Gut microbiota plays a critical role in the treatment of OCA in NAFLD mice
After a 2 weeks of antibiotic-induced microbiome depletion (AIMD), the body weight of high-fat-diet (HFD group) fed mice treated with following antibiotics intervention (HFD + A group) had a significant decrease (P < 0.05) compared with that of HFD-fed mice (HFD group). And HFD-fed mice treated with OCA administration (i.e., HFD + OCA group) also exhibited a significant decrease in body weight (P < 0.05) compared to HFD group. In addition, there was a group of HFD-fed mice that were given oral gavage of antibiotics and following OCA intervention (HFD + A + OCA group). Surprisingly, the body weight of mice in HFD + A + OCA group was continuously increased like HFD group (Fig. 1A, B). Also, no difference was observed in the food intake among the four groups of mice during this experiment (Fig. 1D). Meanwhile, hematoxylin-eosin (HE) staining of liver tissues of mice in HFD, HFD + A, and HFD + A + OCA groups had hepatocellular ballooning, and dramatically increased size of hepatocytes and nuclear marginalization (Fig. 1E). In oil red O staining of mouse liver tissues (Fig. 1F), lipid droplets were obviously visible in hepatocytes of mice in HFD, HFD + A, and HFD + A + OCA groups, indicating that liver fat accumulation was serious in these three groups. Additionally, HE staining of mouse adipose tissues showed that adipocyte size in HFD + A and HFD + A + OCA groups was similar with that in HFD group but was larger than that in HFD + OCA group (Fig. 1G). This phenomenon implied that OCA intervention could effectively ameliorate HFD-induced fat inflammation depending on the presence of gut microbiota. To sum up, gut microbiota played a critical role in the therapeutic effects of OCA administration by reducing hepatic fat accumulation and pathological variations of liver and adipose tissues induced by HFD in mice.
Apart from the improvement of histopathological phenotypes of liver and adipose tissues, HFD fed mice treated with OCA could effectively mitigate glucose intolerance and insulin resistance by decreasing the levels of blood glucose, and homeostatic model assessment for insulin resistance (HOMA-IR) and insulin, respectively (Supplementary Figure S1A-C). Besides, OCA apparently reduced the levels of total triglyceride (TG), total cholesterol (TC), and LDL cholesterol (LDL-C), while increasing the levels of HDL cholesterol (HDL-C) in serum compared with HFD fed mice (Supplementary Figure S1D-G). Moreover, higher levels of alanine aminotransferase (ALT) and aspartic transaminase (AST) in the serum were observed in OCA group compared to HFD group (Supplementary Figure S1H, I). To further confirm the role of gut microbiota played in the treatment of NAFLD by OCA, we performed fecal microbiota transplantation (FMT) from mice of the HFD + OCA group (donors) to the HFD group (recipients). Both body weight and the ratio of liver weight to body weight in FMT group were significantly lower than those in HFD group (P < 0.05) which resembled the effects of OCA treatment (Fig. 2A-C). No significant difference was observed in food intake among the three experimental groups (Fig. 2D). The histopathological examinations of liver and adipose tissues exhibited significant difference between mice of HFD and FMT. Despite some small vacuoles appeared in mouse hepatocytes of FMT group derived from lipid droplets (Fig. 2E), the amounts of large vacuoles and lipid droplets in hepatocytes were obviously decreased in FMT group compared with HFD group (Fig. 2F). And the size of adipocytes in FMT group was smaller than that in HFD group (Fig. 2G). Noticeably, the comparisons of levels of serum TG and LDL-C between FMT and HFD groups reflected that FMT exhibited similar effects in improving liver function like OCA treatment did (Supplementary Figure S2A, B). However, FMT did not exert adequate effects on levels of serum TC, HDL-C, and inflammatory factors like OCA administration (Supplementary Figure S2C-G). These results suggested that gut microbiota could participate in alleviating liver and adipose tissues damages caused by HFD, and improving liver function, thus determine the therapeutic effect of OCA administration on NALFD.
The influence of OCA administration on gut microbiota composition of NAFLD mice
To investigate the specific gut microbes involved in OCA treatment on NALFD, we further implemented metagenomic analysis on the mice fecal microbiota of ND, HFD, and OCA groups. For alpha diversity, Shannon index and Simpson index of gut microbiota had only consistent trends and no significant difference among the three groups of mice (Fig. 3A). However, OCA administration substantially improved the richness of gut microbiota in HFD fed mice. In addition, different Pielou indexes indicated that highest gut microbiota evenness in mice of normal diet (ND) group, while lowest in mice of HFD group. The slightly elevated Pielou index suggested a higher evenness of the gut microbiota in OCA group compared with HFD group. For the gut microbial community structures, both non-metric multidimensional scaling (NMDS) and principal co-ordinates analysis (PCoA) based on Bray-Curtis dissimilarity distance matrix at species level were implemented, the results exhibited significant difference in microbial communities among ND, HFD and HFD + OCA groups (adonis R2 = 0.243, P = 0.001) (Fig. 3B-C). Therefore, gut microbiota structures of mice could be affected by both 20-week HFD feeding and HFD feeding with subsequently 8-week OCA administration.
Further exploration of gut microbial composition at different taxonomic levels, significant differences were found between HFD group and ND group, as well as OCA group and HFD group. At the phylum level, fecal microbiota mainly consisted of four phyla of bacteria: Firmicutes, Bacteroidetes, Proteobacteria, and Verrucomicrobia (Supplementary Figure S3). The comparisons of the proportions of these four kinds of bacteria in feces of mice among three groups demonstrated that HFD substantially increased the proportion of Firmicutes in mouse feces of ND group (P < 0.001, Wilcox test), while it decreased the proportions of Proteobacteria and Verrucomicrobia (P < 0.01, Wilcox test). In contrast, OCA not only profoundly reduced the proportion of Firmicutes, but also improved the proportions of Preteobacteria and Verrucomicrobia in HFD group.
At the genus level, compared with HFD group, the enriched bacteria of mouse gut in ND group were Akkermansia, Mucispirillum, Bifidobacteria, Bacteroides, Helicobacteria, and another three genera (Fig. 3D, 3F). And bacteria enriched in OCA group compared with HFD group were seven genera including Akkermansia, Bifidobacteria, Bacteroides, Escherichia, Lactococcus, and so on (Fig. 3E, 3G). At the species level (Fig. 3H, 3I), Akkermansia miciniphila, Bifidobacterium animalis, Bifidobacterium pseudolongum, Bacteroides massiliensis, Lactobacillus helveticus, Streptococcus thermophilus, Escherichia coli, and another ten species exhibited higher abundance (P < 0.05) in OCA group compared to HFD group. Also, OCA intervention significantly (P < 0.05) reduced the proportions of Lactobacillus murine, Firmicutes bacterium M10-2, Lactobacillus gasseri, and Enterococcus faecium of feces of HFD fed mice. Besides, compared with HFD group, ND group had higher abundance of A. muciniphila, Mucispirillum schaedleri, B. pseudolongum, Bacteroides uniformis, Bacteroides xylanisolvens, and lower abundance of eleven species including the same four species reduced by OCA administration. Thus, OCA administration recovered the gut microbial composition that was altered by HFD, and made it approximate to that in ND group.
The bile acid profiles and correlations between bile acid and gut microbiota
It is well-known that gut microbiota is tightly associated with bile acid metabolism, shifts in gut microbiota might influence host bile acids pool. Indeed, targeted metabolomics analysis of bile acids indicated that both HFD and HFD with following OCA treatment could effectively modified serum bile acid pool in mice. Compared with ND group, HFD feeding profoundly increased (P < 0.05) the level of serum conjugated bile acids, taurochenodeoxycholic acid (TCDCA), taurohyodeoxycholic acid (THDCA), and tauro ursodesoxycholic acid (TUDCA) (HFD vs. ND, 2.8 times higher) in mice (Fig. 4A). In contrast, OCA significantly reduced (P < 0.05) the levels of CA, CDCA, hyodeoxycholic acid (HDCA), and ursodeoxycholic acid (UDCA) (Fig. 4B). Different variations were observed in the levels of fecal bile acids among groups, HFD apparently increased (P < 0.05) the levels of most fecal bile acids in mice compared with ND group (Fig. 4C), while OCA decreased the amounts of most fecal bile acids increased by HFD feeding including the level of fecal conjugated bile acids of glycodeoxycholic acid (GDCA) and glycoursodeoxycholic acid (GUDCA) (Fig. 4D).
To figure out whether bile acids variations in serum and feces were driven by gut microbes, we performed Spearman correlation analysis on abundance of species-level of bacteria and level of bile acids among the ND, HFD and OCA groups. Generally, variations of serum bile acids were stronger correlated with gut microbiota than serum bile acids variations. Bacteria species of B. massiliensis, A. miciniphila, L. johnsonii, and L. reuteri were positively correlated with the levels of serum TCDCA, THDCA, and TUDCA, and were significantly negatively correlated with the level of HDCA except for A. miciniphila (Fig. 4E, F). Besides, E. coli exhibited a significantly negative correlation with the levels of serum HDCA, CDCA, and CA (Fig. 4F). For correlations between mouse gut microbiota and fecal bile acids. The abundance of two species of Lactobacilli (L. murinus, and L. gasseri) exhibited significant positive correlations with the level of fecal tauroursodeoxycholic acid (TDCA), TUDCA, and TCDCA, while A. muciniphila was negatively correlated with fecal TCDCA, THDCA, and TUDCA (Fig. 4G). In addition, B. pseudolongum showed negative correlation with both the serum and fecal TDCA and TUDCA (Fig. 4G). Both A. muciniphila and S. thermophilus displayed positive correlations with fecal THDCA, while L. gasseri was vice versa (Fig. 4H).
Potential of bacterial genomes encoding enzymes involved in bile acids metabolism
To further investigate the functional profiles of gut microbiota influencing bile acids metabolism, we performed enzymes scanning based on SGBs enriched in different groups (HFD vs. ND, HFD vs. OCA). Compared with HFD group, SGBs mainly enriched in ND were species of Bacteroides, Parabacteroides, and Prevotellaceae in Bacteroidetes, Bifidobacterium pseudolongum, Alistipes indistinctus, and Clostridium innocuum (Fig. 5A). And OCA administration to HFD significantly elevated the abundance of two species of Bacteroides, Bifidobacterium pseudolongum, Alistipes indistinctus, two species of Prevotellaceae, and Escherichia_coli and Proteus mirabilis belonging to Proteobacteria (Fig. 5B). Worthy of note was that HFD compared to OCA treatment enriched bacteria exclusively belonging to Firmicutes (Fig. 5B). Intriguingly, some bacteria species were both enriched in ND and OCA, such as Bifidobacterium pseudolongum, Alistipes indistinctus, and species of Bacteroides and Prevotellaceae. The enzymes encoded by SGBs enriched in different groups indicated that bacterial species enriched in ND and OCA groups had adequate potential to encode both the BSHs (EC:3.5.1.24) and 7α-HSDs (EC:1.1.1.159), while HFD enriched bacteria exhibited poor capacity of encoding enzymes of 7α-HSDs, and mainly encoded BSHs responsible for primary bile acids synthesis (Fig. 5A, B). Collectively, HFD enabled the accumulation of bacteria encoding BSHs which accelerated the secretion of bile into gut and the uncoupling of conjugated bile acids into primary bile acids. And OCA administration elevated the abundance bacteria with potential to encode 7α-HSDs which facilitated the degradation of primary bile acids and promote of secondary bile acids bioconversion in the gut (Fig. 5C).