This study was conducted to clarify the role of cytoglobin (Cygb), a globin expressed in hepatic stellate cells (HSCs), in the development of liver fibrosis and cancer in nonalcoholic steatohepatitis (NASH). Cygb expression was assessed in patients with NASH and hepatocellular carcinoma. Mouse NASH model was generated in Cygb-deficient (Cygb(-/-)) or wild-type (WT) mice by giving a choline-deficient amino acid-defined diet and, in some of them, macrophage deletion and N-acetyl cysteine treatment were used. Primary-cultured mouse HSCs isolated from WT (HSCs(Cygb-wild)) or Cygb(-/-) (HSCs(Cygb-null)) mice were characterized. As results, the expression of CYGB was reduced in patients with NASH and hepatocellular carcinoma. Choline-deficient amino acid treatment for 8 weeks induced prominent inflammation and fibrosis in Cygb(-/-) mice, which was inhibited by macrophage deletion. Surprisingly, at 32 weeks, despite no tumor formation in the WT mice, all Cygb(-/-) mice developed liver cancer, which was ameliorated by N-acetyl cysteine treatment. Altered expression of 31 genes involved in the metabolism of reactive oxygen species was notable in Cygb(-/-) mice. Both HSCs(Cygb-null) and Cygb siRNA-transfected-HSCs(Cygb-wild) exhibited the preactivation condition. Our findings provide important insights into the role that Cygb, expressed in HSCs during liver fibrosis, plays in cancer development with NASH.

Cytoglobin (Cygb) was identified in hepatic stellate cells (HSCs) and pericytes of all organs; however, the effects of Cygb on cellular functions remain unclear. Here, we report spontaneous and age-dependent malformations in multiple organs of Cygb−/− mice. Twenty-six percent of young Cygb−/− mice (<1 year old) showed heart hypertrophy, cystic disease in the kidney or ovary, loss of balance, liver fibrosis and lymphoma. Furthermore, 71.3% (82/115) of aged Cygb−/− mice (1–2 years old) exhibited abnormalities, such as heart hypertrophy and cancer development in multiple organs; by contrast, 5.8% (4/68) of aged wild-type (WT) mice had abnormalities (p < 0.0001). Interestingly, serum and urine analysis demonstrated that the concentration of nitric oxide metabolites increased significantly in Cygb−/− mice, resulting in an imbalance in the oxidative stress and antioxidant defence system that was reversed by NG-monomethyl-L-arginine treatment. A senescent phenotype and evidence of DNA damage were found in primary HSCs and the liver of aged Cygb−/− mice. Moreover, compared with HSC+/+, HSC−/− showed high expression of Il-6 and chemokine mRNA when cocultured with mouse Hepa 1–6 cells. Thus, the absence of Cygb in pericytes provokes organ abnormalities, possibly via derangement of the nitric oxide and antioxidant defence system and through accelerated cellular senescence.

Background and Aims Antifibrotic therapy remains an unmet medical need in human chronic liver disease. We report the antifibrotic properties of cytoglobin (CYGB), a respiratory protein expressed in hepatic stellate cells (HSCs), the main cell type involved in liver fibrosis. Approach and Results Cygb‐deficient mice that had bile duct ligation–induced liver cholestasis or choline‐deficient amino acid–defined diet–induced steatohepatitis significantly exacerbated liver damage, fibrosis, and reactive oxygen species (ROS) formation. All of these manifestations were attenuated in Cygb‐overexpressing mice. We produced hexa histidine–tagged recombinant human CYGB (His‐CYGB), traced its biodistribution, 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 through a 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 1 alpha 1 production and α‐smooth muscle actin expression. Replacement the iron center of the heme group with cobalt nullified the effect of His‐CYGB. In addition, His‐CYGB induced interferon‐β secretion by HSCs that partly contributed to its antifibrotic function. Momelotinib incompletely reversed the effect of His‐CYGB. Intravenously injected His‐CYGB markedly suppressed liver inflammation, fibrosis, and oxidative cell damage in mice administered TAA or DDC mice without adverse effects. RNA‐sequencing analysis revealed the down‐regulation of inflammation‐ and fibrosis‐related genes and the up‐regulation of antioxidant genes in both cell culture and liver tissues. The injected His‐CYGB predominantly localized to HSCs but not to macrophages, suggesting specific targeting effects. His‐CYGB exhibited no toxicity in chimeric mice with humanized livers. Conclusions His‐CYGB could have antifibrotic clinical applications for human chronic liver diseases.

This study clarified the role of Cygb, the fourth globin in mammals originally discovered in rat hepatic stellate cells (HSCs), in cholestatic liver disease. Bile duct ligation (BDL) augmented inflammatory reactions as revealed by increased infiltrating neutrophils, CD68+-macrophages, and chemokine expression in Cygb−/− mice. In these mice, impairment of bile canalicular indicated by the loss of CD10 expression, down-regulation of bile salt transporters, increased total bile acid, and massive apoptotic and necrotic hepatocytes occurred with the release of cytochrome c, activation of caspase 3, resulting in reduced animal survival compared to wild-type mice. In Cygb−/− mouse liver, all of NO metabolites and oxidative stress were increased. Treatment with NO inhibitor restrained all above phenotypes and restored CD10 expression in BDL Cygb−/− mice, while administration of NO donor aggravated liver damage in BDL-wild type mice to the same extent of BDL-Cygb−/− mice. N-acetylcysteine administration had a negligible effect in all groups. In mice of BDL for 1–3 weeks, expression of all fibrosis-related markers was significantly increased in Cygb−/− mice compared with wild-type mice. Thus, Cygb deficiency in HSCs enhances hepatocyte damage and inflammation in early phase and fibrosis development in late phase in mice subjected to BDL, presumably via altered NO metabolism.

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