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

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-ß 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.

Newly synthesised oxime and lactone derivatives from Dipterocarpus alatus dipterocarpol as anti-diabetic inhibitors: experimental bioassay-based evidence and theoretical computation-based prediction.

Dipterocarpus alatus-derived products are expected to exhibit anti-diabetes properties. Natural dipterocarpol (1) was isolated from Dipterocarpus alatus collected in Quang Nam province, Vietnam; afterwards, 20 derivatives including 13 oxime esters (2 and 3a-3m) and 7 lactones (4, 5, 6a-6e) were semi-synthesised. Their inhibitory effects towards diabetes-related proteins were investigated experimentally (α-glucosidase) and computationally (3W37, 3AJ7, and PTP1B). Except for compound 2, the other 19 compounds (3a-3m, 4, 5, and 6a-6d) are reported for the first time, which were modified at positions C-3, C-24 and C-25 of the dipterocarpol via imidation, esterification, oxidative cleavage and lactonisation reactions. A framework based on docking-QSARIS combination was proposed to predict the inhibitory behaviour of the ligand-protein complexes. Enzyme assays revealed the most effective α-glucosidase inhibitors, which follow the order 5 (IC50 of 2.73 ± 0.05 µM) > 6c (IC50 of 4.62 ± 0.12 µM) > 6e (IC50 of 7.31 ± 0.11 µM), and the computation-based analysis confirmed this, i.e., 5 (mass: 416.2 amu; polarisability: 52.4 Å3; DS: -14.9 kcal mol-1) > 6c (mass: 490.1 amu; polarisability: 48.8 Å3; DS: -13.7 kcal mol-1) > 6e (mass: 549.2 amu; polarisability: 51.6 Å3; DS: -15.2 kcal mol-1). Further theoretical justifications predicted 5 and 6c as versatile anti-diabetic inhibitors. The experimental results encourage next stages for the development of anti-diabetic drugs and the computational strategy invites more relevant work for validation.

Selective overexpression of cytoglobin in stellate cells attenuates thioacetamide-induced liver fibrosis in mice.

Cytoglobin (CYGB), discovered in hepatic stellate cells (HSCs), is known to possess a radical scavenger function, but its pathophysiological roles remain unclear. Here, for the first time, we generated a new transgenic (TG) mouse line in which both Cygb and mCherry reporter gene expression were under the control of the native Cygb gene promoter. We demonstrated that the expression of Cygb-mCherry was related to endogenous Cygb in adult tissues by tracing mCherry fluorescence together with DNA, mRNA, and protein analyses. Administration of a single dose (50 mg/kg) of thioacetamide (TAA) in Cygb-TG mice resulted in lower levels of alanine transaminase and oxidative stress than those in WT mice. After 10 weeks of TAA administration, Cygb-TG livers exhibited reduced neutrophil accumulation, cytokine expression and fibrosis but high levels of quiescent HSCs. Primary HSCs isolated from Cygb-TG mice (HSCCygb-TG) exhibited significantly decreased mRNA levels of α-smooth muscle actin (αSMA), collagen 1α1, and transforming growth factor ß-3 after 4 days in culture relative to WT cells. HSCsCygb-TG were resistant to H2O2-induced αSMA expression. Thus, cell-specific overexpression of Cygb attenuates HSC activation and protects mice against TAA-induced liver fibrosis presumably by maintaining HSC quiescence. Cygb is a potential new target for antifibrotic approaches.