*4.1. Therapies to Reduce Intestinal Permeability*

Studies examining intestinal permeability in ALD [24,53,54], NAFLD/NASH [67,70,164], and fibrotic liver disease/cirrhosis [86,87,165] have focused primarily on the reduction in TJ proteins such as ZO-1 and Claudin-1 [70,87,94], though the mechanisms by which this loss occurs remains largely unknown. Loss of intestinal mucus or mucosal IgA production [166] can also significantly increase intestinal permeability; however, these mechanisms have been largely overlooked in CLD.

Expansion of pathogenic bacterial species both within the colon and into the SIBO have been documented in CLD [70,167], motivating the examination of antibiotics and prebiotics as potential therapies. Antibiotic studies commonly use a broad spectrum and poorly absorbed antibiotics such as neomycin or rifaximin to achieve a gut-targeted intestinal decontamination. In rodent models of obesity [168], long-term ethanol exposure [169], NASH [170], and fibrosis [105], antibiotic treatment has been shown to reduce intestinal permeability and subsequent liver injury. In humans, a number of clinical trials are underway to assess the efficacy of antibiotics for the treatment of AH, NASH, and cirrhosis (reviewed in [171]). Early data suggests that short-term rifaximin treatment can reduce serum endotoxin and liver inflammation in NAFLD/NASH patients [172], whereas rifamixin prophylaxis over 24 weeks can significantly reduce hospitalization and mortality among cirrhotic patients [173]. These data support previous findings that rifaximin can reduce the risk of complications associated with cirrhosis, including hepatic encephalopathy, variceal bleeding, and SBP [174]. Importantly, antibiotics such as levofloxacin and metronidazole, but not rifaximin, can significantly increase gut proteases, thus contributing to intestinal permeability in both humans [175] and rats [176]. An antibiotic-mediated reduction in anti-proteolytic bacterial richness and abundance in the colon is thought to contribute to gut permeability.

Significant microbial perturbations caused by broad spectrum antibiotics have stimulated interest in probiotics (beneficial microbes) and prebiotics (beneficial microbial substrates) for the improvement of intestinal health. In mouse models of ethanol-induced liver injury, probiotic *Lactobacillus rhamnosus* strains have been shown to reduce serum endotoxin, hepatic oxidative stress, and inflammatory TNFα production [177,178]. While intestinal permeability was not assessed in these studies, Wang et al. demonstrated that *Lactobacillus* probiotics could prevent the alcohol-induced loss of intestinal ZO-1, Caludin-1, and Occludin-1, keeping the intestinal epithelium intact [179]. In rats, a similar reduction in ZO-1, intestinal permeability, steatosis, and fibrosis was observed in the choline-deficient/L-amino-acid-defined NASH diet, and was significantly attenuated by probiotic treatment with *Clostridium butyricum* MIYAIRI 588 strain. Interestingly, in mouse models of CCL4-induced fibrosis, different probiotics achieve improved intestinal permeability via unique mechanisms: *Bifidobacterium* probiotics have been shown to increase intestinal TJ expression [180] whereas *Lactobacillus-paracasei*-fermented milk reduces intestinal permeability by increasing antimicrobial β-defensin expression [181].

The study of prebiotics allows researchers to understand the relationship between a substrate, microbial metabolism, gut health, and permeability. The majority of prebiotics used today are indigestible carbohydrate polymers (fibers) that become fermented by gut bacteria to produce, among other things, short chain fatty acids (SCFAs) consisting of acetate, propionate, and butyrate [182]. SCFAs are significant homeostatic and anti-inflammatory signaling molecules in the gut (reviewed in Reference [183]), but can also significantly alter intestinal permeability. In vitro, butyrate has been shown to increase ZO-1 expression in Caco-2 cells [184], and stimulate mucous secretion in E12 human colon cells [185]. In vivo, rats supplemented with oats rich in fermentable β-glucans were significantly more resistant to alcohol-induced oxidative stress and intestinal permeability [186]. In humans, increased fiber intake has also been associated with improved permeability (reduced circulating ZO-1) and reduced ALT/AST in NALFD patients [187]. These data are supported by two recent meta-analyses finding that both prebiotics and probiotics reduce liver enzymes ALT, AST, and GGT in NAFLD patients [188], with probiotics also reducing serum ammonia and hepatic encephalopathy in cirrhotic patients [189].
