Novel anti-fibrotic properties of heme binding proteins including Neuroglobin, Myoglobin, and Hemoglobin in comparison with Cytoglobin in vitro and in vivo

基盤研究(C)

LE THITHANHTHUY  大阪公立大学, 大学院医学研究科, 准教授 (10572175)

4,290千円 (直接経費: 3,300千円、間接経費: 990千円)

These scientific originality of the anti-fibrotic capacity of rhCYGB protein suggests an investigation whether other members of globin family such as hemoglobin (HB), myoglobin (MB), and neuroglobin (NGB) possess the similar effect. We investigated the role of these proteins in liver fibrosis.

We demonstrated the intracellular translocation of MB, NGB, or CYGB, but not HB, in which they localized to the membranous, cytoplasmic, nuclear, and cytoskeletal fractions of HHSteCs. The proportion of extracellular globins that translocated into HHSteCs was highest for CYGB (566 pg/g total protein), followed by NGB (100 pg/g) and MB (3.4 pg/g). We performed RNA-seq analysis in rhNGB-treated HHSteCs compared with untreated controls which showed upregulated 226 genes, with the most significantly pathway identified as Interferon (IFN) α/β signaling, upregulated IFN-stimulated genes, including IFI27, IFI6, ISG15, IRF 7, IRF9, and OAS2 , which was confirmed by qRT-PCR. At the protein level, NGB treatment induced the phosphorylation of signal transducer and activator of transcription 1 .

現在までの達成度 (区分)

1: 当初の計画以上に進展している

理由

We have shown the effects of MB, NGB, HB, and CYGB on the activation status of a human HSC cell line, HHSteC, and in a carbon tetrachloride (CCl4)-induced mouse model of liver fibrosis. First, we found that extracellular MB, NGB, and CYGB, but not HB, were endocytosed into HHSteCs, exhibited intracellular ROS scavenging activity, and suppressed COL1A1 promoter activation. Second, we demonstrated that MB and NGB deactivated HHSteCs and promoted matrix metalloproteinase (MMP)-1 secretion, which is involved in collagen degradation in the ECM. Third, intravenously injected MB, NGB, or CYGB were delivered to hepatic sinusoidal cells in mice and suppressed liver fibrosis induced by CCl4.

Next step, we need to demonstrate the safety and efficacy of globins for inhibiting collagen production and fibrosis development in vitro and in vivo. Furthermore, it is necessary to verify that ROS production increased COL1A1 promoter transactivation, accelerating collagen production in HHSteCs, and extracellularly administered globins scavenged ROS, thereby subsequently inhibited COL1A1 expression. In an in vivo CCl4-induced liver injury model, after administration of exogenous MB, NGB, or CYGB, the liver tissues will be used for assessment of inflammation and liver fibrosis development, coinciding with the appearance of 4-HNE, the oxidative stress marker with strong pro-fibrogenic activity on HSCs, also investigated on ROS scavenging function of these globin.