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

Abstract

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.

Capacity of extracellular globins to reduce liver fibrosis via scavenging reactive oxygen species and promoting MMP-1 secretion

“…We previously reported the application of rhCYGB as antifibrotic therapy in HHSteCs and in multiple in vivo models of advanced liver fibrosis [ 17 ]. CYGB belongs to the globin family in mammals; therefore, we compared the functions of other globins, including HB, MB, and NGB, with those of CYGB, including antifibrotic properties and capacities for cellular protection against oxidative stress and chemically induced liver fibrosis.…”

“…However, the mechanism through which exogenous MB, NGB, and CYGB enter cells remains unknown. Our previous work showed that rhCYGB enters cells through clathrin-mediated endocytosis [ 17 ]. Since we obtained non-labeled, commercially-available MB, which is unable to be traced under fluorescence microscopy, NGB was employed to clarify the endocytosis mechanism.…”

“…Recombinant human (rh) CYGB was produced as described previously [ 17 ]. Human MB (#30-AM20) and human HB (#30–1134) were obtained commercially (Fitzgerald, Acton, MA).…”

“…CYGB knockdown promoted fibrosis development during bile duct ligation–induced cholestasis [ 15 ], whereas the selective upregulation of CYGB in HSCs attenuated liver injury and collagen accumulation [ 16 ]. Extracellular administration of CYGB attenuated HSC activation by ROS scavenging activity and the induction of interferon β and suppressed liver fibrosis in several models in mice [ 17 ]. These findings prompted us to consider the anti-fibrotic properties of other globins.…”

Novel Therapeutic Targets in Liver Fibrosis

“…Hepatic macrophages also enhance liver fibrosis through the release of IL-1β, TNF-α, CCL2 and PDGF. During liver steatosis, neutrophils and Kupffer cells release reactive oxygen species (ROS), promoting HSC activation and liver fibrosis (Jiang et al, 2012;Dat et al, 2021). Th17 cells produce IL-17, which activates Kupffer cells and express the proinflammatory cytokines IL-6, IL-1β and TNF-α.…”