Mechanistic and therapeutic advances in non-alcoholic fatty liver disease by targeting the gut microbiota

Abstract

Non-alcoholic fatty liver disease (NAFLD) is one of the most common metabolic diseases currently in the context of obesity worldwide, which contains a spectrum of chronic liver diseases, including hepatic steatosis, non-alcoholic steatohepatitis and hepatic carcinoma. In addition to the classical “Two-hit” theory, NAFLD has been recognized as a typical gut microbiota-related disease because of the intricate role of gut microbiota in maintaining human health and disease formation. Moreover, gut microbiota is even regarded as a “metabolic organ” that play complementary roles to that of liver in many aspects. The mechanisms underlying gut microbiota-mediated development of NAFLD include modulation of host energy metabolism, insulin sensitivity, and bile acid and choline metabolism. As a result, gut microbiota have been emerging as a novel therapeutic target for NAFLD by manipulating it in various ways, including probiotics, prebiotics, synbiotics, antibiotics, fecal microbiota transplantation, and herbal components. In this review, we summarized the most recent advances in gut microbiota-mediated mechanisms, as well as gut microbiota-targeted therapies on NAFLD.

Keywords

gut microbiota / NAFLD / obesity / insulin resistance / bile acids / probiotic

Introduction

Gut microbiota serves as a “metabolic organ” and comprise about 1000 species of bacteria with a total weight of approximately 1−2 kg; this value may be much higher according to a recent report [¹,²]. Gut microbiota provides the host with an extensive set of metabolic functions in both health and disease, and approximately 150 times more genes than that present in the human gene complement [³]. Firmicutes and Bacteroidetes are the most important phyla in intestinal bacteria, with a proportion of over 90%; moreover, they play significant roles in nutrient absorption [⁴]. Gut microbiota seem to have the potential capacity of affecting the host metabolic balance through pathophysiological factors [⁵]. In many studies, alterations in the gut microbiota were reflected in metabolic disorders, which involved varieties of metabolic diseases, such as type 2 diabetes (T2D) [⁶], obesity [⁷,⁸], cardiovascular disease [⁹], and non-alcoholic fatty liver disease (NAFLD) [¹⁰,¹¹]. In addition, a meta-analysis has found that gut microbiota may influence the metabolic effects that are relevant to obesity; however, in this report, the relationship between microbiota and obesity was weak, and the individual differences and research lack of sufficient samples [¹²].

The prevalence of NAFLD is increasing worldwide and NAFLD is now being increasingly recognized as a severe threat to health [¹³]. NAFLD is a complex disease with perturbed metabolic processes associated with powerful relationships between environmental and genetic risk factors [¹⁴], and its pathological features are associated with inflammation, hepatocyte injury, steatosis and fibrosis [¹⁵]. About 80 years ago, Hoefert et al. [¹⁶] observed significant changes in the intestinal bacteria of individuals diagnosed with chronic liver disease and identified links between the gut microbiota and the liver. In recent years, evidence has supported the role of the gut–liver axis in the development of NAFLD [¹⁷]. Moreover, the classic “Two-hit hypothesis” theory of NAFLD has become widely recognized [¹⁸]. Furthermore, the gut microbiota plays a crucial role in promoting the processes underlying NAFLD through modification of inflammation, insulin-resistance, bile acids, and energy and choline metabolism. Based on these findings, a novel therapeutic target, intestinal flora, was suggested. The most commonly used methods to manipulate intestinal bacteria include the administration of pre-probiotics and synbiotics. Recently, some herbal medicines and naturally active ingredients have been found to be effective in treating NAFLD through gut microbiota.

In this present review, we describe the current understanding of the mechanistic roles played by the gut microbiota in NAFLD. Further, we briefly summarize the novel gut microbiota-targeted therapy in clinical and experimental studies.

Mechanistic advances in gut microbial-mediated NAFLD development

Gut microbiota regulates energy metabolism of the hosts

Intestinal microbiota have vital actions in the modulation of the energy metabolism of the host, which may contribute to augmentation of obesity and metabolic diseases related to obesity [¹⁹,²⁰]. Obesity is strongly linked with diabetes, particularly T2D, [²¹,²²] and liver diseases [²³–²⁷]. Gut microbiota hosts energy absorption involved in the modulation of signaling mechanisms and the production of microbial metabolites.

Indeed, intestinal flora may have a major impact on sugar and lipid metabolism, leading to excess nutrition of the body, which further leads to obesity. Alterations in human gut microbiota are linked to host energy metabolism [²⁸]. Ley et al. [¹⁹] reported that the proportion of Bacteroidetes is lower in obese people compared with in lean people, and that the restoration of Bacteroidetes population is correlated with weight lost. In 2004, Backhed et al. [²⁹] demonstrated that conventionalization of germ-free (GF) mice with a population of microbiota obtained from normally raised animals led to increased insulin resistance and total body fat content. These findings suggested that microbiota can promote the absorption of monosaccharides from the gut, thereby triggering de novo lipogenesis in the liver. Fasting-induced adipose factor (FIAF), a powerful metabolism and adiposity regulator belonging to the angiopoietin-like protein family, was selectively downregulated in intestinal epithelium of normal mice by conventionalization. Thus, the suppression of FIAF secretion is closely associated with intestinal dysbiosis [³⁰]. Moreover, blockade of the actions of angiopoietin-related protein 4 leads to the continuous expression of lipoprotein lipase, which is responsible for the secretion of triglycerides (TG) from chylomicrons and very low-density lipoprotein, eventually resulting in the augmented uptake of fatty acids and accumulation of TG in the adipocytes [³¹].

In addition, the production of microbial metabolites can, to some extent, contribute to the host energy harvest [³²]. Polysaccharides are converted by bacteria into monosaccharides and short chain fatty acids (SCFAs), including acetate, propionate, and butyrate [³³]. As an energy precursor, SCFAs are implicated in the formation and development of NAFLD, which will accelerate the host energy harvest in the gut [³⁴,³⁵]. SCFAs affect the G protein-coupled receptors GPR41 and GPR43, thus triggering the release of glucagon-like peptide 1 (GLP-1) and peptide YY from neuroendocrine L cells [³⁶]. In a study, the administration of VSL#3 enhanced the release of GLP-1 and resulted in reduced food intake and improved glucose tolerance [³⁷]. Moreover, when energy delivery was increased in the form of SCFAs, adenosine monophosphate-activated protein kinase activity was inhibited in the liver, which in turn increased hepatic free fatty acid accumulation by decreasing b-oxidation [³⁸]. Considerable knowledge has been acquired regarding the mechanisms and roles of gut microbiota in regulating the energy metabolism of the host; however, there is more to learn.

Gut microbiota regulates insulin-sensitivity

Disorders on insulin regulation are intimately connected with metabolic syndrome, with insulin resistance thought to be an important initiating factor in the deleterious effects of obesity and play vital roles in the underlying processes leading to the pathogenesis of NAFLD [³⁹,⁴⁰]. Moreover, insulin resistance may enhance fat accumulation and promote hepatocyte injury, following the triggering of inflammatory reactions [⁴¹]. Perry et al. [⁴²] found that high-fat diets induce acetate production, thereby triggering gut microbiota–brain–b-cell axis positive feedback and increasing ghrelin secretion and energy storage. This result indicated that gut-derived acetate causes insulin resistance.

The intestinal barrier is a complicated structure consisting of gut epithelial cells connected through intestinal mucus layers that covers the intestinal epithelial surface. Many antimicrobial peptides are produced by paneth cells, which are important components of the mechanical barrier of the intestinal mucosa. Small intestinal bacterial overgrowth (SIBO) is characterized by excessive Gram-negative bacteria production in the proximal region of the small bowel [⁴³]. In a study, researchers investigated the effects of SIBO in the pathogenesis of non-alcoholic steatohepatitis (NASH) in rats and found that the levels of tumor-necrosis factor a (TNF-a), alanine aminotransferase (ALT), and serum aspartate aminotransferase (AST) were significantly reduced after cidomycin treatment [⁴⁴]. Moreover, the prevalence of SIBO was investigated in NASH patients, and higher prevalence of SIBO and TNF-a levels were found compared with those in control subjects [⁴⁵]. Cani et al. [⁴⁶] reported that disorders of the gut microbiota inhibits the expression of specific tight junction proteins, namely, occludin and ZO-1, thereby leading to increased intestinal permeability. In 2007, scientists concluded that genetically obese mice displayed increased gut permeability, which led to enhanced portal endotoxemia that rendered hepatic stellate cells more sensitive to the actions of bacterial endotoxins [⁴⁷]. In patients with NAFLD, the increase in intestinal permeability was five times more likely to occur compared with in the controls [⁴⁸]. Similarly, Kessoku et al. [⁴⁹] found that the mechanism of fibrotic progression via endotoxin that is involved in NAFLD may strongly be associated with gut permeability. Lipopolysaccharide (LPS), also known as endotoxin, and an abundant component of the outer cell membrane of Gram-negative bacteria will enter the blood stream when the gut barrier is damaged or destroyed, where it combines with LPS-binding protein (LBP) [⁵⁰]. LPS has direct effects on hepatic stellate and Kupffer cells to push steatohepatitis through the fibrosis condition [⁵¹]. Toll-like receptor 4 (TLR4) is a pattern recognition receptor that acts as an LPS sensor and has important functions in modulating the innate immunity response and the development of insulin resistance [⁵²,⁵³]. Gut-derived endotoxemia induces the development of insulin resistance, especially by its action on the LPS-TLR4-monocyte differentiation on antigen CD14 system, which activates the Jun N-terminal kinase (JNK) and the nuclear factor-kB (NF-kB) pathway [⁵⁴]. This activation induces inflammasomes and proinflammatory cytokines (TNF-a, interleukin-6) [⁵⁵]. In patients with obesity and T2D, the administration of subcutaneous adipocytes with LPS markedly increased IL-6 and TNF-a secretion [⁵⁶]. In obese animal models of NAFLD, the administration of either probiotic or anti-TNF antibodies may improve NAFLD. Thus, endogenous signals induced by intestinal bacteria might play a vital role in biochemical processes underlying hepatic insulin resistance [⁵⁷]. Furthermore, TLR4 activates several pro-inflammatory kinases including IKK, JNK, and p38 that impair insulin signal transduction by inhibiting the phosphorylation of the insulin receptor substrate1 that induces NAFLD progression [⁵³,⁵⁸].

In summary, alterations in intestinal bacteria may contribute to the mechanisms underlying insulin resistance, which suggests potential significance for the restoration of the gut barrier integrity and the potential use of gut microbiota manipulation as a novel therapeutic strategy to treat NAFLD [⁵⁹].

Gut microbiota-regulated choline metabolism

Choline is an important constituent of membrane phospholipids, which plays important roles in biochemical processes, including the enterohepatic bile acid cycle and lipid and cholesterol metabolism. It is supplied either through food intake or endogenous synthesis. The liver is responsible for choline metabolism. Choline can promote fat transport in the form of phospholipids produced by the liver, which can prevent abnormal fat accumulation in the liver; hepatocyte death and hepatic steatosis often occur when cells are deprived of choline [⁶⁰,⁶¹]. Intestinal bacteria produce the enzymes flavin monooxygenases 1 and 3 (FMO1 and FMO3) that metabolize dietary choline into toxic dimethylamine and trimethylamine [⁶²]. GF mice are not capable of excreting trimethylamine, suggesting the vital role of gut microbiota in the conversion of choline [⁶³]. These toxic amines are absorbed into the liver to generate trimethylamine-N-oxide (TMAO) that can cause liver inflammation [⁶⁴], eventually leading to choline deficiency.

Genetic analysis has shown that choline metabolism gene set are found widely distributed in three major bacterial phyla (Proteobacteria, Firmicutes, and Actinobacteria) and are components of the human gut microbiota [⁶⁵]. The importance of these bacteria has been demonstrated in recent years by colonizing GF mice with choline-metabolizing bacteria. A clear decrease in the choline serum concentration and the bioavailability of choline was observed [⁶⁶]. Several years ago, Spencer et al. [⁶⁷] investigated how choline deficiency might influence the composition of intestinal bacteria and potential development of fatty liver under choline-deficient conditions. The study showed that the gut microbiota composition changed according to the choline levels in the diet. Variations in the abundance of Erysipelotrichi and Gammaproteo bacteria were directly correlated with changes in liver fat levels. The relationship between choline metabolism and gut microbiota suggested a new perspective for the development of gut microbiota-targeted therapy for NAFLD.

Gut microbiota-regulated bile acid metabolism

The main components of bile are bile acids (BA), which contribute to homeostasis in the small bowel through the regulation of cholesterol metabolism [⁶⁸]. BAs are steroidal molecules synthesized after cholesterol oxidation by a number of enzymes present in hepatocytes. Enterohepatic circulation is closely controlled by nuclear receptor signaling, which controls the capture of BA and various metabolites of steroids synthesized in the liver and secreted into the intestine, where they are reabsorbed into the circulation and then taken up by liver cells [⁶⁹]. Aside from its well-established role in regulating cholesterol homeostasis and lipid absorption through actions on transporters and various metabolic enzymes, BA also function as signaling molecules that modulate several physiological processes [⁷⁰]. A member of the superfamily of nuclear receptors, farnesoid X receptor (FXR), has emerged as a vital modulator that controls various metabolic pathways [⁷¹]. BA can activate mitogen-activated protein kinase and FXR-a, which are ligands for the G protein-coupled receptor TGR5 [⁷⁰]. When these downstream signaling pathways are activated, BA controls its own synthesis and the enterohepatic circulation, as well as glucose, triglyceride, and cholesterol homeostasis [⁷²–⁷⁴]. The FXR is abundant in the gut and liver relative to other tissues, and FXR-mediated inhibition of the synthesis of BA requires the matching actions of FXR [⁷⁵].

Gut microbiota plays an important role in metabolizing BA, which transforms primary BA into secondary BA: β-muricholic acid (β-MCA) in mice and lithocholic acid, ursodeoxycholic acid, and deoxycholic acid in humans [⁷⁶,⁷⁷]. Interestingly, BA have antibacterial properties [⁷⁸]. The administration of conjugated BA to cirrhotic rats with ascites increased the secretion of BA, an effect suppressed by SIBO, reduced bacterial translocation, and endotoxemia; additionally, findings of this study suggested that orally administered conjugated BA may minimize endotoxemia and bacterial translocation in cirrhotic patients [⁷⁹]. Studies have shown that BA can promote digestion and absorption of fat-soluble foods and prevent bacterial translocation to preserve the intestinal barrier [⁷⁹,⁸⁰]. Scientists found that mice administered with antibiotics reduced the development of NAFLD and altered the composition of BA and the suppression of signaling by FXR. Furthermore, FXR-deficient mice did not develop diet- and genetic-induced obesity. Therefore, FXR is a potential novel drug target for the treatment of NAFLD [⁸¹]. When the hepatic FXR/SHP pathway is activated, that hepatic lipogenesis is inhibited [⁸²,⁸³]. Thus, the gut microbiota might contribute to liver disease via modifying BA and regulating FXR. The interaction between intestinal bacteria and BA has provided good evidence for gut microbiota-targeted therapy for NAFLD. A schematic view summarizing the mechanisms of gut microbiota on the development of NAFLD is shown in Fig. 1.

Gut microbiota-targeted therapy for NAFLD

Based on the intricate relationship between gut microbiota and NAFLD, gut microbiota-targeted therapy for NAFLD has become a promising approach for NAFLD by using probiotics, prebiotics, fecal microbiota transplantation, antibiotics, and some chemical components from herbal medicine [⁸⁴].

Probiotics

The internationally recognized definition of a probiotic is “a live microbial feed supplement” [⁸⁵,⁸⁶]. These probiotic bacteria are usually present in considerable numbers in the intestinal tract; however, their levels are decreased in pathological circumstances. Thus, administration of probiotics will benefit the host metabolism by improving the intestinal balance of these microbes [⁸⁷,⁸⁸].

The Gram-positive genus bacteria Lactobacillus can transform sugars into lactic acid. It has been tested as a probiotic in several studies [⁸⁹,⁹⁰]. For instance, Okubo et al. [⁹¹] found that the administration of Lactobacillus casei strain Shirota (LcS) orally to NASH mice, induced by a methionine-choline-deficient (MCD) diet, increased the amount of Lactobacillus casei subgroup and other lactic acid bacteria. The development of NASH was evaluated at various levels, and the data revealed that intervention of LcS suppressed NASH markedly, with serum LPS concentrations being reduced and inflammation being suppressed in the liver. In fructose-induced steatosis in a mouse model, fructose consumption produced significant increase in hepatic steatosis and plasma ALT levels, and effects of which were ameliorated by the administration of LcS. These findings indicated that LcS intake inhibited the onset of NAFLD by suppressing the TLR-4 signaling cascade in liver hepatocytes [⁹²]. Mice fed with a high-fat diet (HFD) exhibited higher intestinal permeability and concentration of antibodies specific to LPS in the blood compared with the controls. However, in the L. gasseri SBT2055 (LG2055) treatment group, increases in gut permeability and anti-LPS antibody concentrations were significantly lowered, which may improve the inflammation in adipose tissue [⁹³]. Moreover, L. plantarum probiotics were investigated in NAFLD models, including L. plantarum MA2, L. plantarum A7, and L. plantarum NCU116. The results demonstrated ameliorations in serum lipids [⁹⁴], liver function, and reduction in the accumulation of fat in the liver [⁹⁵,⁹⁶]. In addition, Bifidobacterium (Bif), a member of the Bifidobacteria genera found in the gastrointestinal tract of mammals, has often been used as a probiotic [⁹⁷–⁹⁹]. The administration of Bif significantly improved the accumulation of visceral fat and insulin sensitivity in rats fed with HFD [¹⁰⁰]. Cano et al. [¹⁰¹] evaluated the effects of B. pseudocatenulatum CECT 7765 on the metabolism in obese mice. In mice fed with HFD, the administration of this strain reduced the levels of Enterobacteria and the inflammatory properties of the gut content but increased the levels of Bifidobacteria. In another study, scientists compared the effects of two different strains of probiotics on the progression of NAFLD. According to the data, the B. longum supplement was superior to L. acidophilus in attenuating the accumulation of liver fat [¹⁰²]. VSL#3, a combination therapy consisting of eight types of probiotics, has exhibited therapeutic potential for a number of diseases [¹⁰³–¹⁰⁶]. Yadav et al. demonstrated that VSL#3 reduced body weight gain and insulin resistance by regulating the composition of gut microbiota and facilitated the release of GLP-1, resulting in reduced food intake and improved glucose tolerance. Furthermore, the changes induced by VSL#3 were linked to increased butyrate (SCFAs) levels and downregulation of the activity of transcription factor NF-κB [³⁷,¹⁰⁷]. Similarly, there are also other types of probiotics with complex bacteria exhibiting efficiency in NAFLD inventions [¹⁰⁸–¹¹³].

In recent years, clinical trials for probiotics have increased, in which VSL#3 attracted more attention from researchers compared with other probiotics. In a randomized clinical trial, the supplement of VSL#3 improved NAFLD in obese children. These beneficial effects were closely associated with VSL#3-dependent increase in GLP-1 [¹¹⁴]. Other complex bacteria have similar beneficial effects on NAFLD. For example, in a recent study, the effects of a probiotic capsule containing L. acidophilus ATCC B3208, B. lactis DSMZ 32269, B. bifidum ATCC SD6576, and L. rhamnosus DSMZ on sonographic and biochemical NAFLD were evaluated, and the probiotic compound was found to be effective in improving pediatric NAFLD [¹¹⁵]. In addition, the single strains were tested in the clinic. Vajro et al. tested the effects of Lactobacillus rhamnosus strain GG in pediatric liver disease related to obesity. Multivariate analysis showed that the levels of ALT were significantly decreased, which suggested a potential therapeutic tool for the treatment of hyper-transaminasemia in hepatopathic obese children [¹¹⁶]. However, in another study, probiotic supplementations did not contribute to significant changes in markers of lipids, including low-density lipoprotein (LDL)-cholesterol, total cholesterol, TG, and LDL/HDL ratios.

Prebiotics

Roberfroid defined prebiotics as “a selectively fermented ingredient that allows specific changes, both in the composition and/or activity in the gastrointestinal microflora that confers benefits upon host well-being and health or the indigestible food ingredients that beneficially affect the host” [¹¹⁷]. Increasing evidence has demonstrated that modulation of intestinal bacteria by prebiotics is beneficial to NAFLD inventions [¹¹⁸,¹¹⁹]. Lactulose is also considered a prebiotic, and studies have indicated that serum endotoxin levels and subsequent inflammation in the liver were decreased in rats with steatohepatitis, and promotions in the growths of lactic acid and Bifidobacteria were observed [¹²⁰,¹²¹]. Oligofructose (OFS) is a mixture of fermentable dietary fibers. In 2009, Cani et al. [¹²²] demonstrated that the OFS treatment group demonstrated lower levels of plasma LPS and cytokines, and reported a decreased expression of oxidative stress and inflammation markers in the liver, which were associated with the improvement of gut permeability and integrity of tight junctions. In MCD-induced steatohepatitis models, dietary fructo-oligosaccharides restored intestinal epithelial barrier functions and microflora to normal levels, as well as significantly decreased hepatic steatosis, the proportion of CD14-positive Kupffer cells, and the expression of TLR4 [¹²³]. Neyrinck et al. investigated the ability of another fungal source prebiotic, chitin-glucan (CG), to affect the gut microbiota, glucose, and lipid metabolism. The administration of CG limited weight gain, glucose intolerance, hepatic triglyceride accumulation, fasting hyperglycemia, and hypercholesterolemia induced by HFD [¹²⁴].

In the clinic, the effects of OFS, an insulin-type fructan, on the metabolism of glucose and lipids in NASH patients were tested in a randomized, double-blind study. Compared with placebo, OFS administration decreased ALT, AST, and insulin levels [¹¹⁹]. In another intervention study, 30 obese women were given ITF prebiotics (a combination of insulin and oligofructose). An increase in Bif and Faecalibacterium prausnitzii was demonstrated, which was negatively correlated with the serum level of LPS [¹²⁵]. In conclusion, prebiotic-induced gut-mediated changes are likely to involve improvements in the gut epithelial barrier and glycemic control, as well as reductions in bacteria-derived hepatotoxins and the degree of inflammation and de novo lipogenesis [¹¹⁸]. Furthermore, prebiotics exhibit their benefits in various diseases [¹⁰,¹²⁶,¹²⁷]. At present, probiotics and prebiotics have pivotal roles in the treatment and prevention of NAFLD; however, the inconsistent experimental results need rigorous experimental designs to improve the quality of clinical research and verify the therapeutic effects [¹²⁸].

Synbiotic

Synbiotics are the synergistic combination of probiotics and prebiotics [¹²⁹]. In recent years, synbiotics have produced various health benefits in metabolic diseases [¹³⁰,¹³¹]. Scientists have clarified the potential effects of synbiotics in NASH models and found that synbiotics could regulate gut microbiota, reduce the degree of fibrosis, and simultaneously decrease endotoxemia [¹³²]. In another study, the progression of NAFLD was attenuated by a synbiotic composed of L. paracasei B21060 with arabinogalactan and fructo-oligosaccharides in HFD rats. The downregulation of liver inflammatory markers, together with an increase in the expression of downstream target genes and peroxisome proliferator-activated receptors were also demonstrated. Mofidi et al. [¹³³] conducted a clinical double-blind study in patients with normal and low BMIs. Synbiotic supplementation improved the main features of NAFLD, at least partially by reducing inflammatory indices. In another study involving 50 NASH patients proven by biopsy, the administration of synbiotics presented a reduction in steatosis [¹³⁴] but did not improve intestinal permeability or LPS levels. In summary, synbiotic supplementation might be useful in the management of NAFLD, but further trials are needed.

Others

Recently, the therapeutic potential of antibiotics on metabolic diseases have been investigated. For example, combined administration of vancomycin and bacitracin decreased the abundance of Firmicutes and Bacteroidetes in the gut, improving insulin resistance and secretion of glucagon-like peptide-1 [¹³⁵]. Meanwhile, administration of cidomycin attenuated the severity of NASH in rats, which was associated with the reduction in serum levels of TNF-a [⁴⁴]. In the clinic, the efficacy of rifaximin on the circulating levels of cytokines and endotoxins was studied in individuals with confirmed NAFLD, and the investigators concluded that its short-term administration was effective and safe, especially for NASH [¹³⁶].

Fecal microbiota transplantation (FMT) is not a new concept. It re-balances the gut microbiome by transplanting fecal bacteria from healthy to diseased subjects. FMT has been used to treat diarrhea and food poisoning in traditional Chinese medicine [¹³⁷]. Promrat et al. applied FMT by transplanting gut bacteria from lean donors to NASH patients, which proved useful for defining the pathogenesis of NAFLD. Other related studies indicated that FMT may provide further therapeutic modes for these patients [¹³⁸], and further and larger studies are needed to validate that possibility.

Diet and exercise are two factors that affect NAFLD. Fructose is an integral part of the human diet. A fructose-enriched diet can alter liver metabolism and gut barrier function, ultimately leading to NAFLD [¹³⁹]. Chronic fructose consumption activates several key transcription factor expression of lipogenic enzymes via SREBP1c and ChREBP; it contributes to lipogenesis and associated pathologies, including steatosis, dyslipidemia, and hepatic insulin resistance [¹⁴⁰].

Exercise is known for its health benefits, and it reduces the risk of NAFLD. Exercise training increases the Bacteroidetes/Firmicutes ratio and alpha diversity (within the Bacteroidetes phylum) of microbiota [¹⁴¹]. Animal experiments on C57 BL/6 mice by Denou et al. [¹⁴²] showed that exercise training can improve insulin sensitivity and change the gut and fecal microbiota caused by HFD-induced obesity. Matsumoto et al. [¹⁴³] used exercise and sedentary Wistar rats and suggested that the exercised rats showed significantly higher n-butyrate concentration than the sedentary rats, and showed altered gut microbial environment. Despite strong associations among exercise, liver health, gut microbiota, and NAFLD in humans, further research is still needed.

In addition to prebiotics, fecal microbiota transplantation, and synbiotics, other gut microbiota-targeted inventions have been reported, such as antibiotics and herbal medicines or natural active ingredients.

Herbal medicine or natural active ingredients have been shown to regulate intestinal bacteria with minor side effects [¹⁴⁴,¹⁴⁵]. As a typical component of herbal medicine, the effects of berberine on metabolic diseases have increasingly been confirmed [¹⁴⁶,¹⁴⁷]. Currently, berberine has beneficial effects on NASH. In the treatment groups, the administration of berberine restored the proportion of Bacteroidetes and Firmicutes, which led to a significant reduction in body weight and serum lipids levels; moreover, the serum transaminase activity and NAFLD activity score were greatly improved, and normalization of the gut microbiota may be responsible for its effects. Lin et al. [¹⁴⁸] explored the functions of 2, 3, 5, 4′-tetrahydroxy-stilbene-2-O-β-D-glucoside (TSG) in a rat model of NAFLD induced by HFD. The results suggested that the preventive effects of NAFLD provided by TSG were mediated by the modulation of gut microbiota and TLR4/NF-κB pathway, which may be responsible for the alleviation of chronic low-grade inflammation. Moreover, the beneficial effects of some Chinese herbal formula (CHF) might be associated with the modulation of gut microbiota. Recently, researchers have evaluated the anti-obesity properties of Daesiho-tang (DSHT) by using an obese mice model [¹⁴⁹]. The study demonstrated that DSHT could reduce body weight gain and regulate the expression of leptin and adiponectin genes in adipose tissue, with a parallel modulation of gut microbiota. Qushi Huayu Fang is an herbal formulation composed of Curcuma longa L., Gardenia jasminoides Ellis, Artemisia capillaries Thunb, Fallopia japonica, and Hypericum japonicum Thunb. In a study [¹⁵⁰], potential changes in the composition of gut microbiota were analyzed with HFD-induced NAFLD rats. Compared with the control group, the levels of the genera Escherichia/Shigella were significantly enhanced in rats fed with HFD, and the levels decreased to the control values after CHF administration. Furthermore, the profiles of intestinal bacteria in HFD-induced rats could be regulated by CHF administration.

Conclusions

In recent years, interactions between gut dysbiosis and the pathology of NAFLD have been extensively investigated. However, due to the complexity of gut microbiota, determining its precise roles in the genesis of NAFLD remains challenging. It is a key factor contributing to a number of identified metabolic diseases. Thus, gut microbiota-targeted strategies for NAFLD therapy are being increasingly recognized as promising approaches. The most common strategies are probiotics, prebiotics, and synbiotics, wherein probiotics are tested frequently and have reflected good therapeutic advantages. As a kind of complex bacteria, VSL#3 is favored by researchers for showing excellent therapeutic potential, followed by prebiotics, which are also studied widely and may be beneficial in the treatment of NAFLD. Nevertheless, inconsistent results were observed in the clinic, thus, further studies based on rigorous experimental designs are still needed to evaluate the effect of these therapeutic approaches. Antibiotics provide a new strategy for the prevention and treatment of NAFLD, but with the problem on side effects, they are not efficacious choice for humans. Diet modulation and physical exercise can alter gut microbiota and reduce the risk of NAFLD. Currently, studies on naturally active ingredients or Chinese herbal formula have been extensively accomplished, and some of the results have shown their effective potential in NAFLD therapy. Overall, there is a huge potential for gut microbiota-targeted therapy to be developed, which will undoubtedly make a meaningful contribution to human health worldwide.

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