To date, the only effective strategy for the treatment of NAFLD and NASH is the loss of at least 5% of body weight in overweight and/or obese subjects, although many therapeutic approaches have been proposed. However, so far, no drugs have been approved for the treatment of NASH, no specific treatments can be recommended, and any drug treatment is off label [
61].
4.1. Therapeutic Approach to Reducing Lipopolysaccharides in NAFLD
Few studies have been performed to assess whether LPS serum levels could be affected by dietary interventions.
Dietary patterns reflecting healthy food choices seem to be associated with lower serum LPS activity and a Mediterranean diet pattern, rich in unsaturated fats and fiber, has been suggested as a dietary strategy to reduce endotoxemia [
62]. In fact, in a nutritional survey of 668 individuals with type 1 diabetes in the Finn Diane Study, healthy dietary choices, such as the consumption of fish, fresh vegetables, and fruits and berries, were associated with reduced systemic endotoxemia [
63]. Moreover, placing eight healthy subjects on a Western-style diet for 1 month induced a 71% increase in plasma levels of endotoxin activity, whereas a prudent diet reduced levels by 31% [
64]. In a further study, the consumption of a low-fat high-carbohydrate diet enriched in n-3 polyunsaturated fatty acids (PUFAs) for three weeks reduced the LPS fasting plasma (0.24 ± 0.01 EU/mL) levels when compared with a Mediterranean diet (0.38 ± 0.06 EU/mL) and a high saturated fat diet (0.35 ± 0.03 EU/mL) in twenty older adults [
65].
Postprandial endotoxemia, after meals with different fatty acid compositions, has been evaluated in several studies. Overall, these studies show increased LPS serum levels after high saturated fat meals, suggesting the different effects of dietary fats on the regulation of the intestinal epithelial endotoxin transport [
66] and the postprandial low-grade inflammation [
67,
68]. However, the link between dietary patterns, intestinal microbiota, sub-clinical inflammation, and endotoxemia is still debated, although most evidence shows a positive association with high-fat diets [
69]
Regulating gut flora with probiotics or prebiotics has become a new approach used to prevent and treat several metabolic diseases, such as NAFLD. Probiotic bacteria are known to reduce pathogenic bacterial growth and restore the integrity of the intestinal barrier against LPS-induced epithelial toxicity [
70].
Previous data showed that serum LPS, liver TLR4-mRNA, and serum inflammatory cytokines in a probiotics intervention group were all significantly decreased compared to that in the NAFLD model group. Additionally, the degree of liver steatosis and inflammatory cell infiltration in the intervention group was also reduced relative to the model group; based on these results, it was speculated that probiotics may delay the process of NAFLD by inhibiting the LPS–TLR4 signaling pathway [
70]. In a narrative review, Eslamparast and colleagues summarized the studies that show that probiotic supplementation in animal models and human studies improve the inflammatory status and clinical manifestations in NAFLD [
71]. Recently, the dose-dependent effects of multispecies probiotic supplementation on the serum LPS levels and cardiometabolic profile in obese postmenopausal women were demonstrated [
72].
Many antibiotics have regulatory effects on intestinal microbiota and are of benefit to NAFLD [
73]. For example, oral treatment with Cidomycin was found to promote the small intestine transit rate and reduce serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and tumor necrosis factor alpha (TNF-α) in a NASH mouse model, indicating the potential of Cidomycin in alleviating the severity of NASH via intestinal microbiota modulation [
74]. Rifaximin, which is a largely water-insoluble and nonabsorbable (<0.4%) drug, has been shown to exert antimicrobial activity against enteric bacteria, such as
Streptococcus,
Bacteroides, and
Citrobacter [
75]. Gangarapu et al. have demonstrated that a short-term administration of rifaximin (1200 mg/day for 28 days) improved the clinical status of patients with NAFLD/NASH, which was associated with reduced serum transaminases and circulating endotoxins [
76]. Abdel-Razik et al. reported that after rifaximin therapy (1100 mg/day for 6 months), patients with NASH showed significantly reduced levels of proinflammatory cytokines, ALT, and NAFLD-liver fat score [
77]. However, in an open-label clinical trial, rifaximin administration (800 mg/day for 6 weeks) was not effective for humans with NASH [
78]. The inconsistency may be due to the small sample size, the relatively low treatment dose, or the short duration of the clinical study.
4.2. Therapeutic Approach to Reducing Oxidative Stress in NAFLD
Current evidence generally supports the benefits of a healthy diet on oxidative stress and chronic diseases. In fact, oxidative stress can be triggered by a high-level consumption of several macronutrients. Therefore, saturated fatty acids, omega-6 PUFA, and glucose may induce inflammation through nuclear factor NF-κB-mediated pathways [
79]. Furthermore, diets rich in saturated fat, cholesterol, trans-fatty acids, and increased fructose can induce oxidative stress in NAFLD. In contrast, a protective role is played by the high consumption of plant-based foods (whole grains, cereals, seeds, nuts, legumes, vegetables, and fruits), and low consumption of meat, milk, and dairy products.
A Mediterranean diet (MD) is the best suggested dietary approach for the treatment of NAFLD according to guidelines [
61]. Several dietary intervention trials have reported the favorable effects of MD on a variety of oxidative stress biomarkers reflecting different sources of oxidative stress (e.g., 8OHdG, malondialdehyde, and F2-iso-prostanes). Most studies showed reduced levels of oxidative stress in subjects treated with an MD [
80,
81,
82,
83,
84,
85]. In contrast, in a large, cross-sectional study of healthy women, healthy eating patterns were not associated with a lower level of oxidative stress, which was measured using fluorescent oxidation products as global markers of oxidative stress [
86]. A large body of research has also investigated the potential beneficial effect of dietary antioxidants, such as olive oil, a greater ratio of omega-3/omega-6 PUFAs, polyphenols, carotenoids, and high-fiber foods, which may ameliorate oxidative stress in NAFLD [
87,
88,
89].
The mechanism by which oxidative stress could be reduced following n-3 PUFA supplementation is still unresolved. The effect could be related to the immuno-modulatory and anti-inflammatory properties of n-3 PUFAs and to their ability to increase antioxidant enzymes, which could contribute to reducing the generation of ROS and other oxidative stress agents [
90]. However, the differences in the treatment duration of randomized trials, doses, biomarkers for assessing oxidative stress make it difficult to form conclusions about the effectiveness of n-3 PUFAs at reducing oxidative stress. In a recent meta-analysis, data suggest that n-3 PUFA supplementation may be effective in the early stages of NAFLD, but not in patients with more severe NAFLD or NASH [
91].
For patients with less severe NAFLD, several types of nutraceuticals have been suggested, mostly with antioxidant effects. In fact, anti-oxidative therapy using natural antioxidants represents a reasonable therapeutic approach for the prevention and treatment of liver diseases due to the role of oxidative stress in contributing to the initiation and progression of hepatic damage. The results of our previously reported studies showing increased systemic oxidative stress [
50] and decreased vitamin E serum levels [
52] in subjects with NAFLD suggest that early antioxidant treatment is beneficial, even in patients with simple fatty liver disease.
Vitamin E appears to be effective for the treatment of nondiabetic subjects with advanced NASH. In fact, in the “PIoglitazone versus Vitamin E versus placebo for the treatment of non-diabetic patients with Nonalcoholic Steatohepatitis (PIVENS) study”, vitamin E (800 IU/day) improved steatosis, inflammation, and ballooning and induced the resolution of NASH in 36% of patients (21% in the placebo arm) [
92]. However, the high suggested daily dosages are a matter of concern due to the results of different metanalyses showing increased all-cause mortality and increased relative risks for hemorrhagic stroke [
93] and prostate cancer [
94] in those treated with high vitamin E dosages (higher than 400 IU/day). The PIVENS findings were not confirmed in 58 children and adolescents where vitamin E, supplied at 800 IU/day for 96 weeks, was not superior to a placebo, neither in attaining a sustained reduction in ALT levels or improving the liver histology [
95]. Furthermore, Nobili et al. found no benefit due to vitamin E supplementation in a cohort of 53 children and adolescents with NAFLD [
96].
Several other therapeutic approaches have been proposed to reduce oxidative stress. There are insufficient data to support the use of vitamin C in NAFLD patients, although vitamin C intake was reported to be inversely associated with the severity of NAFLD [
97]. Moreover, polyphenols may present hepatoprotective effects because they reduce liver fat accumulation by decreasing oxidative stress, insulin resistance, and inflammation, and increasing fatty acid oxidation [
98]. Silymarin is a valuable treatment option for liver diseases triggered by oxidative stress, such as alcoholic and non-alcoholic fatty liver disease and drug-induced liver disease. Silymarin may improve liver enzymes in the early stages of NAFLD. In a meta-analysis of 8 randomized clinical trials including 587 patients, Silymarin showed positive efficacy toward reducing transaminases levels in NAFLD patients [
99]. Long-term treatment may also decrease fibrosis and slow liver disease progression in patients with NASH [
100].
Thus, the influence of nutraceuticals on gut microbiota and oxidative stress in NAFLD patients has not yet been elucidated and there are insufficient data either to support or refuse the use of nutraceuticals to treat subjects with NAFLD.