Article Cite This: J. Agric. Food Chem. 2019, 67, 6202−6211
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Highly Acylated Anthocyanins from Purple Sweet Potato (Ipomoea batatas L.) Alleviate Hyperuricemia and Kidney Inflammation in Hyperuricemic Mice: Possible Attenuation Effects on Allopurinol Zi-cheng Zhang,† Qing Zhou,‡ Yang Yang,† Yu Wang,† and Jiu-liang Zhang*,†,§ †
College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, People’s Republic of China Department of Pharmacy, Wuhan City Central Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430014, People’s Republic of China § Key Laboratory of Environment Correlative Dietology, Ministry of Education, Wuhan 430070, People’s Republic of China
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‡
S Supporting Information *
ABSTRACT: Allopurinol is the first-line medication for hyperuricemia treatment. However, severe drug-related adverse effects have often been reported among patients who received allopurinol administration. This study is aimed at evaluating the possible attenuation effects of highly acylated anthocyanins from purple sweet potato (HAA-PSP) on hyperuricemia and kidney inflammation in hyperuricemic mice treated with allopurinol. In comparison with 5 mg kg−1 allopurinol used alone, the combination of 25 mg kg−1 HAA-PSP and 2.5 mg kg−1 allopurinol could not only reduce serum uric acid level in hyperuricemic mice but also attenuate the kidney damage, as indicated by the level of serum biomarkers as well as histopathological examination. The inflammatory response was partially mitigated by inhibiting the protein expression of typical cytokines in the kidney. Our findings provide new evidence for the supplementary therapeutic potential of HAA-PSP with allopurinol on hyperuricemia and inflammation-related syndromes. Moreover, this study provides a theoretical basis for assessing the potential of anthocyanin-rich foods in health. KEYWORDS: highly acylated anthocyanins, purple sweet potato (Ipomoea batatas L.), hyperuricemia, anti-inflammatory, allopurinol
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INTRODUCTION Long-term hyperuricemia could cause the deposition of uric acid crystals in and around joints in humans, leading to one of the extensive metabolic diseases, gout. The occurrence and development of hyperuricemia is relevant to the purinemetabolism disorder and persistent increase of serum urate concentrations.1−3 In the past decades worldwide, a growing number of people have been found to suffer from hyperuricemia and gout. In China, the number of patients with hyperuricemia had grown to over 180 million by 2014, making it the second metabolic disease after diabetes.4,5 In addition, hyperuricemia is believed to be closely linked with the development of chronic kidney diseases.6,7 Once xanthine oxidase (XO) with increased catalytic activity produces excessive uric acid (UA) which is beyond the renal excretion capacity, the uric acid that cannot be excreted in vitro will deposit and crystallize in the kidney, directly causing renal injury. Second, the UA crystals deposited in the kidney will induce oxidative stress, inflammatory cell accumulation, and release of inflammatory cytokines by regulating the nuclear factor-κB (NF-κB) pathway, which may together aggravate renal injury.8,9 As an inhibitor of XO, allopurinol can effectively suppress the conversion of xanthine to UA in the purine synthesis pathway, which will further reduce the UA level in humans. For over 50 years, allopurinol has been widely used as the first-line medication for pharmacological urate-lowering therapy of gout and hyperuricemia.10 However, the clinical use of allopurinol © 2019 American Chemical Society
still has its limitations. First, previous research has revealed that the uric acid produced by normal human purine metabolism is the most abundant plasma antioxidant molecule.11 Blindly pursuing the minimum level of uric acid may break the purine metabolic balance, resulting in a series of negative chain reactions, thereby increasing the incidence of other diseases. In particular, allopurinol may cause allopurinol hypersensitivity syndrome (AHS) in patients, a rare but life-threatening disease suspected to be immune-mediated and characterized by Stevens−Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN).12,13 The patients of AHS may suffer from leukocytosis, eosinophilia, fever, acute liver injury, and kidney damage, and AHS can be fatal in up to 32% of cases,14 and even those who have been spared from death are often accompanied by sequelae including renal insufficiency.15 Pharmacogenomic studies have indicated that individuals carrying human leukocyte antigen HLA-B*5801 are more susceptible to AHS, especially for Asians (with a prevalence of 7.4%).16,17 According to the 2012 guidelines of the American College of Rheumatology, before administration of allopurinol, high-risk patients need to undergo the detection of HLAB*5801, a testing marker of AHS, to assess the risk of drug use.18,19 However, due to the limitations of genetic screening Received: Revised: Accepted: Published: 6202
March 22, 2019 May 13, 2019 May 16, 2019 May 16, 2019 DOI: 10.1021/acs.jafc.9b01810 J. Agric. Food Chem. 2019, 67, 6202−6211
Article
Journal of Agricultural and Food Chemistry
bicinchoninic acid (BCA) protein assay kit was purchased from Beyotime Biotechnology Co. (Haimen, Jiangsu, China). Peonidin 3O-(6-O-(E)-caffeoyl-(2-O-(6-O-(E)-feruloyl)-β-D-glucopyranosyl)-βD-glucopyranoside)-D-glucopyranoside (Peo 3-caffeoyl-feruloyl soph5-glc, purity >97%) was obtained from Luo in our laboratory.29 HPLC grade acetonitrile was purchased from Sigma Chemical Co. (Saint Louis, MO, USA). HPLC grade formic acid was purchased from Guangfu Fine Chemical Research Institute (Tianjin, China). Ultrapure water was purified by a Milli-Q Direct 8 System from Millipore Co. (Billerica, MA, USA). All other regular reagents were of analytical grade and were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Preparation and Characterization of Highly Acylated Anthocyanins from Purple Sweet Potato. The extraction, enrichment, and purification of anthocyanins were carried out in accordance with our previously described method.32 Dried purple sweet potato powder was dissolved, whisked by magnetic force in 40% anhydrous ethanol at a temperature of 60 °C, centrifuged, and evaporated. Then, the crude anthocyanin extract, which is the concentrated supernatant, was loaded on an AB-8 macroporous resin column with weak polarity (particle size 0.3−1.25 mm; 25 mm × 100 mm; purchased from Nankai Hecheng Science & Technology Co., Tianjin, China) and washed with 70% acidic ethanol. The sample collected by elution was evaporated, freeze-dried, and finally named as anthocyanin-rich purple sweet potato extract (APSPE). Next, APSPE received a secondary purification using a Daisogel SP-120-30/50̈ ODS-B column (DAISO Co., Osaka, Japan) to separate different enriched fractions. The primary purified product was loaded on the column and then sequentially washed with water and 10% methanol solution (v/v). Subsequently, fractions 1−4 were collected by isocratic elution with 20%, 30%, 40%, and 50% methanol (v/v) in sequence. TFA (0.1%) was added to all elution solvents. Since fraction 3 was found to have a stronger inhibitory effect on XO in vitro according to our previous report,33 it was further evaporated, freeze-dried, named HAA-PSP, and used in this experiment. In our previous study, HAA-PSP was characterized by HPLC-DAD with a C18 column, and 11 individual anthocyanins were separated.33 Meanwhile, all structures of the 11 individual anthocyanins were further compared and identified on the basis of our UPLC-ESI-MS/ MS database, and the results are displayed in Table 1. In addition, the quantification of HAA-PSP was performed by HPLC-DAD at 525 nm using an external standard method with Peo 3-caffeoyl-feruloyl soph-
and detection techniques in China, the complexity of current detection methods, and other influencing factors, the detection of HLA-B*5801 allele still does not have wide clinical application.20 This means that patients with hyperuricemia in China may be a potentially high risk group of AHS in choosing allopurinol for therapy. In addition, the dosage and side effects of allopurinol are more uncertain for the hyperuricemia patients who are detected to carry the HLA-B*5801 allele. Sweet potato (Ipomoea batatas L.), the seventh largest crop in the world, is a root-derived food that originated in Latin America.21 One of its varieties, purple sweet potato (PSP), is mainly planted in Japan and Korea and was introduced into China in the 1980s.22 Because of its high contents of anthocyanins and other phytochemicals beneficial to human health, purple sweet potato is popular among consumers as a functional food. The composition of anthocyanins in PSP is complex, and acylated anthocyanins are the dominant component (93%).23 Anthocyanins in PSP mainly belong to the families of cyanidin and peonidin, with several non-, mono-, or diacylated glucosides and acyl groups such as feruloyl, caffeoyl, hydroxybenzyl, and coumaroyl groups.24 The functional activity and property of anthocyanins vary with their chemical structures. Acylated anthocyanins display higher pH stability25 in comparison to anthocyanidins and have various health benefits, such as cancer prevention,26 visual protection,27 antidiabetes activity,28 hepatoprotective activity,29 antioxidant capacity,30 and protection against oxidative stress damage.31 Our previous research has reported the antihyperuricemic effect of anthocyanin-rich purple sweet potato extracts (APSPE) on potassium oxonate induced hyperuricemic mice.32 Furthermore, our in vitro experiment confirmed that highly acylated anthocyanins from purple sweet potato (HAA-PSP) are the main active components in APSPE that inhibit XO.33 However, HAA-PSP was only presumed to be a potential XO inhibitor, which calls for further in vivo validation. In addition, the mechanism by which anthocyanins relieve organ inflammation caused by hyperuricemia and their supplementary therapeutic effect with allopurinol is not yet clear. In this study, we evaluated the hypouricemic and renal protective effects of HAA-PSP, allopurinol, and their combination on potassium oxonate induced hyperuricemic mice. Important biochemical indicators in serum that reflect renal injury and oxidative stress level, such as creatinine (Cr), blood urea nitrogen (BUN), total superoxide dismutase (TSOD) activity, and malondialdehyde (MDA) level, were accurately measured. Renal histopathological examination and Western blot analysis of inflammatory molecules such as cytokines TNF-α, TGF-β1, IL-6, IL-1β, ICAM-1, COX-2, and NF-κB in kidney were performed to explore the possible mechanisms. Our results provide important insights into the potential attenuation of therapeutic effects of HAA-PSP on hyperuricemia and inflammation-related syndromes treated with allopurinol.
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Table 1. Identification and Quantification of HAA-PSP by HPLC at 525 nma quantification (mg/g)
identification cyanidin 3-(6′,6″-dicaffeoyl sophoroside)-5-glucoside cyanidin 3-(6′-caffeoyl-6′′-p-hydroxybenzoyl sophoroside)-5-glucoside peonidin 3-caffeoyl sophoroside-5-glucoside cyanidin 3-(6′-caffeoyl-6″-feruloyl sophoroside)-5glucoside peonidin 3-(6′,6″-dicaffeoyl sophoroside)-5-glucoside peonidin 3-(6′-caffeoyl-6″-p-hydroxybenzoyl sophoroside)-5-glucoside peonidin 3-(6′-caffeoyl-6″-feruloyl sophoroside)-5glucoside peonidin 3-(6′-caffeoyl-6″-p-coumaryl sophoroside)-5glucoside peonidin 3-(6′-feruloyl-6″-p-hydroxybenzoyl sophoroside)-5-glucoside peonidin 3-(6′-coumaryl-6″-p-hydroxybenzoyl sophoroside)-5-glucoside peonidin 3-(6′,6″-diferuloyl sophoroside)-5-glucoside total
MATERIALS AND METHODS
Plant Materials and Chemicals. The dried purple sweet potato powder (Ipomoea batatas L. cultivar Eshu No. 8) was donated by the Puzetian Food Co. Limited (Wuhan, Hubei, China). Potassium oxonate and allopurinol were purchased from Tokyo Chemical Industry Co. (Tokyo, Japan). Assay kits for determination of serum UA, Cr, BUN, T-SOD, and MDA were obtained from Nanjing Jiancheng Biotechnology Institute (Nanjing, Jiangsu, China). The
34.8 ± 6.5 28.5 ± 1.6 37.6 ± 3.5 45.9 ± 1.1 102.9 ± 5.9 160.8 ± 13.8 165.0 ± 8.9 3.1 ± 0.5 3.2 ± 0.2 6.8 ± 0.8 10.4 ± 0.1 599.1 ± 1.9
a
Peo 3-caffeoyl-feruloyl soph-5-glc was used as the standard sample in quantification. Values are expressed as the means ± SD (n = 3). 6203
DOI: 10.1021/acs.jafc.9b01810 J. Agric. Food Chem. 2019, 67, 6202−6211
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Journal of Agricultural and Food Chemistry 5-glc as the standard sample. The whole quantitative analysis was operated three times under the same conditions as those used for the samples. The anthocyanin content of HAA-PSP was finally determined as 599.1 ± 1.9 mg Peo 3-caffeoyl-feruloyl soph-5-glc equiv g−1. Animals. SPF grade 8-week male Kun-Ming strain mice used in this study were purchased from the Hubei Provincial Laboratory Animal Public Service Center (Certificate No. SCXK (Hubei) 20150019, Wuhan, China), which is under the Hubei Provincial Center for Disease Control and Prevention. The whole animal experiment was carried out at the Center for Laboratory Animals in Huazhong Agricultural University with the approval of the Scientific Ethics Committee (Permission No. HZAUMO-2017-031). All procedures involving animals throughout the experimental period strictly followed the Chinese legislation on the use and care of laboratory animals. During the study, mice were housed eight per cage under external environmental conditions of 25 ± 1 °C, 40 ± 10% humidity, and normal light/dark (12 h/12 h) cycle. The animals had free access to distilled water as well as standard laboratory pellet diet during the whole experimental period, and they had 1 week of acclimation before the start of experiments. Animal Model of Potassium Oxonate Induced Hyperuricemic Mice. At the beginning of the formal experiment, mice were randomly divided into six groups, including normal control group (NC group), hyperuricemia group (HUA group), allopurinol group (AP group), HAA-PSP group (HAA group), HAA-PSP with low allopurinol group (HAA-LAP group), and HAA-PSP with high allopurinol group (HAA-HAP group). To induce hyperuricemia, five groups of mice except for the NC group were orally administered with 250 mg kg−1 bw−1 potassium oxonate solution (dispersed in 0.5% carboxymethyl cellulose sodium (CMC)-Na) once daily for 7 consecutive days. Mice in the NC group were treated with solvent vehicle. The dosages of allopurinol (5 mg kg−1 bw−1) and HAA-PSP (25 mg kg−1 bw−1) used in AP group, HAA group, and HAA-HAP group were determined on the basis of a conversion from human clinical practice reported by the Chinese Pharmacopoeia Committee, our previous report, and our preliminary studies.32 In order to determine whether the combination of low-dosage allopurinol and HAA-PSP could enhance the hypouricemic effect and alleviate kidney inflammation, the allopurinol dose in the HAA-LAP group was set as 2.5 mg kg−1 bw−1, which was half of the dose in the AP group and HAA-HAP group. The test agents (allopurinol and HAA-PSP), alone or in combination, were dispersed in 0.5% CMC-Na solution and were administered by gavage 1 h after the administration of potassium oxonate. Food, but not water, was withdrawn from the cages 1 h before the oral administration. The volume of drug was calculated on the basis of the body mass of the mice measured immediately before the administration. Mice received diet fasting for 12 h before the biological sample collection on the seventh day, with distilled water still available. Sample Collection of Blood and Organ Tissues. The mice were sacrificed 1 h after the final administration on the seventh day. Whole blood samples were collected, kept clotted for 1 h, and centrifuged at 2050g for 10 min. The serum supernatant was collected and stored at −20 °C until analysis. Kidney and liver tissues were separated on an ice plate rapidly and carefully. After they were cleaned with saline, they were weighed quickly. One of the two kidney tissue samples was immediately immersed in 4% phosphate-buffered formalin (pH 7.1) for histopathological analysis, while the other was quickly stored at −80 °C for Western blot analysis. Determination of Organ Indexes and Serum Biochemical Indicators. The liver and renal indexes on a fresh weight basis was calculated according to the formulas liver index (mg/g) =
The levels of UA, BUN, Cr, T-SOD activity, and MDA in the serum were determined using commercially available kits on the basis of the instructions of the manufacturers. Each assay was performed in triplicate. Histopathological Examination of Renal Tissues. Kidney samples fixed with formalin were gradually dehydrated in ethanol, clarified in xylene, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) for microscopic observation at a 400× magnifications. Western Blot Analysis. Kidney samples stored at −80 °C were prepared for Western blot analysis of TNF-α, TGF-β1, IL-6, IL-1β, ICAM-1, COX-2, and NF-κB p65 according to standard procedures. The whole procedure was carried out at 4 °C. Briefly, kidney cortex was intermittently homogenized on ice in RIPA buffer including PMSF for 30 min and then centrifuged at 13200g for 5 min. The supernatant was collected, subpacked, and stored at 20 °C. A BCA protein assay kit was used to determine the protein content of the supernatant, and bovine serum albumin was used as a standard sample. The total proteins were incubated in boiling water for 10 min. Electrophoresis plastic was composed of 5% concentration gel and 12% separation gel. Equal amounts (40 μg) of different samples were separated on gels, respectively, until the target proteins were fully separated. The proteins were then electrophoretically transferred to a polyvinylidene difluoride membrane. The membranes were blocked in TBST (Tris-buffered saline containing 0.1% Tween-20) containing 5% skim milk powder and incubated for 2 h. Then, the membranes were treated individually with the specific antibody diluted in TBST, including GAPDH (1:1000), TNF-α (1:2000), TGF-β1 (1:1000), IL6 (1:1000), IL-1β (1:1000), ICAM-1 (1:200), COX-2 (1:200), and NF-κB p65 (1:1000) antibodies, respectively. The information about the primary antibodies is given in Table 2.
Table 2. Antibodies Used for Western Blot Analysis description
catalog no.
rabbit mGAPDH antibody mouse mTNF-α monoclonal antibody rabbit mIL-6 antibody rabbit mNF-κB P65 rela antibody rabbit mTGF-β1 antibody rabbit mIL-1β antibody rabbit mCOX-2 antibody
AB-P-R 001
rabbit mICAM-1 antibody goat antimouse-IgG-HRP goat antirabbit-IgG-HRP
BA2189
Affinity Biosciences (Cincinnati, OH, USA) Boster Biological Technology (Wuhan, China)
60291-1-Ig 21865-1-AP 10745-1-AP AF1027 DF6251 BA0738
BA1051 BA1054
HRP-conjugated goat antirabbit and antimouse IgG (Table 2) diluted in TBST were used as the secondary antibodies (1:50000) to detect the immunoreactive bands. They were made visible by enhanced chemiluminescence and exposed to X-ray film. The contents of target proteins were analyzed using Glyko BandScan software (Glyko, Novato, CA, USA) and normalized by the respective blotting from mGAPDH. Statistical Analysis. All results are presented as the average of three replications and are expressed as mean ± SD values using IBM SPSS Statistics 22 and Origin version 9.0 for Windows to perform one-way analysis of variance and Duncan multiple tests in order to determine the significant difference at p < 0.05.
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weight of liver (mg) body mass (g)
kidney index (mg/g) =
company Goodhere Biotechnology (Hangzhou, PR China) Proteintech Group (Chicago, IL, USA)
RESULTS Hypouricemic Effect of HAA-PSP, Allopurinol, and Their Combination in Hyperuricemic Mice. As shown in Figure 1, after 7 days of potassium oxonate administration, the
weight of kidney (mg) body mass (g) 6204
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Cr level in the HUA group (Figure 2A) was significantly (p < 0.05) increased. After treatment with allopurinol alone or in combination with HAA-PSP, the Cr levels were significantly decreased (p < 0.05) to approximately the normal value. As shown in Figure 2B, the BUN level of the AP group was increased to 9.08 mmol L−1, while the cotreatment of HAAPSP and allopurinol could restore it to the normal level. The T-SOD activity and MDA level in the serum were measured and illustrated, respectively. When oxidative stress develops, the T-SOD activity will be decreased with an increase observed in the MDA level, as shown in the results of mice in the HUA group (Figure 2C ,D). What is more, the use of allopurinol alone was found to have little effect on both TSOD and MDA levels in the serum. However, when anthocyanins were combined with allopurinol, the T-SOD activity increased while the MDA level showed an obvious decrease. These results suggested that the application of HAAPSP alone or together with low-dosage allopurinol could attenuate the oxidative stress caused by potassium oxonate through upregulation of the T-SOD activity and downregulation of the MDA level in the serum. Histopathological Examination. Histopathological analysis of the renal tissues of hyperuricemic mice is displayed in Figure 3. Mice in the NC group maintained a normal kidney structure, without significant inflammatory responses. The morphology of the glomerulus, renal tubules, and interstitium stayed normal with a compact arrangement of cells. However, the kidneys of mice in the HUA group displayed significant morphological changes in the glomerulus and focal infiltration of inflammatory cells (Figure 3A,B). What is worse, the renal tissues of the AP group showed an obvious proximal convoluted tubular injury characterized by a vacuolar degeneration of tubular cells and swelling of epithelial cells. The glomerular basement membrane was thickened, and the glomerular mesangial matrix was slightly proliferated (Figure 3C) in comparison with those of the NC group. When the same concentration of allopurinol was used in combination with HAA-PSP (HAA-HAP group), the renal injury of mice was slightly attenuated. Although the vacuolar degeneration of tubular cells and swelling of epithelial cells could still be observed, the glomerular injury and inflammatory cell infiltration were alleviated significantly (Figure 3F). The examination results of renal tissues in the HAA group (Figure 3D) and the HAA-LAP group (Figure 3E) suggested that, when the dosage of allopurinol was decreased (2.5 mg kg−1 bw−1 in the HAA-LAP group and none in the HAA group, respectively), the renal injury of mice was alleviated to varying degrees, as indicated by decreased inflammatory cell infiltration, restored morphological structure of glomerulus
Figure 1. Effects of HAA-PSP, allopurinol, and their combination on UA level in the serum of hyperuricemic mice. Values are expressed as the means ± SD (n = 8). Different letters marked above the bars are significantly different by an ANOVA multiple test (p < 0.05).
HUA group had a significantly (p < 0.05) higher level of serum UA in comparison with the NC group, indicating that hyperuricemia was effectively established in mice. After HAA-PSP treatment at 25 mg kg−1 bw−1, the UA level was significantly (p < 0.05) decreased from 91.56 to 52.33 μmol L−1 (by nearly 43%). Meanwhile, the UA level in the AP group showed a dramatic (p < 0.05) decrease and was below the detection limit, which was the same trend as in our previous study.32 When HAA-PSP and allopurinol were applied together, the antihyperuricemic effect was more obvious (p < 0.05) in comparison to the use of HAA-PSP alone. With an increasing proportion of allopurinol in the drug system, the UA levels in two cotreatment groups were decreased in a dosedependent manner by 69.69% in the HAA-LAP group and by 94.31% in the HAA-HAP group, respectively. Effects of HAA-PSP, Allopurinol, and Their Combination on Body and Organ Weight and Serum Biochemical Parameters in Hyperuricemic Mice. The body mass, liver weights, kidney weights, and organ indexes of hyperuricemic mice are presented in Table 3. All of the mice in the six groups showed no significant differences, indicating that the combination use of HAA-PSP and allopurinol did not have severe toxic side effects on organs. As the BUN and Cr levels in serum are effective indicators of renal function,34 and there is a close correlation between Cr level and UA synthesis in gout patients,35 the levels of BUN and Cr were evaluated, and the results are displayed in Figure 2. As expected, in comparison with the NC group, the serum
Table 3. Effects of HAA-PSP, Allopurinol, and Their Combination on Body, Liver, and Kidney Weights in Hyperuricemic Micea group NC HUA AP HAA HAA-LAP HAA-HAP
body mass (g) 33.10 33.29 33.86 33.46 34.90 33.75
± ± ± ± ± ±
1.96 2.04 1.51 1.50 1.28 1.26
liver weight (g) 1.43 1.51 1.47 1.49 1.61 1.60
± ± ± ± ± ±
0.12 0.14 0.15 0.11 0.12 0.10
kidney weight (g) 0.46 0.47 0.45 0.46 0.48 0.48
± ± ± ± ± ±
0.04 0.04 0.06 0.03 0.05 0.07
liver index (mg/g) 43.37 45.28 43.48 44.59 46.00 47.54
± ± ± ± ± ±
3.45 2.15 2.95 2.25 2.90 2.78
kidney index (mg/g) 14.02 14.06 13.15 13.76 13.72 14.26
± ± ± ± ± ±
0.71 0.88 1.61 0.93 1.27 2.25
a
Abbreviations: NC, normal control group; HUA, hyperuricemia group; AP, allopurinol group; HAA, HAA-PSP group; HAA-LAP, HAA-PSP with low allopurinol group; HAA-HAP, HAA-PSP with high allopurinol group. Values are expressed as the means ± SD (n = 8). 6205
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Figure 2. Effects of HAA-PSP, allopurinol, and their combination on (A) Cr level, (B) BUN level, (C) T-SOD activity, and (D) MDA level in the serum of hyperuricemic mice. Values are expressed as the means ± SD (n = 8). Different letters marked above the bars are significantly different by an ANOVA multiple test (p < 0.05).
expression of renal inflammatory cytokines, inhibit the activation of NF-κB p65, alleviate the renal inflammation in hyperuricemic mice, and reduce the adverse effects of allopurinol.
and proximal convoluted tubules, and reduced thickening of glomerular basement membrane and proliferation of glomerular mesangial matrix. These results indicated that HAA-PSP can ameliorate the renal damage caused by hyperuricemia or the application of allopurinol. Effect of HAA-PSP on Renal Inflammation by Suppressing Renal Inflammatory Cytokines and Inhibiting NF-κB Activation. As shown in Figure 4, potassium oxonate significantly (p < 0.05) increased the protein levels of renal inflammatory cytokines including TNF-α, TGF-β1, IL-6, and IL-1β in hyperuricemic mice. Surprisingly, after the treatment with a high dosage of allopurinol (AP group), the protein levels of the above four inflammatory cytokines all significantly (p < 0.05) increased rather than decreased. Instead, HAA-PSP could significantly (p < 0.05) inhibit the expression of TNF-α, TGF-β1, IL-6, and IL-1β protein in the kidney, whether used alone or in combination with allopurinol. Particularly, although the dosage of allopurinol was different between the HAA-LAP and HAA-HAP groups, the determination results were similar. Even so, the cotreatment with HAA-PSP and low-dosage allopurinol still exhibited a good and stable effect in regulating renal inflammatory cytokine levels, which was also observed in the protein levels of ICAM1, COX-2, and NF-κB p65 in renal tissues of mice. These results demonstrated that HAA-PSP could suppress the
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DISCUSSION Allopurinol is the first-line clinical therapy for the treatment of gout and hyperuricemia. However, severe drug-related adverse effects can be observed among patients who receive allopurinol administration, including AHS, SJS, and TEN.36 These adverse effects are far more likely to occur in Asian people than in people from other regions. What is worse, the usage of highdose allopurinol will also reduce serum uric acid level to below normal levels, and long-term use may easily trigger other diseases. Pharmacologically, it is possible to take advantage of other compounds or natural products to decrease the dosage of allopurinol and further reduce its dose-related adverse effects without sacrificing the therapeutic effect. In previous studies, the hypouricemic effect of anthocyanin from purple sweet potato has been reported and evaluated.32,37 However, due to the incomplete sample purification and complexity of the composition, the key active components in APSPE and their effects on hyperuricemia remain unknown. In a subsequent study in vitro, we attempted to conduct a secondary purification of APSPE on a ODS column to separate 6206
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Figure 3. Effects of fraction 3, allopurinol, and their combination on the renal histopathology of hyperuricemic mice: (A) kidney section of normal control group; (B) kidney section of hyperuricemia group; (C) kidney section of 2.5 mg kg−1 allopurinol group; (D) kidney section of 25 mg kg−1 HAA-PSP group; (E) kidney section of 25 mg kg−1 HAA-PSP with 2.5 mg kg−1 allopurinol group; (F) kidney section of 25 mg kg−1 HAA-PSP with 5 mg kg−1 allopurinol group. Magnification 400×, scale bar 50.00 μm.
nins with allopurinol may have a better mitigation effect on kidney damage in hyperuricemic mice. In addition, histopathological analysis results of renal tissues demonstrated that HAA-PSP could attenuate renal injury characterized by morphological change of the glomerulus, inflammatory cell infiltration, proximal convoluted tubular injury, thickening of glomerular basement membrane, and mesangial matrix proliferation. Thus, it can be speculated that HAA-PSP can reduce the renal injury in hyperuricemic mice caused by hyperuricemia or the application of allopurinol. A previous study has pointed out that hyperuricemia patients are more likely to suffer from oxidative stress induced renal injury, due to the long-lasting toxic effects of high serum uric acid level.39 Kidney is one of the primary viscera affected by oxidative stress resulting from excessive accumulation of UA.40 Potassium oxonate is believed to stimulate oxidants, such as O2•− and the product of oxidative stress MDA, while reducing the activity of the antioxidant enzyme SOD.41 In the present study, T-SOD activity was significantly inhibited while the MDA level was increased in hyperuricemic mice. Highly acylated anthocyanins seemed to have a better effect on oxidative stress in comparison with allopurinol, which is consistent with our previous research findings.30 The cotreatment of HAA-PSP and low-dosage allopurinol surprisingly displayed strong effects on serum T-SOD activity, MDA level,
copigments, anthocyanins with low acylation, and antocyanins with high acylation. The results suggested that highly acylated anthocyanins have strong inhibitory activity on XO.33 In the present study, the dosage of HAA-PSP administration was significantly lower than that of APSPE used in the previous study, but the antihyperuricemic bioactivity was much stronger. A comparison of the differences in anthocyanin content and composition between HAA-PSP and APSPE showed that highly acylated anthocyanin in purple sweet potato is the key active component for the antihyperuricemic effect. In addition, the combination of HAA-PSP and 2.5 mg kg−1 bw−1 allopurinol not only can reduce UA level to the normal range but also can avoid the extreme low level of UA caused by the individual usage of high-dose allopurinol. The above results suggest that HAA-PSP has a certain supplementary therapeutic potential in the treatment of hyperuricemia. Since it is widely acknowledged that hyperuricemia is a risk factor for the progression of chronic kidney diseases,38 this study aimed to evaluate the potential of highly acylated anthocyanins as a supplement of allopurinol on renal protection of hyperuricemic mice. Cotreatment with HAAPSP and allopurinol could effectively regulate serum Cr level in hyperuricemic mice and restore the BUN level in serum to normal. Hence, the combination of highly acylated anthocya6207
DOI: 10.1021/acs.jafc.9b01810 J. Agric. Food Chem. 2019, 67, 6202−6211
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Figure 4. continued
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Figure 4. Western blot of HAA-PSP, allopurinol, and their combination on protein expression for TNF-α, TGF-β1, IL-6, IL-1β, ICAM-1, COX-2, and NF-κB p65 in renal tissue of hyperuricemic mice. The contents of target proteins were normalized to GAPDH. Values are expressed as the means ± SD (n = 8). Different letters marked above the bars are significantly different by an ANOVA multiple test (p < 0.05).
and IL-1β was stimulated, and the protein levels of ICAM-1 and COX-2 were increased in hyperuricemic mice. Surprisingly, although we proved that high-dose allopurinol has a significant antihyperuricemic effect, its administration was found to aggravate the renal inflammation of hyperuricemic mice, possibly due to the toxic side effects of allopurinol, which will increase the burden of the kidney and thus enhance the inflammatory response. However, HAA-PSP could effectively downregulate the expression of renal inflammatory molecules in hyperuricemic mice, reduce the additional damage caused by allopurinol, and then alleviate a variety of renal injuries including glomerulosclerosis, renal interstitial fibrosis, and inflammatory cell infiltration. In addition, the activation of NF-κB is also one of the major causes of renal inflammation.51,52 Activation of the NF-κB signaling pathway is frequently observed in kidney dysfunction induced by potassium oxonate administration, indicating its causal role in the pathogenesis of renal inflammatory injury.53 As a rapid response transcription factor, activated NF-κB binds to the specific DNA sequences in the nucleus to initiate and upregulate the transcription of inflammatory mediators and cytokines.54 As a result, proinflammatory cytokines, oxygen free radicals, and other inflammatory mediators are produced and infiltrate into renal tissues in large quantities, further triggering inflammatory responses and kidney dysfunction. In
and oxidative stress, further demonstrating its good prospects for application in the treatment of hyperuricemia. Inflammatory response is known as the pathologic feature of hyperuricemia and contributes to initiating and developing renal injury. 42 The activation and release of various inflammatory molecules in renal tissue, such as cytokines TNF-α, IL-6, IL-1β, and TGF-β1,43 adhesion molecule ICAM1,44 and synthetase COX-2,45 play an important role in the progression of nephritis and hyperuricemia. As important inflammatory cytokines, TNF-α as well as IL-6 and IL-1β of the interleukin family could participate in the inflammatory response and promote the proliferation of glomerular mesangial cells and mesangial matrix.46,47 Meanwhile, TGF-β not only can accelerate the process of glomerulosclerosis but can also regulate the secretion of excessive collagen fibers and promote the formation of renal interstitial fibrosis.48 The adhesion molecule ICAM-1 could increase the adhesion between inflammatory cells and endothelial cells, enhance the recruitment of inflammatory leukocytes, and prolong the infiltration time of inflammatory cells.49 Overexpression of COX-2 in the renal cortex can promote inflammatory cell infiltration and accumulation, which will also stimulate the synthesis of ROS.50 In this study, hyperuricemic mice were found to suffer from inflammatory response. The expression of renal inflammatory cytokines such as TNF-α, TGF-β1, IL-6, 6209
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(3) Pascart, T.; Lioté, F. Gout: state of the art after a decade of developments. Rheumatology 2018, 58 (1), 27−44. (4) Liu, R.; Han, C.; Wu, D.; Xia, X. H.; Gu, J. Q.; Guan, H. X.; Shan, Z. Y.; Teng, W. P. Prevalence of hyperuricemia and gout in mainland China from 2000 to 2014: a systematic review and metaanalysis. Biomed. Res. Int. 2015, 2015, 762820. (5) Song, P. G.; Wang, H.; Xia, W.; Chang, X. L.; Wang, M. L.; An, L. Prevalence and correlates of hyperuricemia in the middle-aged and older adults in China. Sci. Rep. 2018, 8 (1), 4314. (6) Chang, H. Y.; Tung, C. W.; Lee, P. H.; Lei, C. C.; Hsu, Y. C.; Chang, H. H.; Yang, H. F.; Lu, L. C.; Jong, M. C.; Chen, C. Y.; Fang, K. Y.; Chao, Y. S.; Shih, Y. H.; Lin, C. L. Hyperuricemia as an independent risk factor of chronic kidney disease in middle-aged and elderly population. Am. J. Med. Sci. 2010, 339 (6), 509−515. (7) Lee, C. T.; Chang, L. C.; Liu, C. W.; Wu, P. F. Negative correlation between serum uric acid and kidney URAT1 mRNA expression caused by resveratrol in rats. Mol. Nutr. Food Res. 2017, 61 (10), 1601030. (8) Ye, Y.; Zhang, Y.; Wang, B.; Walana, W.; Wei, J.; Gordon, J. R.; Li, F. CXCR1/CXCR2 antagonist G31P inhibits nephritis in a mouse model of uric acid nephropathy. Biomed. Pharmacother. 2018, 107, 1142−1150. (9) Li, Y. W.; Zhang, Y.; Zhang, L.; Li, X.; Yu, J. B.; Zhang, H. T.; Tan, B. B.; Jiang, L. H.; Wang, Y. X.; Liang, Y.; Zhang, X. S.; Wang, W. S.; Liu, H. G. Protective effect of tea polyphenols on renal ischemia/reperfusion injury via suppressing the activation of TLR4/ NF-κB p65 signal pathway. Gene 2014, 542 (1), 46−51. (10) Seth, R.; Kydd, A. S. R.; Buchbinder, R.; Bombardier, C.; Edwards, C. J. Allopurinol for chronic gout. Cochrane. Database Syst. Rev. 2014, 10, CD006077. (11) Ames, B. N.; Cathcart, R.; Schwiers, E.; Hochstein, P. Uric acid provides an antioxidant defense in humans against oxidant-caused and radical-caused aging and cancer: a hypothesis. Proc. Natl. Acad. Sci. U. S. A. 1981, 78 (11), 6858−6862. (12) Ramasamy, S. N.; Korb-Wells, C. S.; Kannangara, D. R.; Smith, M. W.; Wang, N.; Roberts, D. M.; Graham, G. G.; Williams, K. M.; Day, R. O. Allopurinol hypersensitivity: a systematic review of all published cases, 1950−2012. Drug Saf. 2013, 36 (10), 953−980. (13) Stamp, L. K.; Taylor, W. J.; Jones, P. B.; Dockerty, J. L.; Drake, J.; Frampton, C.; Dalbeth, N. Starting dose is a risk factor for allopurinol hypersensitivity syndrome: a proposed safe starting dose of allopurinol. Arthritis Rheum. 2012, 64 (8), 2529−2536. (14) Alerhand, S.; Cassella, C.; Koyfman, A. Stevens-Johnson syndrome and toxic epidermal necrolysis in the pediatric population: a review. Pediatr. Emerg. Care. 2016, 32 (7), 472−476. (15) Gueudry, J.; Roujeau, J. C.; Binaghi, M.; Soubrane, G.; Muraine, M. Risk factors for the development of ocular complications of Stevens-Johnson syndrome and toxic epidermal necrolysis. Arch. Dermatol. 2009, 145 (2), 157−162. (16) Yeo, S. I. HLA-B*5801: utility and cost-effectiveness in the Asia-Pacific Region. Int. J. Rheum. Dis. 2013, 16 (3), 254−257. (17) Jutkowitz, E.; Dubreuil, M.; Lu, N.; Kuntz, K. M.; Choi, H. K. The cost-effectiveness of HLA-B*5801 screening to guide initial urate-lowering therapy for gout in the United States. Semin. Arthritis Rheum. 2017, 46 (5), 594−600. (18) Khanna, D.; Fitzgerald, J. D.; Khanna, P. P.; Bae, S.; Singh, M. K.; Neogi, T.; Pillinger, M. H.; Merill, J.; Lee, S.; Prakash, S.; et al. 2012 American College of Rheumatology guidelines for management of gout. Part 1: systematic nonpharmacologic and pharmacologic therapeutic approaches to hyperuricemia. Arthritis Care Res. 2012, 64 (10), 1431−1446. (19) Yang, C. Y.; Chen, C. H.; Deng, S. T.; Huang, C. S.; Lin, Y. J.; Chen, Y. J.; Wu, C. Y.; Hung, S. I.; Chung, W. H. Allopurinol use and risk of fatal hypersensitivity reactions: a nationwide population-based study in Taiwan. JAMA. Int. Med. 2015, 175 (9), 1550−1557. (20) Zhang, X. J.; Jin, L.; Wu, Z. Y.; Ma, W. Z.; Chen, Y. M.; Chen, G.; Wang, L. X.; Guan, M. Clinical evaluation of a substitute of HLAB*58:01 in different Chinese ethnic groups. Genet. Mol. Biol. 2018, 41 (3), 578−584.
addition, the cytokines TNF-α and IL-1β induced by activated NF-κB could also further activate the NF-κB signaling pathway.55 Our results showed that NF-κB p65 was significantly activated in hyperuricemic mice, and a high dose of allopurinol aggravated the activation of NF-κB p65, which brought about a further increase in the release of inflammatory cytokines in kidneys of hyperuricemic mice. In contrast, application of HAA-PSP alone or in combination with allopurinol could effectively hinder the activation process of NF-κB p65, reducing the release of various inflammatory mediators and relieving kidney damage. In this study, we investigated the possible attenuation effects of HAA-PSP on hyperuricemia and kidney inflammation in potassium oxonate induced hyperuricemic mice treated with allopurinol. The combination of HAA-PSP at 25 mg kg−1 and allopurinol at 2.5 mg kg−1 showed a significantly better hypouricemic effect and toxicity-reducing effects on kidney damage in comparison with the application of HAA-PSP or allopurinol alone. The cotreatment with the two components could not only alleviate the oxidative stress by regulating serum T-SOD activity and MDA level but also downregulate the protein expression of typical cytokines by mediating the NF-κB pathway, thereby reducing the infiltration of inflammatory cells and alleviating kidney damage. Our results provide new evidence for the potential supplementary effect of HAA-PSP with allopurinol on hyperuricemia and inflammation-related syndromes. Moreover, this study provides a theoretical basis for assessing the potential of anthocyanin-rich foods in health.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.9b01810. Supplementary Figure S1 (PDF)
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AUTHOR INFORMATION
Corresponding Author
*J.Z.: e-mail,
[email protected]; tel, +86-027-87282111; fax, +86-027-87282111. ORCID
Jiu-liang Zhang: 0000-0002-1745-846X Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was supported by the Fundamental Research Funds for the Central Universities of China (No. 2662018PY022), the Natural Science Foundation of Hubei Province (No. 2018CFB738), and the Clinical Research Project of Health and Family Planning Commission of Wuhan Municipality (No. WX13A05). The authors thank Mr. Zuo-xiong Liu for his participation in the language modification process.
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