Protection against ischemia-reperfusion injury using a short interfering RNA that inhibits the activity of myeloperoxidase
| Project/Area Number |
18390428
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Anesthesiology/Resuscitation studies
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| Research Institution | Kyoto University |
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Principal Investigator |
ARAI Toshiyuki Kyoto University, Medicine, Associate Professor (80175950)
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| Co-Investigator(Kenkyū-buntansha) |
YAMASHITA Kouhei Kyoto University, Medicine, Assistant Professor (80402858)
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| Project Period (FY) |
2006 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥16,640,000 (Direct Cost: ¥15,500,000、Indirect Cost: ¥1,140,000)
Fiscal Year 2007: ¥4,940,000 (Direct Cost: ¥3,800,000、Indirect Cost: ¥1,140,000)
Fiscal Year 2006: ¥11,700,000 (Direct Cost: ¥11,700,000)
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| Keywords | Neutreopils / Myeronperoxidaes / RNA interference / Short interfering RNA / Real time PCR / Reactive oxygen species / Human umbilical endothelial cells / Interleukin-8 / ヒト臍帯静脈内皮細胞 / インターロイキン-8 / RNA干渉 / siRNA / フローサイトメーター / 蛍光試薬DCFH / ミエロペルオキシダーゼ / 一重項酸素 / スーパーオキシド / 化学発光 |
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| Research Abstract |
In order to investigate the feasibility of protection against ischemia-reperfusion injury (IRI) using a short interfering RNA (siRNA) that inhibits the activity of myeloperoxidase (MPO) of human neutrophils, the following studies were Performed.
The siRNA to diminish the MPO activity was designed and synthesized. Both effective (positive) and ineffective (negative) siRNAs were prepared. Then, the siRNA was electrically transferred into neutrophils using NucleofectorR system. The Suction of the expression of mRNA of MPO by a positive siRNA was successfully performed, which was confirmed using a real-time PCR. However, when the neutrophils were activated by opsonized zymosan, the generation of reactive oxygen species (ROS) was not diminished in the neutrophils incorporated with a positive siRNA compared to that in the neutrophils incorporated with a negative siRNA. That is, the RNA interference was not effective to inhibit the MPO activity of neutrophils.
As an alternative capture, we tried to inhibit the ROS generation in endothelial cells using human umbilical vein endothelial cells (HUVECs). First, it was revealed that some stimulants, such as lipopolysaccaride (LPS), induced the expression of interleukin-8 (IL-8) and that IL-8, in turn, induced ROS generation in HUVECs. The siRNA to diminish the expression of IL-8 was designed and synthesized. Both positive and negative siRNAs were prepared. Then, the siRNA was electrically transferred into HUVECs using NucleofectorR system. The Suction of the expression of mRNA of IL-8 by a positive siRNA was successfully performed, which was confirmed using a real-time PCR. And, when the HUVECs were stimulated by LPS, the generation of ROS was diminished in the HUVECs incorporated with a positive siRNA compared to that in the HUVECs incorporated with a negative siRNA. It was known that the ROS generated by endothelial cells hurt themselves during IRI. So, we can say that protection against WI using a siRNA was achieved.
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Report
(3 results)
Research Products
(32 results)
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[Journal Article] The radical scavenger edaravone (3-methyl-l-phenyl-2-pyrazolin-5-one) reacts with a pterin derivative and produces a cytotoxic substance that induces intracellular ROS generation and cell death2008
Author(s)
Arai, T., Nonogawa, M., Makino, K., Endo, N., Mori, H., Miyoshi, T., Yamashita, K., Sasada, M., Kakuyama, M., Fukuda, K
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Journal Title
J Pharmacol Exp Ther 324
Pages: 529-538
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] Anticancer photodynamic and non-photodynamic effects of pterin derivatives on a pancreatic cancer cell line2008
Author(s)
Miyoshi, T., Arai, T., Nonogawa, M., Makino, K., Mori, H., Yamashita, K., Sasada, M., Uchiyama, T
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Journal Title
J Pharmacol Sci (in press)
NAID
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] Gene expression in enhanced apoptosis of human lymphoma U937 cells treated with the combination of different free radical generators and hyperthermia2007
Author(s)
Wada, S., Tabuchi, Y., Kondo, T., Cui, Z-G., Zhao, Q-L., Takasaki, I., Salunga, TL., Ogawa, R., Arai, T., Makino, K., Furuta, I
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Journal Title
Free Radical Res 41
Pages: 73-81
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] Effects of edaravone on singlet oxygen released from activated human neutrophils2007
Author(s)
Sommani, P., Arai, T., Yamashita, K., Miyoshi, T., Mori, H., Sasada, M., Makino, K
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Journal Title
J Pharmacol Sci 103
Pages: 117-120
NAID
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] Reactive oxygen species generation through NADH oxidation by 6-formylpterin derivatives in the dark2007
Author(s)
Nonogawa, M., Pack, SP., Arai, T., Endo, N., Sommani, P., Kodaki, T., Makino, K
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Journal Title
Biochem Biophys Res Commun 353
Pages: 1105-1110
Description
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[Journal Article] Effects of intravenous anesthesia with propofol on regional cortical blood flow and intracranial pressure in surgery for Moyamoya disease2007
Author(s)
Kikuta, K., Takagi, Y., Nozaki, K., Yamada, K., Miyamoto, S., Kataoka, H., Arai, T., Hashimoto, N
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Journal Title
Surgical Neurology 68
Pages: 421-424
Description
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[Journal Article] Synthesis of 6-formylpterin nucleoside analogs and their ROS generation activities in the presence of NADH in the dark2007
Author(s)
Nonogawa, M., Pack, SP., Arai, T., Endo, N., Sommani, P., Kodaki, T., Kotake, Y., Makino, K
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Journal Title
Org Biomol Chem 5
Pages: 3314-3319
Description
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[Journal Article] Inhibitory effects of 6-formylpterin on HIF-la protein accumulation2007
Author(s)
Sommani, P., Yamashita, K., Miyoshi, T., Tsunemine, H., Kodaki, T., Mori, H., Hirota, K., Arai, T., Sasada, M., Makino, K
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Journal Title
Biol Pharm Bull 30
Pages: 2181-2184
Description
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[Presentation] The new role of interleukin-8 in sepsis (Reactive oxygen production from endothelial cells by IL-B)2007
Author(s)
Miyoshi, T., Yamashita, K., Arai, T., Itoh, H., Uchiyama, T., Sasada, M
Organizer
The 69th Annual Meeting of the Japanese Society of Hematology
Place of Presentation
Yokohama
Description
「研究成果報告書概要(欧文)」より
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