3.1 L. acidophilus administration ameliorated HFD-induced adiposity and fatty liver in mice
To investigate whether L. acidophilus CICC 6075 can ameliorate HFD-induced metabolic disorders, such as obesity and fatty liver. Consequently, 8-week-old male mice were fed a normal diet (NCD) or a HFD (HFD mice) with different concentrations of L. acidophilus CICC 6075 oral supplementation for 12 weeks. Low concentrations of L. acidophilus CICC 6075 administrations to mice fed a HFD (HFD-L mice) inhibited body weight gain (~40% decrease), with a reduction in liver weight (20%), total white adipose tissue (45%), total white fat weight/body weight ratio and liver weight/body weight ratio, compared to mice fed a HFD (Figure2A-E). Mice given high concentrations of L. acidophilus CICC 6075 with a HFD (HFD-H mice) also showed a similar tendency with HFD-L mice, but the effect of HFD-H on total white adipose tissue weight and liver weight was not statistically significant in mice fed a HFD (Figure3A-C).
To determine the effect of L. acidophilus CICC 6075 on lipid deposition in tissues, tissue sections were subjected to histological examination. When the area of lipid droplets in liver sections was analyzed, HFD-L mice showed a significant improvement in liver appearance and a 39% reduction in liver lipid accumulation compared to HFD-fed obese mice (Figure3D). In addition, the number of adipocytes in the epididymal adipose tissue of HFD mice was reduced by 56.4% in the same field of view, compared with control NCD mice. The number of adipocytes was increased by 41.9 and 9% in HFD-L and HFD-H mice, respectively, compared with HFD mice (Figure3E).
Considering intestinal dysbiosis in HFD-fed animals may affect gut permeability and subsequently lead to release of bacterial LPS into the circulation [27], whether L. acidophilus modulates gut integrity was identified. Then, HE results showed that HFD feeding dramatically increased intestinal permeability and damaged the intestinal mucosa (Figure3F), which however were completely restored by the L. acidophilus treatment.
These results suggest that L. acidophilus reduced weight gain and fat accumula-tion and restored the intestinal barrier in HFD-fed mice, while the group of HFD-L had better results.
3.2 L. acidophilus reverses HFD-induced gut microbiota dysbiosis
The gut microbiota of obese humans and HFD-fed mice is characterized by reduced α-diversity, altered β-diversity, increased ratios of Firmicutes to Bacteroides, elevated endotoxin-producing bacteria, and reduced immunostable bacterial species [28-30]. With this in mind, the effect of L. acidophilus on the composition of the intestinal microbiota was examined by MiSeq sequencing-based analysis of bacterial 16S rRNA gene sequences (V3-V4 region) in feces.
3.2.1 Effects of L. acidophilus on gut microbial diversity and richness
Our results showed that all bacterial libraries from our samples represented the bacterial communities well, as the rarefaction curves tended towards saturation (Figure4A). To measure the extent of differences between microbial communities, β-diversity was calculated using principal coordinate analysis (PCoA) was also performed (Figure4B). The differences in the overall composition of the gut flora between the different groups were then statistically significant according to ANOVA. We also calculated the amount of gut microbial α-diversity indicators including richness and diversity, such as Sobs index and Shannon index. HFD feeding dramatically decreased gut microbial α diversity (Figure4C, D), while supplementation with L. acidophilus completely restored the effect.
To better understand the shared richness among each group and the degree of variation in the samples, hierarchical clustering and a Venn diagram showing the overlap between groups were performed. The microbial composition of the samples within groups differed less at the genus level (Figure5A). Then, it was shown that only 152 of the total richness of 995 OTUs were shared among all the groups, and there were more OTUs among three groups, between two groups or in each group (Figure5B). In addition, L. acidophilus treatment significantly increased OTUs in HFD mice.
These data demonstrated that L. acidophilus treatment remarkably improved richness and diversity of intestinal microbiota.
3.2.2 Effects of L. acidophilus on the gut microbiota composition
The ratio of saprophytes to bacteria is a widely used marker of dysbiosis of the gut flora associated with obesity and obesity-related diseases [31]. HFD feeding profoundly affected the relative abundance of Firmicutes and Firmicutes/Bacteroidetes ratio compared with control NCD feeding (Figure6A, B). Interestingly, under the HFD, L. acidophilus treatment, especially HDF-L group, decreased the relative abundance of Firmicutes and Firmicutes/Bacteroidetes ratio, and significantly increased the abundance of Actinobacteria (Figure7A, B).
In the top 15 families, L. acidophilus treatment remarkably increased the abundance of Erysipelotrichaceae and Atopobiaceae, whereas the relative abundance of Oscillospiraceae, Lachnospiraceae and Marinifilaceae decreased. Interestingly, L. acidophilus supplement increased Lactobacillaceae (Figure7C, D). At genus level, L. acidophilus treatment, especially HDF-L group, caused increase in Ileibacterium(P < 0.05), Lactobacillus, norank_f_Muribaculaceae and Helicobacter, and caused reduction in unclassified_f_Lachnospiraceae(P < 0.05), Lachnospiraceae_NK4A136, Lachnospiraceae_UCG-006(P < 0.05), unclassified_f_Oscillospiraceae and Odoribacter compared to that in HFD mice(P < 0.05) (Figure7E, F).
In addition, the bacterial community structure with notable differences among the NCD group, the HFD group, the HFD-L group, and the HFD-H group was further analyzed by adopting the linear discriminant analysis (LDA) effect-size method (LEfSe) (Figure8A, B). Then, it was figured out that taxa in different levels had differential abundance in the four groups. Besides, unclassified_f_Prevotellaceae, Muribaculum, Eubacterium_siraeum_group, Gammaproteobacteria and Proteobacteria played critical roles and could be taken as a biomarker in the NCD group. However, unclassified_f__Lachnospiraceae and Lachnospiraceae_UCG-006 functioned importantly and could be used as a biomarker in the HFD group. In addition, Actinobacteriota, Coriobacteriales, Atopobiaceae, Faecalibaculum, Bifidobacteriaceae, Lachnoclostridium, Coriobacteriia and Dubosiella played a crucial part and could be employed as a biomarker in the HFD-L group, whereas Erysipelotrichales, Ileibacterium, Bacilli, Romboutsia, Peptostreptococcaceae, Turicibacter, Blautia, and Clostridiaceae were vital and could be regarded as a biomarker in the HFD-H group. Furthermore, the number of taxa with differential abundance in the HFD group was lower than that in the HFD-L group and HFD-H groups, which shows that L. acidophilus has a recovery effect on the increase of some specific microbiota in the HFD group.
3.3 Effects of L. acidophilus on the gut microbiota function
The gut microbiota assumes essential physiological functions in the host. Moreover, this huge potential functionality influences whole-body metabolism and is a key factor in the pathology of obesity. Therefore, PICRUSt was employed to predict the functional potential of bacteria in the HFD group, and further analysis was carried out in the context of the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Then, according to the results, the enzyme, ORTHOLOGY (KO), modules and pathways belonging to KEGG functional categories were identified. Besides, the HFD feeding dramatically affected the four functional categories compared with control NCD feeding. However, these effects were completely restored by the L. acidophilus treatment (Figure9A–F). To sum up, L. acidophilus treatment improves gut microbiota functions involving metabolism, immune response, and pathopoiesia.