2019 Fiscal Year Research-status Report
Cytoglobin overexpression inhibits liver fibrosis and cancer development via anti-oxidant function
| Project/Area Number |
19K08428
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| Research Institution | Osaka City University |
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Principal Investigator |
LE THITHANHTHUY 大阪市立大学, 大学院医学研究科, 特任講師 (10572175)
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| Project Period (FY) |
2019-04-01 – 2022-03-31
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| Keywords | Cytoglobin / Hepatic stellate cells / Anti-fibrosis / Liver cancer / Hepatosteatosis |
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| Outline of Annual Research Achievements |
Mice were sacrificed at 1-3 weeks after BDL, 10 weeks after thioacetamide (TAA) injection, 8-32 weeks after CDAA feeding or 12 months after diethylnitrosamine (DEN) treatment. In BDL model, compared to WT, fibrosis was robustly developed in KO mice indicated by markedly increased expression of α-SMA, collagen 1a1, and Sirius-red positive area. TG mice showed suppression of these factors up to 55% compared to WT mice. Similary results were also found in TAA, CDAA, and DEN model. Specially, in DEN model, 100% liver-, and 40% lung-tumours were found in KO mice, but only 40% and 0%, respectively, in WT mice. In contrast, TG mice showed significantly regressed the mean number (4.76 vs 1.51, p<0.001), and maximum size (7.4 vs 2.05 mm, p<0.01) of liver tumours compared to WT ones.
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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
In vivo administration of rhCYGB exhibited no toxicity to mouse organs and their livers. Tail vein-injected Alexa 488-labelled rhCYGB accumulated in multiple organs, of note, in the liver, it was localized in HSCs and sinusoidal endothelial cells, but not in hepatocytes. In vivo mouse liver fibrosis model induced by ten weeks injection of escalation dose of TAA developed severe liver injury, inflammation, oxidative stress and fibrosis, while all of these manifestations were reversed by rhCYGB (2 mg/kg BW, twice/week) treatment during the last 2 weeks or 5 weeks of TAA administration. rhCYGB administration suppressed up to 60% of AST, ALT, 47% of LDH serum levels, 78% of α-SMA, 72% of Col1a1, 43% of Tgf-β1, 53% of Tgf-β3, 79% of Timp-1, 52% of 4-HNE, 44% of HO-1 expression induced by TAA.
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| Strategy for Future Research Activity |
Our goals are pointed out the down-stream targets of Cygb in protecting liver injuries. RNA sequencing are the first necessary analysis to find out the answer. Then, immunoprecipitation experiments are performed using Pull-Down PolyHis Protein:Protein Interaction Kit to find CYGB target. The potential targets will be examined their binding using the label transfer technique by SUlfo-SBED Biotin Label Transfer Kit. Dynabeads His-Tag Isolation and pulldown (Invitrogen) is also used for the alternative method to confirm the results. For rhCYGB protein production and application: we will perform the shortening full-length of rhCYGB protein to be shorten peptides and explore the important portion of CYGB for its effect.
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| Causes of Carryover |
We need more for next fiscal year for the shortening full-length of rhCYGB protein and explore the important portion of CYGB for its effect. Our working flow including: Design primers to amplify targets; PCR amplification of DNA fragments using pRSET-hisCYGB; Double digestion of PCR products and pRSETA vector; Ligation reaction; Transformation into DH5α competent cells, propagation the plasmids; Verify sequencing of the vectors; Choose the corrected vectors and transformation into Ecoli BL21-AI; Produce rhCYGB short-peptides; Purify the peptides; In vitro application if these peptides work.
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[Journal Article] The anti-oxidative role of cytoglobin in podocytes: implications for a role in chronic kidney disease2020
Author(s)
Elisa B. Randi, Vervaet Benjamin, Maria Tsachaki, Elena Porto, Stijn Vermeylen, Maja T. Lindenmeyer, Le Thi Thanh Thuy, Clemens D. Cohen, Olivier Devuyst, Andreas Kistler, Csaba Szabo, Norifumi Kawada, Thomas Hankeln, Alex Odermatt, Sylvia Dewilde, Roland H. Wenger, David Hoogewijs.
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Journal Title
Antioxid Redox Signal
Volume: 7
Pages: -
DOI
Peer Reviewed / Int'l Joint Research
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[Journal Article] TGF-β-driven reduction of cytoglobin leads to oxidative DNA damage in stellate cells during non-alcoholic steatohepatitis2020
Author(s)
Yoshinori Okina, Misako Sato-Matsubara, Tsutomu Matsubara, Atsuko Daikoku, Lisa Longato, Krista Rombouts, Le Thi Thanh Thuy, Hiroshi Ichikawa, Yukiko Minamiyama, Mitsutaka Kadota, Hideki Fujii, Masaru Enomoto, Kazuo Ikeda, Katsutoshi Yoshizato, Massimo Pinzani, Norifumi Kawada
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Journal Title
J Hepatol
Volume: -
Pages: -
DOI
Peer Reviewed / Open Access / Int'l Joint Research
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[Journal Article] Clinical significance of circulating soluble immune checkpoint proteins in sorafenib-treated patients with advanced hepatocellular carcinoma2020
Author(s)
3.Dong MP, Enomoto M, Thuy LTT, Hai H, Hieu VN, Hoang DV, Iida-Ueno A, Odagiri N, Amano-Teranishi Y, Hagihara A, Fujii H, Uchida-Kobayashi S, Tamori A, Kawada N
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Journal Title
Sci Rep
Volume: 10
Pages: 3392
DOI
Peer Reviewed / Open Access / Int'l Joint Research
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