Identification of core active disaccharides in heparin for HGF‑inducing activity

Objectives: To ascertain the positions of sulfated groups for HGF‑inducing activity using differently sulfated heparin disaccharides and to investigate whether the heparin disaccharide elevates HGF levels in plasma in vivo and exerts protective effects on acute liver injury. Materials and Methods: The heparin disaccharides DUA‑GlcNS, DUA (2S)‑GlcN, DUA‑GlcNAc (6S), DUA‑GlcNS (6S), DUA (2S)‑GlcNS, DUA (2S)‑GlcNAc (6S), DUA‑GlcNAc and DUA (2S)‑GlcNS (6S) were added to MRC‑9 fibroblasts and HGF concentrations in culture media were determined by enzyme‑linked immunosorbent assay. Furthermore, DUA‑GlcNS (100 mg/head) was injected into C57BL/6 mice and plasma levels of HGF measured at 12 h. After acute hepatitis was induced by CCl4 (15 mg/kg) in mice, liver specimens were stained with hematoxylin and eosin (H and E). Levels of aspartate aminotransferase and alanine aminotransferase were measured at 24 h. Results: Among the disaccharides investigated, DUA‑GlcNS, DUA‑GlcNAc (6S) and DUA‑GlcNS (6S) stimulated HGF production in MRC‑9 fibroblasts. However, none of the 2‑O‑sulfated disaccharides [DUA (2S)‑GlcNS, DUA (2S)‑GlcNAc (6S) and DUA (2S)‑GlcNS (6S)] showed any activity despite the presence of N‑sulfated and/or 6‑O‑sulfated disaccharides. Thus, 2‑O‑sulfation of hexuronic acid has an inhibitory effect. Moreover, DUA‑GlcNS administration increased plasma levels of HGF in normal mice and prevented CCl4‑induced liver injury in mice. Conclusions: N‑sulfation and/or 6‑O‑sulfation of glucosamine with nonsulfated hexuronic acid provides a structural basis for the HGF‑inducing activity of disaccharides. DUA‑GlcNS increases plasma levels of HGF and protects against CCl4‑induced acute liver injury.