Live Combined B. subtilis and E. faecium Alleviate Liver Inflammation, Improve Intestinal Barrier Function, and Modulate Gut Microbiota in Mice with Non-Alcoholic Fatty Liver Disease

BACKGROUND Non-alcoholic fatty liver disease (NAFLD) is a chronic, progressive liver disease with an increasing incidence rate. This study investigated the protective effects of live combined Bacillus subtilis and Enterococcus faecium (LCBE) on NAFLD, and its possible mechanisms. MATERIAL AND METHODS Five-week-old C57BL/6 mice were randomly divided into 3 groups: chow, HFD, and HFD+LCBE groups. The levels of serum biochemical markers, glucose tolerance, insulin, the inflammatory cytokines IL-1ß, IL-6, and TNF-alpha, LPS, and histological staining were measured using commercial kits. qPCR was used to examine the mRNA expression levels of inflammatory cytokines in the liver. Western blotting was used to determine the protein levels of TLR4, NF-kappaB p65, PPAR-alpha, and CPT-1 in the liver, and occludin and Claudin1 in the intestine. The intestinal flora of the mice was analyzed by high-throughput sequencing of the V3-V4 region of 16S rDNA. RESULTS LCBE significantly lowered the body weight, liver/body weight ratio, and serum glucose level, and increased the serum insulin level in NAFLD mice. In addition, LCBE treatment improved the liver function and lipid profile, decreased the levels of LPS and inflammatory cytokines, and downregulated the expression of TLR4 and NF-kappaB p65. Moreover, LCBE enhanced the intestinal barrier function by increasing the expression of occludin and Claudin1. Furthermore, LCBE modulated the composition of the gut microbiota by reducing the Firmicutes to Bacteroidetes ratio, and the proportion of inflammation-related and LPS-producing bacteria, thus re-arranging the structure of the gut microbiota. CONCLUSIONS LCBE protects against NAFLD by alleviating inflammation, restoring the intestinal barrier, and modulating gut microbiota composition.