Lactobacillus rhamnosus GG combined with inosine ameliorates alcohol-induced liver injury through regulation of intestinal barrier and Treg/Th1 cells.

ABSTRACT

BACKGROUND: Intestinal epithelial barrier disruption and bacterial translocation exacerbates the progression of alcoholic liver disease. Lactobacillus rhamnosus GG (LGG), a probiotic, has been shown benefits in chronic liver disease and in regulating gut dysbiosis. Previous studies showed the protective roles of LGG in ethanol-disrupted gut barrier functions and liver injury. Inosine, a metabolite produced by intestinal bacteria, has the anti-inflammatory and immunregulatory functions. In this study, the synergistic effect of LGG and inosine was investigated in a mouse model of alcohol-induced liver disease (ALD). METHODS: Male C57BL/6 mice were fed with a Lieber-DeCarli diet containing 5% alcohol for four weeks to establish a model of alcohol-induced liver injury. LGG or a combination of LGG and inosine were administrated orally to explore a new therapeutic method for alcohol-induced liver disease and to investigate the underlying mechanisms. Liver damage was evaluated by transaminases and pathological changes. Tight junction proteins, composition of the gut microbiome, cytokines, lipopolysaccharides (LPS), glutathione (GSH), superoxide dismutase (SOD), malondialdehyde (MDA), F4/80+ macrophages, as well as p38, Jun N-terminal kinase (JNK), were determined by qRT-PCR, RNAseq, ELISA, IHC and western blot. Regulatory T (Treg) cells were characterized by positive staining of CD4, CD25 and Foxp3 using flow cytometry. IFN-γ-producing CD4+ T (Th1) cells were examined by intracellular cytokine staining. RESULTS: Alcohol consumption induced elevated liver enzymes, steatosis and inflammation, while LGG combined with inosine treatment was more significant to ameliorate these symptoms compared with LGG alone. When LGG combined with inosine were administered to ALD mice, intestinal microecology significantly improved reflected by intestinal villi and tight junction proteins recovery and the restoration of intestinal flora. Combined therapy inhibited phosphorylation of p38 and JNK to alleviate hepatic inflammation. Moreover, flow cytometry analysis showed that long-term excessive alcohol consumption reduced Tregs population while increased Th1 population, which was restored by a combination of LGG and inosine treatment. CONCLUSIONS: The findings from the study indicate that the combined LGG and inosine treatment ameliorates ALD by improving the gut ecosystem, intestinal barrier function, immune homeostasis and liver injury.