Ameliorative Effect of Ecklonia cava Polyphenol Extract on Renal

May 1, 2017 - Inflammation Associated with Aberrant Energy Metabolism and. Oxidative Stress in High Fat Diet-Induced Obese Mice. Hyeyoon Eo,. †...
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Ameliorative Effect of Ecklonia cava Polyphenol Extract on Renal Inflammation Associated with Aberrant Energy Metabolism and Oxidative Stress in High Fat Diet-Induced Obese Mice Hyeyoon Eo,† Ji Eun Park,† You-jin Jeon,‡ and Yunsook Lim*,† †

Department of Food and Nutrition, Kyung Hee University, 26 Kyunghee-daero, Dongdaemun-gu, Seoul 02447, Republic of Korea Department of Marine Life Sciences, Jeju National University, Jeju 63243, Republic of Korea

‡

ABSTRACT: Immoderate fat accumulation causes both oxidative stress and inflammation, which can induce kidney damage in obesity. Previously, Ecklonia cava has shown anti-inflammatory and antioxidative effects. Our group aimed to investigate whether E. cava polyphenol extract (ECPE) improves renal damage in high fat diet (HFD)-induced obese mice through regulation of not only energy metabolism but also oxidative stress and inflammation. After obesity induction by HFD, the mice were treated with different dosages of ECPE (100 or 500 mg/kg/day) by gavage for 12 weeks. ECPE treatment lowered the protein levels related to lipid accumulation (SREBP1c, ACC & FAS), inflammation (NLRP3 inflammasome, NFκB, MCP-1, TNF-α & CRP), and oxidative stress (Nrf2, HO-1, MnSOD, NQO1, GPx, 4-HNE and protein carbonyls) in HFD induced obese mice. Moreover, ECPE supplementation significantly up-regulated renal SIRT1, PGC-1α, and AMPK, which are associated with renal energy metabolism. Consequently, the results provide novel insights into the anti-inflammatory roles of ECPE in obesity-induced renal inflammation. KEYWORDS: oxidative stress, inflammation, kidney damage, obesity, E. cava polyphenol extract

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including proteinuria.1,2 In addition, overproduction of these adipokines, including leptin, adiponectin, and tumor necrosis factor-α (TNF-α), in adipose tissues can intensify renal inflammation in obesity.5 Moreover, abnormal energy balance, causing ROS overproduction, activates the NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome, including NLRP3, apoptosis associated speck-like protein containing a caspase activation, and recruitment domain (ASC), procaspase-1. NLRP3 inflammasome contributes to te increase in pro-inflammatory cytokines, in particular, interleukin-1β.12 These pro-inflammatory cytokines, such as IL-1β and TNF-α, stimulate transcription of nuclear factor kappa B (NFκB) and overexpression of NFκB, which subsequently induces various inflammatory factors, such as TNF-α, IL-1β, cell adhesion molecules, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2).13 Hence, regulation of NFκB and its related markers would be a good strategy for improvement of renal inflammation in obesity. Furthermore, aberrant energy and lipid metabolism in the kidney is associated with renal damage.4,14 Sirtuin 1 (SIRT1) and AMP-activated kinase (AMPK) are the regulators of energy and lipid metabolism. AMPK regulates lipid metabolism and repairs energy balance.15 When the intracellular AMP/ATP ratio increases, AMPK moderates the production of cholesterol by suppressing HMG-CoA reductase activation, which is the rate-limiting stage of cholesterol synthesis.16 AMPK also inhibits lipogenesis by reducing the expression of the sterol

INTRODUCTION Obesity is a main risk factor for metabolic diseases such as diabetes, cardiovascular disease (CVD), and chronic kidney disease (CKD). Moreover, it has been reported that CKD prevalence is strongly associated with obesity, and the number of obese CKD patients has consistently been rising.1,2 The major causes of CKD in obesity are hyperglycemia and hypertension associated with lipid accumulation.2−4 These pathologic changes in obesity-related kidney diseases are promoted by oxidative stress, inflammation, and renal lipid accumulation.1,4,5 Chronic energy accumulation causes an imbalance between generated reactive oxidative species (ROS) and its defending system, called antioxidant defense system, and thus leads to excessive oxidative stress.1 At the cellular level, enzymes showing antioxidative properties, such as glutathione peroxidases (GPx), superoxide dismutase (SOD), and heme oxygenase-1 (HO-1), are involved in the antioxidant defense system.6 Nuclear factor erythroid-2-related factor-2 (Nrf2) is a crucial modulator of those antioxidative enzymes playing a role in transcription factor.6,7 Obese individuals have showed different expression patterns of Nrf2 and its related enzymes in different tissues.8−10 The kidney is especially sensitive to oxidative stress because it is one of the mitochondria-rich organs and has a higher chance to encounter ROS.11 Thus, Nrf2 and its associated enzymes could be a target mechanism to regulate oxidative stress in kidney under obese condition. Furthermore, cellular oxidative stress in obesity may cause inflammation by recruitment of macrophage in several tissues, such as those of the kidney, liver, and myocardium by secretion of pro-inflammatory cytokines.1,2 This fact is strongly related to the development and progression of types of tissue damage, © 2017 American Chemical Society

Received: Revised: Accepted: Published: 3811

January 23, 2017 May 1, 2017 May 1, 2017 May 1, 2017 DOI: 10.1021/acs.jafc.7b00357 J. Agric. Food Chem. 2017, 65, 3811−3818

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Journal of Agricultural and Food Chemistry

week for 12 weeks to minimize stress that mice could get. During supplementation, body weight and food intake were checked twice a week. At the end of the animal experiment, all the animals were anesthetized by isoflurane (Baxter, Deerfield, IL, USA). A heparincoated syringe was used to collect the blood samples from the heart. Collected blood samples were centrifuged at 3000 rpm at 4 °C for 15 min. Then the kidneys were isolated, weighed, washed by saline, frozen in liquid nitrogen, and stored at −80 °C before the experiment. To evaluate renal hypertrophy, the kidney index was calculated by kidney weight/body weight (mg/g) as previously described.27 All the experimental protocols were accepted by the Institutional Animal Care and Use Committee of Kyung Hee University. Lipid Extraction and Determination in the Kidneys. Total lipids of the kidneys were extracted from 0.05 g of renal tissue. Renal tissues were homogenized with 1 mL of chloroform and methanol (CM) mixture (2:1) and centrifuged at 3000 rpm for 15 min. Then 200 μL of CM mixture and 200 μL of 0.9% NaCl were added to the supernatants, homogenized again, and centrifuged at 3000 rpm for 5 min. The lower layers were obtained and dried in a dry oven at 60 °C. Iso-propyl alcohols (200 μL) were added to an EP-tube in the dried lipid layer and vortexed to dissolve the lipids. The contents of total triacylglyceride (TG) and total cholesterol (TC) in the kidneys were determined through commercial kits (Asan pharmaceutical, Seoul, South Korea). Western Blotting Assay. Each kidney sample was homogenized in the hypotonic lysis buffer (10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.05% NP40 and distilled water) with protease/ phosphatase inhibitors (Bio-Rad). The homogenates were centrifuged at 14000 rpm at 4 °C for 30 min. The supernatants were used as cytosol extract for Western blot analysis. The remaining pellets were resoaked in hypertonic lysis buffer (5 mM HEPES, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 26% glycerol, and distilled water) with 4.6 M NaCl and homogenized on ice. After shaking on ice for 30 min, the homogenates were centrifuged at 14,000 rpm for 20 min at 4 °C, and then supernatants were used as nuclear extract for Western blotting. The total protein amount of the protein extract was quantified by BCA protein assay (Thermo Fisher, Waltham, Massachusetts, USA). Protein samples were isolated by molecular weight through SDSPAGE and transferred onto polyvinylidine fluoride (PVDF) membranes (Millipore, Marlborough, MA, USA). After blocking in 3% BSA in phosphate buffered saline−0.1% Tween 20 (PBS-T), the membranes were incubated at 4 °C with primary antibodies including AMPK (Cell Signaling Technology, Inc., MA, USA), phosphorylated AMPK (p-AMPK; Cell Signaling Technology, Inc., MA, USA), SREBP1-c (Santa Cruz Biotechnology, Inc., CA, USA), FAS (Santa Cruz Biotechnology, Inc., CA, USA), ACC (Cell Signaling Technology, Inc., MA, USA), phosphorylated ACC (p-ACC; Cell Signaling Technology, Inc., MA, USA), SIRT1 (Abcam, Cambridge, UK), PGC1 (Santa Cruz Biotechnology, Inc., CA, USA), NLRP3 (Santa Cruz Biotechnology, Inc., CA, USA), ASC (Cell Signaling Technology, Inc., MA, USA), caspase-1 (Santa Cruz Biotechnology, Inc., CA, USA), NFκB (Cell Signaling Technology, Inc., MA, USA), MCP-1 (Cell Signaling Technology, Inc., MA, USA), TNF-α (Santa Cruz Biotechnology, Inc., CA, USA), IL-1β (Santa Cruz Biotechnology, Inc., CA, USA), C-reactive protein (CRP; Abcam, Cambridge, UK), iNOS (Santa Cruz Biotechnology, Inc., CA, USA), Nrf2 (Cell Signaling Technology, Inc., MA, USA), HO-1 (Stressgen, CA, USA), MnSOD (Stressgen, CA, USA), NAD(P)H quinone dehydrogenase-1 (NQO1; Cell Signaling Technology, Inc., MA, USA), GPx (Abcam, Cambridge, UK), and 4-hydroxy-trans-2-nonenal (4-HNE; BD Biosciences, NJ, USA). Protein carbonyl groups in renal protein were detected with a Oxidized Protein Western Blot Detection Kit (abcam, Cambridge, UK) according to the manufacturer’s instructions. To confirm equal protein loading, α-tubulin (Sigma-Aldrich, St. Louis, MO, USA) for cytosolic extract and Lamin B1 (Abcam, Cambridge, UK) for nuclear fraction were used, respectively. Then the membranes were washed with PBS-T. The blots were incubated with respective horseradish peroxide (HRP)-conjugated secondary antibodies for 1 h and washed with PBS-T again. The chemiluminescent actions were

regulatory element-binding protein (SREBP-1), acetyl-coA carboxylase (ACC), and fatty acid synthase (FAS) to attenuate renal inflammation.16−18 SIRT1 has protective effects on the kidney upon oxidative stress, inflammation, apoptosis, and lipid metabolism disorder.19 The SIRT1 subunits inhibit NFκB transcription by deacetylating the NFκB p65 subunit.20 PPARγ coactivator 1 (PGC-1α), the downstream target of the SIRT1/ AMPK signaling pathway, suppresses the activation of proinflammatory cytokines and ROS production.21 Previously, it was reported that reduction in glomerular lipid accumulation and renal inflammation can induce decreasing glomerulosclerosis and albuminuria on HFD-induced obese mice.22 Therefore, alleviation of inflammatory response, oxidative stress, and lipid accumulation through SIRT1/AMPK signaling in the kidney would be a possible target mechanism for improving obesity and its complications, especially in the case of renal damage. Ecklonia cava was known for anti-inflammatory, antioxidative, and antidiabetic effects.23,24 It was reported that dieckol, the most abundant polyphenol extracted from E. cava, regulates adipogenesis by stimulating AMPK and decreases the differentiation of adipocytes to suppress release of inflammatory cytokines.24−26 Recently, our group reported that E. cava polyphenol extract (ECPE) supplementation improved lipid metabolism, inflammation, and oxidative stress in liver by AMPK/SIRT1 partnership in high-fat diet (HFD)-induced mice.24 However, it is still unclear whether ECPE ameliorates renal inflammation associated with oxidative stress and aberrant energy metabolism in obese models. Therefore, the current research investigated the effect of ECPE on renal inflammation associated with NFκB, SIRT-1, and AMPK in HFD-induced obese mice.

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MATERIALS AND METHODS

Preparation of ECPE. ECPE in the current research was obtained from Aqua Green Tech., Jeju, Korea. The preprocessing of ECPE was described before because the current research is a follow-up study to our previous research.24 Briefly, E. cava was harvested at Jeju Island, Korea. The E. cava was washed with fresh water to remove foreign materials and salts and then was freeze-dried. E. cava powder (500 g) was soaked in 70% ethanol (10 L) and stirred for 24 h at room temperature. Then, the solution was filtered and enriched under reduced pressure. After that, each fraction was taken by treatment with ethyl acetate, and the extract dried up under vacuum. The yield of the extract was 9.57% (47.85 g/500 g). Animals and Obesity Induction. C57BL/6 mice (4-week-old, male, n = 40) were purchased from Orient Bio (Gyeonggi-do, South Korea). All of the mice were housed two or three per cage in a 12 h light/12 h dark cycle, at continuous temperature (22 ± 1 °C) and relative humidity (50 ± 5%). After acclimation for 1 week, the mice were distributed into two experimental groups randomly: a control diet group (n = 10) and a high-fat diet group (n = 30). Each group was fed with either the control diet (CD; 10% kcal fat, Research Diets, New Brunswick, NJ, USA) or the high fat diet (HFD; 45% kcal fat, Research Diets), and distilled water for 10 weeks, ad libitum. Experimental Design. After induction of obesity by HFD for 10 weeks, every mouse was randomly assigned into 4 experimental groups (n = 6 per group) as follows: (A) The CON group, CD-fed nonobese mice group, was supplemented with distilled water by oral gavage; (B) The HF group, HFD-fed obese mice group, was supplemented with distilled water by oral gavage; (C) The LE group, HFD-fed obese mice group, was supplemented with low doses of ECPE (100 mg/kg/day) by oral gavage; and (D) The HE group, HFD-fed obese mice group, was supplemented with high doses of ECPE (500 mg/kg/day) by oral gavage. ECPE freshly dissolved in distilled water or the equivalent volume of distilled water was administrated by oral gavage 5 days a 3812

DOI: 10.1021/acs.jafc.7b00357 J. Agric. Food Chem. 2017, 65, 3811−3818

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Journal of Agricultural and Food Chemistry Table 1. Effect of ECPE on Body Weight and Kidney Weighta Groupb CON Body weight (g) before supplementation after supplementation weight gain Kidney weight (mg) Kidney Index (mg/g)

30.58 37.28 6.70 177.32 4.78

± ± ± ± ±

0.17a 0.59 a 0.45 a 2.25 ab 0.11 a

HF 40.70 49.88 9.18 174.28 3.50

± ± ± ± ±

LE 0.44 0.41 0.28 4.30 0.09

b c b b c

40.77 ± 0.27 b 49.27 ± 0.62 c 8.50 ± 0.50 b 186.18 ± 3.04 a 3.783 ± 0.05 b

HE 40.42 46.05 5.63 172.38 3.74

± ± ± ± ±

0.45 b 0.55 b 0.28 a 2.84b 0.04 b

Values are mean ± SD. Mean values with unlike letters are significantly different (p < 0.05). bCON, mice fed with control diet (negative control); HF, mice fed with the high-fat diet; LE, mice fed with high-fat diet and supplemented with low dose (100 mg/kg/day) of ECPE; HE, mice fed with high-fat diet and supplemented with high dose (500 mg/kg/day) of ECPE. The kidney index was calculated by kidney weight/body weight (mg/g). a

developed using ECL solution (Biorad, CA, USA). Images of the developed bands were captured. The band intensity was quantified through the Syngene G box (Syngene, Cambridge, UK). Total Superoxide Dismutase (SOD) Activity Assay in Kidney. Enzymatic assay of SOD activity in kidney was assessed using 216 mM potassium phosphate buffer, 10.7 mM ethlenediaminetetraacetic acid solution (EDTA), 1.1 mM cytochrome C solution, 0.108 mM xanthine solution, xanthine oxidase enzyme solution, and superoxide dismutase enzyme solution (Sigma-Aldrich, St. Louis, MO, USA). Renal samples were homogenized in 10 mL of cold lysis buffer (10 mM Tris, 150 mM NaCl, and 0.1 mM EDTA) per gram of tissue. The homogenates were centrifuged at 12000 rpm at 4 °C for 10 min and the tissue lysate supernatants were collected. The total protein amount of the protein extract was quantified by Bradford protein assay (Biorad, Hercules, CA, USA). SOD activity was analyzed according to the manufacturer’s instructions. Data were corrected by protein concentration of loaded sample and were converted into a percentage. Statistical Analysis. Data are presented as mean ± standard deviation (S.D.). Significant differences among the groups were examined by one-way ANOVA through SPSS (version 22.0 for Windows, SPSS Inc., IL, USA) following Duncan’s multiple range tests when differences were determined among the groups at p < 0.05.

Figure 1. Effect of ECPE supplementation on renal lipid accumulation in high-fat diet induced obese mice. Levels of triglyceride and total cholesterol were measured in kidney tissues. Values are means ± SD (n = 6). Bars with a different letter are significantly different (p < 0.05).

among HFD-fed groups regardless of ECPE treatment. At the same time, the protein levels of pAMPK were significantly lower in the HF group than in the CON group, while the protein levels of pAMPK in the HE group were significantly higher than those in the HF group. As a result, the ratio between pAMPK and AMPK was significantly increased in the HE group as compared with those of the HF group. Effect of ECPE Supplementation on the Protein Levels of SIRT1 and Its Related Markers. Renal SIRT1 protein levels in the HF group were significantly lower compared to those in the CON group (Figure 2B). However, the ECPE supplemented groups showed higher protein levels of renal SIRT1 than the HF group, regardless of dosages. In addition, the protein levels of both cytosolic and nuclear PGC1α in the HF group were significantly lower than those in the CON group, but the HE group showed higher protein levels of both cytosolic and nuclear PGC1α than the HF group. Effect of ECPE Supplementation on the Protein Levels of Lipid Metabolism-Related Markers. To examine the effect of ECPE supplementation on renal lipid metabolism, protein levels were measured, including SREBP1c, FAS, ACC, and pACC (Figure 3). The protein levels of nuclear SREBP1c in the HF group were significantly higher than those in the CON group. However, the protein levels of nuclear SREBP1c in the ECPE-supplemented groups were lower than those in the HF groups. While there were no significant differences in the protein levels of FAS between the CON group and the HF group, the protein levels of FAS in the LE group were significantly lower than those in the HF group. At the same time, the protein levels of both ACC and phosphorylated ACC in the HF group were significantly lower than those in the

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RESULTS Effect of ECPE Supplementation on Body Weight, Kidney Weight, and Kidney Index. In the current study, body weight and kidney weight were measured in all the groups (Table 1). The body weight of the HF group was significantly increased compared to the CON group after HFD feeding for 10 weeks. After supplementation for 12 weeks, the HE group showed significantly reduced body weight and body weight gain. Kidney weight had no significant difference between the CON group and the HF group, but the LE group had significantly higher kidney weight compared to the HF group. To estimate renal hypertrophy, a kidney index calcuated by kidney weight/body weight (mg/g) was calculated. The kidney index in the HF group was significantly lower than that in the CON group, but the kidney index of the ECPE supplemented group was significantly higher than that in the HF group. Effect of ECPE Supplementation on Renal Lipid Accumulation. The renal contents of TG and TC were measured (Figure 1). The renal contents of TG and TC in the HF group were significantly increased compared to those in the CON group. However, the LE amd HE groups had lower contents of TG whereas the HE group showed lower contents of both TG and TC compared to the HF group. Effect of ECPE Supplementation on AMPK Phosphorylation. The protein levels of AMPK in the HF group were significantly lowered compared to those in the CON group (Figure 2A). However, there were no significant differences 3813

DOI: 10.1021/acs.jafc.7b00357 J. Agric. Food Chem. 2017, 65, 3811−3818

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Journal of Agricultural and Food Chemistry

Figure 2. Effect of ECPE supplementation on renal AMPK phosphorylation and SIRT1-PGC1α activation in high-fat diet induced obese mice. Renal AMPK phosphorylation (A) and SIRT1-PGC1α activation (B). Representative band images of each marker are shown in the left panel. The intensity of the bands was densitometrically measured, normalized to the band levels of α-tubulin for AMPK, p-AMPK, SIRT1, and cytosolic PGC1α or Lamin B1 for nuclear PGC1α (right panel), and presented as arbitray unit. Values are means ± SD (n = 6). Bars with a different letter are significantly different (p < 0.05).

Figure 3. Effect of ECPE supplementation on renal lipid metabolism in high-fat diet induced obese mice. Representative band images of each marker are shown in the left panel. The intensity of the bands was densitometrically measured, normalized to the band levels of α-tubulin for FAS, ACC, and p-ACC or Lamin B1 for nuclear SREBP1c (right panel), and presented as arbitray unit. Values are means ± SD (n = 6). Bars with a different letter are significantly different (p < 0.05).

group and the HF group; the protein levels of iNOS in the HE group were significantly lower than those in the HF group. Effect of ECPE Supplementation on Plasma and Renal Oxidative Stress. As a marker of lipid peroxidation, the current study measured protein levels of 4-HNE in both kidney and plasma (Figure 5A). The HF group showed higher protein levels of 4-HNE in both the kidney and plasma compared to the CON group. The ECPE-supplemented group showed a significant decrease in the protein levels of plasma 4-HNE compared to the HF group. There were no significant differences in the protein expressions of 4-HNE among HFD-fed groups regardless of ECPE supplementation. Other oxidative stress end products, protein carbonyls in kidney, were increased in the HF group and reduced in the ECPE supplemented groups (Figure 5B). To investigate the effect of ECPE treatment on renal oxidative stress at the molecular level, the protein levels of nuclear Nrf2 and its related markers, including HO-1, MnSOD, NQO1, and GPx, were measured (Figure 5C). The protein levels of nuclear Nrf2 in the HF group were significantly increased compared to those in the CON group, but the HE group showed lower protein levels of nuclear Nrf2 than the HF group. In addition, the protein levels of HO-1, MnSOD,

CON group, while the protein levels of ACC and pACC in the HE group were significatly higher than those in the HF group. Effect of ECPE Supplementation on the Protein Levels of NFκB-Related Inflammatory Markers. NLRP3 inflammasome, NFκB, and its related markers were measured at the protein levels to evaluate the effect of ECPE supplementation on renal inflammation. Although the protein levels of NLRP3 did not have significant differences between the ECPE supplemented groups and the HF group, the protein levels of ASC, procaspase-1, caspase-1, pre IL-1β, and cleaved IL-1β in ECPE supplemented groups were significantly lower than those of the HF group (Figure 4A). Morever, the protein levels of nuclear NFκB in the HF group were significantly higher than those in the CON group. However, the ECPE supplemented group showed decreased protein levels of nuclear NFκB compared to the HF group, regardless of dosages. In addition, the HF group had increased protein levels of NFκB-related inflammatory markers including TNFα, ICAM-1, and COX-2 as well as other inflammatory markers, CRP and MCP-1 compared to the CON group. However, the HE group showed a lower degree of protein in CRP, TNF-α, ICAM-1, and COX2 compared to the HF group. While there were no significant differences in the protein levels of iNOS between the CON 3814

DOI: 10.1021/acs.jafc.7b00357 J. Agric. Food Chem. 2017, 65, 3811−3818

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Journal of Agricultural and Food Chemistry

Figure 4. Effect of ECPE supplementation on renal inflammation in high fat diet induced obese mice. Protein levels of (a) NLRP3 inflammasome and the (B) pro-inflammatory response and the NFκB related inflammatory response; representative band images of each marker are shown in the left panel. The intensity of the bands was densitometrically measured, normalized to the band levels of α-tubulin for cellular markers or Lamin B1 for nuclear markers and presented as arbitray unit (right panel). Values are means ± SD (n = 6). Bars with a different letter are significantly different (p < 0.05).

Figure 5. Effect of ECPE supplementation on plasma and renal oxidative stress in high-fat diet induced obese mice. Representative band images of (A) plasma and renal 4HNE, (B) renal protein carbonyl groups, (C) Nrf2 associated antioxidant defense system, and (D) renal % SOD activity were shown. The intensity of the bands was densitometrically measured, normalized to the band levels of α-tubulin for cellular markers or Lamin B1 for nuclear markers and presented as arbitray unit. Values are means ± SD (n = 6). Bars with a different letter are significantly different (p < 0.05).

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NQO1, and GPx in the HF group were significantly higher than those in the CON group. However, the ECPE-supplemented groups showed lower protein levels of HO-1, MnSOD, NQO1, and GPx than the HF group regardless of dosages. Although the protein levels of antioxiant enzymes were increased, total SOD activity was reduced in the HF group and increased in the HE group (Figure 5D).

DISCUSSION

The current study aimed to research the effect of dietary E. cava supplementaion on renal inflammation, oxidative stress, and altered lipid metabolism, which are factors that threaten kidney health in obesity. All of the results of the current study demonstrated that ECPE supplementaion attenuated the renal inflammatory response related to NLRP3 inflammasome, NFκB, oxidative stress, and energy metabolism under obese 3815

DOI: 10.1021/acs.jafc.7b00357 J. Agric. Food Chem. 2017, 65, 3811−3818

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tissues.19,33,34 SIRT1 activation ameliorates the inflammatory response by suppression of transcription factors, NFκB.21 In the current study, the HF group showed a decrease in SIRT1 with an increase in NFκB. In contrast, ECPE supplementation increased in SIRT1 but decreased NFκB at the protein level in obese mice. Moreover, the present study suggests the role of SIRT1 in mitochondrial biogenesis through enhancement of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α).19 Previous studies showed that SIRT1 overexpression or intervention with SIRT1 activators, such as resveratrol and SRT1720, ameliorated renal function and improved kidney damage through stimulation of PGC-1α expression and deacetylation of PGC-1α.35,36 The current study demonstrated that renal protein levels of SIRT1 and nuclear PGC-1α were reduced in HFD-induced obese mice. However, high doses of ECPE supplementation enhanced the protein levels of SIRT1 and PGC-1α along with reduced NFκB activation in the kidney compared to the HF group. Furthermore, our group investigated renal inflammatory markers as well as antioxidative markers to prove another effect of ECPE supplementation on the kidney in conditions of obesity. The current study focused on NFκB-related inflammatory responses and the Nrf2-associated antioxidant defense system in the kidney. NFκB is activated by oxidative stress and proinflammatory cytokines such as TNF-α and IL-1β. In particular, active IL-1β is regulated by NLRP3 inflammasome. The activation of NLRP3 causes oligomerization and recruitment of adaptor protein, ASC. These interactions facilitate the subsequent activation of caspase-1. Activated caspase-1 cleaves the proform of IL-1β, one of the proinflammatory cytokine leading to NFκB activation.21 NFκB activation leads to gene expression of downstream genes, including TNFα, IL-1β, ICAM-1, COX-2, and iNOS, which are considered as inflammatory markers. Several studies have found that expression within those inflammatory markers, including NLRP3 imflammasome, NFκB, and its related inflammatory markers, was higher in diet-induced obese models.37−40 Similarly, the current result demonstrated that obese mice showed higher renal protein levels of NLRP3 inflammasome, NFκB, and its downstream proteins than the normal mice.41 However, ECPE supplementation reduced renal inflammatory responses, possibly through reduction of NLRP3 inflammasome, NFκB activation, and its related inflammatory markers in HFD-induced obese mice. The results suggest that ECPE effectively ameliorated renal hyperinflammation in obesity. Nrf2, one of the redox-associated transcription factors, is a crucial molecule that regulates antioxidant defense systemrelated enzymes, such as SODs, HO-1, NQO1, and GPx.42,43 Hence, Nrf2 is regarded as a potential target in the prevention and amelioration of obesity and obesity-derived oxidative stress. In previous reports, the antioxidant defense system was shown to be damaged under obese condition.44−46 Our results similarly showed that renal Nrf2 and its related proteins were significantly increased in an obese group. In contrast to protein levels of antioxidant enzymes including MnSOD, total SOD activity in the obesity group was lower than that of the normal group. Moreover, renal 4-HNE and protein carbonyls were significantly increased in the obese mice compared to the normal mice. 4-HNE is one of the lipid hydroperoxides that is produced when excessive ROS insult polyunsaturated fatty acids (PUFAs) of plasma membranes.47 Thus, the amount of 4HNE is considered as a well-studied representative biomarker of oxidative stress.47,48 Protein carbonylation is the severe

conditions. Our results particularly showed that ECPE supplementation not only activated AMPK and its downstreams but also stimulated SIRT1 in the kidneys of obese mice. Our previous study demonstrated that ECPE supplementation reduced hepatic oxidative stress and inflammation through upregulation of the antioxidant defense system besides downregulation of the NFκB-related inflammatory response.24 In addition, supplementation with ECPE reduced hepatic lipid accumulation by up-regulating AMPK phosphorylation.24 However, the current research focuses on kidney tissue in terms of oxidative stress, inflammation, and lipid metabolism with respect to the SIRT1/NFκB/Nrf2 pathway. First of all, the current study demonstrated renal lipid accumulation presented by the contents of TG and TC. A few studies have reported that obese subjects showed high lipid contents in the kidney, experimentally14,28 and clinically. In addition, there have been several reports providing evidence to show that fat accumulation is directly involved in the pathogenesis of kidney damage.14,28,29 Thus, inhibition of renal lipid accumulation is a crucial step for prevention or amelioration of obese-related kidney injuries. Compared with previous studies,14,28 the current study similarly showed that TG and TC contents were increased in HFD-induced obese mice. However, ECPE supplementation reduced renal TG and TC levels in obese mice. How could ECPE supplementation reduce the renal lipid accumulation in obesity? AMPK is a energy-sensing kinase designed to regulate cellular energy metabolism and is ubiquitously expressed in all eukaryotic cells.4,30 Higher AMP/ATP ratio stimulates AMPK phosphorylation, and this reaction up-regulates ACC phosphorylation.4 Phosphorylated ACC reduces fatty acid synthesis through cellular malonyl-CoA levels. Moreover, a fall in malonyl-CoA increases mitochondrial oxidation of fatty acids through activation of carnitine palmitoyltransferase 1 (CPT-1).4 Furthermore, activation of AMPK prevents production and transcriptional activity of SREBP-1c, which is necessary for enzymes related to fatty acid synthesis, such as FAS. Hence, AMPK activation is recognized as a target mechanism to reduce renal lipid accumulation by inhibition of anabolism as well as by stimulation of catabolism in lipid metabolism. In the current study, HFD-induced obese mice showed a reduction in AMPK-ACC activation. However, ECPE supplementation increased AMPK-ACC phosphorylation and reduced protein expressions of SREBP-1c and FAS in the kidney. Thus, it might be inferred that ECPE supplementation could increase fatty acid oxidation and decrease fat accumulation in obese kidney tissue. However, the kidney would still have a high chance to be exposed to cellular stress due to excessive ROS production or altered inflammatory response under obese conditions, and this phenomenon is known as lipotoxicity.14,28,31,32 In general, mechanisms of cellular damage from lipotoxicity include oxidative stess, inflammation, fibrosis, altered intracellular signaling pathways, and lipid-induced apoptosis.32 Thus, in addition to lipid metabolism, regulation of oxidative stress and inflammtory response is also accentuated in the obese kidney. Among the several markers related to cellular stress, SIRT1 is another energy-sensing molecule besides AMPK and is stimulated in response to NAD+ concentration.19 It has been reported several times that SIRT1 is involved in oxidative stress, inflammation, and fibrosis, and these three concepts are known as not only one of the renal pathogenic mechanisms but also one of the common results from lipoxicity in various 3816

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Journal of Agricultural and Food Chemistry modification of protein by oxidative stress.49 As protein-bound carbonyl can be induced by lipid peroxidation, metal-catalyzed oxidation, or glycation/glycoxidation, protein carbonylation is one important characteristic of oxidative stress.49 The current results show that the increase in 4-HNE and protein carbonyl adducts corresponds to Nrf2 activation without an increase in antioxidant enzyme activity. This discrepancy among Nrf2, its antioxidant dependent enzyme levels, and SOD activity might be explained by the compensatory effects of Nrf2 and the antioxidant defense system in response to overproduction of ROS under obese conditions.50 In other words, increased ROS production induces activation of Nrf2 and expression of its related antioxidant enzymes. But, this induction might not be enough to increase antioxidant enzyme activities which reduce oxidative stress in the obese condition presented in this study. However, ECPE supplementation with high dose reduced 4HNE, protein carbonyls, Nrf2, and its related markers with higher activities of total SOD in obesity. In particular, the result indicates that ECPE supplementation reduced oxidative stress, resulting in lower levels of Nrf2 and its related markers in both the plasma and the kidney with higher SOD activity under obese conditions. Therefore, from the results it might be inferred that ECPE supplementation reduces renal oxidative stress under obese conditions. Taken together, the current study reported for the first time that ECPE has ameliorative effects on renal inflammation associated with altered lipid metabolism and oxidative stress in vivo obese mice models. ECPE supplementation especially activates the SIRT1/AMPK pathway and consequently reduces renal lipid accumulation in HFD-induced obese mice. Furthermore, ECPE supplementation modulates renal NLRP3 inflammasome, NFκB, and its up-/downstream inflammatory markers and Nrf2 and its related antioxidant defense system in obesity. Therefore, the present study suggests that E. cava can be a functional food for improving renal health in conditions of obesity.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 82-2-961-0262. Fax: 82-2-961-0261. E-mail: ylim@khu. ac.kr. ORCID

Hyeyoon Eo: 0000-0002-1532-613X Funding

This study was supported by IPET (Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries), Ministry of Agriculture, Food and Rural Affairs (115045-03). Notes

The authors declare no competing financial interest.

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