Protective Effect of Eupatilin Against Renal Ischemia-Reperfusion Injury in Mice E.K. Jeonga, H.J. Jangb,*, S.S. Kima, M.Y. Ohb, D.H. Leeb, D.W. Eomc, K.S. Kangd, H.C. Kwane, J.Y. Hame, C.S. Parkb, D.S. Jangf, and D.J. Hang a
Department of Anesthesiology and Pain Medicine, Ulsan University College of Medicine, Gangneung Asan Hospital, Seoul, South Korea; bDepartment of Surgery, Ulsan University College of Medicine, Gangneung Asan Hospital, Seoul, South Korea; cDepartment of Pathology, Ulsan University College of Medicine, Gangneung Asan Hospital, Seoul, South Korea; dCollege of Korean Medicine, Gachon University, Seongnam, South Korea; eNatural Medicine Center, Korea Institute of Science and Technology (KIST), Gangneung, South Korea; fDepartment of Pharmaceutical Science, College of Pharmacy, Kyung Hee University, Seoul, South Korea; and gAsan Medical Center, Seoul, South Korea
ABSTRACT Background. Eupatilin, a pharmacologically active flavone derived from Artemisia species, is known to have antioxidant and anti-inflammatory activities. Ischemia-reperfusion injury (IRI) is a major complication after renal transplantation, with inflammatory responses to IRI exacerbating the resultant renal injury. In the present study, we investigated whether eupatilin exhibits renoprotective activities against ischemia-reperfusion-induced acute kidney injury in mice. Materials and Methods. Renal IRI was induced in male C57BL/6 mice by bilateral renal pedicle occlusion for 30 minutes followed by reperfusion for 48 hours. Eupatilin (10 mg/kg body weight p.o.) was administered 4 days before IRI. Results. Treatment with eupatilin significantly decreased neutrophil gelatinase-associated lipocalin and kidney injury molecule-1 levels in urine, blood urea nitrogen level, and serum creatinine levels, as well as kidney tubular injury. Western blotting indicated that eupatilin significantly increased the levels of heat shock protein 70 and B-cell lymphoma protein, and it attenuated inducible nitric oxide synthase, Bcl-2eassociated X protein, and caspase3 levels 48 hours after IRI. Conclusion. Our findings suggest that eupatilin is a promising therapeutic agent against acute ischemia-induced renal damage.
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ENAL ISCHEMIA-REPERFUSION INJURY (IRI) is a major complication following kidney transplantation. Interruption of blood flow to the kidney along with subsequent reperfusion leads to an acute inflammatory response [1]. Tubular cell apoptosis also contributes to the pathogenesis of renal IRI [2]. The pathogenesis of IRI represents a complex interplay between biochemical, cellular, vascular, endothelial, and tissue-specific factors. Ischemia results in necrosis, apoptosis, and subsequent restoration of blood flow; the latter, although critical to prevent ongoing injury, paradoxically potentiates the inflammatory response, which exacerbates the existing damage [3]. Therapeutic approaches aimed at suppressing the inflammatory response ª 2015 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
Transplantation Proceedings, 47, 757e762 (2015)
and tubular apoptosis are recognized as effective against renal injury along with a better prognosis after IRI. Eupatilin (5,7-dihydroxy-3,4,6-trimethoxyflavone), 1 of the major pharmacologically active compounds contained in Artemisia species, has been reported to possess antioxidative [4], anti-inflammatory, and antiapoptotic activities [5,6]. This study was supported by a grant from the Gangneung Asan Hospital Biomedical Research Center. *Address correspondence to Hyuk Jai Jang, MD, Department of Surgery, Gangneung Asan Hospital, Gangwon-do, Gangneung, Sachean Myeon, Bangdong gil 38, South Korea. E-mail: [email protected] 0041-1345/15 http://dx.doi.org/10.1016/j.transproceed.2014.12.044
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Eupatilin Administration Eupatilin was donated by D. S. Jang (Kyung Hee University, South Korea) and dissolved in vehicle (5% hydroxypropyl methylcellulose). The chemical structure of eupatilin is shown in Fig 1B. Thirty mice were randomly divided into 3 groups with 10 animals per group. The sham and IRI groups (controls) were administered a single dose of vehicle only. The drug-treated groups (eupatilin treatment) were administered a single dose of vehicle with eupatilin (10 mg/kg body weight [BW]) intragastrically 4 days before bilateral IRI.
Biochemical Tests of Serum and Urine Blood samples were obtained from the inferior vena cava. Blood urea nitrogen (BUN) and serum creatinine levels were measured using Cobas C702 analyzer (Roche, Diagnostics, Mannheim, Germany). Urinary neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) levels were measured by enzymelinked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, Minn., United States) as per the manufacturer’s guidelines.
Histological and Immunohistochemical Analyses Fig 1. (A) Experimental design and (B) the chemical structure of eupatilin (5,7-dihydroxy-3,4,6-trimethoxyflavone).
On the basis of these previous findings, we hypothesized that eupatilin could suppress inflammatory activation and apoptosis and therefore could protect against IRI-induced renal injury. Thus, the present study investigated whether eupatilin can abrogate the harmful effects of renal injury in an animal model of renal IRI.
MATERIALS AND METHODS Experimental Animals and Renal IRI Eight-week-old male C57BL/6 mice (weight: 22e25 g) were purchased from Dae Han Bio Link Co., Ltd. (Eumseong, Korea). All experiments conformed to the “Guide for the Care and Use of Laboratory Animals” published by the National Institutes of Health (publication No. 85-23, revised 1985) and were approved by the Ethics Committee of the Korea Institute of Science and Technology (Gangneung, South Korea). The mice were housed in Makrolon cages under standard laboratory conditions (temperature: 22 C 2 C, relative humidity: 55%). The animals were given standard mouse chow and tap water ad libitum. They were subjected to bilateral renal pedicle clamping with microvascular clamps for 30 minutes. Reperfusion commenced when the artery clamps were removed. Occlusion and reperfusion were verified visually by a change in the color of the kidneys to a paler shade and to a blush, respectively. During the procedure, the animals were kept well hydrated with warm saline. The animals were maintained at a constant body temperature (37 C) using a warm pad. After the clamps were removed, reperfusion of the kidneys was observed. A sham operation was performed in a similar manner, except for clamping of the renal pedicles. Blood and renal tissues were harvested 48 hours after reperfusion. Both kidneys were isolated, snap-frozen in liquid nitrogen, and stored at 80 C until further analysis. Blood samples were obtained from the inferior vena cava (Fig 1A).
For the histopathological examination, the kidneys were collected, cut coronally, fixed in 10% formaldehyde, and embedded in paraffin. Five-micrometer sections were prepared and stained with hematoxylin and eosin (H&E). To evaluate the changes in the kidney, the sections were scored 48 hours after IRI by using a semiquantitative scale specifically designed for this purpose [7]. The percentage of tubules in the corticomedullary junction that showed cellular necrosis and loss of brush border were counted and scored on a scale of 1 to 4 blindly: 1 entire deep coronal section was examined under the microscope and graded according to the extent of tubular necrosis based on the percentage of kidney tissue involved. Higher scores represented more severe damage (4 ¼ maximum score); 0 ¼ normal kidney; 1 ¼ minimal necrosis, 75% involvement. Ethanol-fixed, paraffin-embedded tissue sections were stained with a monoclonal antieheat shock protein 70 (HSP70) antibody (Cell Signaling Technology Inc., Danvers, Mass., United States).
Western Blot Analysis The kidneys were crushed in ice-cold buffer (1 M TriseHCl buffer, pH 7.5, with protease inhibitors; 25 mmol/L NaF; 10 mmol/L NaV; 0.5 mol/L EDTA; and 1% Triton X-100) and centrifuged at 14,000 rpm for 20 minutes. The protein concentration was determined using the Bradford protein assay (Bio-Rad, Hercules, Calif., United States). Aliquots of 200 mg of protein extract were separated on 10% to 15% sodium dodecyl sulfate polyacrylamide gels (SDSPAGE; Bio-Rad) and transferred to nitrocellulose membranes (Bio-Rad). The membranes were blocked with 5% milk in Trisbuffered saline and Tween 20 (TBST) buffer (10 mmol/L Trise base, 100 mM NaCl, and 0.1% Tween-20, pH 8.0) and subsequently probed with primary antibodies against HSP70, glyceraldehyde 3phosphate dehydrogenase (GAPHD), B-cell lymphoma (Bcl-2), Bcl-2eassociated X protein (BAX), caspase-3, cleaved caspase-3 (Cell Signaling Technology Inc.), and inducible nitric oxide synthase (iNOS) (1:1,000 dilution, BD Transduction Laboratory, San Diego, Calif., United States) overnight at 4 C. Membranes were then probed with goat anti-rabbit (1:1,000) or goat anti-mouse (1:1,000) horseradish peroxidaseeconjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, Calif., United States). The
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Fig 2. Eupatilin protects renal function in ischemia-reperfusion injury (IRI). Eupatilin (10 mg/kg body weight [BW]) or vehicle was administered orally to mice 4 days before bilateral IRI. Urine samples were collected 6 hours and 1 day post IRI to determine the urine neutrophil gelatinase-associated lipocalin (NGAL) (A) and kidney injury molecule-1 (KIM-1) (B) levels. Blood samples were collected 2 days post IRI to determine the serum creatinine (C) and blood urea nitrogen (BUN) (D) levels. Data are presented as the mean standard error of mean (SEM) (*P < .05, eupatilin vs the vehicle-treated [IRI] group). protein bands were detected using the EZ-Capture ST imaging system (Atto, Japan). The images of the blots were acquired for quantification and analysis.
Statistical Analysis All data are presented as the mean standard error of mean (SEM) and were evaluated by 1-way analysis of variance (ANOVA) with Bonferroni’s post-hoc correction (SPSS software, version 15.0; SPSS Inc., Chicago, Ill., United States). P values of
Department of Anesthesiology and Pain Medicine, Ulsan University College of Medicine, Gangneung Asan Hospital, Seoul, South Korea; bDepartment of Surgery, Ulsan University College of Medicine, Gangneung Asan Hospital, Seoul, South Korea; cDepartment of Pathology, Ulsan University College of Medicine, Gangneung Asan Hospital, Seoul, South Korea; dCollege of Korean Medicine, Gachon University, Seongnam, South Korea; eNatural Medicine Center, Korea Institute of Science and Technology (KIST), Gangneung, South Korea; fDepartment of Pharmaceutical Science, College of Pharmacy, Kyung Hee University, Seoul, South Korea; and gAsan Medical Center, Seoul, South Korea
ABSTRACT Background. Eupatilin, a pharmacologically active flavone derived from Artemisia species, is known to have antioxidant and anti-inflammatory activities. Ischemia-reperfusion injury (IRI) is a major complication after renal transplantation, with inflammatory responses to IRI exacerbating the resultant renal injury. In the present study, we investigated whether eupatilin exhibits renoprotective activities against ischemia-reperfusion-induced acute kidney injury in mice. Materials and Methods. Renal IRI was induced in male C57BL/6 mice by bilateral renal pedicle occlusion for 30 minutes followed by reperfusion for 48 hours. Eupatilin (10 mg/kg body weight p.o.) was administered 4 days before IRI. Results. Treatment with eupatilin significantly decreased neutrophil gelatinase-associated lipocalin and kidney injury molecule-1 levels in urine, blood urea nitrogen level, and serum creatinine levels, as well as kidney tubular injury. Western blotting indicated that eupatilin significantly increased the levels of heat shock protein 70 and B-cell lymphoma protein, and it attenuated inducible nitric oxide synthase, Bcl-2eassociated X protein, and caspase3 levels 48 hours after IRI. Conclusion. Our findings suggest that eupatilin is a promising therapeutic agent against acute ischemia-induced renal damage.
R
ENAL ISCHEMIA-REPERFUSION INJURY (IRI) is a major complication following kidney transplantation. Interruption of blood flow to the kidney along with subsequent reperfusion leads to an acute inflammatory response [1]. Tubular cell apoptosis also contributes to the pathogenesis of renal IRI [2]. The pathogenesis of IRI represents a complex interplay between biochemical, cellular, vascular, endothelial, and tissue-specific factors. Ischemia results in necrosis, apoptosis, and subsequent restoration of blood flow; the latter, although critical to prevent ongoing injury, paradoxically potentiates the inflammatory response, which exacerbates the existing damage [3]. Therapeutic approaches aimed at suppressing the inflammatory response ª 2015 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
Transplantation Proceedings, 47, 757e762 (2015)
and tubular apoptosis are recognized as effective against renal injury along with a better prognosis after IRI. Eupatilin (5,7-dihydroxy-3,4,6-trimethoxyflavone), 1 of the major pharmacologically active compounds contained in Artemisia species, has been reported to possess antioxidative [4], anti-inflammatory, and antiapoptotic activities [5,6]. This study was supported by a grant from the Gangneung Asan Hospital Biomedical Research Center. *Address correspondence to Hyuk Jai Jang, MD, Department of Surgery, Gangneung Asan Hospital, Gangwon-do, Gangneung, Sachean Myeon, Bangdong gil 38, South Korea. E-mail: [email protected] 0041-1345/15 http://dx.doi.org/10.1016/j.transproceed.2014.12.044
757
758
JEONG, JANG, KIM ET AL
Eupatilin Administration Eupatilin was donated by D. S. Jang (Kyung Hee University, South Korea) and dissolved in vehicle (5% hydroxypropyl methylcellulose). The chemical structure of eupatilin is shown in Fig 1B. Thirty mice were randomly divided into 3 groups with 10 animals per group. The sham and IRI groups (controls) were administered a single dose of vehicle only. The drug-treated groups (eupatilin treatment) were administered a single dose of vehicle with eupatilin (10 mg/kg body weight [BW]) intragastrically 4 days before bilateral IRI.
Biochemical Tests of Serum and Urine Blood samples were obtained from the inferior vena cava. Blood urea nitrogen (BUN) and serum creatinine levels were measured using Cobas C702 analyzer (Roche, Diagnostics, Mannheim, Germany). Urinary neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) levels were measured by enzymelinked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, Minn., United States) as per the manufacturer’s guidelines.
Histological and Immunohistochemical Analyses Fig 1. (A) Experimental design and (B) the chemical structure of eupatilin (5,7-dihydroxy-3,4,6-trimethoxyflavone).
On the basis of these previous findings, we hypothesized that eupatilin could suppress inflammatory activation and apoptosis and therefore could protect against IRI-induced renal injury. Thus, the present study investigated whether eupatilin can abrogate the harmful effects of renal injury in an animal model of renal IRI.
MATERIALS AND METHODS Experimental Animals and Renal IRI Eight-week-old male C57BL/6 mice (weight: 22e25 g) were purchased from Dae Han Bio Link Co., Ltd. (Eumseong, Korea). All experiments conformed to the “Guide for the Care and Use of Laboratory Animals” published by the National Institutes of Health (publication No. 85-23, revised 1985) and were approved by the Ethics Committee of the Korea Institute of Science and Technology (Gangneung, South Korea). The mice were housed in Makrolon cages under standard laboratory conditions (temperature: 22 C 2 C, relative humidity: 55%). The animals were given standard mouse chow and tap water ad libitum. They were subjected to bilateral renal pedicle clamping with microvascular clamps for 30 minutes. Reperfusion commenced when the artery clamps were removed. Occlusion and reperfusion were verified visually by a change in the color of the kidneys to a paler shade and to a blush, respectively. During the procedure, the animals were kept well hydrated with warm saline. The animals were maintained at a constant body temperature (37 C) using a warm pad. After the clamps were removed, reperfusion of the kidneys was observed. A sham operation was performed in a similar manner, except for clamping of the renal pedicles. Blood and renal tissues were harvested 48 hours after reperfusion. Both kidneys were isolated, snap-frozen in liquid nitrogen, and stored at 80 C until further analysis. Blood samples were obtained from the inferior vena cava (Fig 1A).
For the histopathological examination, the kidneys were collected, cut coronally, fixed in 10% formaldehyde, and embedded in paraffin. Five-micrometer sections were prepared and stained with hematoxylin and eosin (H&E). To evaluate the changes in the kidney, the sections were scored 48 hours after IRI by using a semiquantitative scale specifically designed for this purpose [7]. The percentage of tubules in the corticomedullary junction that showed cellular necrosis and loss of brush border were counted and scored on a scale of 1 to 4 blindly: 1 entire deep coronal section was examined under the microscope and graded according to the extent of tubular necrosis based on the percentage of kidney tissue involved. Higher scores represented more severe damage (4 ¼ maximum score); 0 ¼ normal kidney; 1 ¼ minimal necrosis, 75% involvement. Ethanol-fixed, paraffin-embedded tissue sections were stained with a monoclonal antieheat shock protein 70 (HSP70) antibody (Cell Signaling Technology Inc., Danvers, Mass., United States).
Western Blot Analysis The kidneys were crushed in ice-cold buffer (1 M TriseHCl buffer, pH 7.5, with protease inhibitors; 25 mmol/L NaF; 10 mmol/L NaV; 0.5 mol/L EDTA; and 1% Triton X-100) and centrifuged at 14,000 rpm for 20 minutes. The protein concentration was determined using the Bradford protein assay (Bio-Rad, Hercules, Calif., United States). Aliquots of 200 mg of protein extract were separated on 10% to 15% sodium dodecyl sulfate polyacrylamide gels (SDSPAGE; Bio-Rad) and transferred to nitrocellulose membranes (Bio-Rad). The membranes were blocked with 5% milk in Trisbuffered saline and Tween 20 (TBST) buffer (10 mmol/L Trise base, 100 mM NaCl, and 0.1% Tween-20, pH 8.0) and subsequently probed with primary antibodies against HSP70, glyceraldehyde 3phosphate dehydrogenase (GAPHD), B-cell lymphoma (Bcl-2), Bcl-2eassociated X protein (BAX), caspase-3, cleaved caspase-3 (Cell Signaling Technology Inc.), and inducible nitric oxide synthase (iNOS) (1:1,000 dilution, BD Transduction Laboratory, San Diego, Calif., United States) overnight at 4 C. Membranes were then probed with goat anti-rabbit (1:1,000) or goat anti-mouse (1:1,000) horseradish peroxidaseeconjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, Calif., United States). The
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Fig 2. Eupatilin protects renal function in ischemia-reperfusion injury (IRI). Eupatilin (10 mg/kg body weight [BW]) or vehicle was administered orally to mice 4 days before bilateral IRI. Urine samples were collected 6 hours and 1 day post IRI to determine the urine neutrophil gelatinase-associated lipocalin (NGAL) (A) and kidney injury molecule-1 (KIM-1) (B) levels. Blood samples were collected 2 days post IRI to determine the serum creatinine (C) and blood urea nitrogen (BUN) (D) levels. Data are presented as the mean standard error of mean (SEM) (*P < .05, eupatilin vs the vehicle-treated [IRI] group). protein bands were detected using the EZ-Capture ST imaging system (Atto, Japan). The images of the blots were acquired for quantification and analysis.
Statistical Analysis All data are presented as the mean standard error of mean (SEM) and were evaluated by 1-way analysis of variance (ANOVA) with Bonferroni’s post-hoc correction (SPSS software, version 15.0; SPSS Inc., Chicago, Ill., United States). P values of
