These results showed that 12 weeks of probiotic treatment significantly reduced hepatic fat and BMI in obese NAFLD patients. To the best of our knowledge, this is the first randomized controlled trial to examine the effects of probiotics on hepatic fat content and hepatic fibrosis using a reliable technique.

Two randomized controlled trials examining the effects of probiotics in NAFLD have previously been published5,9. The primary endpoint of both studies was liver ALT levels; changes in hepatic fat content and fibrosis were not evaluated. Moreover, changes in intestinal microbiota were not evaluated after probiotic treatment in either of these studies.

We administered a mixture of probiotics (L. acidophilus, L. rhamnosus, L. paracasei, P. pentosaceus, B. lactis, and B. breve) for 12 weeks. To select probiotic strains, we performed in vitro and in vivo studies using 3T3-L1 cells and a high-fat diet animal model, respectively (data not shown). We screened more than 100 strains using our own platform, and finally selected 6 probiotic bacterial species. All but L. paracasei increased in stool samples, based on quantitative polymerase chain reaction testing. However, there was no direct association between these 6 probiotic species and clinical parameters (decrease in body weight and hepatic fat). Agathobaculum, Dorea (OTU 527923), Dorea (OTU 195044), Blautia, Ruminococcus, and Dorea (OTU 470168) were significantly increased after probiotic treatment, but not in the placebo group, and were associated with a decreased hepatic FF. Eubacterium, Fusicatenibacter, Dorea (OTU 195044), Oscillibacter, and Faecalibacterium were increased after probiotic treatment, but not in the placebo group, and were associated with decreased BMI. Dorea (OTU 195044) showed a positive correlation with improvement in NAFLD and obesity (Table 5). These data suggested that some probiotic agents may have a priming or triggering effect in the correction of dysbiosis rather than a direct effect related to decreased hepatic fat and weight loss, in association with a dramatic increase in the amount of probiotic species in the intestinal bacterial community. Probiotic species inhibit pathogens by producing lactic acids, promoting innate and adaptive molecular immunity, and causing changes in the host gut environment14,15. This may contribute to modulation of the intestinal flora through an increase in specific bacteria such as Agathobaculum, Dorea (OTU 527923), Dorea (OTU 195044), Blautia, Ruminococcus, and Dorea (OTU 470168). Future research should investigate the interaction between probiotic species and these strains.

Table 5 Changes in gut microbiome related to fatty liver decrease or increase by probiotic intervention. Full size table

Some findings of this study conflict with those of previous reports16,17. A recent study showed that Ruminococcus, Blautia, and Dorea were increased in pediatric nonalcoholic steatohepatitis (NASH) patients, relative to those in controls, and Oscillospira, Rikenellaceae, Parabacteroides, Bacteroides fragilis, Sutterella, and Lachnospiraceae were increased in NAFLD17. These results were similar to those in a subsequent “meta-omics” study in adult NASH patients, suggesting that Bacteroides and Ruminococcus were independently associated with NASH and significant fibrosis, respectively16. However, Zhu et al. reported that Blautia, Coprococcus, and Ruminococcus were significantly decreased in NASH patients1. These data suggest that environmental factors and age also affect changes in the microbiome. In addition, different species/subspecies may have a variable range of functions. Faecalibacterium prausnitzii is an anti-inflammatory commensal agent that is associated with obesity. Foditsch et al. and Balamurugan et al. reported that F. prausnitzii was associated with weight gain18,19. F. prausnitzii phylotypes are important in homeostasis in humans20. In this study, increases in 2 different strains of Dorea (OTU 527923 and 195044) were associated with decreased hepatic FF and BMI, and an increase in 1 strain of Dorea (OTU 1076587) was associated with weight gain. This suggests a wide range of effects associated with the same genus, depending on the species. Whether the increase in these strains is secondary to correction of intestinal dysbiosis or directly associated with fatty liver and obesity requires further research.

This study used MRI-PDFF, which is widely used in research on NAFLD, to quantify IHF21,22. This technique can accurately measure the distribution of IHF across the liver in seconds, and similar to magnetic resonance spectroscopy is considered very accurate. The decrease in IHF was 40.0% and 17.1% in the probiotic and placebo groups, respectively. There is no consensus on cut-off values for defining a change in hepatic fat. We assumed that a change in the MRI-PDFF value of more than 1.7% was significant. Nouredin et al. used MRI-PDFF to show that the actual change was 1%23. We applied stricter criteria by assuming that the changes in IHF measured with MRI-PDFF in several other studies represented actual tissue changes if the values were 1.6–1.8%24,25,26.

Patients received exercise and diet instruction. Caloric intake was slightly reduced in both groups, but no statistical significance was observed. Moreover, a difference in the number of dietary calories was not associated with a decrease in hepatic fat in the probiotic group. The association between diet and hepatic fat was thought to be small in this study. There was no difference in physical activity level between the 2 groups.

This study had some limitations. First, IHF was measured accurately using MRI-PDFF, but whether hepatic inflammation improved could not be determined. For this determination, liver biopsy was needed, but could not be performed. Second, IHF measured with MRI-PDFF in the probiotic group was decreased, while the controlled attenuation parameter (CAP) measured with transient elastography was not decreased. CAP is useful for measuring hepatic steatosis. However, MRI-PDFF is superior to CAP in diagnosing and measuring steatosis in patients with fatty liver27. Moreover, while baseline CAP and MRI-PDFF values are strongly correlated in assessments of hepatic steatosis, a weak correlation has been observed between CAP changes after treatment and intrahepatic fat changes (%) using MRI-PDFF in a longitudinal setting27,28. CAP measurement was performed with the M probe of a FibroScan instrument, but not the XL probe. Patients with BMI ≥ 28 kg/m2 should be assessed with an XL probe to reduce scan failures and to increase the reliability of hepatic steatosis measurement29. Eight patients in this study were excluded owing to unreliable measurement results29,30. Because of the small sample size, further large-scale investigation is needed; moreover, 12 weeks is insufficient for evaluation of hepatic fibrosis. More convincing MR elastography or liver biopsy and long-term follow-up data are needed. In addition, as the current microbiome database and 16S rRNA sequencing are limited in the ability to distinguish among gut bacteria when more than 1,000 species are present, culturomics and whole-genome sequencing will be necessary to identify specific bacteria associated with disease and to investigate the association between probiotic and other bacteria.

This study showed that administration of probiotics in obese patients with NAFLD for 12 weeks was effective in reducing triglycerides as well as IHF. However, with adjustment for weight changes, the IHF level was not different between treated and control groups. This means that a decrease in hepatic fat is mainly due to changes in body weight. However, the exact mechanism is not known. Further studies involving an analysis of intestinal flora and the metabolomics of intestinal bacteria are expected to identify the exact role of probiotics in patients with NAFLD.