Hexa Histidine–Tagged Recombinant Human Cytoglobin Deactivates Hepatic Stellate Cells and Inhibits Liver Fibrosis by Scavenging Reactive Oxygen Species

BACKGROUND & AIMS: Anti-fibrotic therapy remains an unmet medical need in human chronic liver disease. We report the anti-fibrotic properties of cytoglobin (CYGB), a respiratory protein expressed in hepatic stellate cells (HSCs), the main cell type involved in liver fibrosis. APPROACH & RESULTS: Cygb-deficient mice which had bile duct ligation (BDL)-induced liver cholestasis or choline-deficient L-amino acid-defined (CDAA) diet-induced steatohepatitis significantly exacerbated liver damage, fibrosis and reactive oxygen species (ROS) formation. All these manifestations were attenuated in Cygb-overexpressing mice. We produced 6His-tagged recombinant human CYGB (His-CYGB), traced its bio-distribution and assessed its function in HSCs or in mice with advanced liver cirrhosis using thioacetamide (TAA) or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). In cultured HSCs, extracellular His-CYGB was endocytosed and accumulated in endosomes via clathrin-mediated pathway. His-CYGB significantly impeded ROS formation spontaneously or in the presence of ROS inducers in HSCs, thus leading to the attenuation of collagen type I alpha 1 production and alpha-smooth muscle actin expression. Replacement the iron centre of the heme group with cobalt nullified the effect of His-CYGB. In addition, His-CYGB induced interferon-β secretion by HSCs which partly contributed to its anti-fibrotic function. Momelotinib incompletely reversed the effect of His-CYGB. Intravenously injected His-CYGB markedly suppressed liver inflammation, fibrosis and oxidative cell damage in TAA- or DDC-administered mice without adverse effects. RNA-seq analysis revealed the downregulation of inflammation and fibrosis-related genes and the upregulation of antioxidant genes in both cell culture and liver tissues. The injected His-CYGB predominantly localised to HSCs but not to macrophages, suggesting specific targeting effects. His-CYGB exhibited no toxicity in humanised liver chimeric PXB mice. CONCLUSIONS: His-CYGB could have anti-fibrotic clinical applications for human chronic liver diseases

Hexa Histidine–Tagged Recombinant Human Cytoglobin Deactivates Hepatic Stellate Cells and Inhibits Liver Fibrosis by Scavenging Reactive Oxygen Species

BACKGROUND & AIMS: Anti-fibrotic therapy remains an unmet medical need in human chronic liver disease. We report the anti-fibrotic properties of cytoglobin (CYGB), a respiratory protein expressed in hepatic stellate cells (HSCs), the main cell type involved in liver fibrosis. APPROACH & RESULTS: Cygb-deficient mice which had bile duct ligation (BDL)-induced liver cholestasis or choline-deficient L-amino acid-defined (CDAA) diet-induced steatohepatitis significantly exacerbated liver damage, fibrosis and reactive oxygen species (ROS) formation. All these manifestations were attenuated in Cygb-overexpressing mice. We produced 6His-tagged recombinant human CYGB (His-CYGB), traced its bio-distribution and assessed its function in HSCs or in mice with advanced liver cirrhosis using thioacetamide (TAA) or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). In cultured HSCs, extracellular His-CYGB was endocytosed and accumulated in endosomes via clathrin-mediated pathway. His-CYGB significantly impeded ROS formation spontaneously or in the presence of ROS inducers in HSCs, thus leading to the attenuation of collagen type I alpha 1 production and alpha-smooth muscle actin expression. Replacement the iron centre of the heme group with cobalt nullified the effect of His-CYGB. In addition, His-CYGB induced interferon-β secretion by HSCs which partly contributed to its anti-fibrotic function. Momelotinib incompletely reversed the effect of His-CYGB. Intravenously injected His-CYGB markedly suppressed liver inflammation, fibrosis and oxidative cell damage in TAA- or DDC-administered mice without adverse effects. RNA-seq analysis revealed the downregulation of inflammation and fibrosis-related genes and the upregulation of antioxidant genes in both cell culture and liver tissues. The injected His-CYGB predominantly localised to HSCs but not to macrophages, suggesting specific targeting effects. His-CYGB exhibited no toxicity in humanised liver chimeric PXB mice. CONCLUSIONS: His-CYGB could have anti-fibrotic clinical applications for human chronic liver diseases