Human-Induced Hepatocytes-Derived Extracellular Vesicles Ameliorated Liver Fibrosis in Mice Via Suppression of TGF-β1/Smad Signaling and Activation of Nrf2/HO-1 Signaling

Liver fibrosis is a wound-healing response caused by persistent liver injury and often occurs in chronic liver diseases. Effective treatments for liver fibrosis are still pending. Recent studies have revealed that extracellular vesicles (EVs) derived from primary hepatocytes (Hep-EVs) have therapeutic potential for multiple liver diseases. However, Hep-EVs are difficult to manufacture in bulk because of the limited sources of primary hepatocytes. Human-induced hepatocytes (hiHep) are hepatocyte-like cells that can expand in vitro, and their cell culture supernatant is thus an almost unlimited resource for EVs. This study aimed to investigate the potential therapeutic effects of EVs derived from hiHeps. hiHep-EVs inhibited the expression of inflammatory genes and the secretion of inflammation-related cytokines, and suppressed the activation of hepatic stellate cells by inhibiting the transforming growth factor (TGF)-β1/Smad signaling pathway. The anti-inflammatory and antifibrotic effects of hiHep-EVs were similar to those of mesenchymal stem cell-EVs. Furthermore, the administration of hiHep-EVs ameliorated oxidative stress, inflammation, and fibrosis in a CCl₄-induced liver fibrosis mouse model. The expression of α smooth muscle actin, collagen I, and collagen III was reduced, which may be attributed to the regulation of matrix metalloproteinase (MMP)-9, tissue inhibitor of metalloproteinases (TIMP)-1, and TIMP-2 by hiHep-EVs, and the protein expression of Nrf2, HO-1, and NQO1 was increased. Taken together, our results suggested that hiHep-EVs alleviated liver fibrosis by activating the Nrf2/HO-1 signaling pathway and inhibiting the TGF-β1/Smad signaling pathway. This study revealed the hepatoprotective effect of hiHep-EVs, and provided a new approach to treating liver fibrosis.