Abstract
Hepatic fibrosis occurs when liver tissue becomes scarred from repetitive liver injury and inflammatory responses; it can progress to cirrhosis and eventually to hepatocellular carcinoma. Previously, we reported that neoagarooligosaccharides (NAOs), produced by the hydrolysis of agar by β-agarases, have hepatoprotective effects against acetaminophen overdose-induced acute liver injury. However, the effect of NAOs on chronic liver injury, including hepatic fibrosis, has not yet been elucidated. Therefore, we examined whether NAOs protect against fibrogenesis in vitro and in vivo. NAOs ameliorated PAI-1, α-SMA, CTGF and fibronectin protein expression and decreased mRNA levels of fibrogenic genes in TGF-β-treated LX-2 cells. Furthermore, downstream of TGF-β, the Smad signaling pathway was inhibited by NAOs in LX-2 cells. Treatment with NAOs diminished the severity of hepatic injury, as evidenced by reduction in serum alanine aminotransferase and aspartate aminotransferase levels, in carbon tetrachloride (CCl4)-induced liver fibrosis mouse models. Moreover, NAOs markedly blocked histopathological changes and collagen accumulation, as shown by H&E and Sirius red staining, respectively. Finally, NAOs antagonized the CCl4-induced upregulation of the protein and mRNA levels of fibrogenic genes in the liver. In conclusion, our findings suggest that NAOs may be a promising candidate for the prevention and treatment of chronic liver injury via inhibition of the TGF-β/Smad signaling pathway.
Highlights
Liver fibrosis is a highly conserved wound healing response to chronic liver injury [1]
When we used isolated primary hepatic stellate cells (HSCs) from mice, we observed increased plasminogen activator inhibitor-1 (PAI-1), α-SMA, connective tissue growth factor (CTGF) and fibronectin expression; TGF- was antagonized by NAOs (Figure 1D)
RT-PCR analysis confirmed that transforming growth factor-β (TGF-β) markedly increased mRNA levels of PAI-1, α-SMA and collagen 1A1 (COL1A1), which were almost completely blocked by pretreatment with NAOs (Figure 1E–G)
Summary
Liver fibrosis is a highly conserved wound healing response to chronic liver injury [1]. Inhibition of the TGF-β/Smad signaling pathway attenuates the progression of liver fibrosis in vitro and in vivo [11,12]. We reported that NAOs inhibit metabolic liver disease and acute liver injury in mice administered a high cholesterol diet or acetaminophen overdose, respectively [13,14]. NAOs present no signs of toxicity up to 5,000 mg/kg body weight/day in acute, repeated 14-day and 91-day oral toxicity tests [15] These results strongly support the potential application of NAOs in dietary supplements and medications. NAOs antagonized TGF-β-induced profibrotic gene expression through inhibition of the Smad signaling pathway in cultured immortalized human semi-activated HSCs. NAOs reduced the progression of carbon tetrachloride(CCl4)-induced liver fibrosis as evidenced by serum liver enzymes, histopathological changes, and production of ECM proteins. Our results indicate that NAOs have hepatoprotective effects in TGF-βtreated HSCs and CCl4-induced liver fibrosis, and have potential applications in clinical interventions
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