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Contents lists available at ScienceDirect
Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox 6 7
Purple sweet potato color ameliorates kidney damage via inhibiting oxidative stress mediated NLRP3 inflammasome activation in high fat diet mice
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Qun Shan a, Yuan-lin Zheng a,⇑, Jun Lu a, Zi-Feng Zhang a, Dong-mei Wu a, Shao-hua Fan a, Bin Hu a, Xiang-jun Cai a, Hao Cai b, Pei-long Liu b, Fan Liu b a b
Key Laboratory for Biotechnology on Medicinal Plants of Jiangsu Province, School of Life Science, Jiangsu Normal University, Xuzhou 221116, Jiangsu Province, PR China Kewen College, Jiangsu Normal University, Xuzhou 221116, Jiangsu Province, PR China
a r t i c l e
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Article history: Received 1 February 2014 Accepted 22 April 2014 Available online xxxx Keywords: PSPC HFD NLRP3 inflammasome Oxidative stress Kidney damage
a b s t r a c t Inflammation plays a crucial role in the pathogenesis of obesity. Purple sweet potato color (PSPC) has potential anti-inflammation efficacy. We evaluated the effect of PSPC on kidney injury induced by high fat diet (HFD) and explored the mechanism underlying these effects. The results showed that PSPC (700 mg/kg per day) reduced body weight, ratio of urine albumin to creatinine, inflammatory cell infiltration, and Collagen IV accumulation in mice fed an HFD (60% fat food) for 20 weeks. PSPC significantly reduced the expression level of kidney NLRP3 inflammasome including NLRP3 and ASC and Caspase-1, and resulted in decline of IL-1b. Moreover, PSPC inhibited the activation of I kappa B kinase b (IKKb) and the nuclear translocation of nuclear factor kappa beta (NF-jB). Additionally, PSPC decreased the expression level of oxidative stress-associated AGE receptor (RAGE) and thioredoxin interacting protein (TXNIP) in the upstream of NLRP3 inflammasome. These data imply that the beneficial effects of PSPC on HFD-induced kidney dysfunction and damage are mediated through NLRP3 signaling pathways, suggesting a potential target for the prevention of obesity. Ó 2014 Published by Elsevier Ltd.
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1. Introduction
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Inflammation, as a major cause of organ damage, plays a crucial role in a variety of pathological processes of metabolic disorder diseases, such as gout, scurvy, and diabetic ketoacidosis. Emerging evidence suggests that inflammation-mediated kidney damage is involved in pathogenesis of obesity disease. Recently, several
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Abbreviations: ACR, urine albumin/creatinine ratio; AGEs, advanced glycation end products; ASC, apoptosis associated speck-like protein containing a CARD; Col IV, Collagen IV; COX-2, cyclooxygenase-2; HFD, high fat diet; IL-1b, interleukin-1b; iNOS, induce nitric oxide synthase; IjBa, I kappa B a; IKKb, I kappa B kinase b; PSPC, purple sweet potato color; NF-jB, nuclear factor kappa beta; NLRP3, NOD-like receptor 3; PAI-1, plasminogen activator inhibitor-1; ROS, reactive oxygen species; RAGE, the receptor of advanced glycation end products; T2D, type 2 diabetes; TNFa, tumor necrosis factor-a; TXNIP, thioredoxin interacting protein. ⇑ Corresponding author. Tel./fax: +86 51683500348. E-mail addresses: [email protected] (Q. Shan), [email protected], [email protected] (Y.-l. Zheng), [email protected] (J. Lu), zhangzifengsuper@ jsnu.edu.cn (Z.-F. Zhang), [email protected] (D.-m. Wu), fshfl[email protected] (S.-h. Fan), [email protected] (B. Hu), [email protected] (X.-j. Cai), [email protected] (H. Cai), [email protected] (P.-l. Liu), liufan_001@hotmail. com (F. Liu).
studies have shown chronic overfeeding, such as a high fat diet (HFD), leads to immune cell infiltration in different tissues, resulting in the production of inflammatory gene expression including cytokines, chemokines, and other mediators (Dong et al., 2014; Stemmer et al., 2012), which are implicated in oxidative stress and inflammatory response associated with obesity. HFD-induced activation of NOD-like receptor 3 (NLRP3) inflammasome and the alteration of its upstream or downstream signaling molecules, such as nuclear factor kappa beta (NF-jB) and the receptor of advanced glycation end products (RAGE), have also been demonstrated to be involved in obesity process, resulting in kidney injury and insulin resistance (Solini et al., 2013; Vandanmagsar et al., 2011; Harcourt et al., 2011). Furthermore, manipulation of inflammation-related genes can prevent and reverse kidney damage induced by HFD. For example, traditional fermented soybean productsdoenjang and cheonggukjang blunted the kidney inflammatory response by suppressing NF-jB-related activities of inflammatory proteins in rats fed a HFD (Choi et al., 2011). Medical plant Magnolia extract (BL153) improved urine protein and kidney structure by reducing the expression of inflammation markers tumor necrosis factor-a (TNF-a) and plasminogen activator inhibitor-1 (PAI-1) in
http://dx.doi.org/10.1016/j.fct.2014.04.033 0278-6915/Ó 2014 Published by Elsevier Ltd.
Please cite this article in press as: Shan, Q., et al. Purple sweet potato color ameliorates kidney damage via inhibiting oxidative stress mediated NLRP3 inflammasome activation in high fat diet mice. Food Chem. Toxicol. (2014), http://dx.doi.org/10.1016/j.fct.2014.04.033
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HFD-treated mice (Cui et al., 2013). Collectively, the natural productions contribute to attenuating kidney damage induced by HFD; however, the novel natural products and their protective mechanisms still need to be determined. Purple sweet potato (Ipomoea batatas) color (PSPC), as a kind of anthocyanins, is a natural stable polyphenolic pigment and could be directly absorbed to the blood without cytoxicity (Kano et al., 2005). Emerging studies demonstrate that PSPC possesses potential efficacy in the prevention of different inflammation-induced diseases via its in vivo effective anti-oxidant activities. The previous data in our lab have showed that PSPC could significantly ameliorate learning memory or liver injury by improving oxidative damage and inflammatory response of brain or liver in D-galactosemediated aging mice (Shan et al., 2009; Zhang et al., 2009). Recently, PSPC is found to accelerate improvement in the prevention and treatment of HFD-mediated mouse obesity by reducing liver inflammation via AMPK signaling inhibition (Hwang et al., 2011a, 2011b) or by raising liver insulin sensitivity via blocking NF-jB signaling (Zhang et al., 2013). These results suggest that exploring the protective mechanism of PSPC on the metabolic tissues associated with obesity disease, such as kidney, may accelerate the progress in the treatment of obesity disease. In the present study, we studied kidney response of PSPC-administrated mice to HFD feeding by testing oxidative stress and inflammatory response, and the regulatory mechanism of PSPC’ preventing kidney damage by measuring the role of NLRP3 inflammasome in the protective process.
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2. Materials and methods
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2.4. Histological evaluations
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The mice were perfused transcardially with 100 ml of 0.9% sterile saline, and kidney tissues were removed immediately and fixed in a fresh solution of 4% paraformaldehyde (PH 7.4) at 4 °C for 4 h, then incubated overnight at 4 °C in 100 mM sodium phosphate buffers (PH 7.4) containing 15%, 20%, and 30% sucrose, respectively; and embedded in optimal cutting temperature (OCT) compound (Leica, CA, Germany). 12 lM cryosections were collected on 3-animopropyltrimethoxysilane-coated slides (Sigma–Aldrich). The kidney sections were stained with hematoxylin and eosin, and measured by an expert in kidney pathology (S.M.) blinded to the type of treatment received by the animals. Immunohistochemical staining was performed following a commercial kit (Zhongshan Golden Bridge of Biotechnology, Beijing, China). Firstly, endogenous peroxidase activity in the tissue sections was blocked with 3% H2O2 for 10 min, and nonspecific binding sites were blocked with 5% goat serum for 1 h. Then the sections were incubated with rabbit anti-Collagen IV (Col IV, 1:200, Santa Cruz Biotechnology) at 4 °C overnight. And, a biotinylated goat anti-rabbit IgG secondary antibody was added for incubation of 20 min. Subsequently, the avidin–biotin– horseradish peroxidase was applied for 20 min. Horseradish peroxidase was reacted with DAB and H2O2 for 5 min to produce the yellow deposit. The stained sections were washed in distilled water and sealed by water or neutral resins. The stained sections were captured using a leica 200 microscope (leica 4000, German).
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2.5. Tissue homogenates
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For biochemical analysis, mice were deeply anaesthetized and sacrificed. The kidney tissues were immediately separated and homogenized in ice cold 1/10 (w/v) 50 mM phosphate buffer saline solution (PBS, pH 7.2) with 10 strokes at 1200 rpm in a Potter homogenizer (Kontes, Vineland. NJ, USA). Homogenates were centrifuged at 12,000g for 10 min to obtain the supernatants for detecting the level of advanced glycation end products (AGEs) and reactive oxygen species (ROS). For ROS assay, The kidney tissue homogenates were diluted at 1:20 (v:v) with ice-cold Locke’s buffer (154 mM NaCl, 5.6 mM KCl, 3.6 mM NaHCO3, 2.0 mM CaCl2, 10 mM D-glucose, and 5 mM 4-(hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, PH 7.4) to obtain the concentration of 5 mg tissue/ml. For western blotting analysis, the kidneys were homogenized in 1/5 (w/v) icecold lysis buffer (25 mM HEPES, PH 7.4, 125 mM NaCl, 25 mM NaF, 1 mM EDTA, 1 mM ethylene glycol tetraacetic acid, 1% NP-40, 1 mM Na3VO4, and the protease inhibitor mixture described above). The tissue homogenates were sonicated ten times for 10 s with 30 s intervals using a sonicator and centrifuged at 14,000g for 40 min at 4 °C, and then the supernatants were collected and stored at 70 °C for western blotting analysis. The expression levels of NF-jB p65 in cytoplasm and nuclear extracts of kidney tissues were assessed by western blotting, which was obtained by a nuclear/cytoplasm fractionation kit (BioVision, Inc., USA). Protein contents in the supernatants were detected by the bicinchoninic acid assay kit (Pierece Biotechnology, Inc., Rockford, IL, USA).
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2.6. Determination of redox status
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2.6.1. ROS assay ROS was measured as described (Zhang et al., 2013). In brief, the reaction mixture (1 ml) containing Locke’s buffer (pH 7.4), 0.2 ml of homogenate, and 10 ll of DCFH-DA (5 mM) was incubated for 15 min at room temperature to allow incorporation of DCFH-DA into any membrane-bound vesicles and cleavage of the diacetate group by esterases. After 30 min of further incubation, the conversion of DCFH-DA to the fluorescent product DCF was measured using a spectrofluorometer with excitation at 484 nm and emission at 530 nm. Blanks were included to correct for background fluorescence (conversion of DCFH-DA in the absence of homogenate). ROS formation was quantified from a DCF standard curve. Data are expressed as pmol of DCF formed per minute per mg of protein.
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2.1. Animal and treatment
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Eight-week-old male ICR strain mice (32.12 ± 2.14 g) were purchased from the Branch of National Breeder Center of Rodents (Beijing, China). The mice were housed in a room under the conditions of constant temperature (23 ± 1 °C) and humidity (60%), and a 12 h light/dark schedule (lights on 8:00–20:00), and the mice were given free access to food and water. After one week of acclimatization to the conditions, mice were randomly divided into four groups including Control group, HFD, HFD/PSPC, PSPC. Mice were fed on either normal diet containing 11.4% fat, or HFD of 60% fat. In addition, mice were either administrated with PSPC [including two major components, cyanidin acyl glucosides and peonidin acyl glucosides (>90%), Qingdao Pengyuan Natural Pigment Research Institute, China] at dose of 700 mg/kg per day or its solvent (0.1% Tween 80 water solution) by oral gavage for 20 weeks as the described previously (Zhang et al., 2013). All procedures in the experiment consisted with Chinese legislation on the use and care of laboratory animals and were approved by the respective university committee for animal experiments. After 20 weeks, mice were sacrificed and kidney tissues were used for experiments or stored at 80 °C for later use. Body weight of mice was measured every four weeks after 6 h fasting. Urine samples were collected from the mice housed in metabolic cages for 24 h to detect urine creatinine and urine protein content.
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2.2. Determination of albumin and creatinine in urine
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After the drug treatment, the excretion of urine protein was detected using urine albumin-to-creatinine ratio (ACR) in 24 h urine collections. The concentration of urine creatinine and albumin was assessed using the commercial kits (Jiancheng Institute of Biotechnology, Nanjing, China). The absorbed value was detected by UV2501.
2.6.2. AGEs assay Advanced glycation end products (AGEs) are formed due to nonenzymatic glycation of macromolecules, especially proteins leading to their irreversible oxidation is increased under the hyperglycemia of diabetes mellitus and obesity. Therefore, AGEs content could reflect the degree of tissue oxidative damage. AGEs content was measured according to the protocols of AGEs assay kit (Lu et al., 2010).
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2.3. Glucose and insulin tolerance tests
2.7. Western blotting analysis
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After 20-week dietary intervention, glucose tolerance test and insulin tolerance test were carried out respectively. Glucose tolerance test was performed in fasted mice (12 h) with oral treatment of 1.5 g/kg glucose, then blood glucose values were detected immediately before and 30, 60, 90 and 120 min. For insulin resistance test was performed in fasted mice with intraperitoneal injection of insulin (1 U/kg body weight), and blood glucose values were detected immediately before and 30, 60, 90 and 120 min. Blood samples were obtained by tail venipuncture, and blood glucose values were detected by the Ascensia Elite glucose meter (Bayer Corporation, Mishawaka, IN, USA).
Western blotting analysis was performed as previously described (Shan et al., 2009). The supernatants were then separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and analyzed by western blotting. For western blotting, samples (30 lg protein) were separated by denaturing SDS– PAGE and transferred to a polyvinylidene difluoride membrane (Roche Diagnostics Corporation, Indianapolis, IN) by electrophoretic transfer. The membrane was blocked with 5% nonfat milk or 5% BSA and 0.1% Tween-20 in TBS and incubated overnight with one of the following primary antibodies: rabbit anti-RAGE and rabbit anti-NLRP3; rabbit anti-I kappa B kinase b (IKKb), rabbit anti-p-IKKb and
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Please cite this article in press as: Shan, Q., et al. Purple sweet potato color ameliorates kidney damage via inhibiting oxidative stress mediated NLRP3 inflammasome activation in high fat diet mice. Food Chem. Toxicol. (2014), http://dx.doi.org/10.1016/j.fct.2014.04.033
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rabbit anti-IjBa, rabbit anti-thioredoxin interacting protein (TXNIP), rabbit antipro-Caspase-1, mouse anti-cleaved Caspase-1, rabbit anti-interleukin-1b (IL-1b), rabbit anti-apoptosis associated speck-like protein containing a CARD (ASC), rabbit anti-cyclooxygenase-2 (COX2), mouse anti-induce nitric oxide synthase (iNOS), and mouse anti-NF-jB, (Santa Cruz Biotechnology, Inc., California, USA; Cell Signaling Technology, Inc., Beverly, MA and Abcam, Cambridge, UK). Protein bands were detected using horseradish peroxidase–conjugated anti-rabbit, anti-goat, or antimouse secondary antibodies. The mean optical density (OD) values of detected bands were measured with Scion image analysis software (Scion Corp., Frederick, MD, USA) and were normalized to mouse anti-b-actin (Chemicon International Inc., Temecula, CA), or rabbit anti-b-tublin (Cell Signaling Technology, Inc.) as internal controls (OD detected protein/OD internal control).
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2.8. Statistic analysis
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All the data were analyzed by the software SPSS 15.0 statistically. ROS level, AGEs and western blotting results were analyzed with Newman–Keuls or Tukey’s HSD post hoc test. Data are expressed as mean ± standard deviation (SD). Statistical significance is set at P < 0.05.
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3. Results
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3.1. PSPC ameliorates obesity, glucose intolerance and insulin resistance in HFD-treated mice
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Chronic overfeeding such as a high fat diet leads to obesity, glucose metabolism dysregluation and insulin resistance. To identify the effect of PSPC on metabolism syndrome caused by HFD, we firstly tested the changes in body weight, blood glucose and insulin tolerance. We found that body weight significantly increased from the beginning of HFD treatment for 4 weeks [4 weeks: F (3, 36) = 7.150, P < 0.001; 8 weeks: F (3, 36) = 13.191, P < 0.001; 12 weeks: F (3, 36) = 22.151, P < 0.001; 16 weeks: F (3, 36) = 27.992, P < 0.001; 20 weeks: F (3, 36) = 32.409, P < 0.001]. And PSPC administration for HFD mice substantially inhibited the increase of body weight. Glucose and insulin tolerance tests are
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taken to evaluate glucose metabolism and insulin secretion in HFD treatment mice (Fig. 1). After 20-week dietary intervention, HFD treatment showed an obvious increase of fasting blood glucose value compared to Control group [F (3, 28) = 78.651, P < 0.001]. Blood glucose level during glucose tolerance tests and insulin tolerance tests was found to be enhanced in HFD group compared to Control group. The data of glucose and insulin tolerance tests revealed that 20-week HFD treatment markedly impaired glucose tolerance and insulin sensitivity. However, PSPC administration for HFD mice significantly suppressed blood glucose level, improved glucose intolerance and insulin resistance. There were no difference among PSPC/HFD, PSPC and Control groups.
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3.2. PSPC reduces urine albumin and kidney tissue damage in HFD-treated mice
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Then, considering urine albumin-to-creatinine ratio (ACR) is an important marker of urine protein and associated with glomerular damage and progressive renal dysfunction in obesity and diabetes (Praga and Morales, 2010), we tested the changes of urine albumin-to-creatinine ratio and Collagen IV protein in the kidneys of PSPC treated HFD-mice, The results showed that HFD treatment dramatically enhanced the ACR ratio [F (3, 36) = 58.188, P < 0.001], while PSPC reduced the enhancement in urinary albumin caused by HFD (Fig. 2). Western blotting analysis and immunohistochemical staining also showed that HFD treatment exhibited higher extracellular matrix accumulation [F (3, 16) = 20.016, P < 0.001], and PSPC inhibited the accumulation of Collagen IV protein (P < 0.001 vs. HFD group). Further, inflammatory cell infiltration and tissue damage were demonstrated by HE staining in the kidneys of HFD-treated mice. Comfortingly, PSPC treatment markedly ameliorated the histopathologic changes, however there
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Fig. 1. PSPC ameliorates obesity, glucose intolerance and insulin resistance in HFD-treated mice. (A) Weight body gain in different treatment groups at different time points (n = 10). (B) Effects of PSPC on fasting blood glucose of HFD mice after 20-week treatment (n = 8). (C) Data of glucose tolerance tests after oral gavage of 1.5 g/kg glucose in different treatment groups (n = 8). (D) Data of insulin tolerance tests after i.p. injection of 1 U/kg insulin in different treatment groups (n = 8). All values represent the mean ± SD. P < 0.01, P < 0.001 vs. HFD group; #p < 0.05, ###p < 0.001 vs. control group.
Please cite this article in press as: Shan, Q., et al. Purple sweet potato color ameliorates kidney damage via inhibiting oxidative stress mediated NLRP3 inflammasome activation in high fat diet mice. Food Chem. Toxicol. (2014), http://dx.doi.org/10.1016/j.fct.2014.04.033
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Fig. 2. PSPC reduces urine albumin and kidney tissue damage in HFD-treated mice. (A) The ratio of urine creatinine to albumin concentration (mg/g, ACR) in 24 h urine collections in different treatment groups (n = 10). (B) Western-blotting analysis of Col IV (Collagen IV) protein in different treatment groups for 20 weeks (n = 5). The relative density is expressed as the ratio of Col IV/b-actin. (C) Representative photomicrographs of kidney HE staining in different treatment groups for 20 weeks. The arrowheads indicate kidney inflammatory cells. (Scale bar = 100 lm). (D) Immunohistochemical detection of kidney Col IV in different treatment groups for 20 weeks. The arrowheads show positive signals. All values are expressed as mean ± SD, P < 0.001 vs. HFD group; ###p < 0.001 vs. control group.
Q3 was no significant among Control, HFD/PSPC and PSPC groups. (See Fig. 3).
However, there were no notable difference among HFD/PSPC group, PSPC group and vehicle Control.
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3.3. PSPC attenuates oxidative stress in the kidney of HFD-treated mice
3.4. PSPC inhibits the activation of IKKb/NF-jB signaling in the kidney of HFD-treated mice
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To assess whether PSPC reduces oxidative stress which could induce inflammatory response, we measured the ROS level and the AGEs content. The results showed that ROS and AGEs significantly increased in HFD group [F (3, 16) = 23.695, P < 0.001; F (3, 16) = 26.589, P < 0.001] compared with Control group. Our data showed that HFD induced oxidative stress in the kidneys of mice, while oral administration of PSPC to HFD mice significantly decreased the level of ROS (P < 0.001) and AGEs (P < 0.001).
To clarify the regulatory mechanism underlying the protective effect of PSPC on the mouse kidney damage induced by HFD treatment, we firstly analyzed the expression of IKKb/NF-jB signaling, which plays a key role in the regulation of various genes involved in proliferation, apoptosis, immune and inflammatory response. The expression levels of IKKb, IjBa, NF-jB, iNOS and COX-2 were measured by western blotting. As shown in Fig. 4, HFD treatment
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Please cite this article in press as: Shan, Q., et al. Purple sweet potato color ameliorates kidney damage via inhibiting oxidative stress mediated NLRP3 inflammasome activation in high fat diet mice. Food Chem. Toxicol. (2014), http://dx.doi.org/10.1016/j.fct.2014.04.033
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Fig. 3. PSPC attenuates oxidative stress in the kidney of HFD-treated mice (n = 5). (A) The effect of PSPC on ROS generation in the kidney of HFD-treated mice for 20 weeks. ROS contents were measured by DCFH-DA method. (B) Effects of PSPC on AGEs content in the kidney of HFD-treated mice. Quantitative measurement of AGEs was performed by enzyme-linked immunosobent assay. All values are expressed as means ± S.D. P < 0.001 vs. HFD group; ###p < 0.001 vs. control group.
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induced the activation of IKKb/NF-jB pathway by enhancing the phosphorylation of IKKb [p-IKKb (Ser181): F (3, 16) = 42.939, P < 0.001, vs. Control group], leading to the degradation of IjBa [F (3, 16) = 20.255, P < 0.001, vs. Control group], accelerating NF-jB from the cytoplasm into the nucleus [cytoplasm NF-jB F (3, 16) = 12.744, nuclear NF-jB F (3, 16) = 10.844], and increasing the expression of inflammatory markers COX-2 and iNOS [F (3, 16) = 25.521, P < 0.001; F (3, 16) = 16.136, P < 0.001]. Oral administration of PSPC to HFD mice for 20 weeks significantly reversed these changes (P < 0.05, vs. the HFD group). There were no significant differences among Control group, PSPC group and HFD/PSPC group. 3.5. PSPC reduces the protein expression of NLRP3 inflammasome in the kidney of HFD-treated mice The recent discoveries suggest a potential role of NOD-like receptors (NLRs) in innate immune triggered interest in the studies of chronic diseases such as obesity and type 2 diabetes (T2D) (Grant and Dixit, 2013). NLRP3, as one important component of NLRs, is a crucial protein of inflammasome complex including NLRP3, ASC, pro-Caspase-1. Activated NLRP3 interacts with ASC, and recruits pro-Caspase-1, resulting in the secretion of proinflammatory cytokines IL-1b. As shown in Fig. 5, compared with Control group, HFD treatment significantly increased the expression level of NLRP3 and ASC [respectively F (3, 16) = 12.155, P < 0.001; F (3, 16) = 12.526, P < 0.001], and activated Caspase-1 (cleaved Caspase-1: F (3, 16) = 11.988) and IL-1b [F (3, 16) = 15.737, P < 0.001], while the content of pro-caspase-1 was no significant change among four groups. Our results showed that PSPC activated NLRP3 inflammasome pathway in the mouse kidney. Similarly, PSPC administration in the HFD-treated mice reversed these abnormal changes, made the above parameters restore to a near normal level (no significant vs. Control group). However, PSPC treatment did not substantially change these parameters in the kidneys of mice (P > 0.05 vs. Control group).
Fig. 4. PSPC inhibits the activation of IKKb/NF-jB signaling in the kidney of HFD-treated mice (n = 5). (A) Western-blotting results of p-IKKb, IKKb, IjBa, C-NFjB (C represents cytoplasm), N-NF-jB (N represents nucleus), iNOS and COX-2. (B) Relative density analysis of p-IKKb, IjBa, C-NF-jB, N-NF-jB, iNOS and COX-2 protein bands. The relative density is expressed as the ratio [p-IKKb/ (p-IKKb + IKKb), IjBa/b-actin, iNOS/b-actin, COX-2/b-actin, C-NF-jB/b-actin and N-NF-jB/b-tublin]. All values are expressed as means ± S.D. P < 0.01, P < 0.001 vs. HFD group; ###p < 0.001 vs. control group.
3.6. PSPC decreases the protein expression of RAGE and TXNIP in the kidney of HFD-treated mice
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TXNIP is ubiquitously expressed in a variety of cells, and acts an endogenous suppressor of ROS scavenging protein thioredoxin. TXNIP and RAGE are identified as the molecular nutrient sensors in the regulation of energy metabolism, linked oxidative stress and inflammation (Chutkow et al., 2010; Leuner et al., 2012). As shown in Fig. 6, HFD administration increased the expression level of TXNIP and RAGE in the kidneys of mice [F (3, 16) = 18.185, P < 0.001; F (3, 16) = 10.612, P < 0.001]. While PSPC suppressed the increase of renal TXNIP and RAGE induced by HFD-treatment, no significant change was found among Control group, PSPC group and PSPC/HFD group.
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4. Discussion
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The incidence of obesity has been climbing dramatically worldwide. It is a key risk factor for many metabolic diseases, including diabetes, hypertension, and heart disease. Recent studies indicate that obesity is accompanied by chronic inflammation in important metabolic tissues including adipose, liver and kidney, resulting in the release of pro-inflammatory cytokines and chemokines that contribute to the development of metabolic syndrome
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Fig. 5. PSPC reduces the expression levels of NLRP3 inflammasome in the kidney of HFD-treated mice (n = 5). (A) Western-blotting results of NLRP3, ASC, P-Caspase-1(P represents pro) C-Caspase-1(cleaved-Caspase-1) and IL-1b protein. (B) Relative density analysis of NLRP3, ASC, P-Caspase-1, C-Caspase-1 and IL-1b protein bands. The relative density is expressed as the ratio (NLRP3/b-actin, ASC/b-actin, P-Caspase1/b-actin, C-Caspase-1/b-actin and IL-1b/b-actin). All values are expressed as means ± S.D. P < 0.01, P < 0.001 vs. HFD group; ###p < 0.001 vs. control group.
Fig. 6. PSPC decreases the protein expression of RAGE and TXNIP in the kidney of HFD-treated mice (n = 5). (A) Western-blotting results of RAGE and TXNIP protein. (B) Relative density analysis of RAGE and TXNIP protein bands. The relative density is expressed as the ratio of RAGE/b-actin and TXNIP/b-actin. All values are expressed as means ± S.D. P < 0.01, P < 0.001 vs. HFD group; ###p < 0.001 vs. control group.
(Lu et al., 2011; Yakala et al., 2012; Zhang et al., 2013). Long-term high fat diet could induce obesity and T2D, causing the occurrence of metabolic syndrome (Wang et al., 2012;Wang et al., 2009). HFD administration could induce oxidative stress by down-regulating GSH, decreasing antioxidant enzyme activities (Noeman et al., 2011), and accelerating the production of AGEs and ROS (Leuner et al., 2012; Ruggiero et al., 2011), subsequently cause the serious damage of kidney. In the present study, HFD treatment for 20 weeks led to fasting hyperglycemia, insulin resistance, renal Collagen IV accumulation and urine protein. Anthocyanins are naturally unique polyphenolic compounds in higher plants. PSPC, as a kind of easily absorbed anthocyanins (Suda et al., 2002), has displayed special physiological efficacy such as anti-oxidant, antiinflammatory and anti-hyperglycemic effects (Matsui et al., 2002; Zhao et al., 2013). Recent researches show that acylated cyanidin and peonidin are two major components in PSPC (Qiu et al., 2009; Montilla et al., 2010), they may have a key role in PSPC’s pharmacological effect. Notably, we found PSPC effectively eliminated the metabolic disorders induced by HFD; the efficacy is achieved by inhibiting oxidative stress and inflammatory response, suggesting that PSPC effectively prevented kidney injury induced by HFD feeding. The sustained oxidative stress is involved in many important signaling pathways, IKKb/NF-jB and NLRP3/IL-1b are reported to be closely linked to the development of inflammatory response (Salminen et al., 2012). Consistently, we found kidney IKKb was significantly activated after HFD administration, and this alteration was accompanied with the increase of nuclear NF-jB, a nuclear transcription factor. The result is in accord with the activated NF-jB in the kidneys of HFD fed mice (Choi et al.,2011). In the present study, PSPC administration significantly reduced the activities of kidney NF-jB. Additionally, we previously found that PSPC also reduced activities of liver NF-jB in HFD-fed mice (Zhang et al., 2013). Because NF-jB regulates the expression of various genes, including cytokines, iNOS and COX-2, which are used to be major inflammatory mediators and pro-inflammatory markers, it plays critical roles in apoptosis, various autoimmune diseases and inflammation. Our data showed that HFD treatment increased the expression of iNOS and COX-2 in the kidneys, indicating PSPC’s effective preventive function for the kidney inflammation via NF-jB regulating the expression of iNOS and COX-2. Most recently, the NLRP3 inflammasome, which consists of NLRP3 molecule, adaptor protein ASC and pro-Caspase-1 that catalytically activates Caspase-1, causing the release of IL-1b and IL-18, was demonstrated to prevent obesity-induced inflammation and insulin resistance (Vandanmagsar et al., 2011). Consequently, NLRP3 inflammasome has emerged as an unexpected sensor for the pathogenesis of auto-inflammatory, metabolic danger and stress. Moreover, NLRP3 inflammasome is increasingly suspected of playing a major role in other human pathologies such as cancer, asbestosis and Alzheimer’s disease. NLRP3 is an important component of the inflammasome complex, and NLRP3 gene is activated by IKKb/NF-jB. Deleting NLRP3 in mice can prevent inflammasome activation in adipose tissue and liver as well as increase insulin sensitivity (Stienstra et al., 2011), demonstrating that NLRP3 plays an important role in the development of obesity and T2D (Pejnovic et al., 2013). Interestingly, elevated ROS can activate NLRP3 inflammasome, resulting in the secretion of bioactive IL-1b, triggering the activation of NFjB which in turn promotes the secretion of bioactive IL-1b (Grant and Dixit, 2013). We also found the related triggering regulatory results among ROS, NLRP3 inflammasome (NLRP3, ACS and Caspase-1), IKKb and NF-jB in the kidneys of HFD-treated mice. However, PSPC reduced the production of ROS and AGEs; these changes are closely related to the alleviation of kidney oxidative damage and associated urine protein in PSPC-treated mice. Furthermore, PSPC reduced HFD-induced renal inflammation by
Please cite this article in press as: Shan, Q., et al. Purple sweet potato color ameliorates kidney damage via inhibiting oxidative stress mediated NLRP3 inflammasome activation in high fat diet mice. Food Chem. Toxicol. (2014), http://dx.doi.org/10.1016/j.fct.2014.04.033
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dampening NLRP3/IL-1b and IKKb/NF-jB pathways similar to recent reports (Dragano et al., 2013; Zhang et al., 2013). NLRP3 inflammasome is also activated by TXNIP and RAGE and promotes the secretion of IL-1b (Zhou et al., 2010). The expression of TXNIP, as a hallmark of cell oxidative stress, is enhanced in obesity, T1D and T2D (Wang et al., 2013; Koenen et al., 2011). In the present study, HFD consumption was found to substantially stimulate the production of TXNIP, while, PSPC-treatment dampened the activation to ameliorate renal damage. Additionally, RAGE, a AGEs receptor, is promoted to express by the increase of AGEs under the condition of oxidative stress and hyperglycemia in obesity disease (Tomino et al., 2011). In contrary, deleted RAGE reduces the cardiac inflammation and protects against atherosclerosis in HFD feeding mice (Soro-Paavonen et al., 2008; Tikellis et al., 2008). Consistently, the accumulation of AGEs and RAGE is exhibited in the kidneys of HFD mice in our experiment; PSPC significantly decreased their contents in kidneys. The result is consistent with the previous findings from another lab that the proanthocyanidins from grape seed ameliorates diabetic nephropathy by regulation of the levels of kidney AGEs and RAGE (Li et al., 2009). RAGE and high glucose can induce TXNIP expression in retinal endothelial cells (Perrone et al., 2009), while TXNIP silencing abolishes RAGE-induced IL-1b secretion (Sbai et al., 2010), suggesting they may play the regulatory role in the development of obesity through mutually reinforcing expression. Combined our results with the previously reported ones, we speculated that PSPC prevented kidney damage by blocking endogenous oxidative cues and inflammatory reaction in HFD-induced obesity. This preventive pharmacologic efficacy is possibly obtained by PSPC’ inhibiting the expression of RAGE and of TXNIP, resulting in the inactivating of NLRP3 inflammasome, and causing the fall of NF-jB entranced into the nuclear. Although we have clearly established the importance of PSPC’ protective function on the HFD-induced kidney injury during the development of obesity in animal models, additional studies focused on unraveling the importance of PSPC’ preventing against the inflammation induced by HFD diet in human kidney tissue are needed. In conclusion, PSPC showed a significant amelioration on kidney injury in HFD-fed mice by reducing the production of AGEs and ROS, and improving the sensitivity of insulin, this protection function on the kidney is played by suppressing the expression of TXNIP and RAGE, further inhibiting the activation of NLRP3 inflammasome and IKKb/NF-jB pathways. Therefore, PSPC represents a potential natural product in the prevention of nephropathy induced by obesity.
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The authors declare there are no conflicts of interest.
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Acknowledgements This work was supported by the Priority Academic Program Q4 Development of Jiangsu Higher Education Institutions (PARD), Q5 the National Natural Science Foundation of China (81171012, 31200873), the 2010 ‘‘Qinglan Project’’ Scientific and Technological Innovation Team Training Program of Jiangsu College and University, the Major Fundamental Research Program of the Natural Science Foundation of the Jiangsu Higher Education
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Institutions of China (07KJA36029), grants from the Key Laboratory of Jiangsu Province, PR, China, Jiangsu Province postgraduate innovation projects (2008, CX10S-043Z and 2012JSSPITP3192) and from the Natural Science Foundation of Jiangsu Normal University (11XLA08).
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Purple sweet potato color ameliorates kidney damage via inhibiting oxidative stress mediated NLRP3 inflammasome activation in high fat diet mice
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Qun Shan a, Yuan-lin Zheng a,⇑, Jun Lu a, Zi-Feng Zhang a, Dong-mei Wu a, Shao-hua Fan a, Bin Hu a, Xiang-jun Cai a, Hao Cai b, Pei-long Liu b, Fan Liu b a b
Key Laboratory for Biotechnology on Medicinal Plants of Jiangsu Province, School of Life Science, Jiangsu Normal University, Xuzhou 221116, Jiangsu Province, PR China Kewen College, Jiangsu Normal University, Xuzhou 221116, Jiangsu Province, PR China
a r t i c l e
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Article history: Received 1 February 2014 Accepted 22 April 2014 Available online xxxx Keywords: PSPC HFD NLRP3 inflammasome Oxidative stress Kidney damage
a b s t r a c t Inflammation plays a crucial role in the pathogenesis of obesity. Purple sweet potato color (PSPC) has potential anti-inflammation efficacy. We evaluated the effect of PSPC on kidney injury induced by high fat diet (HFD) and explored the mechanism underlying these effects. The results showed that PSPC (700 mg/kg per day) reduced body weight, ratio of urine albumin to creatinine, inflammatory cell infiltration, and Collagen IV accumulation in mice fed an HFD (60% fat food) for 20 weeks. PSPC significantly reduced the expression level of kidney NLRP3 inflammasome including NLRP3 and ASC and Caspase-1, and resulted in decline of IL-1b. Moreover, PSPC inhibited the activation of I kappa B kinase b (IKKb) and the nuclear translocation of nuclear factor kappa beta (NF-jB). Additionally, PSPC decreased the expression level of oxidative stress-associated AGE receptor (RAGE) and thioredoxin interacting protein (TXNIP) in the upstream of NLRP3 inflammasome. These data imply that the beneficial effects of PSPC on HFD-induced kidney dysfunction and damage are mediated through NLRP3 signaling pathways, suggesting a potential target for the prevention of obesity. Ó 2014 Published by Elsevier Ltd.
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1. Introduction
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Inflammation, as a major cause of organ damage, plays a crucial role in a variety of pathological processes of metabolic disorder diseases, such as gout, scurvy, and diabetic ketoacidosis. Emerging evidence suggests that inflammation-mediated kidney damage is involved in pathogenesis of obesity disease. Recently, several
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Abbreviations: ACR, urine albumin/creatinine ratio; AGEs, advanced glycation end products; ASC, apoptosis associated speck-like protein containing a CARD; Col IV, Collagen IV; COX-2, cyclooxygenase-2; HFD, high fat diet; IL-1b, interleukin-1b; iNOS, induce nitric oxide synthase; IjBa, I kappa B a; IKKb, I kappa B kinase b; PSPC, purple sweet potato color; NF-jB, nuclear factor kappa beta; NLRP3, NOD-like receptor 3; PAI-1, plasminogen activator inhibitor-1; ROS, reactive oxygen species; RAGE, the receptor of advanced glycation end products; T2D, type 2 diabetes; TNFa, tumor necrosis factor-a; TXNIP, thioredoxin interacting protein. ⇑ Corresponding author. Tel./fax: +86 51683500348. E-mail addresses: [email protected] (Q. Shan), [email protected], [email protected] (Y.-l. Zheng), [email protected] (J. Lu), zhangzifengsuper@ jsnu.edu.cn (Z.-F. Zhang), [email protected] (D.-m. Wu), fshfl[email protected] (S.-h. Fan), [email protected] (B. Hu), [email protected] (X.-j. Cai), [email protected] (H. Cai), [email protected] (P.-l. Liu), liufan_001@hotmail. com (F. Liu).
studies have shown chronic overfeeding, such as a high fat diet (HFD), leads to immune cell infiltration in different tissues, resulting in the production of inflammatory gene expression including cytokines, chemokines, and other mediators (Dong et al., 2014; Stemmer et al., 2012), which are implicated in oxidative stress and inflammatory response associated with obesity. HFD-induced activation of NOD-like receptor 3 (NLRP3) inflammasome and the alteration of its upstream or downstream signaling molecules, such as nuclear factor kappa beta (NF-jB) and the receptor of advanced glycation end products (RAGE), have also been demonstrated to be involved in obesity process, resulting in kidney injury and insulin resistance (Solini et al., 2013; Vandanmagsar et al., 2011; Harcourt et al., 2011). Furthermore, manipulation of inflammation-related genes can prevent and reverse kidney damage induced by HFD. For example, traditional fermented soybean productsdoenjang and cheonggukjang blunted the kidney inflammatory response by suppressing NF-jB-related activities of inflammatory proteins in rats fed a HFD (Choi et al., 2011). Medical plant Magnolia extract (BL153) improved urine protein and kidney structure by reducing the expression of inflammation markers tumor necrosis factor-a (TNF-a) and plasminogen activator inhibitor-1 (PAI-1) in
http://dx.doi.org/10.1016/j.fct.2014.04.033 0278-6915/Ó 2014 Published by Elsevier Ltd.
Please cite this article in press as: Shan, Q., et al. Purple sweet potato color ameliorates kidney damage via inhibiting oxidative stress mediated NLRP3 inflammasome activation in high fat diet mice. Food Chem. Toxicol. (2014), http://dx.doi.org/10.1016/j.fct.2014.04.033
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HFD-treated mice (Cui et al., 2013). Collectively, the natural productions contribute to attenuating kidney damage induced by HFD; however, the novel natural products and their protective mechanisms still need to be determined. Purple sweet potato (Ipomoea batatas) color (PSPC), as a kind of anthocyanins, is a natural stable polyphenolic pigment and could be directly absorbed to the blood without cytoxicity (Kano et al., 2005). Emerging studies demonstrate that PSPC possesses potential efficacy in the prevention of different inflammation-induced diseases via its in vivo effective anti-oxidant activities. The previous data in our lab have showed that PSPC could significantly ameliorate learning memory or liver injury by improving oxidative damage and inflammatory response of brain or liver in D-galactosemediated aging mice (Shan et al., 2009; Zhang et al., 2009). Recently, PSPC is found to accelerate improvement in the prevention and treatment of HFD-mediated mouse obesity by reducing liver inflammation via AMPK signaling inhibition (Hwang et al., 2011a, 2011b) or by raising liver insulin sensitivity via blocking NF-jB signaling (Zhang et al., 2013). These results suggest that exploring the protective mechanism of PSPC on the metabolic tissues associated with obesity disease, such as kidney, may accelerate the progress in the treatment of obesity disease. In the present study, we studied kidney response of PSPC-administrated mice to HFD feeding by testing oxidative stress and inflammatory response, and the regulatory mechanism of PSPC’ preventing kidney damage by measuring the role of NLRP3 inflammasome in the protective process.
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2. Materials and methods
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2.4. Histological evaluations
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The mice were perfused transcardially with 100 ml of 0.9% sterile saline, and kidney tissues were removed immediately and fixed in a fresh solution of 4% paraformaldehyde (PH 7.4) at 4 °C for 4 h, then incubated overnight at 4 °C in 100 mM sodium phosphate buffers (PH 7.4) containing 15%, 20%, and 30% sucrose, respectively; and embedded in optimal cutting temperature (OCT) compound (Leica, CA, Germany). 12 lM cryosections were collected on 3-animopropyltrimethoxysilane-coated slides (Sigma–Aldrich). The kidney sections were stained with hematoxylin and eosin, and measured by an expert in kidney pathology (S.M.) blinded to the type of treatment received by the animals. Immunohistochemical staining was performed following a commercial kit (Zhongshan Golden Bridge of Biotechnology, Beijing, China). Firstly, endogenous peroxidase activity in the tissue sections was blocked with 3% H2O2 for 10 min, and nonspecific binding sites were blocked with 5% goat serum for 1 h. Then the sections were incubated with rabbit anti-Collagen IV (Col IV, 1:200, Santa Cruz Biotechnology) at 4 °C overnight. And, a biotinylated goat anti-rabbit IgG secondary antibody was added for incubation of 20 min. Subsequently, the avidin–biotin– horseradish peroxidase was applied for 20 min. Horseradish peroxidase was reacted with DAB and H2O2 for 5 min to produce the yellow deposit. The stained sections were washed in distilled water and sealed by water or neutral resins. The stained sections were captured using a leica 200 microscope (leica 4000, German).
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2.5. Tissue homogenates
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For biochemical analysis, mice were deeply anaesthetized and sacrificed. The kidney tissues were immediately separated and homogenized in ice cold 1/10 (w/v) 50 mM phosphate buffer saline solution (PBS, pH 7.2) with 10 strokes at 1200 rpm in a Potter homogenizer (Kontes, Vineland. NJ, USA). Homogenates were centrifuged at 12,000g for 10 min to obtain the supernatants for detecting the level of advanced glycation end products (AGEs) and reactive oxygen species (ROS). For ROS assay, The kidney tissue homogenates were diluted at 1:20 (v:v) with ice-cold Locke’s buffer (154 mM NaCl, 5.6 mM KCl, 3.6 mM NaHCO3, 2.0 mM CaCl2, 10 mM D-glucose, and 5 mM 4-(hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, PH 7.4) to obtain the concentration of 5 mg tissue/ml. For western blotting analysis, the kidneys were homogenized in 1/5 (w/v) icecold lysis buffer (25 mM HEPES, PH 7.4, 125 mM NaCl, 25 mM NaF, 1 mM EDTA, 1 mM ethylene glycol tetraacetic acid, 1% NP-40, 1 mM Na3VO4, and the protease inhibitor mixture described above). The tissue homogenates were sonicated ten times for 10 s with 30 s intervals using a sonicator and centrifuged at 14,000g for 40 min at 4 °C, and then the supernatants were collected and stored at 70 °C for western blotting analysis. The expression levels of NF-jB p65 in cytoplasm and nuclear extracts of kidney tissues were assessed by western blotting, which was obtained by a nuclear/cytoplasm fractionation kit (BioVision, Inc., USA). Protein contents in the supernatants were detected by the bicinchoninic acid assay kit (Pierece Biotechnology, Inc., Rockford, IL, USA).
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2.6. Determination of redox status
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2.6.1. ROS assay ROS was measured as described (Zhang et al., 2013). In brief, the reaction mixture (1 ml) containing Locke’s buffer (pH 7.4), 0.2 ml of homogenate, and 10 ll of DCFH-DA (5 mM) was incubated for 15 min at room temperature to allow incorporation of DCFH-DA into any membrane-bound vesicles and cleavage of the diacetate group by esterases. After 30 min of further incubation, the conversion of DCFH-DA to the fluorescent product DCF was measured using a spectrofluorometer with excitation at 484 nm and emission at 530 nm. Blanks were included to correct for background fluorescence (conversion of DCFH-DA in the absence of homogenate). ROS formation was quantified from a DCF standard curve. Data are expressed as pmol of DCF formed per minute per mg of protein.
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2.1. Animal and treatment
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Eight-week-old male ICR strain mice (32.12 ± 2.14 g) were purchased from the Branch of National Breeder Center of Rodents (Beijing, China). The mice were housed in a room under the conditions of constant temperature (23 ± 1 °C) and humidity (60%), and a 12 h light/dark schedule (lights on 8:00–20:00), and the mice were given free access to food and water. After one week of acclimatization to the conditions, mice were randomly divided into four groups including Control group, HFD, HFD/PSPC, PSPC. Mice were fed on either normal diet containing 11.4% fat, or HFD of 60% fat. In addition, mice were either administrated with PSPC [including two major components, cyanidin acyl glucosides and peonidin acyl glucosides (>90%), Qingdao Pengyuan Natural Pigment Research Institute, China] at dose of 700 mg/kg per day or its solvent (0.1% Tween 80 water solution) by oral gavage for 20 weeks as the described previously (Zhang et al., 2013). All procedures in the experiment consisted with Chinese legislation on the use and care of laboratory animals and were approved by the respective university committee for animal experiments. After 20 weeks, mice were sacrificed and kidney tissues were used for experiments or stored at 80 °C for later use. Body weight of mice was measured every four weeks after 6 h fasting. Urine samples were collected from the mice housed in metabolic cages for 24 h to detect urine creatinine and urine protein content.
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2.2. Determination of albumin and creatinine in urine
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After the drug treatment, the excretion of urine protein was detected using urine albumin-to-creatinine ratio (ACR) in 24 h urine collections. The concentration of urine creatinine and albumin was assessed using the commercial kits (Jiancheng Institute of Biotechnology, Nanjing, China). The absorbed value was detected by UV2501.
2.6.2. AGEs assay Advanced glycation end products (AGEs) are formed due to nonenzymatic glycation of macromolecules, especially proteins leading to their irreversible oxidation is increased under the hyperglycemia of diabetes mellitus and obesity. Therefore, AGEs content could reflect the degree of tissue oxidative damage. AGEs content was measured according to the protocols of AGEs assay kit (Lu et al., 2010).
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2.3. Glucose and insulin tolerance tests
2.7. Western blotting analysis
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After 20-week dietary intervention, glucose tolerance test and insulin tolerance test were carried out respectively. Glucose tolerance test was performed in fasted mice (12 h) with oral treatment of 1.5 g/kg glucose, then blood glucose values were detected immediately before and 30, 60, 90 and 120 min. For insulin resistance test was performed in fasted mice with intraperitoneal injection of insulin (1 U/kg body weight), and blood glucose values were detected immediately before and 30, 60, 90 and 120 min. Blood samples were obtained by tail venipuncture, and blood glucose values were detected by the Ascensia Elite glucose meter (Bayer Corporation, Mishawaka, IN, USA).
Western blotting analysis was performed as previously described (Shan et al., 2009). The supernatants were then separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and analyzed by western blotting. For western blotting, samples (30 lg protein) were separated by denaturing SDS– PAGE and transferred to a polyvinylidene difluoride membrane (Roche Diagnostics Corporation, Indianapolis, IN) by electrophoretic transfer. The membrane was blocked with 5% nonfat milk or 5% BSA and 0.1% Tween-20 in TBS and incubated overnight with one of the following primary antibodies: rabbit anti-RAGE and rabbit anti-NLRP3; rabbit anti-I kappa B kinase b (IKKb), rabbit anti-p-IKKb and
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Please cite this article in press as: Shan, Q., et al. Purple sweet potato color ameliorates kidney damage via inhibiting oxidative stress mediated NLRP3 inflammasome activation in high fat diet mice. Food Chem. Toxicol. (2014), http://dx.doi.org/10.1016/j.fct.2014.04.033
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rabbit anti-IjBa, rabbit anti-thioredoxin interacting protein (TXNIP), rabbit antipro-Caspase-1, mouse anti-cleaved Caspase-1, rabbit anti-interleukin-1b (IL-1b), rabbit anti-apoptosis associated speck-like protein containing a CARD (ASC), rabbit anti-cyclooxygenase-2 (COX2), mouse anti-induce nitric oxide synthase (iNOS), and mouse anti-NF-jB, (Santa Cruz Biotechnology, Inc., California, USA; Cell Signaling Technology, Inc., Beverly, MA and Abcam, Cambridge, UK). Protein bands were detected using horseradish peroxidase–conjugated anti-rabbit, anti-goat, or antimouse secondary antibodies. The mean optical density (OD) values of detected bands were measured with Scion image analysis software (Scion Corp., Frederick, MD, USA) and were normalized to mouse anti-b-actin (Chemicon International Inc., Temecula, CA), or rabbit anti-b-tublin (Cell Signaling Technology, Inc.) as internal controls (OD detected protein/OD internal control).
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2.8. Statistic analysis
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All the data were analyzed by the software SPSS 15.0 statistically. ROS level, AGEs and western blotting results were analyzed with Newman–Keuls or Tukey’s HSD post hoc test. Data are expressed as mean ± standard deviation (SD). Statistical significance is set at P < 0.05.
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3.1. PSPC ameliorates obesity, glucose intolerance and insulin resistance in HFD-treated mice
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Chronic overfeeding such as a high fat diet leads to obesity, glucose metabolism dysregluation and insulin resistance. To identify the effect of PSPC on metabolism syndrome caused by HFD, we firstly tested the changes in body weight, blood glucose and insulin tolerance. We found that body weight significantly increased from the beginning of HFD treatment for 4 weeks [4 weeks: F (3, 36) = 7.150, P < 0.001; 8 weeks: F (3, 36) = 13.191, P < 0.001; 12 weeks: F (3, 36) = 22.151, P < 0.001; 16 weeks: F (3, 36) = 27.992, P < 0.001; 20 weeks: F (3, 36) = 32.409, P < 0.001]. And PSPC administration for HFD mice substantially inhibited the increase of body weight. Glucose and insulin tolerance tests are
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taken to evaluate glucose metabolism and insulin secretion in HFD treatment mice (Fig. 1). After 20-week dietary intervention, HFD treatment showed an obvious increase of fasting blood glucose value compared to Control group [F (3, 28) = 78.651, P < 0.001]. Blood glucose level during glucose tolerance tests and insulin tolerance tests was found to be enhanced in HFD group compared to Control group. The data of glucose and insulin tolerance tests revealed that 20-week HFD treatment markedly impaired glucose tolerance and insulin sensitivity. However, PSPC administration for HFD mice significantly suppressed blood glucose level, improved glucose intolerance and insulin resistance. There were no difference among PSPC/HFD, PSPC and Control groups.
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3.2. PSPC reduces urine albumin and kidney tissue damage in HFD-treated mice
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Then, considering urine albumin-to-creatinine ratio (ACR) is an important marker of urine protein and associated with glomerular damage and progressive renal dysfunction in obesity and diabetes (Praga and Morales, 2010), we tested the changes of urine albumin-to-creatinine ratio and Collagen IV protein in the kidneys of PSPC treated HFD-mice, The results showed that HFD treatment dramatically enhanced the ACR ratio [F (3, 36) = 58.188, P < 0.001], while PSPC reduced the enhancement in urinary albumin caused by HFD (Fig. 2). Western blotting analysis and immunohistochemical staining also showed that HFD treatment exhibited higher extracellular matrix accumulation [F (3, 16) = 20.016, P < 0.001], and PSPC inhibited the accumulation of Collagen IV protein (P < 0.001 vs. HFD group). Further, inflammatory cell infiltration and tissue damage were demonstrated by HE staining in the kidneys of HFD-treated mice. Comfortingly, PSPC treatment markedly ameliorated the histopathologic changes, however there
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Fig. 1. PSPC ameliorates obesity, glucose intolerance and insulin resistance in HFD-treated mice. (A) Weight body gain in different treatment groups at different time points (n = 10). (B) Effects of PSPC on fasting blood glucose of HFD mice after 20-week treatment (n = 8). (C) Data of glucose tolerance tests after oral gavage of 1.5 g/kg glucose in different treatment groups (n = 8). (D) Data of insulin tolerance tests after i.p. injection of 1 U/kg insulin in different treatment groups (n = 8). All values represent the mean ± SD. P < 0.01, P < 0.001 vs. HFD group; #p < 0.05, ###p < 0.001 vs. control group.
Please cite this article in press as: Shan, Q., et al. Purple sweet potato color ameliorates kidney damage via inhibiting oxidative stress mediated NLRP3 inflammasome activation in high fat diet mice. Food Chem. Toxicol. (2014), http://dx.doi.org/10.1016/j.fct.2014.04.033
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Fig. 2. PSPC reduces urine albumin and kidney tissue damage in HFD-treated mice. (A) The ratio of urine creatinine to albumin concentration (mg/g, ACR) in 24 h urine collections in different treatment groups (n = 10). (B) Western-blotting analysis of Col IV (Collagen IV) protein in different treatment groups for 20 weeks (n = 5). The relative density is expressed as the ratio of Col IV/b-actin. (C) Representative photomicrographs of kidney HE staining in different treatment groups for 20 weeks. The arrowheads indicate kidney inflammatory cells. (Scale bar = 100 lm). (D) Immunohistochemical detection of kidney Col IV in different treatment groups for 20 weeks. The arrowheads show positive signals. All values are expressed as mean ± SD, P < 0.001 vs. HFD group; ###p < 0.001 vs. control group.
Q3 was no significant among Control, HFD/PSPC and PSPC groups. (See Fig. 3).
However, there were no notable difference among HFD/PSPC group, PSPC group and vehicle Control.
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3.3. PSPC attenuates oxidative stress in the kidney of HFD-treated mice
3.4. PSPC inhibits the activation of IKKb/NF-jB signaling in the kidney of HFD-treated mice
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To assess whether PSPC reduces oxidative stress which could induce inflammatory response, we measured the ROS level and the AGEs content. The results showed that ROS and AGEs significantly increased in HFD group [F (3, 16) = 23.695, P < 0.001; F (3, 16) = 26.589, P < 0.001] compared with Control group. Our data showed that HFD induced oxidative stress in the kidneys of mice, while oral administration of PSPC to HFD mice significantly decreased the level of ROS (P < 0.001) and AGEs (P < 0.001).
To clarify the regulatory mechanism underlying the protective effect of PSPC on the mouse kidney damage induced by HFD treatment, we firstly analyzed the expression of IKKb/NF-jB signaling, which plays a key role in the regulation of various genes involved in proliferation, apoptosis, immune and inflammatory response. The expression levels of IKKb, IjBa, NF-jB, iNOS and COX-2 were measured by western blotting. As shown in Fig. 4, HFD treatment
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Fig. 3. PSPC attenuates oxidative stress in the kidney of HFD-treated mice (n = 5). (A) The effect of PSPC on ROS generation in the kidney of HFD-treated mice for 20 weeks. ROS contents were measured by DCFH-DA method. (B) Effects of PSPC on AGEs content in the kidney of HFD-treated mice. Quantitative measurement of AGEs was performed by enzyme-linked immunosobent assay. All values are expressed as means ± S.D. P < 0.001 vs. HFD group; ###p < 0.001 vs. control group.
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induced the activation of IKKb/NF-jB pathway by enhancing the phosphorylation of IKKb [p-IKKb (Ser181): F (3, 16) = 42.939, P < 0.001, vs. Control group], leading to the degradation of IjBa [F (3, 16) = 20.255, P < 0.001, vs. Control group], accelerating NF-jB from the cytoplasm into the nucleus [cytoplasm NF-jB F (3, 16) = 12.744, nuclear NF-jB F (3, 16) = 10.844], and increasing the expression of inflammatory markers COX-2 and iNOS [F (3, 16) = 25.521, P < 0.001; F (3, 16) = 16.136, P < 0.001]. Oral administration of PSPC to HFD mice for 20 weeks significantly reversed these changes (P < 0.05, vs. the HFD group). There were no significant differences among Control group, PSPC group and HFD/PSPC group. 3.5. PSPC reduces the protein expression of NLRP3 inflammasome in the kidney of HFD-treated mice The recent discoveries suggest a potential role of NOD-like receptors (NLRs) in innate immune triggered interest in the studies of chronic diseases such as obesity and type 2 diabetes (T2D) (Grant and Dixit, 2013). NLRP3, as one important component of NLRs, is a crucial protein of inflammasome complex including NLRP3, ASC, pro-Caspase-1. Activated NLRP3 interacts with ASC, and recruits pro-Caspase-1, resulting in the secretion of proinflammatory cytokines IL-1b. As shown in Fig. 5, compared with Control group, HFD treatment significantly increased the expression level of NLRP3 and ASC [respectively F (3, 16) = 12.155, P < 0.001; F (3, 16) = 12.526, P < 0.001], and activated Caspase-1 (cleaved Caspase-1: F (3, 16) = 11.988) and IL-1b [F (3, 16) = 15.737, P < 0.001], while the content of pro-caspase-1 was no significant change among four groups. Our results showed that PSPC activated NLRP3 inflammasome pathway in the mouse kidney. Similarly, PSPC administration in the HFD-treated mice reversed these abnormal changes, made the above parameters restore to a near normal level (no significant vs. Control group). However, PSPC treatment did not substantially change these parameters in the kidneys of mice (P > 0.05 vs. Control group).
Fig. 4. PSPC inhibits the activation of IKKb/NF-jB signaling in the kidney of HFD-treated mice (n = 5). (A) Western-blotting results of p-IKKb, IKKb, IjBa, C-NFjB (C represents cytoplasm), N-NF-jB (N represents nucleus), iNOS and COX-2. (B) Relative density analysis of p-IKKb, IjBa, C-NF-jB, N-NF-jB, iNOS and COX-2 protein bands. The relative density is expressed as the ratio [p-IKKb/ (p-IKKb + IKKb), IjBa/b-actin, iNOS/b-actin, COX-2/b-actin, C-NF-jB/b-actin and N-NF-jB/b-tublin]. All values are expressed as means ± S.D. P < 0.01, P < 0.001 vs. HFD group; ###p < 0.001 vs. control group.
3.6. PSPC decreases the protein expression of RAGE and TXNIP in the kidney of HFD-treated mice
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TXNIP is ubiquitously expressed in a variety of cells, and acts an endogenous suppressor of ROS scavenging protein thioredoxin. TXNIP and RAGE are identified as the molecular nutrient sensors in the regulation of energy metabolism, linked oxidative stress and inflammation (Chutkow et al., 2010; Leuner et al., 2012). As shown in Fig. 6, HFD administration increased the expression level of TXNIP and RAGE in the kidneys of mice [F (3, 16) = 18.185, P < 0.001; F (3, 16) = 10.612, P < 0.001]. While PSPC suppressed the increase of renal TXNIP and RAGE induced by HFD-treatment, no significant change was found among Control group, PSPC group and PSPC/HFD group.
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4. Discussion
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The incidence of obesity has been climbing dramatically worldwide. It is a key risk factor for many metabolic diseases, including diabetes, hypertension, and heart disease. Recent studies indicate that obesity is accompanied by chronic inflammation in important metabolic tissues including adipose, liver and kidney, resulting in the release of pro-inflammatory cytokines and chemokines that contribute to the development of metabolic syndrome
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Fig. 5. PSPC reduces the expression levels of NLRP3 inflammasome in the kidney of HFD-treated mice (n = 5). (A) Western-blotting results of NLRP3, ASC, P-Caspase-1(P represents pro) C-Caspase-1(cleaved-Caspase-1) and IL-1b protein. (B) Relative density analysis of NLRP3, ASC, P-Caspase-1, C-Caspase-1 and IL-1b protein bands. The relative density is expressed as the ratio (NLRP3/b-actin, ASC/b-actin, P-Caspase1/b-actin, C-Caspase-1/b-actin and IL-1b/b-actin). All values are expressed as means ± S.D. P < 0.01, P < 0.001 vs. HFD group; ###p < 0.001 vs. control group.
Fig. 6. PSPC decreases the protein expression of RAGE and TXNIP in the kidney of HFD-treated mice (n = 5). (A) Western-blotting results of RAGE and TXNIP protein. (B) Relative density analysis of RAGE and TXNIP protein bands. The relative density is expressed as the ratio of RAGE/b-actin and TXNIP/b-actin. All values are expressed as means ± S.D. P < 0.01, P < 0.001 vs. HFD group; ###p < 0.001 vs. control group.
(Lu et al., 2011; Yakala et al., 2012; Zhang et al., 2013). Long-term high fat diet could induce obesity and T2D, causing the occurrence of metabolic syndrome (Wang et al., 2012;Wang et al., 2009). HFD administration could induce oxidative stress by down-regulating GSH, decreasing antioxidant enzyme activities (Noeman et al., 2011), and accelerating the production of AGEs and ROS (Leuner et al., 2012; Ruggiero et al., 2011), subsequently cause the serious damage of kidney. In the present study, HFD treatment for 20 weeks led to fasting hyperglycemia, insulin resistance, renal Collagen IV accumulation and urine protein. Anthocyanins are naturally unique polyphenolic compounds in higher plants. PSPC, as a kind of easily absorbed anthocyanins (Suda et al., 2002), has displayed special physiological efficacy such as anti-oxidant, antiinflammatory and anti-hyperglycemic effects (Matsui et al., 2002; Zhao et al., 2013). Recent researches show that acylated cyanidin and peonidin are two major components in PSPC (Qiu et al., 2009; Montilla et al., 2010), they may have a key role in PSPC’s pharmacological effect. Notably, we found PSPC effectively eliminated the metabolic disorders induced by HFD; the efficacy is achieved by inhibiting oxidative stress and inflammatory response, suggesting that PSPC effectively prevented kidney injury induced by HFD feeding. The sustained oxidative stress is involved in many important signaling pathways, IKKb/NF-jB and NLRP3/IL-1b are reported to be closely linked to the development of inflammatory response (Salminen et al., 2012). Consistently, we found kidney IKKb was significantly activated after HFD administration, and this alteration was accompanied with the increase of nuclear NF-jB, a nuclear transcription factor. The result is in accord with the activated NF-jB in the kidneys of HFD fed mice (Choi et al.,2011). In the present study, PSPC administration significantly reduced the activities of kidney NF-jB. Additionally, we previously found that PSPC also reduced activities of liver NF-jB in HFD-fed mice (Zhang et al., 2013). Because NF-jB regulates the expression of various genes, including cytokines, iNOS and COX-2, which are used to be major inflammatory mediators and pro-inflammatory markers, it plays critical roles in apoptosis, various autoimmune diseases and inflammation. Our data showed that HFD treatment increased the expression of iNOS and COX-2 in the kidneys, indicating PSPC’s effective preventive function for the kidney inflammation via NF-jB regulating the expression of iNOS and COX-2. Most recently, the NLRP3 inflammasome, which consists of NLRP3 molecule, adaptor protein ASC and pro-Caspase-1 that catalytically activates Caspase-1, causing the release of IL-1b and IL-18, was demonstrated to prevent obesity-induced inflammation and insulin resistance (Vandanmagsar et al., 2011). Consequently, NLRP3 inflammasome has emerged as an unexpected sensor for the pathogenesis of auto-inflammatory, metabolic danger and stress. Moreover, NLRP3 inflammasome is increasingly suspected of playing a major role in other human pathologies such as cancer, asbestosis and Alzheimer’s disease. NLRP3 is an important component of the inflammasome complex, and NLRP3 gene is activated by IKKb/NF-jB. Deleting NLRP3 in mice can prevent inflammasome activation in adipose tissue and liver as well as increase insulin sensitivity (Stienstra et al., 2011), demonstrating that NLRP3 plays an important role in the development of obesity and T2D (Pejnovic et al., 2013). Interestingly, elevated ROS can activate NLRP3 inflammasome, resulting in the secretion of bioactive IL-1b, triggering the activation of NFjB which in turn promotes the secretion of bioactive IL-1b (Grant and Dixit, 2013). We also found the related triggering regulatory results among ROS, NLRP3 inflammasome (NLRP3, ACS and Caspase-1), IKKb and NF-jB in the kidneys of HFD-treated mice. However, PSPC reduced the production of ROS and AGEs; these changes are closely related to the alleviation of kidney oxidative damage and associated urine protein in PSPC-treated mice. Furthermore, PSPC reduced HFD-induced renal inflammation by
Please cite this article in press as: Shan, Q., et al. Purple sweet potato color ameliorates kidney damage via inhibiting oxidative stress mediated NLRP3 inflammasome activation in high fat diet mice. Food Chem. Toxicol. (2014), http://dx.doi.org/10.1016/j.fct.2014.04.033
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dampening NLRP3/IL-1b and IKKb/NF-jB pathways similar to recent reports (Dragano et al., 2013; Zhang et al., 2013). NLRP3 inflammasome is also activated by TXNIP and RAGE and promotes the secretion of IL-1b (Zhou et al., 2010). The expression of TXNIP, as a hallmark of cell oxidative stress, is enhanced in obesity, T1D and T2D (Wang et al., 2013; Koenen et al., 2011). In the present study, HFD consumption was found to substantially stimulate the production of TXNIP, while, PSPC-treatment dampened the activation to ameliorate renal damage. Additionally, RAGE, a AGEs receptor, is promoted to express by the increase of AGEs under the condition of oxidative stress and hyperglycemia in obesity disease (Tomino et al., 2011). In contrary, deleted RAGE reduces the cardiac inflammation and protects against atherosclerosis in HFD feeding mice (Soro-Paavonen et al., 2008; Tikellis et al., 2008). Consistently, the accumulation of AGEs and RAGE is exhibited in the kidneys of HFD mice in our experiment; PSPC significantly decreased their contents in kidneys. The result is consistent with the previous findings from another lab that the proanthocyanidins from grape seed ameliorates diabetic nephropathy by regulation of the levels of kidney AGEs and RAGE (Li et al., 2009). RAGE and high glucose can induce TXNIP expression in retinal endothelial cells (Perrone et al., 2009), while TXNIP silencing abolishes RAGE-induced IL-1b secretion (Sbai et al., 2010), suggesting they may play the regulatory role in the development of obesity through mutually reinforcing expression. Combined our results with the previously reported ones, we speculated that PSPC prevented kidney damage by blocking endogenous oxidative cues and inflammatory reaction in HFD-induced obesity. This preventive pharmacologic efficacy is possibly obtained by PSPC’ inhibiting the expression of RAGE and of TXNIP, resulting in the inactivating of NLRP3 inflammasome, and causing the fall of NF-jB entranced into the nuclear. Although we have clearly established the importance of PSPC’ protective function on the HFD-induced kidney injury during the development of obesity in animal models, additional studies focused on unraveling the importance of PSPC’ preventing against the inflammation induced by HFD diet in human kidney tissue are needed. In conclusion, PSPC showed a significant amelioration on kidney injury in HFD-fed mice by reducing the production of AGEs and ROS, and improving the sensitivity of insulin, this protection function on the kidney is played by suppressing the expression of TXNIP and RAGE, further inhibiting the activation of NLRP3 inflammasome and IKKb/NF-jB pathways. Therefore, PSPC represents a potential natural product in the prevention of nephropathy induced by obesity.
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Conflict of Interest
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The authors declare there are no conflicts of interest.
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Transparency Document
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The Transparency document associated with this article can be found in the online version.
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Acknowledgements This work was supported by the Priority Academic Program Q4 Development of Jiangsu Higher Education Institutions (PARD), Q5 the National Natural Science Foundation of China (81171012, 31200873), the 2010 ‘‘Qinglan Project’’ Scientific and Technological Innovation Team Training Program of Jiangsu College and University, the Major Fundamental Research Program of the Natural Science Foundation of the Jiangsu Higher Education
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Institutions of China (07KJA36029), grants from the Key Laboratory of Jiangsu Province, PR, China, Jiangsu Province postgraduate innovation projects (2008, CX10S-043Z and 2012JSSPITP3192) and from the Natural Science Foundation of Jiangsu Normal University (11XLA08).
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