2023 Fiscal Year Research-status Report
Novel anti-fibrotic properties of heme binding proteins including Neuroglobin, Myoglobin, and Hemoglobin in comparison with Cytoglobin in vitro and in vivo
| Project/Area Number |
22K08083
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| Research Institution | Osaka Metropolitan University |
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Principal Investigator |
LE THITHANHTHUY 大阪公立大学, 大学院医学研究科, 准教授 (10572175)
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| Project Period (FY) |
2022-04-01 – 2025-03-31
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| Keywords | Globin / Cytoglobin / Neuroglobin / Myoglobin / Hemoglobin / Antioxidant / Anti-fibrosis |
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| Outline of Annual Research Achievements |
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 .
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| Current Status of Research Progress |
Current Status of Research Progress
1: Research has progressed more than it was originally planned.
Reason
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.
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| Strategy for Future Research Activity |
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.
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| Causes of Carryover |
We need to further investigate the binding target of these globins to inhibit liver fibrosis by suppressing the intracellular factors of TFGB/SMAD, STATs, etc., pathways. We plan to perform the liposome-encapsulated rhCYGB, NGB to promote the intracellular penetration.
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[Presentation] Soluble PD-1 and BTLA proteins as biomarkers for predicting the anti-HBV treatment responsiveness2023
Author(s)
Hoang Hai, Masaru Enomoto, Le Thi Thanh Thuy, Naoshi Odagiri, Kohei Kotani, Hiroyuki Motoyama, Ritsuzo Kozuka, Atsushi Hagihara, Etsushi Kawamura, Hideki Fujii, Sawako Uchida-Kobayashi, and Norifumi Kawada
Organizer
Asian Pacific Association for the Study of the Liver (APASL)
Int'l Joint Research
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