COMPOSITION FOR PREVENTING OR TREATING ISCHEMIA REPERFUSION INJURY COMPRISING NADPH OXIDASE 1 INHIBITOR AS ACTIVE INGREDIENT

Abstract

The present invention relates to a composition for preventing or treating ischemia-reperfusion injury, the composition comprising an NADPH oxidase 1 (NOX1) inhibitor as an active ingredient, and more specifically, to a composition exhibiting prophylactic or therapeutic effects on ischemia reperfusion injury by suppressing the signaling of extracellular-signal-regulated kinase (ERK) by means of reactive oxygen species (ROS).

Claims

1. A pharmaceutical composition for preventing or treating ischemia-reperfusion injury, comprising an NADPH oxidase 1 (NOX1) inhibitor as an active ingredient.

2. The pharmaceutical composition according to claim 1, wherein the ischemia-reperfusion injury is kidney injury.

3. The pharmaceutical composition according to claim 1, wherein the NOX1 inhibitor is a compound of Formula 1 below or a pharmaceutically acceptable salt thereof. ##STR00002##

4. The pharmaceutical composition according to claim 1, wherein the NOX1 inhibitor is at least one selected from the group consisting of antisense nucleotides, siRNA, shRNA, and ribozyme for NOX1 gene.

5. The pharmaceutical composition according to claim 1, wherein the NOX1 inhibitor exhibits an ischemia-reperfusion injury preventing or therapeutic effect through inhibition of Reactive Oxygen Species (ROS)—mediated Extracellular-signal-Regulated Kkinase (ERK) signal transduction.

6. A food composition for preventing or improving ischemia-reperfusion injury, comprising an NADPH oxidase 1 (NOX1) inhibitor as an active ingredient.

7. The food composition according to claim 6, wherein the ischemia-reperfusion injury is kidney injury.

8. Use of a NADPH oxidase 1 (NOX1) inhibitor for the manufacture of a preparation for the treatment of ischemia-reperfusion injury.

9. The use according to claim 8, wherein the ischemia-reperfusion injury is a kidney injury.

10. The use according to claim 8, wherein the NOX1 inhibitor is a compound of Formula 1 below or a pharmaceutically acceptable salt thereof. ##STR00003##

11. The use according to claim 8, wherein the NOX1 inhibitor is at least one selected from the group consisting of antisense nucleotides, siRNA, shRNA, and ribozyme for the NOX1 gene.

12. A method for treating ischemia-reperfusion injury comprising administering to a subject in need thereof an effective amount of a composition comprising an NADPH oxidase 1 (NOX1) inhibitor as an active ingredient.

13. The method according to claim 12, wherein the ischemia-reperfusion injury is a kidney injury.

14. The method according to claim 12, wherein the NOX1 inhibitor is a compound of Formula 1 below or a pharmaceutically acceptable salt thereof. ##STR00004##

15. The method according to claim 12, wherein the NOX1 inhibitor is at least one selected from the group consisting of antisense nucleotides, siRNA, shRNA, and ribozyme for the NOX1 gene.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0051] FIG. 1a to FIG. 1c is the result of confirming kidney damage through tissue staining and kidney function test of mice treated with ML171 in an animal experiment induced by oxidative stress.

[0052] FIG. 2a to FIG. 2e are the results of confirming the mRNA expression of NOX1 and 4 in the mouse, the expression of NOX4 in the tissue, and the generation of ROS by oxidative stress induction.

[0053] FIG. 3A to FIG. 3C are results of confirming apoptosis in a mouse model.

[0054] FIG. 4A to FIG. 4D are results of confirming renal fibrosis due to oxidative stress.

[0055] FIG. 5A and FIG. 5B are results of confirming the MAPK pathway gene by western blot to confirm the mechanism of renal cell death and fibrosis caused by oxidative stress.

[0056] FIG. 6A to FIG. 6F are results of confirming the ROS generation and NOX expression patterns of kidney cells by oxidative stress using MDCK cells.

[0057] In FIG. 7a to FIG. 7c, it was confirmed that ML171 blocks apoptosis of kidney cells by oxidative stress.

[0058] FIG. 8A and FIG. 8B are results of finding out the mechanism by which ML171 effectively blocks oxidative stress induction by H.sub.2O.sub.2 in cells.

MODE FOR CARRYING OUT INVENTION

[0059] Hereinafter, the present invention will be described in detail by way of Examples. However, the following examples are only for illustrating the present invention, and the present invention is not limited thereto.

[0060] Experimental Method

[0061] C57BL/6 mice were intraperitoneally injected with 60 mg/kg of ML171, a selective NOX inhibitor or excipient at a time, renal ischemia-reperfusion injury (IRI) was induced by clamping bilateral renal vessels for 30 min. MDCK cells were incubated with H.sub.2O.sub.2 (1.4 mM) for 1 h to induce oxidative stress and treated with ML171 (1 and 2.5 μM). Renal damage was estimated using renal function tests and histology. NOX expression, oxidative stress markers, apoptosis assays and MAPK pathways were also evaluated in renal tissue and MDCK cells.

[0062] Experiment Result

[0063] FIG. 1a to FIG. 1c show the results of confirming the kidney damage through the renal function test and tissue staining of mice treated with ML171 in an animal experiment induced by oxidative stress.

[0064] Referring to FIG. 1a and FIG. b, as a result of confirming the level of kidney damage in the blood, a significant decrease was confirmed in the mouse group treated with ML171, and referring to FIG. 1c, it was confirmed that ML171 reduced damage to renal tubular epithelial cells caused by oxidative stress even in histological analysis through PAS staining.

[0065] FIG. 2a to FIG. 2e are the results of confirming the mRNA expression of NOX1 and 4 in the mouse, the expression of NOX4 in the tissue, and the generation of ROS by oxidative stress induction.

[0066] Referring to FIG. 2a and FIG. 2b, as a result of examining the mRNA expression level in mouse kidney, it was confirmed that NOX increased by oxidative stress was significantly reduced by ML171. Referring to FIG. 2c, it was confirmed that the expression level of NOX4 was increased in the IRI animal model even in the results of NOX4 immunostaining. Referring to FIG. 2d and FIG. 2e, as a result of measuring reactive oxygen species (ROS) in the kidney, it was confirmed that ML171 effectively blocked ROS, which significantly increased in the IRI group.

[0067] FIG. 3a to FIG. 3c are results confirming apoptosis in a mouse model.

[0068] Referring to FIG. 3a, as a result of confirming Caspase-3 activity in the kidney, a significant apoptosis alleviation effect by ML171 was confirmed, and referring to FIG. 3b, it was confirmed that ML171 alleviated apoptosis caused by oxidative stress in the results of TUNEL assay histological staining. Referring to FIG. 3c, as a result of examining the protein expression levels in the kidneys of the apoptosis markers BCL-2 and BAX, it was confirmed that ML171 effectively inhibited apoptosis by IRI.

[0069] FIG. 4a to FIG. 4d are results of confirming renal fibrosis due to oxidative stress.

[0070] Referring to FIG. 4a and FIG. 4b, as a result of confirming renal fibrosis through Trichrome stain, it was confirmed that fibrosis was significantly reduced by ML171, and referring to FIG. 4c and Referring to 4d, as a result of confirming the fibrosis markers Fibronectin and a-SMA markers, it was confirmed that the fibrosis progression was significantly increased by oxidative stress.

[0071] FIG. 5a and FIG. 5b are results of confirming the MAPK pathway gene by western blot to confirm the mechanism of renal cell death and fibrosis caused by oxidative stress.

[0072] As a result of confirming the gene of the MAPK pathway in FIG. 5a and FIG. 5b, it was confirmed that phosphorylation of ERK was significantly increased, and it was confirmed that ML171 effectively reduced it.

[0073] FIG. 6a to FIG. 6f are results of confirming the ROS generation and NOX expression patterns of kidney cells by oxidative stress using MDCK cells.

[0074] Referring to FIG. 6a to FIG. 6e, as a result of confirming the expression pattern of NOX-related genes after inducing oxidative stress through H.sub.2O.sub.2, it was confirmed that oxidative stress markers significantly increased by H.sub.2O.sub.2 were effectively decreased by ML171. Referring to FIG. 6f, it was confirmed that ML171 significantly reduced ROS, which was increased in cells by H.sub.2O.sub.2, even when ROS production was confirmed.

[0075] In FIG. 7a to FIG. 7c, it was confirmed that ML171 blocks apoptosis of kidney cells by oxidative stress.

[0076] Referring to FIG. 7a, it was confirmed that when oxidative stress by H.sub.2O.sub.2 was induced through Caspase-3 activity, apoptosis was significantly increased, and referring to FIG. 7b, the protein expression levels of BCL-2 and BAX, which are markers of apoptosis, were increased by H.sub.2O.sub.2, and it was confirmed that apoptosis was significantly inhibited by ML171. As a result of intracellular TUNEL assay staining of FIG. 7c, it was confirmed that the apoptosis pattern was increased by H.sub.2O.sub.2, and it was confirmed that ML171 effectively blocked it.

[0077] In FIG. 8a and In FIG. 8b are results of identifying the mechanism by which ML171 effectively blocks the induction of oxidative stress by H.sub.2O.sub.2 in cells.

[0078] As a result of confirming the MAPK pathway genes in MDCK cells, it was confirmed that oxidative stress-induced kidney damage was induced by ERK phosphorylation as in the in vivo experiment, and it was confirmed that ML171 significantly reduced and regulated this.

[0079] In conclusion, IRI worsened renal function and increased ROS production such as H.sub.2O.sub.2 and DCFDA in renal tissue, whereas treatment with ML171 significantly attenuated IRI-mediated damage. Intraperitoneal ML171 reversed a decrease in Bcl-2 and an increase in Caspase-3 activity. ML171 also reduced the expression of NOX1, NOX2 and p40 induced by H.sub.2O.sub.2 treatment in MDCK cells. H.sub.2O.sub.2 caused changes in oxidative stress-related enzymes in SOD and GXP production that were alleviated by ML171 treatment. ML171 caused a significant increase in Bcl-2 levels and a decrease in Caspase-3 activity. Among the MAPK pathways, ML171 was identified to affect ERK signaling by phosphorylation of ERK in kidney tissue and tubular cells.

INDUSTRIAL APPLICABILITY

[0080] The composition of the present invention comprising a NOX1 inhibitor as an active ingredient exhibits an effect of alleviating tissue damage caused by ischemia-reperfusion through inhibition of reactive oxygen species (ROS)—mediated ERK signal transduction in an organ damage environment caused by ischemia-reperfusion, and thus can be very usefully utilized for the prevention or development of therapeutic agents for organ damage due to ischemia-reperfusion.