Review in the Treatment of the Liver Diseases

Irina A. Kirpich, PhD, Craig J. McClain, MD Division of Gastroenterology, Hepatology, and , Department of Medicine (I.A.K., C.J.M.), Department of Pharmacology and Toxicology (C.J.M.), University of Louisville School of Medicine, University of Louisville Alcohol Research Center (I.A.K., C.J.M.), Robley Rex Veterans Medical Center (C.J.M.), Louisville, Kentucky Key words: nonalcoholic and alcoholic liver disease, microflora, probiotics

The concept that interactions between the gut, the liver, and the immune system play an important role in liver diseases is an old concept that has recently seen a resurgence in interest. Altered intestinal bacterial flora and gut- associated endotoxemia are increasingly recognized as critical components in both nonalcoholic fatty liver disease (NAFLD) and alcoholic liver disease (ALD). Probiotics have been proposed in the treatment and prevention of many conditions, including the liver diseases. Probiotics are live microorganisms that, when consumed in adequate amounts, confer a health benefit to the host. There are many mechanisms by which probiotics enhance intestinal health and influence the gut-liver axis, including modulation of the intestinal microflora, modification of intestinal barrier function, and immunomodulation. The present review summarizes the recent studies highlighting the role of the intestinal microflora in the development of NAFLD and ALD and the potential efficacy of probiotics as a therapeutic strategy for liver diseases.

Key teaching points: Interactions between the gut, the liver, and the immune system are involved in pathogenesis of the liver diseases. Gut microflora plays a significant role in the development of nonalcoholic and alcoholic fatty liver disease. Probiotics are live microorganisms that, when consumed in adequate amounts, confer a health benefit to the host. Probiotics have potential as prevention strategies or therapies for multiple gastrointestinal diseases, including liver diseases of different etiology.

INTRODUCTION It is impossible to understand the gut-liver axis without understanding our gut microbial ecology. Valuable studies The concept that interactions between the gut, the liver, and performed over the past few decades using 16S ribosomal the immune system are involved in pathogenesis of the liver RNA-based molecular technologies have revealed that the diseases has been recognized for at least a half century, but it intestinal microbiota is composed of 500-1000 different has recently seen a resurgence in investigator attention. There is species, with the most abundant bacterial groups belonging to a strong correlation between the health/function of the Bacteroidetes and [4]. Our comprise intestinal tract and that of the liver. Altered intestinal bacterial at least 1013–1014 microbial cells; and the microbiome flora is increasingly recognized to play an important role in represents overall more than 100 times the human genome nonalcoholic fatty liver disease (NAFLD), and is a critical [5] and is called the metagenome. Comprehensive molecular component of alcoholic liver disease (ALD) [1–3]. phylogenetic characterization has revealed the spatial distribu-

Address reprint requests to: Craig J. McClain, MD, Division of Gastroenterology, Hepatology and Nutrition, Department of Medicine, University of Louisville, Louisville, KY 40202. E-mail: [email protected] Irina A. Kirpich has no conflict of interest to declare; Craig J. McClain serves as an advisory consultant and/or a speaker for Abbott, Ocera, Gilead, Baxter, Genentech, Merck, GlaxoSmith-Kline, and Nestle. Abbreviations: ALD ¼ alcoholic liver disease, ALT ¼ alanine aminotransferase, AST ¼ aspartate aminotransferase, IL-1 ¼ interleukin 1, IL-6 ¼ interleukin 6, LGG ¼ rhamnosus Gorbach-Golding, LPS ¼ lipopolysaccharide, MCD diet ¼ methionine- deficient diet, MHE ¼ minimal hepatic encephalopathy, NAFLD ¼ nonalcoholic fatty liver disease, NASH ¼ nonalcoholic steatohepatitis, NF-kB ¼ nuclear factor kB, NKT cells ¼ natural killer T cells, PAMPs ¼ pathogen-associated molecular patterns, SIBO ¼ small intestinal bacterial overgrowth, TJ ¼ tight junctions, TLRs ¼ toll-like receptors, TLR-4 ¼ toll-like receptor-4, TNF-a ¼ tumor necrosis factor-alpha, WHO/FAO ¼ World Health Organization/Food and Agriculture Organization of the United Nations, ZO-1 ¼ zonula occludens-1.

Journal of the American College of Nutrition, Vol. 31, No. 1, 14–23 (2012) Published by the American College of Nutrition

14 Probiotics in the Treatment of the Liver Diseases

Fig. 1. Gut-liver axis. Under the certain circumstances gut-derived, pathogen-associated molecular patterns (PAMP), including lipopolysaccharides, lipopeptides, unmethylated DNA, and double-stranded RNA, translocate from the gut into the portal vein and to the liver, where they are recognized by specific recognition receptors, the toll-like receptors (TLRs), resulting in initiation of innate immune response. LPS ¼ lipopolysaccharide. tion of the intestinal microbiota along the healthy gut, and this can, bacterial DNA), also termed pathogen-associated molec- is being actively studied in humans with different lifestyles, ular patterns (PAMPs), into the circulation. The intestinal ages, and diseases [4,6,7]. microbiota comprise more than 99% of the bacterial mass in the The commensal microflora are involved in several biolog- body and are the principal source of bacterially derived PAMPs ical functions, including defense against pathogens and in health and in many disease states. The liver is the major maintaining mucosal immune homeostasis and intestinal target of PAMPs, which are recognized by specific receptors, barrier integrity. There is a growing appreciation for the the toll-like receptors (TLRs). The healthy liver contains low potential of microorganisms to influence host health. Normally, messenger RNA (mRNA) levels of TLRs and their adaptor- commensal microbes and their host benefit from a mutually molecules (e.g., CD14, MD-2, and MyD88) as compared with symbiotic relationship. However, intestinal microbiota may other organs, suggesting that the low expression of TLR play an essential role in a variety of diseases, including liver signaling molecules may contribute to the high tolerance of the pathology. An extensive body of evidence, mostly derived liver to the gut-derived TLR ligands [8]. Therefore, TLRs from animal studies, suggests that disruption of the equilibrium represent a critical link between the changes in intestinal of the otherwise symbiotic three-way partnership among gut microflora, blood endotoxemia, and liver damage (Fig. 1). microflora, intestinal epithelium, and the immune system is involved in the development and progression of both NAFLD NAFLD: Intestinal Microflora and Low-Grade and ALD. Inflammation Nonalcoholic fatty liver disease is currently the most common liver disease worldwide, in both adults and in GUT-LIVER AXIS IN THE children, and is considered to be the hepatic manifestation of PATHOGENESIS OF THE LIVER the metabolic syndrome. NAFLD includes a spectrum of DISEASES pathologies from simple steatosis to nonalcoholic steatohepa- titis (NASH), with potential progression to cirrhosis over time. Intestine Pathogen-Associated Molecular Pattern: NAFLD generally is associated with obesity, insulin resistance, Hepatic Toll-Like Receptor System and diabetes. The most recent studies have suggested that the Gut flora alterations consist of changed microbiota contribution of the intestinal microflora to the development of equilibrium, bacterial overgrowth, and release of gut-derived obesity and obesity-associated fatty liver is, at least in part, products (e.g., bacterial lipopolysaccharide [LPS], peptidogly- based on the involvement of gut microbiota in the regulation of

JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION 15 Probiotics in the Treatment of the Liver Diseases energy homeostasis [9] and generation of low-grade metabolic flammatory cytokines. In addition, severe intestinal mucosa endotoxemia [10,11]. Microflora also play a role in the damage was observed in the progression from fatty liver to development of NAFLD via increased production of endoge- steatohepatitis in a rat model of NASH [17]. nous ethanol, observed in ob/ob mice, and a subsequent Small intestinal bacterial overgrowth (SIBO) and bacterial increase in hepatic oxidative stress [12]. translocation, as well as increased and An elegant series of experiments from Gordon’s group [9] endotoxemia are well-recognized factors contributing to the have demonstrated that the gut microbiota are involved in progression from liver steatosis to steatohepatitis and liver harvesting energy from the diet and ectopic fat deposition, cirrhosis in clinical settings. Wigg et al. [18] reported a evidencing its role in metabolic diseases. A 2-week conven- significantly higher prevalence of SIBO accompanied by tionalization of germ-free mice with ‘‘normal’’ gut microbiota increased TNF-a levels in NASH patients compared with harvested from the cecum of ‘‘normal’’ mice produced a 60% age- and sex-matched controls. However, this study did not increase in body fat content, insulin resistance, and a twofold demonstrate a statistically significant difference in intestinal increase in hepatic triglyceride content [9]. Administration of permeability or endotoxin levels in NASH patients with or the cecal microbiota from ob/ob mice to germ-free wild-type without SIBO [18]. The susceptibility to gut leakiness in recipients resulted in modest fat gain by these mice, and NASH patients with elevated endotoxin levels was observed in extraction of more calories from their food compared to the the study by Farhadi et al. [19] using the aspirin-induced lean mice having received the gut microbiota from lean donors intestinal barrier dysfunction test. In a recent study by Miele et [13]. Ley et al. [14] found that ob/ob mice have a 50% al. [20], NAFLD subjects demonstrated both increased gut reduction in Bacteroidetes and a proportional increase in permeability and increased prevalence of SIBO. In addition, the Firmicutes. These changes indicate that, in this model, obesity levels of bacterial overgrowth were correlated with the severity alters gut microbial ecology and suggest that manipulation of of steatosis, supporting the theory that such disturbances may intestinal microflora may be useful for regulating energy facilitate an increased absorption of endotoxin from the gut balance in an obese individual. [20]. Harte et al. [21] reported considerably increased Several recent studies from Belgium (Cani et al. [15]) have endotoxin levels in NAFLD patients, with a marked increase demonstrated that high-fat feeding also changes the intestinal in the early stage of fibrosis compared with healthy controls. microflora composition. The population levels of Bifidobacte- The authors [21] suggest that endotoxin levels may represent rium spp and Eubacterium rectale/Clostridium coccoides an important early marker of potential liver disease. group were significantly reduced in animals fed a high fat diet vs mice receiving the standard high carbohydrate diet. These ALD: Gut Endotoxin (LPS)-Mediated Liver Injury events were accompanied by a significant increase in plasma Alcohol abuse is one of the leading causes of alcoholic ‘‘ LPS levels, which the authors defined as a metabolic liver diseases and a major cause of morbidity and mortality in ’’ endotoxemia, increased liver fat accumulation, and expression the United States and worldwide. The gut-liver axis and the of the hepatic inflammatory factors, including tumor necrosis role of gut-derived endotoxin, LPS, are well recognized in a factor-alpha (TNF- ), interleukin 1 (IL-1), interleukin 6 (IL-6), ALD. One of the first experimental studies demonstrating the and plasminogen activator inhibitor-1 (PAI-1) [11]. The involvement of the microflora and the gut-liver interactions in negative correlation between metabolic endotoxemia and ALD was the study by Nanji et al. [22], which showed that Bifidobacterium spp observed by the authors suggests that Lactobacillus (species GG) treatment reduced endotoxemia the modifying gut microbiota may prevent diet-induced and severity of experimental alcoholic liver disease. Further- endotoxemia, obesity, and obesity-associated liver steatosis. more, Adachi et al. [23] reported that treatment with antibiotics Normally, the intestinal epithelium acts as a continuous to sterilize the gut and eliminate the source of endotoxin could barrier to prevent LPS translocation; however, under certain prevent alcohol-induced liver injury in rats. It is now generally conditions intestinal barrier integrity can be altered, resulting in accepted that LPS-mediated hepatocellular damage plays a increased gut permeability to LPS and other gut-associated crucial role in the pathogenesis of ALD [1,2,24]. Numerous endotoxins. Studies by Cani et al. [15] demonstrate that dietary clinical studies [25–27] have demonstrated that the plasma fat can significantly modulate gut barrier integrity via reduced endotoxin levels are significantly elevated in patients with mRNA expression of the intestinal tight junction (TJ) proteins, different stages of ALD—fatty liver, hepatitis, and cirrhosis— mainly zonula occludens-1 (ZO-1) and occludin. Studies from when compared with healthy control subjects. Ethanol-induced our group suggest specific types of fat induce these responses endotoxemia observed in experimental rodent models of ALD [16]. Furthermore, in the study by Brun et al. [3] ob/ob and db/ also supports the essential role of LPS in alcoholic liver injury db mice displayed a low intestinal resistance and profoundly [2,28–30]. modified distribution of TJ proteins (ZO-1 and occludin) in the Alcoholic endotoxemia is a multifactorial event, with intestinal mucosa, increased intestinal permeability, elevated altered microflora and impaired intestinal integrity among the portal endotoxemia, and elevated levels of circulating proin- causal factors. It has been shown that alcohol promotes the

16 VOL. 31, NO. 1 Probiotics in the Treatment of the Liver Diseases overgrowth of gram-negative in the intestines of To summarize, the intestinal microflora and the gut- patients with chronic alcohol abuse [31,32]. Our group [33] has associated endotoxemia play an important role in the patho- recently also demonstrated in a clinical study that chronic genesis of both NAFLD and ALD (Fig. 2). The presence of LPS alcohol consumption altered microflora composition, with in the systemic circulation results in the activation of the innate decreased numbers of bifidobacteria and lactobacilli in human immune system and a massive secretion of proinflammatory alcoholics. The most recent study by Yan et al. [34] cytokines, particularly TNF-a. In the liver, LPS stimulates demonstrated intestinal bacterial overgrowth of both aerobic different cell types to release cytokines, chemokines, and and anaerobic bacteria after 3 weeks of intragastric alcohol reactive oxygen species via toll-like receptor-4 (TLR-4)- feeding in an animal model of ALD. The authors observed that mediated mechanisms [47]. Kupffer cells are among the first hepatic steatosis and steatohepatitis occur at a similar time as cells that respond to LPS and are considered to be the primary translocation of live bacteria to the systemic circulation. macrophages involved in the clearance of gut-derived bacteria Qualitative changes of the microbiota following alcohol or bacterial toxins. Increased production of TNF-a and other administration, characterized by a decrease in Firmicutes, and proinflammatory cytokines/chemokines by Kupffer cells plays a an increase in Bacteroidetes and Verrucomicrobia was noted crucial role in the hepatic immune response to gut-derived also. Lactobacillus was strongly suppressed and almost absent endotoxins and liver pathology [48]. Although the focus has in mice fed alcohol compared with control-fed animals. It is been on LPS and TLR4, clearly other gut-derived toxins/ligands interesting that the lowest levels of Reg3b and Reg3g, host and other TLRs likely play a role in ALD [16,49]. antimicrobial proteins, were observed in the proximal small intestine, where the bacterial overgrowth was most pronounced and luminal alcohol concentrations were highest. Recent data PROBIOTICS, PREBIOTICS, AND from our group show marked downregulation of the critical SYMBIOTICS mucous stabilizing protein—intestinal trefoil factor—in mice chronically fed alcohol [35]. This deregulation of the mucosal The concept of probiotics, a term derived from the Greek innate immune system demonstrates a novel link between and meaning ‘‘for life,’’ was introduced at the beginning of 20th alcohol and enteric dysbiosis. Alterations of a mucosa- century. Russian Nobel laureate Elie Metchnikoff, in his 1907 associated microbiota in the colon (intestinal dysbiosis) were work The Prolongation of Life: Optimistic Studies, proposed also found in a study by Muflu et al. [36] after daily alcohol the idea that ingesting microbes could have beneficial effects consumption for 10 weeks. for human beings, especially to treat digestive diseases [50]. Alcohol and its metabolite, acetaldehyde, induce intestinal The World Health Organization and the Food and Agriculture permeability to various macromolecules including LPS in both Organization of the United Nations (WHO/FAO, 2001) defines human subjects and animal models of ALD [26,37–39]. probiotics as live microorganisms that, when consumed in Translocation of LPS across the gut epithelial barrier has adequate amounts, confer a health benefit to the host. By this recently been attributed to the disruption of the intestinal barrier definition, a native bacteria species is not a until the integrity. Recently, Keshavarzian’s group has demonstrated bacteria is isolated, purified, and proven to have a health benefit decreased ZO-1 protein levels from sigmoid colon biopsies of when administered. human alcoholics compared with healthy controls. This was Probiotics have potential as prevention strategies or attributed to an increase in miRNA-212 expression observed in therapies for multiple gastrointestinal diseases, including liver alcoholic subjects compared with controls [40]. This group [41– diseases of different etiologies. Current evidence indicates that 44] also reported that alcohol-induced oxidative stress and varying probiotic strains mediate their effects by a variety of generation of nitric oxide in the intestine of experimental different mechanisms (carefully reviewed in several recent animals led to loss of TJ integrity, gut leakiness, endotoxemia, publications [51,52]). Although the exact mechanisms of hepatic inflammation, and liver injury. Results from our group beneficial effects of probiotics on the gut-liver axis are not [30,45] have also shown increased intestinal permeability in yet fully elucidated, many of the favorable therapeutic effects experimental animal models of ALD due to redistribution and of probiotics may result from (1) modulation of the intestinal decreased expression of intestinal TJ proteins. The study by microflora composition and antibacterial factor production; (2) Ferrier et al. [24] demonstrated that an acute and moderate modification of intestinal epithelial permeability and function; ethanol intake can alter the epithelial barrier through ethanol and/or (3) modulation of the immune system at both local and oxidation to acetaldehyde by the colonic microflora and systemic levels. downstream mast cell activation. Increased intestinal production The most commonly used microorganisms are within the of proinflammatory cytokines, such as TNF-a and IL-6, can also genera Bifidobacteria and Lactobacillus due, at least in part, to contribute to alcoholic endotoxemia by altering TJ morphology their traditional role in fermentable dairy products such as and distribution, thereby creating a self-perpetuating vicious and their resistance to gastric acid, bile salts, and cycle that can amplify bacteria translocation [46]. pancreatic enzymes in the upper gastrointestinal tract. A

JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION 17 Probiotics in the Treatment of the Liver Diseases

Fig. 2. The mechanism of alcohol and high-fat diet-induced liver injury via TLR-4-mediated recognition of LPS. Alcohol and high fat diet lead to changes in bacterial species and overgrowth of gram-negative bacteria in the intestine. These factors contribute to increased gut permeability to LPS and increased endotoxemia in the portal circulation. LPS is taken up by Kupffer cells via a TLR4-mediated mechanism. In response to LPS, Kupffer cells produce a significant amount of proinflammatory cytokines, resulting in liver injury. probiotic preparation may contain one or several different models of NAFLD. The most studied probiotic used in high-fat strains of microorganisms. Given that probiotic strains exert diet-induced obesity, fatty liver, and insulin resistance animal their effects through a variety of mechanisms that are unique models is a VSL#3 mixture (a multi-strain preparation for each strain, the choice of strain or combination of strains is composed of Bifidobacterium longum, Bifidobacterium breve, crucial for therapeutic success. Bifidobacterium infants, , Lactobacillus Of relevance, prebiotics and symbiotics can also be used as plantarum, Lactobacillus acidophilus, Lactobacillus del- tools to modulate the gut microbiota. Prebiotics are non- brueckii subsp bulgaricus, and Streptococcus thermophilus). digestible food ingredients that beneficially affect the host by Several mechanisms for the beneficial effects of VSL#3 on selectively stimulating growth and/or modifying the metabolic NAFLD have been proposed. VSL#3 supplementation for 4 activity of selected intestinal bacteria [53]. Inulin and fructo- weeks improved high-fat diet-induced liver steatosis and oligosaccharides are well studied dietary fibers, and insulin resistance in mice by modulation of hepatic natural they stimulate growth of health-promoting Bifidobacterium spp killer T cells (NKT cells) and suppression of the TNF-a/IKK-b and Lactobacillus spp species [54]. A probiotic can be signaling pathway. Moreover, VSL#3 exhibited anti-inflam- combined with a prebiotic to form a symbiotic. The concept matory activity through a reduction of nuclear factor kB (NF- of prebiotics and symbiotics has primarily the same aim as kB) activation, decreasing hepatic TNF-a production, and probiotics—to improve host health via modulation of the cyclooxygenase and inductible nitric oxide synthase expression intestinal microflora. [55]. Four weeks of VSL#3 treatment attenuated high-fat diet- induced oxidative and inflammatory liver damage in the experimental rat model of NASH [56]. It has been shown that PROBIOTIC TARGETS: WHAT WE VSL#3 treatment decreased serum alanine aminotransferase (ALT) levels, increased insulin sensitivity, and improved LEARN FROM EXPERIMENTAL hepatic inflammation by reducing the activity of Jun N- ANIMAL MODELS OF LIVER DISEASES terminal kinase and decreasing the DNA-binding activity of NF-kB in ob/ob mice [57]. In a methionine-choline deficient Probiotics and Experimental NAFLD (MCD) diet-induced mouse model of NASH, 9 weeks of Evidence of the beneficial effects of probiotics, prebiotics, VSL#3 supplementation prevented liver fibrosis without and symbiotics on the liver is extensively derived from rodent significant attenuation of the hepatic steatosis. However,

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VSL#3 did not reduce MCD diet-induced endotoxemia in this and ameliorate alcoholic steatohepatitis, and these events were model [58]. associated with partial restoration of host antimicrobial Reg3g Several strains of Lactobacillus have shown protective protein levels. effects on NAFLD. Eight weeks of oral administration of Lactobacillus rhamnosus PL60 showed antiobesity effects and improved liver steatosis in a mouse model of diet-induced PROBIOTICS IN THE TREATMENT OF obesity [59]. Lactobacillus acidophilus and Lactobacillus casei THE LIVER DISEASES administration for 8 weeks demonstrated antioxidative effects in the liver and pancreatic tissues in high-fructose diet-fed mice Despite substantial experimental data demonstrating bene- [60]. Wang et al. [61] have reported that 5 weeks’ ficial effects of probiotics in the animal models of NAFLD and administration of Lactobacillus plantarum MA2 decreased ALD, only a few clinical studies have reported favorable both liver cholesterol and triglycerides in rats fed a cholesterol- effects of probiotics in human liver diseases. There is some enriched diet, and their regimen also increased the number of promising evidence that probiotics might have efficacy in the fecal lactobacilli and bifidobacteria. clinical settings, but more studies are needed. F19 significantly attenuated liver injury induced by ischemia- reperfusion and a methionine/choline-deficient diet in rats by Probiotics and Clinical NAFLD and ALD restoring gut microbiota and reducing inflammation and steatosis [62]. Of note, administration of different Lactobacil- A preliminary study by Loguercio et al. [67] using lactic lus and Bifidobacterium strains in an acute liver injury rat acid bacteria in 10 patients with NASH and 10 patients with model has shown different effects on bacterial translocation alcoholic cirrhosis demonstrated statistically significant im- and hepatocellular damage [63], further supporting the provement of liver function, as assessed by reduction of ALT a importance of careful evaluation of probiotic strains in levels, and a reduction in TNF- levels was also observed. This therapeutic use. group, in an uncontrolled study using VSL#3 preparation, demonstrated improvement in serum liver tests (reduction in ALT and AST) and reduction of proinflammatory cytokines in Probiotics and Experimental ALD patients with NAFLD and alcoholic cirrhosis. This study also Our current knowledge about the favorable effects of demonstrated reduction in the lipid peroxidation markers, probiotics, prebiotics, and symbiotics in ALD is mostly malondialdehyde, and 4-hydroxynonenal in NAFLD and obtained from experimental studies. Lactobacillus rhamnosus alcoholic cirrhotic patients as compared with noncirrhotic Gorbach-Golding (LGG) is the most frequently used probiotic chronic hepatitis C virus patients [68]. in the animal models of ALD. One of the first studies An open-label study by Stadlbauer et al. [69] showed that demonstrating favorable effects of LGG in ALD was the study administration of Lactobacillus casei Shirota for 4 weeks by Nanji et al. [22] showing significantly reduced endotoxemia restored neutrophil function in alcoholic cirrhotic patients and and severity of experimental ALD by LGG supplementation. suggested that this bacteria is safe to treat patients with Pretreatment with LGG attenuated the suppressive actions of defective immunity, as shown by the lack of any adverse ethanol on the mucus-secreting layer and transmucosal effects and the fact that markers of inflammation/infection resistance [64]. Another study [65], using the same probiotics, remained unchanged over the study period. Lata et al. [70], in a demonstrated that LGG treatment significantly ameliorated double-blind randomized study, reported an improvement of liver injury in a rat model of alcoholic steatohepatitis, reduced liver function, intestinal (colonic) colonization and a trend alcohol-induced gut leakiness, and blunted oxidative stress and toward reduced endotoxin levels in blood in patients with inflammation in both intestine and liver. The same group [36] alcoholic cirrhosis after probiotic Escherichia coli Nissle also reported that daily alcohol consumption for 10 weeks intervention for 42 days. altered colonic mucosa-associated bacterial microbiota compo- Our group [33] also recently demonstrated that after 5 days sition in rats, which was prevented by both oats and LGG of probiotic therapy with Bifidobacterium bifidum and supplementation. Notably, the beneficial effects of probiotics Lactobacillus plantarum 8PA3, alcoholic patients had signif- were achieved not only by live bacteria but also by heat- icantly increased numbers of both bifidobacteria and lactoba- inactivated, nonviable bacteria, as demonstrated in a study by cilli compared with the standard therapy arm. Despite similar Segawa et al. [66]. Thus, oral administration of heat-killed values at study initiation, patients treated with probiotics had Lactobacillus brevis SBC8803 ameliorated ethanol-induced significantly lower AST and ALT activity at the end of liver injury. The suggested mechanism involves induction of treatment than those treated with standard therapy alone. In a cytoprotective heat shock proteins and enhancement of subgroup of patients with well-characterized mild alcoholic intestinal barrier function. hepatitis, probiotic therapy was associated with a significant Prebiotics (fructo-oligosaccharides) have recently been end-of-treatment reduction in ALT, AST, lactate dehydroge- reported by Yan et al. [34] to reduce bacterial overgrowth nase, and total bilirubin. In this subgroup, there was a

JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION 19 Probiotics in the Treatment of the Liver Diseases significant end-of-treatment mean ALT reduction in the probiotic arm versus the standard therapy arm (Fig. 3). Therefore, short-term oral supplementation with Bifidobacte- rium bifidum and Lactobacillus plantarum 8PA3 was associ- ated with restoration of the bowel flora and greater improvement in alcohol-induced liver injury than standard therapy alone [33].

Probiotics and Liver Diseases of Different Etiologies A study by Liu et al. [71] demonstrated beneficial effects of a symbiotic preparation (known as Cocktail 2000) consisting of 4 types of bacteria (Pediacoccus pentoseceus, Leuconostoc mesenteroides, Lactobacillus paracasei, and Lactobacillus plantarum) along with fermentable fibers (glucan, inulin, pectin, resistant starch) on hepatic inflammation and overall liver function in patients with cirrhosis (viral hepatitis, mostly Fig. 3. Probiotic therapy plus alcohol cessation, but not alcohol hepatitis B). Symbiotic treatment was also associated with a cessation alone, was associated with an end of treatment ALT reduction significant reduction in endotoxemia [71]. In a clinical trial in patients with mild alcoholic hepatitis. * p , 0.05. conducted by Zhao et al. [72], two different probiotic preparations (Bifidobacterium þ Lactobacillus acidophilus þ patients with baseline ammonia levels .50 mmol/L, the Enterococcus and Bacillus subtilis þ Enterococcus faecium) administration of probiotics significantly reduced the ammonia given for 14 days to patients, with mainly viral hepatitis B and levels starting after 1 month of treatment. However, this effect C, increased the bifidobacteria count and reduced fecal pH, as diminished and lost its significance compared with the placebo well as fecal and blood ammonia. group. Recently, Bajaj et al. [73] in a randomized, controlled trial, demonstrated that probiotic yogurt supplementation containing Streptococcus thermophilus, Lactobacillus bulgaricus, Lacto- bacillus acidophilus, Bifidobacteria, and Lactobacillus casei CONCLUSION over 60 days can reverse minimal hepatic encephalopathy (MHE) in patients with nonalcoholic cirrhosis. The data In conclusion, the gut-liver axis plays a critical role in the suggest that, in addition to lactulose and antibiotics, dietary pathogenesis of liver diseases. Although the potential patho- intervention with probiotic yogurt can be an effective strategy genic role of the intestinal microflora and the therapeutic effects for MHE therapy [73]. A randomized, double-blind, placebo- of probiotics in liver pathology are still debatable and require controlled study conducted by Malaguarnera et al. [74] further investigation, the growing evidence suggests that demonstrated improvement in biochemical and neuropsycho- restoration of bowel flora is a rational novel target in the logical tests in 30 patients with MHE treated for 3 months with treatment of both NAFLD and ALD and possibly liver diseases Bifidobacterium longum and fructo-oligosaccharide compared of other etiologies. with the placebo group. This group [75] also reported that the same probiotics administered for 2 months resulted in reduced blood ammonia levels and improved psychometric tests in 35 ACKNOWLEDGMENTS patients with hepatitis B virus, 70 patients with hepatitis C virus, and 20 patients with cryptogenetic cirrhosis. This work was supported by National Institutes of Health Nevertheless, it is important to note that probiotic grants P01 AA017103 (C.J.M.), R01 AA0015970 (C.J.M.), administration in liver diseases is not always beneficial. For R01 AA018016 (C.J.M.), R01 DK071765 (C.J.M.), R37 example, Pereg et al. [76], in a double-blind placebo-controlled AA010762 (C.J.M.), R01 AA018869 (C.J.M.), P30 study that included patients with compensated liver cirrhosis (a AA019360 (C.J.M.), RC2AA019385 (C.J.M.), and the De- total of 36 patients distributed equally between the probiotic partment of Veterans Affairs (C.J.M.). and placebo groups), reported that administration of probiotic capsules containing Lactobacillus acidophilus, Lactobacillus bulgaricus, Bifidobacterium lactis, and Streptococcus thermo- REFERENCES philus for a period of 6 months was not associated with significant differences in either clinical or laboratory parame- 1. Purohit V, Bode JC, Bode C, Brenner DA, Choudhry MA, ters between the two groups. Of note, in the subgroup of Hamilton F, Kang YJ, Keshavarzian A, Rao R, Sartor RB,

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