Advances in Research of Gut Microbiota and Nonalcoholic Fatty Liver Disease

Jing YANG, Weibo DAI, Wenxia ZHANG, Xiwen ZHONG

Zhongshan Hospital of Traditional Chinese Medicine, Affiliated to Guangzhou University of Chinese Medicine, Zhongshan 528400, China

Abstract The "gut-liver axis" theory is the theoretical basis for the influence of gut microbiota on the occurrence and development of chronic liver diseases, and it mainly elaborates the relationship between the gut and chronic liver diseases. With the emergence of high-throughput sequencing metagenomic technology, it is feasible to conduct in-depth research on the diversity and population structure of gut microbes in nonalcoholic fatty liver disease (NAFLD), so as to provide more ideas for the research on the pathogenesis and therapeutic targets of NAFLD. This paper reviewed the bidirectional effects of gut microbiota and NAFLD. In addition, it summarized the relevant content of regulating gut microbiota in the treatment of NAFLD, in the hope of providing more reference for the treatment of NAFLD from the perspective of gut microbiota.

Key words Gut microbiota, Nonalcoholic fatty liver disease (NAFLD), Review

1 Introduction

Nonalcoholic fatty liver disease (NAFLD) refers to a series of symptoms caused by the accumulation of more than 5% fat in liver cells, including nonalcoholic simple steatosis and nonalcoholic steatohepatitis (elevated serum alanine aminotransferase and aspartate aminotransferase), and it is a hepatic manifestation of disorders of lipid and glucose metabolism[1-2]. In recent years, NAFLD has gradually become a serious public health concern with a global incidence of about 25%-45%[3], and has become the leading cause of liver transplantation and liver cancer[4]. The pathogenesis of NAFLD is complex. At present, it is generally believed that genetics, metabolic abnormalities, environmental factors, obesity, insulin resistance, dietary intake, hormone secretion and other pathogenic factors jointly promote the occurrence and development of NAFLD[5]. In terms of treatment strategies, there is currently no specific drug for NAFLD. Studies have shown that a 10% weight loss can contribute to the alleviation of the NAFLD[6]. Other treatment methods include: improving gut microbial diversity and abundance (antibiotic treatment and probiotic treatment), targeting the "gut-liver" axis, targeting bile acid metabolism, targeting gut hormones, adsorption and fecal microbiome transplantation[7]. The gut is the place where many microorganisms live, including bacteria, archaea, eukaryotes, fungi and viruses, which are collectively referred to as gut microbes[8]. In a healthy organism, two types of bacteria, Firmicutes and Bacteroidetes, dominate the gut. In addition, there are other relatively small numbers of Actinobacteria, Proteobacteria, Verrucomicrobia and Fusobacteria[9]. The composition of the gut microbiota is often regulated by host genes and the environment. Naturally born babies inherit about 40% of their mother’s gut flora during delivery. After birth, differences in diet are the main cause of differences in gut microbiota. With increasing age, gender, race, geographical environment, immune system, antibiotic use, body mass index (BMI),etc.can become factors influencing the gut flora[10]. Under normal circumstances, the gut flora is in a stable state, including the composition, function and metabolites of the gut flora, as well as the stability of the intestinal physical and immune barriers. Gut microbiota compete for various resources (such as various carbon resources, trace metals, vitamins) by evolving unique nutrients, using pathways or by adhering to the surface of intestinal epithelial cells, thereby keeping a competitive advantage[11]. By secreting antibacterial components and inhibitory metabolites, gut microbiota resists the colonization, growth and invasion of gut epithelial cells by pathogens and maintains the homeostasis of the entire gut microbiota and gut health. Furthermore, the gut microbiota can also absorb nutrients from carbohydrates that cannot be digested by the host to generate vitamins, which has a profound impact on maintaining human health[9].

2 The "gut-liver axis" theory

The "gut-liver axis" theory proposed by Marshall in 1998 mainly elaborates the relationship between the gut and the occurrence of chronic liver diseases. The portal circulation is the anatomical basis of the "gut-liver axis" theory. On the basis of the portal circulation, metabolic, immune and neuroendocrine interactions between the gut microbiota and the liver are carried out[12]. An indication of dysregulation of the "gut-liver" axis is abnormal bile acid metabolism. Bile acid metabolism is an important bridge between the liver and the gut. Under normal circumstances, bile acids are secreted by the liver and excreted from the common bile duct into the intestinal lumen to maintain the pH in the intestinal tract and ensure the homeostasis of gut microbiome. Disorder of bile acid metabolism will lead to gut dysbiosis, excessive proliferation of enterogenic pathogens, and damage to the gut barrier function, allowing Gram-negative bacteria and their products, lipopolysaccharides, to enter the liver through the portal vein, causing liver inflammation[13]. Another hallmark of disorder of the "gut-liver" axis is disruption of the gut barrier. Once the gut barrier is disrupted, gut permeability increases, and microorganisms and microbial products are transplanted into the liver through the portal circulation, accordingly triggering liver disease. Lipopolysaccharide (LPS), a component of the cell wall of Gram-negative bacteria, is the most studied pathogenic signal of gut origin. LPS is sensed by TLR4 receptors on hepatic Kupffer cells and hepatic stellate cells by binding to the co-receptors CD14 or MD-2. The MyD88-dependent TLR4 signaling pathway ultimately leads to NF-κB activation, phosphorylation, nuclear translocation and activation of pro-inflammatory molecular genes, resulting in a series of inflammations. In addition to causing liver cell damage, it also accelerates the process of liver fibrosis[14].

3 Gut microbiota in NAFLD

3.1 Effects of NAFLD on gut microbiotaAccording to a report issued by Wiggetal.[15], NAFLD patients had a higher prevalence of small intestinal bacterial overgrowth (SIBO) than healthy subjects. Through research, Mieleetal.believed that the increased prevalence of SIBO was associated with increased intestinal permeability in NAFLD patients. They detected that the level of endotoxin in peripheral serum of NAFLD patients was higher than that of healthy controls. In addition to serum endotoxin levels, Boursieretal.[16]conducted a multivariate analysis of the gut microbiota of NAFLD patients, and the results showed that the abundance of Bacteroidetes was positively correlated with nonalcoholic steatohepatitis. In a related study of children with nonalcoholic hepatitis, Zhu Lixinetal.[17]found that compared with healthy controls, children with obesity or NAFLD had higher numbers of Prevotella and Bacteroides in the gut bacteria. These indicate that the gut microbiota of NAFLD patients has changed, and the reasons for the changes in the gut microbiota may be related to the following two aspects.

3.1.1Changes in bile acid metabolism. The physiological functions of bile acids include promoting the digestion and absorption of fat, regulating energy balance, and bile acids are mainly synthesized by the liver. The effects of bile acids and gut microbiota are bidirectional. Bile salt hydrolases (BSHs) produced by Bacteroides, Bifidobacterium, Clostridium and other bacteria can decompose bile acids, the decomposed primary bile acids are processed into secondary bile acids by 7α-dehydrogenase, and 7α-dehydrogenase is mainly synthesized by Clostridium and Eubacterium in Firmicutes[18]. Besides, the gut microbiota also participates in various reactions such as esterification, desulfurization, and oxidation/epimerization in bile acid metabolism, and plays an important role in the enterohepatic circulation of bile acids[19]. However, in NAFLD patients, due to the decline in the synthesis capacity of the liver, the bile acids excreted into the gut also decrease, and the number of gut microbiota involved in bile acid metabolism also decreases accordingly, resulting in changes in the composition of the gut microbiota. Furthermore, bile acids also play an important role in maintaining the diversity of gut microbiota. Bile acids can inhibit the growth of certain types of bacteria while promoting the growth of certain types of bacteria, and the secretion of bile acids in NAFLD patients is reduced, resulting in a decline in the diversity of gut microbiota.

3.1.2Short-chain fatty acids. Short-chain fatty acids are metabolites of gut microbiota, including acetate, propionate, and butyrate,etc.Many studies have proved their anti-inflammatory properties and their ability to modulate host inflammatory responses, and butyrate is most effective[20-21]. The main mechanism of butyrate anti-inflammatory effect is that butyrate can regulate tight junction function and maintain gut integrity, and provide an energy source for colon cells, while activating regulatory T cells in the gut, thereby inhibiting Th17 cells and T cells, ultimately reducing pro-inflammatory signaling pathways[22]. For NAFLD patients, the decreased diversity of the gut microbiota leads to a decline in the number of butyrate-producing bacteria, and the decreased butyrate leads to an increase in gut permeability and an increased risk of bacteria and LPS entering the systemic circulation[6].

Based on the above findings, more and more researchers hope to develop gut microbial markers for detecting liver fibrosis by finding the association between specific microbiota and NAFLD.

3.2 Effects of gut microbiota on NAFLD

3.2.1Lipopolysaccharide (LPS). LPS is the major outer membrane component of Gram-negative bacteria, also known as endotoxin. It can activate the host’s innate immune system. A study by Harteetal.[23]indicated that in NAFLD patients, serum LPS levels were elevated, which was mainly related to the increased gut permeability of patients and the entry of gut bacteria into the blood circulation. LPS can activate Toll-like receptor 4 distributed on hepatocytes, Kupffer cells and stellate cells, leading to the massive secretion of inflammatory factors IL-6, IL-1 and TNF-α, which aggravates liver damage in NAFLD patients[24].

3.2.2Bacterial DNA. Bacterial DNA can directly activate a variety of immune cells including macrophages, NK cells, B cells and dendritic cells, and can enter endolysosome through endocytosis, activate Toll-like receptor 9 (TLR9), which can activate the NF-κB/MAPK pathway and secrete inflammatory factors such as IL-12 and TNF-α, which aggravate the hepatic inflammatory response in NAFLD patients[25-26].

3.2.3Indole. Prevotella and Bacteroides can degrade tryptophan to indole by tryptophanase[27]. Indole can enhance tight junctions in epithelial cells and reduce intestinal inflammation and damage[28]. Beaumontetal.[29]demonstrated that indole-treated mice were resistant to LPS-induced liver inflammation and altered cholesterol metabolism. When gut microbiota is dysregulated, indole levels in the gut are reduced, which will accelerate the NAFLD progression.

3.2.4Abnormal glucose metabolism. The disorder of gut microbiota can cause abnormal glucose metabolism in the body. The gut microbiota can absorb more energy from food into the body, and can also metabolize polysaccharides that cannot be digested by the human body into monosaccharides and short-chain fatty acids, and activate the carbohydrate response element binding protein (ChREBP) in the liver to promote the uptake of fatty acids in the liver, resulting in the accumulation of fat in the liver[30].

3.2.5Short-chain fatty acids. Short-chain fatty acids can act on and promote the expression of G protein-coupled receptors in the gut and adipose tissue. These receptors can reduce intestinal peristalsis and allow the body to absorb more fatty acids. They also inhibit the breakdown of fat cells, activate intestinal neutrophils, and alter gut permeability, thereby promoting the progression of NAFLD. Acetic acid is the most easily absorbed short-chain fatty acid and a substrate for the synthesis of fat and cholesterol in the liver and adipose tissue[31]. When gut microbiota is dysregulated, production of butyrate and propionate decreases and production of acetic acid increases, leading to increased production of fat and cholesterol, accordingly accelerating the progression of NAFLD.

3.2.6Ethanol. Under normal circumstances, gut microbiota can convert pyruvate produced during glycolysis into acetaldehyde, which is further metabolized to form ethanol. It can influence the gut barrier function, increase gut permeability to bacterial toxins, and induce TLR-dependent signaling cascades in the liver[32]. Changes in the structure and quantity of Proteobacteria, Enterobacteriaceae, and Escherichia coli may be involved in the amount of endogenous alcohol production[33]. Increased ethanol and acetaldehyde can directly damage liver tissue, and by initiating related enzyme activities in the body, lead to increased production of oxygen free radicals and lipid peroxidation in the body, and aggravate liver inflammation.

3.3 Gut microbiota and the treatment of NAFLD

3.3.1Probiotics. The most commonly used probiotics come from the genera Lactobacillus and Bifidobacterium. In recent years, flora related to butyrate production has also received great attention. Through animal experiments, Okuboetal.[34]proved that oral administration of Lactobacillus can increase the content of lactic acid in the intestinal tract of NAFLD mice, reduce the level of serum LPS, and recall various liver indicators (ALT, AST,etc.) changed by NAFLD. Wagnerbergeretal.[35]also confirmed that Lactobacillus can improve liver steatosis and reduce serum ALT levels mainly by inhibiting TIR4 signaling pathway and up-regulating PPARγ. Lactobacillus rhamnosus GG (LGG) has been confirmed to strengthen the gut barrier function and reduce the level of LPS entering the systemic circulation due to increased gut permeability. It can inhibit the NF-κB signaling pathway to reduce intestinal inflammation, but also reduce the mRNA levels of TNF-α, IL-8R, IL-1β and other inflammatory factors, and reduce the accumulation of fat in liver cells[36].

3.3.2Prebiotics. Prebiotics refer to some substances that cannot be digested by the body, but can be fermented and utilized by certain gut microbiota, thereby promoting gut health and homeostasis, such as fructooligosaccharides. Fructooligosaccharides are present in many fruits and vegetables and cannot be hydrolyzed by human digestive enzymes. Takaietal.[37]found that fructooligosaccharides can improve the pathological changes of liver steatosis, inflammatory cell infiltration and hepatocyte ballooning in NAFLD model mice, and can also reduce the expression of fatty acid synthase and glycerol-3-phosphate acyltransferas.

3.3.3Synbiotics. Synbiotics refer to the combined use of probiotics and prebiotics to maximize the effectiveness of both. In an NAFLD rat experiment using a high-sugar diet model, Aguayoetal.[38]administered a synbiotic formula composed of Lactobacillus CECT5716 and fructooligosaccharides to rats, and found that liver steatosis and insulin resistance in rats were significantly improved. Alvesetal.[39]found that synbiotics improved hepatic steatosis mainly by upregulating PPARα to increase lipid β oxidation while inhibiting lipid production.

3.3.4Fecal microbiome transplantation (FMT). Compared with probiotics and prebiotics, FMT can provide a greater variety of beneficial gut microbes more quickly, thereby promoting gut homeostasis and health more effectively. Through experiment, Zhou Daetal.[40]demonstrated that compared with the model group, the serum inflammatory factor IL-17 level was decreased in the mice receiving fecal transplantation, while the level of serum inflammatory factor IL-17 was decreased, while the level of serum anti-inflammatory factor IL-4 and IL-22 was increased, and the hepatic steatosis induced by high-fat diet was also lower than that in the model group.

3.3.5Butyrate. Butyrate is a metabolite of gut microbiota. The level of glucagon-like peptide-1 receptor (GLP-1R) in the liver of NAFLD patients is lower than that of healthy subjects, while butyrate can up-regulate the expression level of GLP-1R. Relevant animal experiments also showed that administration of sodium butyrate to NAFLD model mice increased the expression of GLP-1R in the liver of mice, and ultimately reduced hepatic steatosis[41].

3.3.6Antibiotics. Antibiotics are used to treat NAFLD patients based on gut disturbances in NAFLD patients to reduce the health effects of overgrown gut bacteria and their metabolites. There are two main types of commonly used antibiotics. One type is absorbable antibiotics, which can directly enter the blood through the gut barrier to kill pathogens that have left the intestinal tract, such as metronidazole; the other type is non-absorbable antibiotics, which cannot pass through the gut barrier and are confined to the intestine to eliminate excessively proliferating gut microbes, such as polymyxin B,etc.[42-44]. Wu Wanchunetal.[45]applied cidomycin to NAFLD mice and found that cidomycin could reduce serum ALT, AST and TNF-α levels. Gangarapuetal.[46]demonstrated that short-term administration of rifaximin can reduce serum ALT and LPS levels in NAFLD patients and improve the clinical status of patients. Although the above studies have proved the effectiveness of antibiotics in NAFLD, long-term use of antibiotics will bring many adverse reactions, especially in patients with low immunity. Therefore, it is necessary to make effort to avoid the increased risk of bacteremia due to antibiotics.

4 Traditional Chinese medicines participating in the treatment of NAFLD by regulating the gut microbiota

Many scholars have found that traditional Chinese medicine can treat NAFLD by regulating the gut microbiota. Song Lietal.[47]found that the Hand-Foot-Twelve-Needle combined with Jiawei Yinchenhao Decoction can improve the levels of ALT, AST and blood lipids in NAFLD patients by increasing the levels of Lactobacillus and Aerobic bacteria, and accordingly reducing the levels of Enterococcus and Escherichia. Fang Jingetal.[48]found that the proportion of pathogenic bacteria such as Firmicutes, Desulfovibrionaceae, and Desulfovibrio in the intestine of rats with NAFLD induced by high-fat diet were increased to varying degrees, while Dahuang Zexie Decoction can reduce the proportion of these pathogenic bacteria, and at the same time increase the proportion of beneficial bacteria such as Bacteroidetesother, Ruminococcaceae, Oscillibacter,etc., and reduce the level of LPS in the systemic circulation, and alleviate the liver inflammation. Qiu Bangdongetal.[49]found that compared with NAFLD model rats, Biejiajian Pills can reduce the gut permeability of rats, reduce the number of Bacteroides in the gut increase the number of Lactobacillus, and reduce serum ALT, AST, and ALP, TC, LDL-C and other indicators, improve inflammatory cell infiltration.

5 Discussion

The anatomical and physiological connection between the gut microbiota and the liver determines their interdependence. They are highly integrated with a mutual stake. With the advent of high-throughput sequencing metagenomic technology, the relationship between gut microbiota and NAFLD has gradually been revealed. More and more researchers have found that the relationship between gut microbiota and NAFLD is bidirectional. For NAFLD patients, changes in bile acid metabolism can reduce the diversity of gut microbiota, and the reduced diversity of gut microbiota further changes the composition of bacterial metabolites in the intestinal tract, resulting in intestinal ecological imbalance. Unbalanced gut bacteria can damage the intestinal epithelial barrier and shift the gut microbiota through various pathogenic factors and metabolites such as lipopolysaccharide, bacterial DNA, indole, abnormal glucose metabolism, short-chain fatty acids, and ethanol, and further aggravates the progression of NAFLD. In terms of treatment, a variety of treatment strategies related to gut microbiota have also been developed, such as probiotics, prebiotics, fecal transplantation, antibiotics,etc.Traditional Chinese medicines have also shown considerable advantages in the research on the treatment of NAFLD from the perspective of gut microbiota. The main mechanism is to improve the diversity of gut microbiota and increase the abundance of beneficial bacteria. However, there is still a lack of effective drugs for the treatment of NAFLD. Although the study of gut microbiota can provide more ideas, it has not solved the root cause of abnormal glucose and lipid metabolism in NAFLD patients. Therefore, it is necessary to take gut microbiota therapy as an adjuvant treatment method, and to conduct in-depth study and obtain more clinical support data.