Increase of Akkermansia muciniphila by a Diet Containing Japanese Traditional Medicine Bofutsushosan in a Mouse Model of Non-Alcoholic Fatty Liver Disease

Abstract

Non-alcoholic fatty liver disease (NAFLD) is considered a worldwide healthcare problem that mirrors the increased prevalence of obesity. Gut microbiota plays a crucial role in the progression and treatment of NAFLD. Bofutsushosan (BTS), a pharmaceutical-grade Japanese traditional medicine, has long been prescribed in Japan for obesity and obesity-related syndrome. Although BTS has been reported to exert an anti-obesity effect in obese patients as well as various obesity-model animals, its effect on gut microbiota is unknown. Here, the effects of BTS on obesity, liver damage, and the gut microbiome in genetically obese mice, ob/ob, were studied. Seven-week-old ob/ob mice were fed a standard diet with (BTS group) or without (CONT group) 5% BTS for 4 weeks. By comparison to the CONT group, the BTS group showed reduced body weight gain and hyperlipidemia as well as improved liver function. Moreover, gut microbiota in the CONT and BTS group formed a significantly different cluster. Specifically, the genera Akkermansia, Bacteroides and an unknown genus of the family Enterobacteriaceae expanded dramatically in the BTS group. Noteworthy, the population of Akkermansia muciniphila, which is reported to elicit an anti-obesity effect and improve various metabolic abnormalities, was markedly increased (93-fold) compared with the CONT group. These results imply that BTS may be a promising agent for treating NAFLD.

Keywords: Akkermansia muciniphila; Bofutsushosan; Kampo; NAFLD; obesity.

Figure 1

Study design.

Figure 2

Effects of dietary administration of Bofutsushosan (BTS) on body weight and food intake. Seven-week-old ob/ob and C57BL/6J mice were administered a standard diet with or without 5% (w/w) BTS for 4 weeks. (A) Change of relative body weight based on the value at Week 0. Closed circle, C57BL/6 mice (WILD group); open circle, ob/ob mice fed a standard diet (CONT group); gray triangle, ob/ob mice fed a standard diet supplemented with 5% BTS (BTS group). Data are shown as mean ± SE (n = 6). (B) Mice were housed two per cage. Food intake was measured per cage. Closed, open, and gray columns represent WILD, CONT, and BTS groups, respectively. Data are shown as mean ± SE (n = 3). *; p < 0.05 by Student’s t-test with Bonferroni’s correction (CONT vs. BTS).

Figure 3

Representative microscopic images of the liver of mice treated with or without BTS. The liver was taken from mice fed with or without 5% (w/w) BTS for 4 weeks. The specimens were stained with hematoxylin and eosin and examined by light microscopy. (A,D) WILD group, (B,E) CONT group, (C,F) BTS group, (A–C) × 20 magnitude, (D–F) × 40 magnitude.

Figure 4

Microbiota composition in stool samples of mice fed a diet with or without BTS. Stools were collected weekly from mice fed with or without 5% (w/w) BTS. Relative abundance of gut microbiota was determined by 16S metagenome sequence analysis. Average relative abundance at each sampling point is shown as bar charts of phylum (A) and genus (B) levels (n = 6). B6, C57BL/6J mice (seven-week-old); ob, ob/ob mice (seven-week-old); CONT, ob/ob mice fed a standard diet; BTS, ob/ob mice fed a standard diet with 5% BTS.

Figure 5

Non-metric multidimensional scaling of stool microbiota in ob/ob mice fed a diet with or without BTS. Similarity of microbiota between CONT and BTS was assessed by non-metric multidimensional scaling (NMDS). Circle, stool sample; Cross, detected genera; Bold cross, genera showing significant differences with 10-fold changes between CONT and BTS groups in Table 2. The limb of each group is shown by a dotted line. The dissimilarity test between CONT and BTS was performed by permutational multivariant analysis of variance test.

Figure 6

Scattered plot of relative abundance of stool bacteria versus relative increase of body weight. Genera Akkermansia (A), Bacteroides (B), unknown genus in the family Enterobacteriaceae (C), and unknown genus in the family Helicobacteraceae (D), were analyzed by 16S metagenome sequencing and plotted versus relative increase of body weight. Only Akkermansia muciniphila was analyzed by qPCR (E). Open triangle, CONT group; closed circle, BTS group. Increase of body weight (%) was calculated as follows: 100 × ((body weight at Week 4) − (body weight at Week 0))/(body weight at Week 0).