PHARMACEUTICAL COMPOSITION, FOR PREVENTING OR TREATING HEPATIC FIBROSIS, COMPRISING 8-OHDG

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating liver fibrosis or liver cirrhosis, comprising 8-OHdG or a pharmaceutically acceptable salt thereof, a method for preventing or treating liver fibrosis or liver cirrhosis using the same, and a food composition for ameliorating liver fibrosis or liver cirrhosis, comprising 8-hydroxydeoxyguanosine (8-OHdG) or a pharmaceutically acceptable salt thereof. As use of the pharmaceutical composition for preventing or treating liver fibrosis or liver cirrhosis can reduce levels of various markers whose expression level increases due to induced liver fibrosis, the pharmaceutical composition can be widely used in the effective prevention or treatment of liver fibrosis or liver cirrhosis induced thereby.

Claims

1. A pharmaceutical composition for preventing or treating liver fibrosis, comprising 8-hydroxydeoxyguanosine (8-OHdG) or a pharmaceutically acceptable salt thereof.

2. The pharmaceutical composition of claim 1, wherein the 8-OHdG inhibits expression of a liver fibrosis marker selected from the group consisting of hydroxyproline, transforming growth factor β (TGF-β), tissue inhibitor of metalloproteinase 1 (TIMP-1), collagen, α-smooth muscle actin (α-SMA), NADPH oxidase 1 (NOX1), NADPH oxidase 2 (NOX2), ras-related C3 botulinum toxin substrate 1 (Rac1), and a combination thereof in a liver tissue.

3. The pharmaceutical composition of claim 1, further comprising a pharmaceutically acceptable carrier, excipient, or diluent.

4. A method for preventing or treating liver fibrosis or liver cirrhosis, comprising administering the pharmaceutical composition of any one of claims 1 to 3 to a subject having or at risk of developing liver fibrosis.

5. A food composition for ameliorating liver fibrosis or liver cirrhosis, comprising 8-hydroxydeoxyguanosine (8-OHdG) or a pharmaceutically acceptable salt thereof.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0043] FIG. 1 is a graph showing the effect of 8-OHdG on the level of hydroxyproline measured in the liver fibrosis animal model.

[0044] FIG. 2a is a graph showing the effect of 8-OHdG on the level of mRNA of TGF-β measured in the liver fibrosis animal model.

[0045] FIG. 2b is a graph showing the effect of 8-OHdG on the level of mRNA of TIMP-1 measured in the liver fibrosis animal model.

[0046] FIG. 2c is a graph showing the effect of 8-OHdG on the level of mRNA of collagen measured in the liver fibrosis animal model.

[0047] FIG. 2d is a graph showing the effect of 8-OHdG on the level of mRNA of α-SMA measured in the liver fibrosis animal model.

[0048] FIG. 2e is a graph showing the effect of 8-OHdG on the level of mRNA of NOX1 measured in the liver fibrosis animal model.

[0049] FIG. 2f is a graph showing the effect of 8-OHdG on the level of mRNA of NOX2 measured in the liver fibrosis animal model.

[0050] FIG. 3a is a graph showing the effect of 8-OHdG on the level of protein of collagen measured in the liver fibrosis animal model.

[0051] FIG. 3b is a photo of western blot results showing the effect of 8-OHdG on the level of protein of α-SMA measured in the liver fibrosis animal model.

[0052] FIG. 3c is a photo showing the effect of 8-OHdG on the level of protein of immunostained NOX1, measured in the liver fibrosis animal model.

[0053] FIG. 3d is a photo showing the effect of 8-OHdG on the level of protein of immunostained NOX2, measured in the liver fibrosis animal model.

DETAILED DESCRIPTION OF THE INVENTION

[0054] Hereinafter, the present invention will be described in details with reference to the following Examples. However, these Examples are for illustrative purposes only, and the scope of the present invention is not limited to these Examples.

Example 1: Preparation of Sample

[0055] 250 g to 300 g male Sprague-Dawley rats were subject to bile duct ligation (BDL) and atresia of bile duct to prepare liver fibrosis animal models.

[0056] The prepared animal models were used to prepare one control group and two experimental groups.

[0057] Seven of the animal models were laparotomized, ligated, and recovered to be used for the control group.

[0058] Six of the animal models were laparotomized, and the proximal and distal bile ducts were ligated and then cut in the middle to separate the bile duct. The animal models were then sutured and allowed to recover to be used for Experimental Group 1.

[0059] Six animal models treated in the same manner as experimental group 1 were sutured and administered with 8-OHdG in the amount of 60 mg/kg/day for 3 weeks to be used for Experimental Group 2.

[0060] The animal models of each of the control and experimental groups were sutured and raised for 3 weeks, followed by taking blood and liver tissues therefrom to be used for subsequent experimental procedures.

Example 2: Effect of 8-OHdG on the Level of Hydroxyproline in Liver Tissue

[0061] The effect of 8-OHdG on the level of hydroxyproline, known as a biomarker of liver fibrosis, in the liver tissue was to be verified.

[0062] Specifically, 100 μL of distilled water was added to 10 mg of liver tissues of the control and experimental groups prepared in Example 1 and homogenized, and 100 μL of 12 N HCL was added to 100 μL of the homogenized sample and allowed to react for 3 hours at 120° C. Upon termination of the reaction, 10 μL of the reaction solution was placed in each well of a 96-well plate and dried. After adding 100 μL of chloramine T, the reaction solution was reacted for 90 minutes at 60° C. After the reaction, absorbance at 560 nm was measured to calculate the level of hydroxyproline in the liver tissue and compared (FIG. 1).

[0063] FIG. 1 is a graph showing the effect of 8-OHdG on the level of hydroxyproline measured in the liver fibrosis animal model. As shown in the figure, the hydroxyproline level in the liver tissue of the liver fibrosis animal model was remarkably increased (Experimental Group 1), whereas the increased level of hydroxyproline was reduced by 8-OHdG treatment (Experimental Group 2).

Example 3: Effect of 8-OHdG on the mRNA Level of the Liver Fibrosis Biomarkers in Liver Tissue

[0064] The effect of 8-OHdG on the mRNA level of TGF-β,l TIMP-1, collagen, α-SMA, NOX1, and NOX2, known biomarkers of liver fibrosis and ROS formation process, in liver tissues were to be verified.

Example 3-1: Change of TGF-β on mRNA Level

[0065] Total RNA was extracted from the control and experimental groups prepared in Example 1 using RNeasy Mini Kit (Qiagen, Hilden, Germany), and the extracted total RNA was reverse translated using high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, Calif.) to obtain cDNA.

[0066] qPCR was performed using thus-obtained cDNA as a template, as well as the primers (below), the Bio-Rad CFX96 real-time PCR detection system (Bio-Rad, Hercules, Calif.), and an SYBR Premix Ex Taq II kit (Takara Biotechnology) to measure and compare the mRNA levels of TGF-β in the control and experimental groups (FIG. 2a). The mRNA of GAPDH was used as an internal control.

TABLE-US-00001 (SEQ ID NO: 1) TGF-β F: 5′-AGAAGTCACCCGCGTGCTAA-3′ (SEQ ID NO: 2) TGF-β R: 5′-TCCCGAATGTCTGACGTATTGA-3′

[0067] FIG. 2a is a graph showing the effect of 8-OHdG on the level of mRNA of TGF-β measured in the liver fibrosis animal model. As shown in the figure, the mRNA level of TGF-β in the liver tissue of the liver fibrosis animal model was remarkably increased (Experimental Group 1), whereas the increased level of mRNA was reduced by 8-OHdG treatment (Experimental Group 2).

Example 3-2: Change of TIMP-1 on mRNA Level

[0068] Except that the primers below were used instead, the same method was performed as in Example 3-1 to measure and compare the mRNA levels of TIMP-1 in the liver tissues of the control and experimental groups (FIG. 2b).

TABLE-US-00002 (SEQ ID NO: 3) TIMP-1 F: 5′-TCCTCTTGTTGCTATCATTGATAGCTT-3′ (SEQ ID NO: 4) TIMP-1 R: 5′-CGCTGGTATAAGGTGGTCTCGAT-3′

[0069] FIG. 2b is a graph showing the effect of 8-OHdG on the level of mRNA of TIMP-1 measured in the liver fibrosis animal model. As shown in the figure, the mRNA level of TIMP-1 in the liver tissue of the liver fibrosis animal model was remarkably increased (Experimental Group 1), whereas the increased mRNA level of TIMP-1 was reduced by 8-OHdG treatment (Experimental Group 2).

Example 3-3: Changeof Collagen on mRNA Level

[0070] Except that the primers below were used instead, the same method was performed as in Example 3-1 to measure and compare the mRNA levels of collagen in the liver tissues of the control and experimental groups (FIG. 2c).

TABLE-US-00003 (SEQ ID NO: 5) Collagen Iα1 F: 5′-TGCCGATGTCGCTATCCA-3′ (SEQ ID NO: 6) Collagen Iα1 R: 5′-TCTTGCAGTGATAGGTGATGTTCTG-3′

[0071] FIG. 2c is a graph showing the effect of 8-OHdG on the level of mRNA of collagen measured in the liver fibrosis animal model. As shown in the figure, the mRNA level of collagen in the liver tissue of the liver fibrosis animal model was remarkably increased (Experimental Group 1), whereas the increased mRNA level of collagen was reduced by 8-OHdG treatment (Experimental Group 2).

Example 3-4: Change of α-SMA on mRNA Level

[0072] Except that the primers below were used instead, the same method was performed as in Example 3-1 to measure and compare the mRNA levels of α-SMA in the liver tissues of the control and experimental groups (FIG. 2d).

TABLE-US-00004 (SEQ ID NO: 7) α-SMA F: 5′-GCTGACAGGATGCAGAAGGA-3′ (SEQ ID NO: 8) α-SMA R: 5′-GCCGATCCAGACAGAATATTTG-3′

[0073] FIG. 2d is a graph showing the effect of 8-OHdG on the level of mRNA of α-SMA measured in the liver fibrosis animal model. As shown in the figure, the mRNA level of α-SMA in the liver tissue of the liver fibrosis animal model was remarkably increased (Experimental Group 1), whereas the increased mRNA level of α-SMA was reduced by 8-OHdG treatment (Experimental Group 2).

Example 3-5: Change of NOX1 on mRNA Level

[0074] Except that the primers below were used instead, the same method was performed as in Example 3-1 to measure and compare the mRNA levels of NOX1 in the liver tissues of the control and experimental groups (FIG. 2e).

TABLE-US-00005 (SEQ ID NO: 9) NOX1 F: 5′-CTACAGTAGAAGCCAACAGGCCAT-3′ (SEQ ID NO: 10) NOX1 R: 5′-ACTGTCACGTTTGGAGACTGGATG-3′

[0075] FIG. 2e is a graph showing the effect of 8-OHdG on the level of mRNA of NOX1 measured in the liver fibrosis animal model. As shown in the figure, the mRNA level of a-SMA in the liver tissue of the liver fibrosis animal model was remarkably increased (Experimental Group 1), whereas the increased mRNA level of NOX1 was reduced by 8-OHdG treatment (Experimental Group 2).

Example 3-6: Change of NOX2 on mRNA Level

[0076] Except that the primers below were used instead, the same method was performed as in Example 3-1 to measure and compare the mRNA levels of NOX2 in the liver tissues of the control and experimental groups (FIG. 2f).

TABLE-US-00006 (SEQ ID NO: 11) NOX2 F: 5′-CTACAGTAGAAGCCAACAGGCCAT-3′ (SEQ ID NO: 12) NOX2 R: 5′-ACTGTCACGTTTGGAGACTGGATG-3′

[0077] FIG. 2f is a graph showing the effect of 8-OHdG on the level of mRNA of NOX2 measured in the liver fibrosis animal model. As shown in the figure, the mRNA level of α-SMA in the liver tissue of the liver fibrosis animal model was remarkably increased (Experimental Group 1), whereas the increased mRNA level of NOX2 was reduced by 8-OHdG treatment (Experimental Group 2).

[0078] In light of the experimental results of Examples 3-1 to 3-6, the mRNA levels of TGF-β, TIMP-1, collagen, α-SMA, NOX1, and NOX2, known biomarkers of liver fibrosis and ROS formation process, increased in the liver tissues of the liver fibrosis animal models; however, upon administration with 8-OHdG, the increased mRNA levels of the biomarkers were reduced.

Example 4: Effect of 8-OHdG on the Level of Protein of Liver Fibrosis Marker in the Liver Tissue

[0079] From the result of Example 3, the mRNA levels of TGF-β, TIMP-1, collagen, α-SMA, NOX1, and NOX2, known biomarkers of liver fibrosis and ROS formation process, in the liver tissues were confirmed to be reduced by administration of 8-OHdG. In this regard, whether the same effect would be exhibited in terms of protein was to be verified.

Example 4-1: Change of Collagen on Protein Level

[0080] Collagen of the liver tissues of the control and experimental groups prepared in Example 1 was stained with Picro Sirius Red Stain kit (Abcam Company Ltd. China), and the level of the collagen labeled in red was analyzed using ImageJ software (NIH, USA) (FIG. 3a).

[0081] FIG. 3a is a microscopic image and a graph showing the effect of 8-OHdG on the level of protein of immunostained NOX1, measured in the liver fibrosis animal model. As shown in the figure, the protein level of collagen in the liver tissue of the liver fibrosis animal model was remarkably increased (Experimental Group 1), whereas the increased protein level of collagen was reduced by 8-OHdG treatment (Experimental Group 2).

Example 4-2: Change of α-SMA on Protein Level

[0082] The protein level of α-SMA, expressed in the liver tissues of the control and experimental groups prepared in Example 1, was measured by western blot using anti-α-SMA antibodies (FIG. 3b). GAPDH protein was used as an internal control.

[0083] FIG. 3b is a photo of western blot results showing the effect of 8-OHdG on the level of protein of α-SMA measured in the liver fibrosis animal mode. As shown in the figure, the protein level of α-SMA in the liver tissue of the liver fibrosis animal model was remarkably increased (Experimental Group 1), whereas the increased protein level of α-SMA was reduced by 8-OHdG treatment (Experimental Group 2).

Example 4-3: Change of NOX1 on Protein Level

[0084] Immunofluorescent staining of NOX1 was performed on the liver tissues of the control and experimental groups prepared in Example 1 using anti-NOX1 antibodies, secondary antibody conjugated with Alexa Fluor 594, and secondary antibody conjugated with Alexa Fluor 488, and the results were compared (FIG. 3c). α-SMA was used as a hepatic stellate cell activity index.

[0085] FIG. 3c is a photo showing the effect of 8-OHdG on the level of protein of immunostained NOX1, measured in the liver fibrosis animal model. As shown in the figure, the protein level of NOX1 in the liver tissue of the liver fibrosis animal model was remarkably increased (Experimental Group 1), as was that of α-SMA, used as a hepatic stellate cell activity index, whereas the increased protein levels of α-SMA and NOX1 were reduced by 8-OHdG treatment (Experimental Group 2).

Example 4-4: Change of NOX2 on Protein Level

[0086] Immunofluorescent staining of NOX2 was performed on the liver tissues of the control and experimental groups prepared in Example 1 using anti-NOX2 antibodies, secondary antibody conjugated with Alexa Fluor 594, and secondary antibody conjugated with Alexa Fluor 488, and the results were compared (FIG. 3d). α-SMA was used as a hepatic stellate cell activity index.

[0087] FIG. 3d is a photo showing the effect of 8-OHdG on the level of protein of immunostained NOX2, measured in the liver fibrosis animal model. As shown in the figure, the protein level of NOX2 in the liver tissue of the liver fibrosis animal model was remarkably increased (Experimental Group 1), as was that of α-SMA used as a hepatic stellate cell activity index, whereas the increased protein levels of α-SMA and NOX2 were reduced by 8-OHdG treatment (Experimental Group 2).

[0088] In light of the results of Examples 4-1 to 4-4, the protein levels of TGF-β, TIMP-1, collagen, α-SMA, NOX1, and NOX2, known biomarkers of liver fibrosis and ROS formation process, increased in the liver tissues of the liver fibrosis animal models; however, upon administration with 8-OHdG, the increased protein levels of the biomarkers were reduced.

[0089] In conclusion, the levels of the biomarkers of liver fibrosis and ROS formation process, which were increased due to induced liver fibrosis, are reduced by administration of 8-OHdG in liver fibrosis animal model. Accordingly, the 8-OHdG can be used for the treatment of liver fibrosis.

[0090] From the foregoing, a skilled person in the art to which the present disclosure pertains will be able to understand that the present disclosure may be embodied in other specific forms without modifying the technical concepts or essential characteristics of the present disclosure. In this regard, the exemplary embodiments disclosed herein are only for illustrative purposes and should not be construed as limiting the scope of the present disclosure. On the contrary, the present disclosure is intended to cover not only the exemplary embodiments but also various alternatives, modifications, equivalents, and other embodiments that may be included within the spirit and scope of the present disclosure as defined by the appended claims.