Bifidobacterium longum-Derived Extracellular Vesicles Prevent Hepatocellular Carcinoma by Modulating the TGF-β1/Smad Signaling in Mice.

Abstract

Background: The involvement of gut microbiota in carcinogenesis has gradually been highlighted in past decades. Bacteria could play its role by the secretion of extracellular vesicles (EVs); however, interrelationship between bacterial EVs and hepatocellular carcinoma (HCC) development has not been investigated much.

Methods: Diethylnitrosamine (DEN) was utilized to produce HCC model in mice, of which fecal was collected for detecting Bifidobacterium longum (B.longum) with real-time polymerase chain reaction (PCR). EV isolated from B.longum (B.longum-EV) with ultracentrifugation were stained with PKH26 to investigate the cellular uptake of murine hepatocytes (AML12). After treatment with B.longum-EV, TGF-β1-induced AML12 cells were subjected to morphological observation, fibrosis- and apoptosis-related marker detection with western blot, apoptotic ratio and reactive oxygen species (ROS) level analysis with flow cytometry, and oxidative stress biomarker assessment with enzyme-linked immunosorbent assay (ELISA); meanwhile, animal studies including liver function, tumor formation rate, and histological analysis, were also performed to investigate the role of B.longum-EV in the fibrosis, apoptosis, oxidative stress, and carcinogenesis of the liver in vivo.

Results: The levels of B.longum were significantly reduced in HCC model mice. B.longum-EV could enter AML12 cells and effectively attenuate TGF-β1-induced fibrosis, apoptosis, and oxidative stress in AML12 cells. In vivo studies showed that B.longum-EV administration alleviated DEN-induced liver fibrosis, apoptosis, and oxidative stress at the early stage. Moreover, B.longum-EV administration also effectively reduced the tumor formation rate and liver function injury in DEN-induced mice and down-regulated TGF-β1 expression and Smad3 phosphorylation of mouse liver.

Conclusions: B.longum-EVs protect hepatocytes against fibrosis, apoptosis, and oxidative damage, which exert a potential of preventing HCC development.