiliated Hospital of Kunming Medical University, Kunming 650032, China

2.Infectious diseases Department of the Third People's Hospital of Yunnan Province, Kunming 650011, China

Keywords:

ABSTRACT In recent years, the relationship between intestinal flora and liver disease has become an important research direction of liver diseases.A growing body of evidence indicates that gut bacteria play a key role in the pathophysiology of liver disease, this article combed the at home and abroad in recent years, the changes of intestinal flora and autoimmune liver disease, alcoholic liver disease, fatty liver disease related to metabolism, hepatitis b viral hepatitis, cirrhosis and liver cancer occurrence and progress of relationship of related research, And the new progress of regulating intestinal microecology in the treatment of liver diseases.Dysregulation of intestinal flora plays an important role in the occurrence and development of liver diseases.Regulating intestinal flora to improve the prognosis of liver diseases will be an important development direction in the future.

There are 500 to 11,500 species of bacteria in the human gut,numbering over 100 trillion[1], forming a complex and diverse gut flora.The human intestinal flora is mainly composed of Firmicutes,Bacteroidetes, actinobacteria and Proteobacteria[2].According to their effects on the human body, they can be roughly divided into beneficial bacteria, harmful bacteria and neutral bacteria, which are closely related to human metabolism, immunity, aging and disease occurrence.Intestinal flora, on the one hand, provides energy for the activities of intestinal epithelial cells, participates in the synthesis of vitamins and the metabolism of bile salts; On the other hand, the intestinal mucosal immune function canbe activated through competition for living space, thus preventing the invasionof pathogens and helping the human body to complete a series of physiologicalfunctions such as nutrition, immunity and metabolism.Imbalance of intestinalflora refers to that, due to some reason, the balance between normal intestinalflora is broken,resulting in location transfer, proportion imbalance and selfinfection[3].

With the development of metagenomics, macrotranscriptomics and other technologies, especially the establishment of the concept of “enteric-liver axis”[4] in recent years, people have gained a new understanding of the relationship between intestinal flora and liver diseases.In addition to receiving the blood from portal vein reflux and nutrients absorbed by the intestine, the liver also acts as a firewall to prevent pathogenic bacteria and metabolites from spreading to the whole body.Bacterial overgrowth, bacterial species change and bacterial translocation in the intestinal tract will change the permeability and immune state of the intestinal tract, and bacteria and their products will more easily enter the liver through the portal vein, affecting the normal physiological function of the liver and leading to the occurrence and development of a series of liver diseases[5].Dysbiosis of intestinal flora is an important common pathway for the oc-currence and progression of autoimmune liver disease, alcoholic liver disease, metabolizationrelated fatty liver disease, viral hepatitis, cirrhosis and hepatocellular carcinoma.

At present, in the diagnosis and treatment of liver-related diseases,most clinicians have insufficient understanding of the correlation between intestinal flora and liver diseases, and pay insufficient attention to the treatment of liver diseases by regulating intestinal flora imbalance.In order to help clinicians to further understand the relationship between intestinal microflora and the occurrence and development of liver diseases, this paper reviewed the main relevant literatures in recent years, summarized the relationship between intestinal microflora imbalance and liver diseases and the new progress in regulating intestinal microecology in the treatment of liver diseases, in order to bring new reference for clinical treatment.

1.Intestinal flora and autoimmune liver disease

Autoimmune liver disease (AILD) is a disease in which the immune system of the human body attacks its own liver tissue, causing liver tissue damage, abnormal liver function and corresponding clinical symptoms.Epicardial hepatitis (AIH), primary biliary cholangitis(PBC), primary sclerosing cholangitis, PSC, and so on[6].At present,the etiology of AILD is not clear, and the diagnosis and treatment of patients are very challenging.In recent years, part of the research on AILD has focused on the interaction between intestinal flora and human body.The diversity of intestinal flora in most AILD patients has decreased sig-nificantly, and some intestinal bacterial components or metabolites can trigger the autoimmune response of liver cells, and intestinal flora may have an important influence on the occurrence and development of AILD[7].

1.1 Intestinal flora and AIH

AIH is a chronic idiopathic hepatocellular immune injury syndrome, and its pathogenesis is usually associated with autoantibodies.Chinese scholars Wei et al.[8] found that compared with the healthy control group, the number of Klebsiella,Streptococcus and Lactobacillus in AIH patients increased,especially veillon.However, there is currently no data to support a causal relationship between Veillon and AIH.An Egyptian study[9] also confirmed that the intestinal flora diversity of AIH patients decreased, and the decrease in the relative abundance specificity of Lactobacillus and bifidobacteria was associated with the increased disease activity of AIH[10].AIH patients present impaired intestinal barrier and imbalance of intestinal flora, which induce bacterial translocation and portal endotoxemia, and activate innate immune receptors, namely toll-like receptors, leading to liver inflammation and tissue destruction[11], and these changes are related to the severity of the disease.

In the treatment of AIH, Zhang et al[12] reported that bifidobacterium lactate 420 has a beneficial effect in alleviating AIH.Experimental study of Liang et al.[13] on AIH model mice showed that fecal group transplantation significantly alleviated liver injury and bacterial translocation in AIH mice, improved the imbalance between follicular regulatory T cells (TFR) and helper T cells (TFH), and controlled the progression of hepatitis in AIH mice.These studies suggest that regulating intestinal flora can help reduce liver injury in AIH patients.

1.2 Intestinal flora and PBC

PBC is a chronic insidious disease caused by progressive destruction of intrahepatic bile duct and bile secretion disorder caused by inflammatory reaction.Patients with PBC suffer from liver dysfunction and reduced detoxification ability, leading to the disorder of intestinal flora, which triggers a sustained inflammatory response of the liver through the “hepato-intestinal axis”[14].Lammert et al.[15] found that intestinal flora is related to PBC fibrosis and cirrhosis.Lv et al.[16] analyzed the gene sequencing of intestinal flora in PBC patients and healthy people, and found that the abundance of intestinal flora in PBC patients changed compared with the normal group.The faecal microbiota of patients with early PBC has been depleted of potentially beneficial bacteria such as acidobacteria, Bacteroides, trichospira and ruminococcus.However,stool is rich in opportunistic pathogenic bacteria, such as gammaproteus, Veillon coccus, Haemophilus, Pseudomonas, Klebsiella,enterobacter bacteria.The changes in intestinal flora in PBC patients were demonstrated.The liver function of PBC patients is impaired and detoxification ability is reduced, leading to the disorder of intestinal flora, which induces a sustained inflammatory response of the liver through the “hepato-intestinal axis” [17].

In the treatment of PBC, ursodeoxycholic acid (URSOdeoxycholic acid) is currently the first-line drug for PBC treatment, and its main mechanism of action may include the reconstruction of intestinal flora.Tang et al[18] observed 60 PBC patients and found that there was a positive correlation between the number of Klebsiella and serum bilirubin level, and the abundance of some PBC related bacteria returned to normal half a year after ursodeoxycholic acid treatm-ent.

All the above studies have confirmed the intestinal microflora imbalance in PBC patients, which can be partially alleviated by ursodeoxycholic acid.Whether other drugs can regulate intestinal microflora to treat PBC more effectively remains to be explored.

1.3 Intestinal flora and PSC

PSC is an autoimmune liver disease characterized by multifocal bile duct stenosis caused by idiopathic intrahepatic bile duct inflammation and fibrosis, with chronic cholestatic lesions as the main clinical manifestation[19], and its pathogenesis is still largely unknown.The relationship between Inflammatory bowel disease(IBD) and PSC has been known for about 50 years.The researchers found that about 70% of patients also had IBD[20], suggesting that intestinal inflammation and bile duct inflammation may share a common cause.Sabino et al[21] found that the intestinal flora of PATIENTS with PSC was characterized by decreased diversity of bacteria, with significantly more enterococcus, Clostri-dia,Lactobacillus and Streptococcus than that of healthy controls.This situation existed in PSC patients with or without IBD, and was not related to IBD and ursodeoxycholic acid treatment.This is consistent with the research results of Ruhleman[22].These results suggest that intestinal microflora disorder may be rel-ated to the pathogenesis of PSC.More and more evidence indicates that reduced alkaline phosphatase (ALP) level is associated with improved prognosis of PSC[23, 24].Sabino et al.[21] found that enterococcus was positively correlated with serum ALP level, indicating a potential link between intestinal flora and disease severity.

Currently, the only proven effective treatment for PSC is liver transplantati-on.However, recent studies have shown[25] that the abundance of Velonococcus decreased in patients with sufficient ursodeoxycholic acid response after treatment, while the abundance of Velonococcus increased in patients with poor ursodeoxycholic acid treatment.Veillon was strongly correlated with glutamyl transpeptidase (GGT) (P＜0.01).This suggests that targeting the abundance expression of Veron coccus in intestinal flora may become another research direction for the treatment of PSC.

2.Intestinal flora and alcoholic liver disease

Alcoholic liver disease (ALD) is a toxic liver injury caused by long-term he-avy drinking.Alcoholism will cause intestinal flora disorders: first, alcohol will cause intestinal movement disorders,may lead to intestinal bacterial proliferation; Second, alcohol induces mucus erosion and ulcers mainly by changing the glycosylation of mucus, resulting in high incidence of superficial and atrophic gastritis in alcoholics, decreased production of hypochloric acid or gastric acid, resulting in small intestinal bacterial overgrowth(SIBO).Third the long-term alcohol environment in intestinal mucosa would destroy the integrity of intestinal mucosal barrier,and the increase of its permeability would cause the loss of the function of blocking the invasion of pathogenic microorganisms,and easily lead to bacterial migration, leading to the increase of plasma level of intestinal microbial products lipopolysaccharide(LPS) or endotoxin[26].Fourth, after alcohol abuse, the secretion of antibacterial proteins Reg3b and Reg3g is reduced, which affects the immune and epidemic prevention mechanism and leads to the imbalance of intestinal flora[27].The imbalance of intestinal flora can aggravate liver injury: first, the increased level of enteric-derived endotoxin leads to the increase of CD4+T cells, liver T lymphocytes and natural killer cells in mesenteric lymph nodes, resulting in liver inflammation and injury; Second, overgrown bacteria can also produce endogenous ethanol, destroy the intestinal barrier, and eventually lead to liver inflammation[17].Some studies[28] found that in jejunum of patients with long-term alcoholism, the abundance of Bacteroides and Firmicutes decreased, especially lactobacillus significantly decreased, while proteus bacteria increased.

In treatment of ALD, Peng[29] research shows that alcohol feeding mouse model of saturated long chain fatty acid synthesis, long chain saturated fatty acids by symbiotic lactobacilli metabolism and promote its growth, after the use of prebiotics can increase the number of the genus lactobacillus symbiosis, stability of the intestinal barrier and reduce the liver injury in mice induced by ethanol, Dietary supplementation of saturated long-chain fatty acids prevents changes in intestinal microbiota composition after long-term ethanol exposure.The research and development of diets or drugs derived from intestinal flora has great feasibility and extensibility for the treatment of alcohol-induced liver diseases.

3.Intestinal flora and metabolic fatty liver disease

Metabolic associated fatty liver disease (MAFLD), formerly known as nonalc-oholic fatty liver disease, refers to the presence of fatty liver by liver imaging or histological examination.Liver disease accompanied by obesity or overweight, type 2 diabetes, and metabolic dysfunction: one of the three[30].MAFLD is the main cause of chronic liver disease in the world.Currently, the prevalence of MAFLD is increasing worldwide, and the trend of globalization and younger age is developing.

Dysregulation of intestinal flora is closely related to the incidence of MAFLD[31].Signals from dietary intake and environmental factors can cause dysbiosis in the gut.Changes in intestinal flora can promote energy intake through the synthesis of shortchain fatty acids.This excess energy is converted into free fa-tty acids by anabolism in intestinal cells.At the same time, ethanolproducing bacteria increase endogenous levels of this metabolite,which leads to mucus erosion and increased intestinal permeability,leading to bacterial translocation.Bacteria, associated antigens,and free fatty acids are transported to the liver thro-ugh the portal vein, where lipotoxicity occurs in liver cells, inflammation due to pathogen-associated molecular pattern recognition, and ultimately immune cell recruitment.It causes apoptosis and fibrosis,leading to deterioration of MAFLD[32].Le et al[33] found in the experimental study that NORMAL mice also developed MAFLD after the intestinal bacteria of the modeled MAFLD mice were transplanted into normal mice, confirming the relationship between intestinal flora and the incidence of MAFLD.A meta-analysis of MAFLD[34] showed elevated levels of Escherichia, Prevotella, and Streptococcus in PATIENTS with MAFLD, while reduced levels of Faecalis, Faecalis, and ruminococcus.Escherichia coli rich in MAFLD patients can synthesize ethanol, increase intestinal mucosal permeability, induce oxidative stress, and promote the occurrence of MAFLD.

Current interventions in the management of MAFLD focus on medication management to control lipid and glucose levels.Multiple animal-based studies[35, 36] have shown that the use of probiotics can prevent liver steatosis, improve ste-atohepatitis and fibrosis.The mechanisms behind these protective effects may be to reduce hepatic lipid accumulation, reduce endotoxemia, and avoid the activation of anti-inflammatory pathways by regulating inflammatory factors.Meta- analysis of clinical studies[37] also showed that probiotic therapy reduced liver transaminase, total cholesterol,TNF-α and improved insulin resistance in PATIENTS with MAFLD.

4.Intestinal flora and viral hepatitis B

Viral hepatitis B is a liver disease caused by hepatitis B virus(HBV) infecti-on.During the progression of HBV infection to chronic hepatitis B (CHB).int-estinal flor-a changes to varying degrees.Lu,et al.[38] by gene sequencing of HBV infec-ted droppings found that the main microbes in the detection,compared with healthy controls, asymptomatic carriers, patients with CHB, decompensated liver cirrhosis patients with HBV intestinal bifidobacterium,lactobacillus, etc beneficial intestinal flora population decline,dung enterococcus, harmful gut bacteria such as e.coli number increase, The percentage of decrease or increase varied with the degree of liver injury, and the diversity of intestinal flora decreased significantly.HBV alters the composition of intestinal flora in patients and increases the colonization of opportunistic pathogens,and this change in intestinal flora is associated with the progression of viral hepatitis type B.Chou et al.[39] conducted serological tests on mice modeled with HBV infection and found that adult mice with mature intestinal flora could clear HBV in a short term, but young mice with immature intestinal flora and adult mice cleared with antibiotics were prone to develop chronic infection after infection with HBV.

The regulation of intestinal flora has an obvious auxiliary effect on the treatment of HBV.Studies[40, 41] confirmed that on the basis of conventional treatme-nt, combined probiotics can improve the relative contents of enterococcus, bifidobacteria and lactobacillus to improve the imbalance of intestinal flora, reduce the content of serum endotoxin and inflammation, and improve the liver function of patients.A Korean study[42] found that cell extract SPM0212 of bifidobacterium youth limited HBV growth and HBsAg secretion in HepG2 cells by 50% by inhibiting HBV surface antigen (HBsAg)gene expression at the transcriptional level.In recent years, the treatment of hepatitis B by fecal bacteria transplantation is a hot research topic.Small sample studies[43] have shown that fecal bacteria transplantation can induce HBeAg clearance in hBeAgpositive patients with CHB.

5.Intestinal flora and cirrhosis

Academician Li Lanjuan’s research team[44] collected stool samples from 181 patients with cirrhosis and conducted intestinal microflora sequencing and analy-sis using metagenomic research methods.Other related studies also suggest that the number of enterobacter is closely related to cirrhosis.Studies[45] have shown that with the progression of cirrhosis, the proportion of Proteus and Clostridium increases significantly, and the diversity of intestinal flora decreases, proving that cirrhosis can cause changes in intestinal flora.Chinese scholar Tang Yuan-lin et al[46] found that there was imbalance of bacterial flora in patients with cirrhosis,which was manifested as the number of enterobacter, enterococcus,staphylococcus and yeast significantly increased, while the number of Bacteroides, bifidobacteria, Lactobacillus and Clostridium significantly decreased.The number of enterobacter was positively correlated with the severity and poor prognosis of cirrhosis, while the number of lactobacillus and Clostridium was negatively correlated with the condition and prognosis.Foreign studies[47] also showed that the incidence of SIBO in patients with cirrhosis was closely related to child-pugh grade, and the incidence of SIBO increased significantly with the increase of child-pugh grade of liver function.It has been proved that regulating intestinal flora can treat the complications of cirrhosis.At present, the use of antibiotics,probiotics and other measures to regulate intestinal flora is a common method to prevent and treat decompensated cirrhosis complications, fecal group transplantation therapy is a new treatment plan.Take the common complications of Hepatic encephalopathy(HE) and Spontaneous bacterial peritonitis (SBP) for example:

HE is an end-stage manifestation of various liver diseases.Studies have proved[48] that in cirrhosis, intestinal Gram-negative enterobacter significantly increases, which produces urease and leads to elevated blood ammonia, and easily causes HE.At present,there is no specific drug for the treatment of HE, and the principle of treatment is to remove the inducement in time and reduce the generation and absorption of intestinal toxic substances.Regulating intestinal microflora imbalance has obvious curative effect on the prevention and treatment of HE in liver cirrhosis.A meta-analysis by Zhe Zhang et al[49] showed that probiotics combined with lactudrop in HE patients can more effectively reduce blood ammonia and improve liver function compared with lactudrop alone.Yu et al.[50] found that rifaximin can maintain the overall composition and diversity of intestinal flora and reduce hyperammonemia in HE patients with cirrhosis.Meanwhile, faecal transplantation therapy has also made encouraging progress.Bajaj et al.[51] found in the experiment that compared with traditional antibiotic treatment, the cognitive function of HE patients treated with faecal transplantation was significantly improved, and the hospitalization rate, mortality rate and infection rate were significantly reduced.

SBP is a common complication in patients with decompensated cirrhosis.According to literature[52], the incidence of SBP in patients with decompensated cirrhosis within 1 year is about 20%, and the short-term mortality after SBP is about 15% to 40%.

Decompensated cirrhosis patients have intestinal microflora imbalance, increased intestinal mucosal permeability, decreased immunity, resulting in intestinal bacterial translocation, resulting in infection.A study[53] reported that nearly 50% of the ascites of patients with alcoholic cirrhosis contained bacterial DNA, and lactobacillus, chlamydia and Enterobacter were found among the bacteria measured.In clinical practice, SBP patients are often treated with antibiotics to inhibit intestinal microflora disorder and effectively reduce the recurrence rate.Logna et al.[54] conducted a meta-analysis of antibiotic treatment for SBP in patients with decompensated cirrhosis and found that about 75% of patients improved, but there is still a great deal of uncertainty about which antibiotic treatment is better.Liu YULing et al.[55] found that compared with patients treated with conventional antibiotics,patients treated with probiotics for cirrhosis complicated with SBP had a higher ascites regression rate, and inflammatory factors such as TNF-α, CRP and PCT decreased significantly.

6.Intestinal flora and liver cancer

Hepatocellular carcinoma (HCC) is a malignant epithelial tumor originating from liver.The morbidity and mortality of HCC are high in the world.Lei et al.[56] and Ren et al[57] found that intestinal flora of HCC patients was mainly enterobacter and lactic acid bacteria,and the proportion of Firmicute and Bacteroidetes in HCC patients decreased significantly compared with healthy controls.There are many risk factors for THE occurrence of HCC, including viral infection, alcoholism, non-alcoholic fatty liver disease, aflatoxins,etc., which can stimulate the imbalance of intestinal flora, increase intestinal permeability, induce endotoxemia, and induce synthesis of prostaglandin E2 PGE2 by hepatic stellate cells, thereby reducing the anti-tumor activity of CD8+ lymphocytes[58].Intestinal bacterial metabolites lipoteichoic acid (LTA) and secondary bile acid deoxycholic acid (DCA) can increase the expression of cyclooxygenase 2 (COX-2) and senescence related secretion phenotype (SASP) in hepatic stellate cells.Increased expression of COX-2 and SASP can promote the occurrence of liver infla-mmation and up-regulate the expression of growthregulated oncogene -α [59].Regulation of intestinal flora has the potential of adjuvant therapy for HC-C.By promoting the growth of beneficial bacteria, antiinflammatory metabolites can be produced to relieve the liver oxidative stress response in HCC, thereby alleviating liver injury.Li et al.[60] found that probiotics can inhibit tumor growth in HCC mouse models.In this model, mice were injected with tumor cells subcutaneously and continuously fed a novel probiotic diet containing Prohep, and tumor growth was significantly inhibited in mice fed Prohep compared to a control group (normal diet).The size and weight of the tumor decreased by about 40 percent, the area of blood vessels decreased by 52 percent, and the buds of blood vessels decreased by 54 percent.They suggest that probiotics inhibit the recruitment of T-cofactor 17(Th17) cells from the gut and peripheral blood to the tumor site, which leads to a decrease in angiogenic interleukin (IL-17) and ultimately inhibits angiogenesis and tumor growth.

7.Conclusion

Recent studies at home and abroad show that intestinal flora and liver diseases interact.Regulating the relative content of intestinal flora is helpful to delay the development of liver disease and control the occurrence of related complic-ations.Studies have proved that the use of antibiotics, probiotics, fecal group transplantation and other measures or methods to regulate the unblocked flora, the treatment of alcoholic liver disease, MAFLD has obvious efficacy.It has significant preventive and therapeutic effect on HE and SBP of cirrhosis com-plications.The therapeutic effect on autoimmune liver disease, HBV and HCC remains to be further confirmed.Of course, as an emerging treatment method for liver disease, it is not a panacea.It needs to be further verified through more rigorous and thorough experimental design and multi-center, large-sample randomized controlled clinical studies, so as to find valuable biomarkers and find potential treatment directions for effective prevention and treatment of liv-er diseases.

Author’s Contribution

Zhao Jingfang, the first author, mainly carried out literature search,article writing and article revision; You Jing is the corresponding author, mainly responsible for the overall control of articles, article content review, etc.

Description of Conflict of interest

No potential conflict of interest was found.