# Gut Microbiota and Drug-Related Liver Injury: Challenges and Perspectives

Authors: Yuanyuan Su; Ning Lu; Qian Li; Hua Wen; Xiao Qin Zhang; Mingxin Zhang
Journal: Advanced Gut & Microbiome Research
 (2023)

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## Abstract

Drug-related liver injury (DILI) is increasing in the incidence of liver injury due to nonviral liver disease and has become a health problem that should not be underestimated. As a hot research topic in recent years, gut microbiota have been studied in various tumors, cardiovascular metabolic diseases, and human immunity. However, there is still a lack of research related to gut microbiota and DILI. It is known that they can influence each other through the regulation of the “gut-liver axis,” and the relationship between them and the possible mechanisms of action are still at the research stage. Human leukocyte antigen (HLA) gene polymorphisms are closely related to the development of DILI, and the gene can also affect changes in the composition of gut microbes. In this paper, we review the possible relationships and mechanisms between DILI and gut microbiota in order to provide new research directions for the prevention and treatment of DILI in the future. In the future, untargeted therapies using antibiotics, probiotics, or FMT will be replaced by personalised and precision medicine approaches, such as bioengineered bacterial strains or drugs that modulate specific bacterial enzymes and metabolic pathways.


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## Body

## 1. Background

Drug-related liver injury (DILI) occurs in 23.8 per 100,000 people in China [1] and 2.7-14 per 100,000 people in Europe and the United States [2, 3]. This shows that the incidence of DILI in China is much higher than that in Western countries, and the incidence of DILI has been increasing year by year. DILI has become a major cause of drug development interruptions during clinical studies and drug withdrawals from the pharmaceutical market [4]. In the United States, DILI accounts for more than 50% of patients with acute liver failure (ALF), and acute DILI can evolve into chronic liver injury and liver failure, which requires liver transplantation and even leads to death [5]. Thus, DILI has become a health problem that should not be ignored.DILI is divided into two types, which includes DILI and specific DILI [6]. DILI is usually attributed to the toxicity of the drug itself and the drug dose, but the mechanism and the pathogenesis of specific DILI are not known. An article published in Cell in 2016 stated that each adult had 3 billion of its own cells and 4 billion of bacteria [7]. The gut microbiota affects the metabolism of the drug and can also be affected by the drug [8, 9]. There is growing evidence that shows that gut microbes play an important role in DILI, and the two are linked in the “gut-liver axis” [10, 11], a theory first proposed by Marshall in 1998 [12]. Under normal circumstances, the intestinal barrier is the first line to protect the body from exogenous substances. The liver provides a second line against antigens and inflammatory factors that escape gastrointestinal mucosal immune surveillance [13, 14]. When the intestine is in a pathological state, the barrier is damaged. It increases the intestinal permeability, and then bacteria and its metabolites in the intestine will enter the liver from the portal vein. Then, it will activate Kupffer cells in the liver and release a series of inflammatory factors, which aggravate liver injury and disease progression [15, 16]. In turn, decreased phagocytosis of Kupffer cells, reduced synthesis of immune proteins, and the presence of altered hemodynamics during cirrhosis contribute to impaired intestinal function. These have been demonstrated in nonalcoholic fatty liver disease, cirrhosis, and liver fibrosis, but little is known in DILI [17, 18]. In this review, we will discuss gut microbiota changes after DILI, and gut microbiota affects individual susceptibility to DILI.


## 2. Gut Microbiota Changes after DILI

### 2.1. Gut Microbiota Changes in DILI due to Acetaminophen (APAP)

In APAP-induced liver injury in rats, APAP is absorbed from the intestine and broken down by the liver. After hepatic monophasic metabolism, most of APAP was eliminated by binding with glucuronic acid or sulfuric acid. While a small part was oxidized to N-acetyl-p-benzoquinone imine (NAPQI) by cytochrome P450 proteins1A2 (CYP1A2) and cytochrome P450 proteins2E1 (CYP2E1), then NAPQI was eliminated by binding to reduced glutathione (GSH) [19, 20]. However, when there is excessive intake of APAP, GSH cannot completely eliminate the metabolic production of NAPQI. Excessive NAPQI will cause mitochondrial dysfunction, lead to oxidative damage, and promote inflammatory promotion, apoptosis, and necrosis of hepatocytes [21]. Another study on APAP-induced liver injury in rats found that the gut microbiota metabolite 1-phenyl-1,2-propanediol (PPD) was involved. It resulting in a decrease in liver GSH levels, suggesting that gut microbiota may be a potential target for alleviating acute liver injury induced by APAP [22]. This may be due to the change of gut microbiota in mice after DILI, resulting in an increase in the production of PPD and a decrease in the level of GSH in the liver, which leads to liver damage. Further, given oral enzyme, bacteria to mice with liver injury result a decrease in PPD and a reduction in liver injury [23].


### 2.2. Gut Microbiota Changes in DILI due to Antituberculosis Drugs

In the study of DILI caused by antituberculosis drugs, it showed that the gut microbiota of pulmonary tuberculosis mice changed only slightly before antituberculosis drug treatment [24]. However, gut microbiota has changed after antituberculosis drug treatment (isoniazid, rifampin, pyrazinamide, and ethambutol). Anti-TB therapy caused a rapid, significant alteration in the community structure. The relative abundance of members of genus Clostridiales of the phylum Firmicutes significantly decreased during anti-TB treatment, while many members of genus Bacteroides, including Bacteroides OTU230 and Bacteroides fragilis, were among the taxa that increased. OTU8 and OTU2972 assigned to family Erysipelotrichaceae of the phylum Firmicutes showed a dramatic increase 1 week after the start of therapy, while the other members of this family decreased [25]. Some studies have shown that the changes of liver function and morphology are closely related to the abundance of clostridium [25]. Antituberculosis drugs will lead to decrease the abundance of clostridium and long-term imbalance of gut microbiota. It leads to the development of DILI, but the exact mechanism of DILI is now unclear [26].


### 2.3. Gut Microbiota Changes in DILI due to Iron

It is estimated that approximately 2 billion people worldwide suffer from iron-deficiency anemia (IDA) [27]. Oral iron is an effective treatment, but excessive iron can cause damage to human and animal health. Some studies have shown that free iron can catalyze the formation of reactive oxygen species (ROS) in the gut, which can damage the intestinal mucosal barrier [28]. Recent studies have shown that oral excessive iron can elevate ALT and AST in mice, which are higher than those of the control group. And excessive iron can damage the intestinal mucosa of mice and destroy the intestinal barrier. The toxins were produced by the liver through the intestinal-liver axis, and then, the gut microbiota changed. The gut microbiota of mice overdosed with iron was analyzed in some studies. Results also showed that the abundance of two kinds of probiotics in Lachnospiraceae (Clostridium) and mycoplasma (Allobaculum) decreased significantly [29].


### 2.4. Gut Microbiota Changes in DILI due to Other Drugs

The drugs that can cause DILI are far more than paracetamol, anti-tuberculosis drugs and iron drugs. Many studies have shown that there are changes in gut microbiota in liver injury caused by triclosan [30], carbon tetrachloride [31], copper [32], and D-galactosamine [33]. The characteristics of gut microbiota changes are summarized in Table 1. More and more evidences find that the gut microbiota of DILI patients will change. It is not only that the drug can cause changes in gut microbiota but also that the gut microbiota can cause different responses to the drug in individuals. But these changes can only be specific to a genus, not to a specific bacterium, so we need to meet a bigger challenge.Table 1
Changes of gut microbiota after DILI.
DrugsResearch methodCharacteristics of gut microbiota changeReferencesAPAPAnimal experimentThe ratio of thick-walled bacteria to Bacillus pseudomallei decreased[22]Antituberculosis drugsCase-control studyReduced abundance of Clostridium perfringens spp. in thick-walled phylumIncreased abundance of Bacillus spp. and Bacteroides fragilis spp.[25]Liquid ironAnimal experimentThe abundance of Lachnospiraceae and mycoplasma decreased[29]TriclosanAnimal experimentIncreased abundance of Proteobacteria and EnterobacteriaceaeReduced abundance of Firmicutes and Bacteroidetes[30]Carbon tetrachlorideAnimal experimentsReduced abundance ofAdlercreutzia, Flavonifractor, Lactobacillus, and TuricibacterIncreased abundance of Enterococcus, Enterorhabdus, Oscillibacter, and Oscillospira[31]CopperAnimal experimentsReduced the abundance of probiotics and the ratio of Firmicutes to Bacteroidetes[32]D-GalactosamineAnimal experimentsReduced the abundance of gut Actinobacteria and Firmicutes[33]


## 2.1. Gut Microbiota Changes in DILI due to Acetaminophen (APAP)

In APAP-induced liver injury in rats, APAP is absorbed from the intestine and broken down by the liver. After hepatic monophasic metabolism, most of APAP was eliminated by binding with glucuronic acid or sulfuric acid. While a small part was oxidized to N-acetyl-p-benzoquinone imine (NAPQI) by cytochrome P450 proteins1A2 (CYP1A2) and cytochrome P450 proteins2E1 (CYP2E1), then NAPQI was eliminated by binding to reduced glutathione (GSH) [19, 20]. However, when there is excessive intake of APAP, GSH cannot completely eliminate the metabolic production of NAPQI. Excessive NAPQI will cause mitochondrial dysfunction, lead to oxidative damage, and promote inflammatory promotion, apoptosis, and necrosis of hepatocytes [21]. Another study on APAP-induced liver injury in rats found that the gut microbiota metabolite 1-phenyl-1,2-propanediol (PPD) was involved. It resulting in a decrease in liver GSH levels, suggesting that gut microbiota may be a potential target for alleviating acute liver injury induced by APAP [22]. This may be due to the change of gut microbiota in mice after DILI, resulting in an increase in the production of PPD and a decrease in the level of GSH in the liver, which leads to liver damage. Further, given oral enzyme, bacteria to mice with liver injury result a decrease in PPD and a reduction in liver injury [23].


## 2.2. Gut Microbiota Changes in DILI due to Antituberculosis Drugs

In the study of DILI caused by antituberculosis drugs, it showed that the gut microbiota of pulmonary tuberculosis mice changed only slightly before antituberculosis drug treatment [24]. However, gut microbiota has changed after antituberculosis drug treatment (isoniazid, rifampin, pyrazinamide, and ethambutol). Anti-TB therapy caused a rapid, significant alteration in the community structure. The relative abundance of members of genus Clostridiales of the phylum Firmicutes significantly decreased during anti-TB treatment, while many members of genus Bacteroides, including Bacteroides OTU230 and Bacteroides fragilis, were among the taxa that increased. OTU8 and OTU2972 assigned to family Erysipelotrichaceae of the phylum Firmicutes showed a dramatic increase 1 week after the start of therapy, while the other members of this family decreased [25]. Some studies have shown that the changes of liver function and morphology are closely related to the abundance of clostridium [25]. Antituberculosis drugs will lead to decrease the abundance of clostridium and long-term imbalance of gut microbiota. It leads to the development of DILI, but the exact mechanism of DILI is now unclear [26].


## 2.3. Gut Microbiota Changes in DILI due to Iron

It is estimated that approximately 2 billion people worldwide suffer from iron-deficiency anemia (IDA) [27]. Oral iron is an effective treatment, but excessive iron can cause damage to human and animal health. Some studies have shown that free iron can catalyze the formation of reactive oxygen species (ROS) in the gut, which can damage the intestinal mucosal barrier [28]. Recent studies have shown that oral excessive iron can elevate ALT and AST in mice, which are higher than those of the control group. And excessive iron can damage the intestinal mucosa of mice and destroy the intestinal barrier. The toxins were produced by the liver through the intestinal-liver axis, and then, the gut microbiota changed. The gut microbiota of mice overdosed with iron was analyzed in some studies. Results also showed that the abundance of two kinds of probiotics in Lachnospiraceae (Clostridium) and mycoplasma (Allobaculum) decreased significantly [29].


## 2.4. Gut Microbiota Changes in DILI due to Other Drugs

The drugs that can cause DILI are far more than paracetamol, anti-tuberculosis drugs and iron drugs. Many studies have shown that there are changes in gut microbiota in liver injury caused by triclosan [30], carbon tetrachloride [31], copper [32], and D-galactosamine [33]. The characteristics of gut microbiota changes are summarized in Table 1. More and more evidences find that the gut microbiota of DILI patients will change. It is not only that the drug can cause changes in gut microbiota but also that the gut microbiota can cause different responses to the drug in individuals. But these changes can only be specific to a genus, not to a specific bacterium, so we need to meet a bigger challenge.Table 1
Changes of gut microbiota after DILI.
DrugsResearch methodCharacteristics of gut microbiota changeReferencesAPAPAnimal experimentThe ratio of thick-walled bacteria to Bacillus pseudomallei decreased[22]Antituberculosis drugsCase-control studyReduced abundance of Clostridium perfringens spp. in thick-walled phylumIncreased abundance of Bacillus spp. and Bacteroides fragilis spp.[25]Liquid ironAnimal experimentThe abundance of Lachnospiraceae and mycoplasma decreased[29]TriclosanAnimal experimentIncreased abundance of Proteobacteria and EnterobacteriaceaeReduced abundance of Firmicutes and Bacteroidetes[30]Carbon tetrachlorideAnimal experimentsReduced abundance ofAdlercreutzia, Flavonifractor, Lactobacillus, and TuricibacterIncreased abundance of Enterococcus, Enterorhabdus, Oscillibacter, and Oscillospira[31]CopperAnimal experimentsReduced the abundance of probiotics and the ratio of Firmicutes to Bacteroidetes[32]D-GalactosamineAnimal experimentsReduced the abundance of gut Actinobacteria and Firmicutes[33]


## 3. Gut Microbiota Affects Individual Susceptibility to DILI

### 3.1. Human Leukocyte Antigen (HLA) Contributes to Individual Susceptibility to DILI by Gut Microbiota Affecting Drug Metabolism

When using the same drug, only a small number of susceptible individuals will develop obvious DILI, which may be related to the mechanism of liver immune tolerance and the single nucleotide polymorphism of HLA gene [34]. The HLA gene may have made the choice for us; in the genome-wide association study of a large number of cases of DILI, it was found that most of the DILI caused by flucloxacillin [35], amoxicillin-clavulanic acid [36], fenofibrate [37], terbinafine [38], lampatinib [39], and trimethoprim-sulfamethoxazole [40] were located in the major histocompatibility complex (MHC) region. And DILI is mainly associated with HLA gene. These associations may not be reliable enough to screen HLA alleles before using drugs, but it suggests immunological pathogenesis. Some studies have shown that specific CD4+T cells can be detected in patients with DILI by amoxicillin-clavulanic acid. Further studies have shown that amoxicillin can combine with lysine residues to form protein adducts and then activate T cells [41].HLA may indirectly select people with DILI by gut microbiota to regulate the host’s immune system, affecting the content of sex hormones and changing the expression of drug metabolic genes. The association of MHC class II genes with the risk of developing autoimmune diseases has been well-described. MHC class II molecules play a central role in the maturation of T-cells and thus can affect the autoreactivity of an individual’s T-cell population [42]. In the genome-wide association study of a large number of cases of DILI, Indeed, multiple HLA genes in humans such as HLA-B∗57 : 01, HLA-A∗02 : 01, and HLA-DRB1∗07 : 01 have been associated with DILI susceptibility. It has been shown that the HLA can alter the gut microbiota of the population, and a significant reduction in the abundance of Coprococcus and Enterorhabdus was found in AH8.1 haplotype carriers [43]. A study shows that HLA class II molecules influence the composition of the gut microbiome [44]. Gut microbiota can directly or indirectly influence drug metabolism, which involved in drug reductive metabolism and hydrolysis, which includes demethylation, deamination, dehydroxylation deacylation, decarboxylation, and oxidation reactions, and may have an impact on drug metabolism [45]. Some studies have confirmed that gut microbiota may also affect drug metabolism by changing the expression of drug metabolism genes [46]. Later, some people have compared the mRNA transcription level in the liver of aseptic mice and conventional mice. It has been proved that gut microbiota affect the development of various drug metabolism genes in the liver in a sex-specific way [47]. The HLA gene alters the gut microbiota and thus affects drug metabolism. So, the HLA gene largely determines who will develop DILI.


### 3.2. Gut Microbiota Affects Liver Immunity Leading to Individual Susceptibility to DILI

More and more studies have shown that gut microbiota plays an important role in the maturation of the human immune system, and dynamic changes in gut microbiota have a fine-tuning effect on the human immune system [48]. In the genome-wide association study of a large number of cases of DILI, the liver is a specific immune organ, and antigens entering the liver cause an immune response in the liver. To avoid this, the immune tolerance mechanism of the liver is particularly important, and immune tolerance is a necessary adaptation to protect hepatocytes from damage caused by a persistent inflammatory state in the liver [49]. A good example here is liver transplantation, where 20% of liver transplant patients are completely free from immunosuppression, which may be related to hepatic immune tolerance [50]. It is known that the liver and gut microbiota are linked to the enterohepatic axis, and the excretion of bile acids from the liver to the intestine can affect the gut microbiota. Conversely, the metabolites of the gut microbiota can be reintroduced to the liver through the enterohepatic cycle, and the flora products can be used as antigens to activate immune cells and produce cytokines, which can then initiate the host immune response [51–53]. It has been shown in several papers that gut microbiota can promote the host immune system, and the immune system can also influence the composition of the gut microbiota. And it has been shown that germ-free mice have impaired development and maturation of gut-associated lymph node tissue compared to normal mice [54, 55]. This suggests that gut microbiota can indeed influence the immune system of the host, when the venous blood carries intestinal bacteria and their products back to the liver. They will be neutralized by the immune cells of the liver, if the number of intestinal bacteria and their products is relatively small, so that the immune cells of the liver will produce immune tolerance to specific gut microbiota. However, when the number and composition of intestinal microorganisms change, intestinal permeability can be increased, and the number of bacteria and their products returning to the liver will increase. And more foreign antigens will be exposed to the immune cells of the liver, and it will break the established immune tolerance. Then, it will lead to liver injury, and people with changes in gut microbiota may break the established immune tolerance [56]. Previous studies have shown that people who take long-term acid-suppressing drugs disrupted their gut microbiota and more susceptible to liver failure [57]. It has been hypothesized that gut microbiota may be a potential mechanism for individual susceptibility to DILI, the above evidence provides evidence for our hypothesis, and we expect more results to confirm [58].


### 3.3. Gut Microbiota Contributes to Individual Susceptibility to DILI by Affecting the Level of Sex Hormones

Recent animal experiments have shown that gut microbiota may be related to gender differences in diseases, and gut microbiota may affect the content of sex hormones, which leads to gender differences in diseases. For example, the gender difference in the incidence of rheumatoid arthritis and multiple sclerosis decreases with the increase of age, which is consistent with the decrease of testosterone [59, 60]. Women are more likely to suffer from autoimmune hepatitis [61]. In the study of liver injury caused by inhaled anesthetic halothane, it was found that all the patients with severe liver injury were women, and the same pattern was found in subsequent animal experiments. When the mice were treated with estradiol and progesterone, it was found that the sex difference disappeared [62]. Therefore, we hypothesize that gut microbiota may be involved in the development of DILI by affecting sex hormones.HLA can indirectly select people with DILI by gut microbiota to regulate the host’s immune system, affecting the content of sex hormones and changing the expression of drug metabolic genes, as shown in Figure1. But this is only at a theoretical stage, and we need more clinical trials to confirm.Figure 1
The role of gut microbiota in susceptibility of the DILI population. Drug metabolism, sex hormone expression, and hepatic immune tolerance will lead to individual differences in DILI, such as that women are more likely to develop DILI in the population.


## 3.1. Human Leukocyte Antigen (HLA) Contributes to Individual Susceptibility to DILI by Gut Microbiota Affecting Drug Metabolism

When using the same drug, only a small number of susceptible individuals will develop obvious DILI, which may be related to the mechanism of liver immune tolerance and the single nucleotide polymorphism of HLA gene [34]. The HLA gene may have made the choice for us; in the genome-wide association study of a large number of cases of DILI, it was found that most of the DILI caused by flucloxacillin [35], amoxicillin-clavulanic acid [36], fenofibrate [37], terbinafine [38], lampatinib [39], and trimethoprim-sulfamethoxazole [40] were located in the major histocompatibility complex (MHC) region. And DILI is mainly associated with HLA gene. These associations may not be reliable enough to screen HLA alleles before using drugs, but it suggests immunological pathogenesis. Some studies have shown that specific CD4+T cells can be detected in patients with DILI by amoxicillin-clavulanic acid. Further studies have shown that amoxicillin can combine with lysine residues to form protein adducts and then activate T cells [41].HLA may indirectly select people with DILI by gut microbiota to regulate the host’s immune system, affecting the content of sex hormones and changing the expression of drug metabolic genes. The association of MHC class II genes with the risk of developing autoimmune diseases has been well-described. MHC class II molecules play a central role in the maturation of T-cells and thus can affect the autoreactivity of an individual’s T-cell population [42]. In the genome-wide association study of a large number of cases of DILI, Indeed, multiple HLA genes in humans such as HLA-B∗57 : 01, HLA-A∗02 : 01, and HLA-DRB1∗07 : 01 have been associated with DILI susceptibility. It has been shown that the HLA can alter the gut microbiota of the population, and a significant reduction in the abundance of Coprococcus and Enterorhabdus was found in AH8.1 haplotype carriers [43]. A study shows that HLA class II molecules influence the composition of the gut microbiome [44]. Gut microbiota can directly or indirectly influence drug metabolism, which involved in drug reductive metabolism and hydrolysis, which includes demethylation, deamination, dehydroxylation deacylation, decarboxylation, and oxidation reactions, and may have an impact on drug metabolism [45]. Some studies have confirmed that gut microbiota may also affect drug metabolism by changing the expression of drug metabolism genes [46]. Later, some people have compared the mRNA transcription level in the liver of aseptic mice and conventional mice. It has been proved that gut microbiota affect the development of various drug metabolism genes in the liver in a sex-specific way [47]. The HLA gene alters the gut microbiota and thus affects drug metabolism. So, the HLA gene largely determines who will develop DILI.


## 3.2. Gut Microbiota Affects Liver Immunity Leading to Individual Susceptibility to DILI

More and more studies have shown that gut microbiota plays an important role in the maturation of the human immune system, and dynamic changes in gut microbiota have a fine-tuning effect on the human immune system [48]. In the genome-wide association study of a large number of cases of DILI, the liver is a specific immune organ, and antigens entering the liver cause an immune response in the liver. To avoid this, the immune tolerance mechanism of the liver is particularly important, and immune tolerance is a necessary adaptation to protect hepatocytes from damage caused by a persistent inflammatory state in the liver [49]. A good example here is liver transplantation, where 20% of liver transplant patients are completely free from immunosuppression, which may be related to hepatic immune tolerance [50]. It is known that the liver and gut microbiota are linked to the enterohepatic axis, and the excretion of bile acids from the liver to the intestine can affect the gut microbiota. Conversely, the metabolites of the gut microbiota can be reintroduced to the liver through the enterohepatic cycle, and the flora products can be used as antigens to activate immune cells and produce cytokines, which can then initiate the host immune response [51–53]. It has been shown in several papers that gut microbiota can promote the host immune system, and the immune system can also influence the composition of the gut microbiota. And it has been shown that germ-free mice have impaired development and maturation of gut-associated lymph node tissue compared to normal mice [54, 55]. This suggests that gut microbiota can indeed influence the immune system of the host, when the venous blood carries intestinal bacteria and their products back to the liver. They will be neutralized by the immune cells of the liver, if the number of intestinal bacteria and their products is relatively small, so that the immune cells of the liver will produce immune tolerance to specific gut microbiota. However, when the number and composition of intestinal microorganisms change, intestinal permeability can be increased, and the number of bacteria and their products returning to the liver will increase. And more foreign antigens will be exposed to the immune cells of the liver, and it will break the established immune tolerance. Then, it will lead to liver injury, and people with changes in gut microbiota may break the established immune tolerance [56]. Previous studies have shown that people who take long-term acid-suppressing drugs disrupted their gut microbiota and more susceptible to liver failure [57]. It has been hypothesized that gut microbiota may be a potential mechanism for individual susceptibility to DILI, the above evidence provides evidence for our hypothesis, and we expect more results to confirm [58].


## 3.3. Gut Microbiota Contributes to Individual Susceptibility to DILI by Affecting the Level of Sex Hormones

Recent animal experiments have shown that gut microbiota may be related to gender differences in diseases, and gut microbiota may affect the content of sex hormones, which leads to gender differences in diseases. For example, the gender difference in the incidence of rheumatoid arthritis and multiple sclerosis decreases with the increase of age, which is consistent with the decrease of testosterone [59, 60]. Women are more likely to suffer from autoimmune hepatitis [61]. In the study of liver injury caused by inhaled anesthetic halothane, it was found that all the patients with severe liver injury were women, and the same pattern was found in subsequent animal experiments. When the mice were treated with estradiol and progesterone, it was found that the sex difference disappeared [62]. Therefore, we hypothesize that gut microbiota may be involved in the development of DILI by affecting sex hormones.HLA can indirectly select people with DILI by gut microbiota to regulate the host’s immune system, affecting the content of sex hormones and changing the expression of drug metabolic genes, as shown in Figure1. But this is only at a theoretical stage, and we need more clinical trials to confirm.Figure 1
The role of gut microbiota in susceptibility of the DILI population. Drug metabolism, sex hormone expression, and hepatic immune tolerance will lead to individual differences in DILI, such as that women are more likely to develop DILI in the population.


## 4. Regulating Gut Microbiota Can Be a New Target for Liver Protection

### 4.1. Reduce Liver Injury by Regulating Gut Microbiota through Dietary Modifications

Recent studies have shown that Western diet (high in saturated fat and sucrose and low in fiber) aggravates CCl4-induced chronic liver injury by disrupting intestinal microflora and affecting bile acid metabolism, including reducing the richness of Salmonella and increasing the richness of Streptococcus [63]. A retrospective analysis of DILI in the United States shows that African-Americans are more likely to develop DILI than Caucasians [64]. It is also indicated in another literature that the probability of DILI is also different among people of different races [65]. It is possible that different diets lead to different gut microbiota, resulting in different metabolic abilities of drugs. Some studies have shown that in cirrhosis and nonalcoholic fatty liver disease, liver preservation can be achieved through dietary regulation of intestinal flora, which may be related to bile acid metabolism [66–69]. In the mouse model of liver injury induced by carbon tetrachloride, it was found that mice fed with goat milk could reduce the damage DILI caused by carbon tetrachloride. And it was found that the damage caused by carbon tetrachloride could even be prevented by taking goat milk for 7 days in advance. Sequencing of the gut microbiota of mice showed that the flora of mice after taking goat milk was closer to that of normal mice [31], although some studies have confirmed that whether changing the diet or supplementing certain dietary ingredients can reduce liver injury. And the foothold of these studies will eventually come back to the gut microbiota. But the gut microbiota is a huge whole, and we only know that through diet can change the gut microbiota and then achieve the goal of reducing liver damage. However, we do not know what happened, what bacteria are affected by these dietary regulation, and what role the gut microbiota play in this, all of which are still a huge challenge.


### 4.2. Regulating Gut Microbiota through Probiotic Supplementation to Reduce Liver Injury

Compared with dietary regulation, the supplement of live bacteria is more controllable. In recent years, more and more attention has been paid to probiotics as auxiliaries in the prevention or treatment of gastrointestinal diseases. Saccharomyces boulardii is a probiotics yeast that is often used to treat gastrointestinal disorders [70]. An animal study showed that oral administration of Saccharomyces boulardii decelerates the progress of liver fibrosis in rats with carbon tetrachloride-induced cirrhosis, as evidenced by reductions in liver fibrosis markers such as alpha smooth muscle actin (SMA), transforming growth factor beta (TGF), serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and malondialdehyde (MDA) [71]. Decreased numbers of Bacteroidetes and Aspergillus have been reported to be associated with a variety of liver diseases, including cirrhosis [72] and nonalcoholic fatty liver disease [73], and Saccharomyces boulardii can reverse the aforementioned changes in the flora [74]. Many studies in recent years have shown that supplementation with live bacteria can indeed improve DILI as shown in Table 2[75–81]. Supplementation of probiotics can not only improve gut microbiota and strengthen intestinal barrier to improve liver injury but also affect host immune status to improve liver injury. Study also found that Lactobacillus can activate intestinal intrinsic lymphocytes to produce IL-22 and increase the level of systemic IL-22. Intestinal-derived IL-22 can enhance mucosal barrier function and promote regulatory dendritic cells (CDC) to recruit to the liver. These CDC are activated by TLR9 to produce IL-10 and TGF, thereby preventing further liver inflammation and reducing the level of liver injury [82]. A recent meta-analysis indicated that supplementation with probiotics or prebiotics has a significant ameliorative effect on acute liver injury through the enterohepatic axis. It can upregulate tight junction proteins, correct ileal flora abundance, reduce endotoxin invasion, and inhibit oxidative stress and proinflammatory mediators, thereby in turn improving liver biochemical parameters [83]. In the model of liver injury caused by carbon tetrachloride in mice, the degree of liver injury was reduced by supplementation with Lactobacillus fermentum and Lactobacillus plantarum [84]. Probiotic supplementation can alleviate liver injury by regulating gut microbiota, strengthening intestinal barrier, and affecting host immunity. But it only stays at the level of animal experiments. After all, there are inherent differences in gut microbiota between humans and animals. This still requires a large number of clinical trials to verify its efficacy.Table 2
Improvement of liver injury after supplementation of probiotics.
DrugsProbioticsResearch methodSupplementary methodsImprovementsReferencesD-GalactosamineSaccharomyces boulardiiAnimal experimentsOralDecreased ALT and AST levels, reduced inflammatory response, and reduction of Mycobacterium-like bacteria and Aspergillus[75]D-GalactosamineLactobacillus helveticus R0052Animal experimentsOralThe levels of ALT and AST are decreased, and the inflammatory reaction is alleviated[76]D-GalactosamineBifidobacterium longum R0175Animal experimentsOralThe levels of ALT and AST decreased, alleviated the inflammatory reaction, and improved the bile acid metabolism of intestinal microorganisms[77]D-GalactosamineBifidobacterium pseudocarinii LI09Bifidobacterium chain LI10Animal experimentsOralReduction of plasma M-CSF, MIP-1, and MCP-1 and reduction of bacterial translocation[78]D-GalactosaminePorphyromonas OTU170_ and Bacteroides OTU12_Animal experimentsOralImprove the composition of intestinal flora[79]CCl4Probiotic Clostridium typhimuriumAnimal experimentsGavageDecreasing ALT and AST, reducing the inflammatory response of the liver, and increased the abundance of thick-walled Porphyromonas and the mimicry proportion decreases[80]Antituberculosis drugsLactobacillusRandomized controlled trialOralIts modification on blood lipopolysaccharide, intestinal barrier function and gut microbiota.[81]


### 4.3. Fecal Microbiota Transplantation (FMT) Reduces Liver Injury by Regulating Gut Microbiota

If supplementation with live bacteria is using one type of bacteria to affect the entire gut microbiota, FMT replicates the entire intestinal microenvironment, which may be more convincing. It has now been shown that in ulcerative colitis, the nonalcoholic fatty liver disease mouse model, regulating gut microbiota has been achieved to reduce colonic inflammation and liver injury [85–87]. And now, studies have confirmed that FMT could change the diversity of gut microbiota in patients with liver cirrhosis and change the cognition of patients with hepatic encephalopathy [88]. In the D-galactose amine-induced liver injury mice model, it has been shown that FMT can increase butyrate-producing bacteria, improve liver inflammation, and reduce the extent of liver injury [89]. Another study showed that FMT improved acute liver failure caused by D-galactosamine by regulating the expression of Treg/Th17 cytokines [90].At present, there are not many studies on FMT in the treatment of DILI and the published result of microbiota of the host after FMT. These changes may improve the permeability of intestinal mucosal barrier, which remain at the level of “phenomenon.” What we know is that significant changes have taken place in the gut microeffect bile acid metabolism and affect host immune function, thus alleviating liver injury. If we focus our efforts on the gut microbiota and change the gut microbiota by changing the diet, oral supplementation of live bacteria or FMT to treat DILI or to reduce the degree of liver injury, this may be a new mechanism to protect the liver, but it is undoubtedly also a great challenge.


## 4.1. Reduce Liver Injury by Regulating Gut Microbiota through Dietary Modifications

Recent studies have shown that Western diet (high in saturated fat and sucrose and low in fiber) aggravates CCl4-induced chronic liver injury by disrupting intestinal microflora and affecting bile acid metabolism, including reducing the richness of Salmonella and increasing the richness of Streptococcus [63]. A retrospective analysis of DILI in the United States shows that African-Americans are more likely to develop DILI than Caucasians [64]. It is also indicated in another literature that the probability of DILI is also different among people of different races [65]. It is possible that different diets lead to different gut microbiota, resulting in different metabolic abilities of drugs. Some studies have shown that in cirrhosis and nonalcoholic fatty liver disease, liver preservation can be achieved through dietary regulation of intestinal flora, which may be related to bile acid metabolism [66–69]. In the mouse model of liver injury induced by carbon tetrachloride, it was found that mice fed with goat milk could reduce the damage DILI caused by carbon tetrachloride. And it was found that the damage caused by carbon tetrachloride could even be prevented by taking goat milk for 7 days in advance. Sequencing of the gut microbiota of mice showed that the flora of mice after taking goat milk was closer to that of normal mice [31], although some studies have confirmed that whether changing the diet or supplementing certain dietary ingredients can reduce liver injury. And the foothold of these studies will eventually come back to the gut microbiota. But the gut microbiota is a huge whole, and we only know that through diet can change the gut microbiota and then achieve the goal of reducing liver damage. However, we do not know what happened, what bacteria are affected by these dietary regulation, and what role the gut microbiota play in this, all of which are still a huge challenge.


## 4.2. Regulating Gut Microbiota through Probiotic Supplementation to Reduce Liver Injury

Compared with dietary regulation, the supplement of live bacteria is more controllable. In recent years, more and more attention has been paid to probiotics as auxiliaries in the prevention or treatment of gastrointestinal diseases. Saccharomyces boulardii is a probiotics yeast that is often used to treat gastrointestinal disorders [70]. An animal study showed that oral administration of Saccharomyces boulardii decelerates the progress of liver fibrosis in rats with carbon tetrachloride-induced cirrhosis, as evidenced by reductions in liver fibrosis markers such as alpha smooth muscle actin (SMA), transforming growth factor beta (TGF), serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and malondialdehyde (MDA) [71]. Decreased numbers of Bacteroidetes and Aspergillus have been reported to be associated with a variety of liver diseases, including cirrhosis [72] and nonalcoholic fatty liver disease [73], and Saccharomyces boulardii can reverse the aforementioned changes in the flora [74]. Many studies in recent years have shown that supplementation with live bacteria can indeed improve DILI as shown in Table 2[75–81]. Supplementation of probiotics can not only improve gut microbiota and strengthen intestinal barrier to improve liver injury but also affect host immune status to improve liver injury. Study also found that Lactobacillus can activate intestinal intrinsic lymphocytes to produce IL-22 and increase the level of systemic IL-22. Intestinal-derived IL-22 can enhance mucosal barrier function and promote regulatory dendritic cells (CDC) to recruit to the liver. These CDC are activated by TLR9 to produce IL-10 and TGF, thereby preventing further liver inflammation and reducing the level of liver injury [82]. A recent meta-analysis indicated that supplementation with probiotics or prebiotics has a significant ameliorative effect on acute liver injury through the enterohepatic axis. It can upregulate tight junction proteins, correct ileal flora abundance, reduce endotoxin invasion, and inhibit oxidative stress and proinflammatory mediators, thereby in turn improving liver biochemical parameters [83]. In the model of liver injury caused by carbon tetrachloride in mice, the degree of liver injury was reduced by supplementation with Lactobacillus fermentum and Lactobacillus plantarum [84]. Probiotic supplementation can alleviate liver injury by regulating gut microbiota, strengthening intestinal barrier, and affecting host immunity. But it only stays at the level of animal experiments. After all, there are inherent differences in gut microbiota between humans and animals. This still requires a large number of clinical trials to verify its efficacy.Table 2
Improvement of liver injury after supplementation of probiotics.
DrugsProbioticsResearch methodSupplementary methodsImprovementsReferencesD-GalactosamineSaccharomyces boulardiiAnimal experimentsOralDecreased ALT and AST levels, reduced inflammatory response, and reduction of Mycobacterium-like bacteria and Aspergillus[75]D-GalactosamineLactobacillus helveticus R0052Animal experimentsOralThe levels of ALT and AST are decreased, and the inflammatory reaction is alleviated[76]D-GalactosamineBifidobacterium longum R0175Animal experimentsOralThe levels of ALT and AST decreased, alleviated the inflammatory reaction, and improved the bile acid metabolism of intestinal microorganisms[77]D-GalactosamineBifidobacterium pseudocarinii LI09Bifidobacterium chain LI10Animal experimentsOralReduction of plasma M-CSF, MIP-1, and MCP-1 and reduction of bacterial translocation[78]D-GalactosaminePorphyromonas OTU170_ and Bacteroides OTU12_Animal experimentsOralImprove the composition of intestinal flora[79]CCl4Probiotic Clostridium typhimuriumAnimal experimentsGavageDecreasing ALT and AST, reducing the inflammatory response of the liver, and increased the abundance of thick-walled Porphyromonas and the mimicry proportion decreases[80]Antituberculosis drugsLactobacillusRandomized controlled trialOralIts modification on blood lipopolysaccharide, intestinal barrier function and gut microbiota.[81]


## 4.3. Fecal Microbiota Transplantation (FMT) Reduces Liver Injury by Regulating Gut Microbiota

If supplementation with live bacteria is using one type of bacteria to affect the entire gut microbiota, FMT replicates the entire intestinal microenvironment, which may be more convincing. It has now been shown that in ulcerative colitis, the nonalcoholic fatty liver disease mouse model, regulating gut microbiota has been achieved to reduce colonic inflammation and liver injury [85–87]. And now, studies have confirmed that FMT could change the diversity of gut microbiota in patients with liver cirrhosis and change the cognition of patients with hepatic encephalopathy [88]. In the D-galactose amine-induced liver injury mice model, it has been shown that FMT can increase butyrate-producing bacteria, improve liver inflammation, and reduce the extent of liver injury [89]. Another study showed that FMT improved acute liver failure caused by D-galactosamine by regulating the expression of Treg/Th17 cytokines [90].At present, there are not many studies on FMT in the treatment of DILI and the published result of microbiota of the host after FMT. These changes may improve the permeability of intestinal mucosal barrier, which remain at the level of “phenomenon.” What we know is that significant changes have taken place in the gut microeffect bile acid metabolism and affect host immune function, thus alleviating liver injury. If we focus our efforts on the gut microbiota and change the gut microbiota by changing the diet, oral supplementation of live bacteria or FMT to treat DILI or to reduce the degree of liver injury, this may be a new mechanism to protect the liver, but it is undoubtedly also a great challenge.


## 5. Prospect

DILI is associated with changes in the gut microbiota, reducing the degree of liver injury of DILI via probiotic supplementation, dietary modifications, and FMT, in which dysbiotic changes seem to contribute to hepatic disease. Although we have gained some insight into the interactions between gut microbial communities and host, a comprehensive understanding of the gut microbiota’s function during DILI is lacking. In fact, we need more clinical trials to confirm that restoring disturbed gut microbiota can reduce DILI. A better characterisation of the intestinal microbiome, metabolome, and host response using different preclinical models, stages of liver disease, and larger cohorts of patients is required. This will allow us to determine subtypes or groups of patients that would benefit most from therapies targeting the gut microbiota. Microbiome and metabolome analyses in patients with DILI might become a routine diagnostic test to stratify patients for tailored microbiome treatment approaches. A study shows that recent advances in sequencing technologies and computational tools have allowed an increasing number of metagenomic studies to be performed [91]. These technological advances enable a more thorough study of the genomics of the gut. In the future, untargeted therapies using antibiotics, probiotics, or FMT will be replaced by personalised and precision medicine approaches, such as bioengineered bacterial strains or drugs that modulate specific bacterial enzymes and metabolic pathways as shown in Figure 2.Figure 2
The table of contents. Gut microbiota will change after DILI, human leukocyte antigen (HLA), liver immunity, and sex hormones contribute to individual susceptibility to DILI, regulating gut microbiota by dietary modifications, and probiotic supplementation and FMT can be a new target for liver protection. In the future, by analyzing the gut microbiota of people prone to DILI, RNA sequencing of their gut microbiota was performed to summarize the sequencing characteristics. Screening for DILI-prone individuals and to achieve precision medicine.



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