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Preventive effects of rutin on the development of experimental diabetic nephropathy in rats
Life Sciences 91 (2012) 959–967
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Preventive effects of rutin on the development of experimental diabetic nephropathy in rats Hui-hui Hao a, Zhu-min Shao b, Dao-quan Tang a, c,⁎, Qian Lu a, Xu Chen a, c, Xiao-xing Yin a,⁎⁎, Jing Wu a, c, Hui Chen a, c a b c
Key Laboratory of New Drug and Clinical Application, Xuzhou Medical College, Xuzhou 221004, China Department of Pharmacy, Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, China Department of Pharmaceutical Analysis, School of Pharmacy, Xuzhou Medical College, Xuzhou 221004, China
a r t i c l e
i n f o
Article history: Received 29 March 2012 Accepted 12 September 2012 Keywords: Rutin Diabetic nephropathy CTGF Smads TGF-β1 Oxidative stress
a b s t r a c t Aims: Diabetic nephropathy (DN) is an important microvascular complication and one of the main causes of end-stage renal disease. In this study, the preventive effect and mechanism of rutin on the development of DN in streptozotocin (STZ)-induced diabetic rats were investigated. Main methods: After an early DN model was induced by STZ, rats were orally administered rutin at 3 doses for 10 weeks. Fasting blood glucose, creatinine (Cr), blood urea nitrogen (BUN), urine protein, kidney index, antioxidase, advanced glycosylation end products (AGEs), extracellular matrix (ECM) including collagen IV and laminin, connective tissue growth factor (CTGF), phosphorylated Smad 2/3 (p-Smad 2/3) and Smad 7 (p-Smad 7), and transforming growth factor-β1 (TGF-β1) were determined by different methods, respectively. The ultrastructural morphology was observed by a transmission electron microscope. Key findings: Compared with the DN group, rutin decreased the levels of fasting blood glucose, Cr, BUN, urine protein, the intensity of oxidative stress and p-Smad 7 significantly. The expression of AGEs, collagen IV and laminin, TGF-β1, p-Smad 2/3 and CTGF was inhibited by rutin significantly. Moreover, rutin was observed to inhibit proliferation of mesangial cells and decrease thickness of glomerular basement membrane (GBM) by electron microscopy. Significance: The preventive effect of rutin on the development of DN is closely related to oxidative stress and the TGF-β1/Smad/ECM and TGF-β1/CTGF/ECM signaling pathways. Those results suggest that rutin can prevent the development of experimental DN in rats. © 2012 Elsevier Inc. All rights reserved.
Introduction The early stage of diabetic nephropathy (DN) is characterized by renal hypertrophy, glomerular hypertrophy, glomerular hyperfiltration, and microalbuminuria (Wolf and Ziyadeh, 1999; Makino et al., 1996; Raptis and Viberti, 2001). These changes are related to the subsequent development of glomerular morphological abnormalities and the prognosis of DN. One of the characteristic pathological changes in DN is the accumulation of extracellular matrix (ECM) components including collagens, fibronectin and laminin in the glomeruli and the interstitium of kidney (Kanwar et al., 2008). Hyperglycemia is usually considered as the main determinant factor of the initiation and progression of DN which increases the formation of advanced glycosylation end products (AGEs), oxidative stress and transforming growth factor-β1 (TGF-β1) ⁎ Correspondence to: D.Q. Tang, Key Laboratory of New Drug and Clinical Application, Xuzhou Medical College, Xuzhou 221004, China. Tel./fax: +86 516 83262136. ⁎⁎ Corresponding author. Tel./fax: +86 516 83262009. E-mail addresses: [email protected] (D. Tang), [email protected] (X. Yin). 0024-3205/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.lfs.2012.09.003
mRNA expressions. TGF-β1 is the most potent growth factor contributing to ECM accumulation, which it does by stimulating ECM production and suppressing its degradation (Border et al., 1996). Smad proteins are transcription factors that mediate TGF-β/activin signaling by forming complexes with each other. Smads 2 and 3 are specific for TGF-β signaling, and they are phosphorylated by TGF-β receptor type 1 after binding with TGF-β (Wang et al., 2005; Gagliardini and Benigni, 2006). Either Smad 2 or 3 forms a heterodimer with Smad 4, and the complex immediately translocates into the nucleus and induces transcription of various genes by combining specific binding sites of genomic DNA. Smad 7 can be an inhibitory Smad protein that blocks the phosphorylation of Smad 2/3, counterbalancing the TGF-β signaling (Stopa et al., 2000). Connective tissue growth factor (CTGF) has been described as a growth factor that acts downstream of TGF-β1 and is a potent inducer of ECM in the fibrotic process (Hishikawa et al., 2001). TGF-β1/Smad/ ECM or TGF-β1/CTGF/ECM seems to be a linked response in the pathogenesis of DN. Rutin is found in many foods and many traditional Chinese medicines, whose treatment on diabetic mellitus has been proved by the Stanely Mainzen Prince group. Their innovative achievements have
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published that rutin can improve glucose homeostasis of diabetic rats by decreasing fasting plasma glucose and increasing insulin levels, increasing the content of glycogen in the liver and muscle whereas decreasing that in the kidney, increasing the activity of hexokinase and decreasing the activities of glucose-6-phosphatase and fructose1,6-bisphosphatase in the tissues (Stanley Mainzen Prince and Kamalakkannan, 2006). This research group also proved that rutin can decrease the levels of lipids, low density and very low density lipoprotein cholesterol in plasma, increase the levels of plasma high density lipoprotein cholesterol and the activity of plasma lipoprotein lipase and lecithin cholesterol acyltransferase and decrease the levels of lipids and glycoproteins and the activity of 3-hydroxy 3-methylglutaryl coenzyme A reductase in the liver and in the kidney in diabetic rats basing on its antioxidant property (Stanely Mainzen Prince and Kannan, 2006). Moreover, Stanely Mainzen Prince group also reported that rutin can protect the diabetic rats kidney by decreasing the content of hydroxyproline and collagen and the levels of tissue inhibitors of metalloproteinases, increasing the activity of matrix metalloproteinases in the kidney (Kamalakkannan and Stanely Mainzen Prince, 2006). Other scholars also proved that rutin can inhibit the formation of AGEs (Cervantes-Laurean et al., 2006) in STZ-induced rats. All of these offer us a potential pharmacological foundation of rutin for DN therapy. We previously reported that rutin can decrease the accumulation of ECM mediated by TGF-β1/Smads in high glucose cultured-rat mesangial cells (Tang et al., 2011). Although Kamalakkannan and Prince have reported that rutin can decrease fasting plasma glucose, glycosylated hemoglobin thiobarbituric acid reactive substances and lipid hydroperoxides, increase insulin, C-peptide, hemoglobin, protein levels and non-enzymic antioxidants in diabetic rats induced by streptozotocin (STZ), their work haven't focused on the creatinine (Cr), blood urea nitrogen (BUN), and urine protein in DN rats (Kamalakkannan and Prince, 2006). Moreover, the protective effects and mechanism of rutin on early experiment DN in vivo have not been studied by Stanley Mainzen Prince group and other scholars to date, which may restrict the clinical application of rutin. Thus, the present study was designed to investigate the effect of rutin on the kidney of STZ-induced type 1 diabetic rats by observing the changes of renal morphology and some signaling including TGF-β1, phosphorylated Smad 2/3 (p-Smad 2/3), phosphorylated Smad 7 (p-Smad 7), CTGF, collagen IV, and laminin. The blood glucose, Cr, BUN, urine protein, kidney index, antioxidases, and AGEs in DN rats were simultaneously observed. Captopril (CAP) was used as a positive control drug in this study. Materials and methods Animals Adult male Sprague–Dawley rats (Certificate No. SYXK 2010-0011), weighing 180 g–220 g, procured from the Laboratory Animal Center of Xuzhou Medical College were used in this study. The rats were maintained in a controlled environment (12 h light/dark cycle) and temperature (21 ±2 °C). All animals were fed on a standard pellet diet and water was freely available. The animals were acclimatized to the laboratory conditions before starting the experiment. All the experiments were approved by Institutional Animal Ethics Committee and followed the Guiding Principles for Care and Use of Laboratory Animals of Xuzhou Medical College. Chemicals Rutin and STZ were purchased from Sigma Chemicals Company (St. Louis, MO, USA). Captopril (CAP, Lot No. 050050), serving as a positive control drug, was kindly provided by Changzhou Pharmaceutical Factory (Changzhou, China).
Induction and assessment of diabetes STZ was freshly dissolved in citrate buffer (0.01 mol/L, pH 4.4) and maintained on ice before use. The overnight fasted rats were made diabetic with a single intraperitoneal injection of STZ (60 mg/kg). Age-matched control normal standard rats (NS group, n = 10) received an equivalent amount of citrate buffer. Diabetes was confirmed in the STZ-injected rats by measuring the fasting plasma glucose levels 72 h post injection. After an overnight fast, rats with blood glucose levels above 13.89 mmol/L (250 mg/dL) were considered as diabetic and were used in this study. Treatment with rutin was started on the third day after STZ-injection. Experimental design Diabetic rats were randomly allotted into 5 groups as follows: diabetic rats were treated with 1% sodium carboxymethyl cellulose (CMC) solution (DN group, n =8); diabetic rats with 10, 30, and 90 mg/kg rutin for the RL group (low dose, n=8), the RM group (moderate dose, n= 9), and the RH group (high dose, n =9), respectively; the diabetic rats were treated with 10 mg/kg of CAP (CAP group, n=9). The low dose of rutin was calculated according to its content in the moderate effective dose of Ginkgo biloba extract for therapy of DN published by our laboratory (Ginkgo biloba extract employed in that experiment (Lu et al., 2007) contains 11.4% rutin (Tang et al., 2010)). Drugs were suspended in 1% CMC solution and orally administered to rats using an intragastric tube for a period of 10 weeks. The same volume of CMC solution was administered to the NS group (n= 10). Normal and diabetic rats were housed 5 per cage in the barrier environment referred to as breed specific pathogen-free grade animals. At the end of the experiments, after a 24 h urine sample collection, the blood samples of the rats were withdrawn from the abdominal aorta. Serum was collected to assess the renal function by measuring plasma levels of Cr and urea nitrogen. Kidneys were removed, after being weighed, parts of fresh kidney cortexes were stored in 10% formaldehyde solution for immunohistochemical assays, and 1 mm× 1 mm× 1 mm cubes of renal cortices were fixed in 2.5% glutaraldehyde for electron microscopic measurement. The rest of the kidneys were stored at −80 °C for the later analysis. Measurement of blood glucose and renal function Blood glucose was measured by blood glucose meter (OneTouch UltraEasy, USA). The 24 h urinary albumin was measured by ELISA kit purchased from R&D Systems Company (R&D, USA). BUN and Cr were determined by the automatic biochemistry analyzer (Olympus2000, Tokyo, Japan). The kidney index was expressed as 1000 × kidney weight/body weight. Assessment of renal oxidative stress and determination of AGEs, collagen IV and laminin The kidney tissue was minced and a homogenate was prepared with 10% (w/v) phosphate buffered saline (0.1 mol/L, pH 7.4) using a homogenizer. The kidney homogenate was centrifuged at 15,000 rpm for 15 min at 4 °C. The supernatant was collected to determine the content of protein by bicinchoninic acid (BCA) protein assay kit purchased from Beyotim Institute of Biotechnology (Nantong, China). The total antioxidative capability (T-AOC), catalase (CAT), total superoxidase dismutase (T-SOD) and glutathione-per-oxidase (GSH-Px) in the renal cortex and the level of malonaldehyde (MDA) were measured by using the commercially available kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). AGEs play a critical role in DN by stimulating type IV collagen and laminin, the main component of ECM. The quantity of type IV collagen,
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laminin and AGEs in the renal cortex was determined by ELISA kit purchased from R&D Systems Company (R&D, USA). Immunohistochemical measurements of CTGF Kidney cortexes, fixed in 10% formaldehyde previously, were embedded in paraffin and 4 μm thick sections were prepared for immunohistochemistry analysis of CTGF. The renal tissue sections were incubated with goat polyclonal anti-CTGF (Lot No. B1030, Zhongshan Golden Bridge Biological Technology, Beijing, China) at a dilution of 1:100 at 37 °C for 2 h. After washing in PBS, the sections were incubated with rabbit anti-goat IgG horseradish peroxidase (Lot K115214D, Beijing, China) for 30 min at room temperature. After washing, to visualize CTGF, the renal tissue sections were stained with 3,3′-diaminobenzidine (DAB) for 10 min and then washed in tap water for 10 min. After the nuclei were stained with hematoxylin for 20 s, the sections were evaluated by a light microscope (×400) and were photographed. Photomicrographs of at least 20 fields in each rat were quantified for the intensity of positive staining in the renal tissue with IPLab software (Scanalytics, Fairfax, VA). Western blot analysis for Smads and TGF-β1 in the renal cortex For detection of p-Smad 2/3, p-Smad 7 and TGF-β1 in the renal cortex, gel electrophoreses and subsequent western blotting was performed. Firstly, renal cortex tissues were homogenized in icecold RIPA buffer containing protease inhibitors. The homogenates were centrifuged at 15,000 rpm for 15 min at 4 °C and an aliquot of the supernatant was kept for protein determination. The content of protein in the supernatant was determined by BCA protein assay kit. All samples were boiled for 5 min in Laemmli sample buffer before loading. A 60 μg sample of total protein was separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to nitrocellulose membranes (Pall Corporation, USA). After being blocked with 5% nonfat dry milk in TBS-Tween (0.1%) for 2 h at room temperature, the membranes were incubated separately with the following antibodies: rabbit anti-phospho-Smad 2/3 (Santa Cruz Biotechnology, USA), rabbit anti-total-Smad 2/3 (Bioworld Technology, USA), rabbit anti-phospho-Smad 7 (Santa Cruz Biotechnology, USA), rabbit anti-total-Smad 7 (Abcam, UK), rabbit anti-TGF-β1 (Santa Cruz Biotechnology, USA) and rabbit anti-GAPDH (Beyotim Institute of Biotechnology, China) antibodies. The membranes were probed with the primary antibodies overnight at 4 °C and then washed three times in TBS-Tween for 5 min, followed by incubation with the appropriate secondary horseradish peroxidase conjugate antibody for 1 h at room temperature. After extensive washing, the signals were visualized by the enhanced chemiluminescence system (ECL; Santa Cruz Biotechnology, USA) and the density of each band was determined with Image J analysis software version 1.34s (Wayne Rasband National Institutes of Health, USA).
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RT-PCR for the determination of relative quantities of TGF-β1 mRNA A reverse transcription polymerase chain reaction (RT-PCR) procedure was performed to determine the relative quantities of TGF-β1 mRNA in the renal cortex, while β-actin mRNA, a housekeeping gene, being used as an internal control. The total RNA was extracted from the renal cortex with RNA simple Total RNA Kit (Lot No. J9117, TIANGEN Biotechnology Company, Beijing, China). The upstream and downstream primers for rat TGF-β1 mRNA were: 5′-CCCGCATC CCAGGAC CTCTCT-3′ and 5′-CGGGGGACTGGCGAGCCTTAG-3′, yielding a 519 bp product; whereas those for β-actin were: 5′-GCTGCGT GTGGCCCCTGAG-3′ and 5′-ACGCA-GGATGGCATGAGGGA-3′, yielding a 252 bp product. Equal amounts (1 μg) of each total RNA sample were added in a 50 μL reaction mixture exerting one-step amplification with Quant One Step RT-PCR Kit (Lot No. J9104, TIANGEN Biotechnology Company, Beijing, China). RT-PCR conditions were set to reverse transcription for 30 min at 50 °C, initial denaturation for 2 min at 94 °C, 40 cycles at 94 °C for 30 s, 55 °C for 45 s, 72 °C for 1 min, and final elongation at 72 °C for 10 min. The RT-PCR products were separated by 1.5% agarose gel electrophoresis, and the band densities were analyzed using Image J analysis software version 1.34s. The relative quantities of TGF-β1 mRNA in the renal cortex were represented by the ratio of band density of TGF-β1 versus that of β-actin. Electronmicroscopy for morphological observation Renal tissues, fixed in glutaraldehyde, were postfixed with 1% OsO4 for 2 h and dehydrated with graded ethanol. Samples were embedded with epoxy resin and polymerized at 37 °C for 24 h, 45 °C for 24 h, and 60 °C for 24 h. The renal samples were cut into ultrathin sections and then stained with plumbum citrate for ultrastructural observation under a transmission electron microscope (H600A-2, Hitachi, Japan). The images were amplified 10,000 and the photos were scanned into an image analysis system (Leica Qwin Standard V2.6, Leica Microsystems, Germany). Statistical analysis All the grouped data were analyzed by ANOVA and Dunnett's t-test (2-side) for different groups using SPSS 16.0 software. The values are expressed as mean ± SD. P value b 0.05 was considered significant and included in the study. Results Effect of rutin on blood glucose, kidney index and the renal function of rats In our experiment, the fasting blood glucose levels, urine protein, Cr, BUN, and the kidney index in rats of the DN group were significantly higher than those of the NS group (P b 0.01), which suggested
Table 1 Effects of rutin on blood glucose, urine protein, Cr, BUN and kidney index of rats (mean ± SD). Group
n
Fasting blood glucose (mmol/L)
24 h urine protein (μm/24 h)
Kidney index ×1000
BUN (mmol/L)
Cr (μmol/L)
NS DN RL RM RH CAP
10 8 8 9 9 9
6.16 ± 2.16 20.91 ± 2.02# 17.83 ± 3.14⁎ 13.92 ± 2.64⁎⁎ 12.54 ± 1.75⁎⁎
3.42 ± 0.24 38.71 ± 3.73# 27.15 ± 2.46⁎ 20.18 ± 1.96⁎⁎ 15.14 ± 1.69⁎⁎ 14.50 ± 2.63⁎⁎
7.88 ± 0.69 14.96 ± 1.89# 13.26 ± 0.87⁎ 12.84 ± 1.10⁎⁎ 11.57 ± 1.32⁎⁎ 11.34 ± 1.32⁎⁎
7.53 ± 1.27 26.69 ± 5.90# 22.72 ± 2.56⁎ 18.78 ± 1.97⁎⁎ 16.74 ± 1.92⁎⁎ 17.44 ± 3.48⁎⁎
37.17 ± 5.17 67.44 ± 9.21# 60.42 ± 5.91⁎ 50.13 ± 7.12⁎⁎ 46.14 ± 6.52⁎⁎ 44.63 ± 4.50⁎⁎
18.54 ± 4.47
Mean ± SD. NS: normal standard group. DN: diabetic nephropathy. RL, RM, RH: rutin 10, 30, 90 mg/kg. CAP: captopril 10 mg/kg. # P b 0.01 vs NS group. ⁎ P b 0.05 vs DN group. ⁎⁎ P b 0.01 vs DN group.
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Table 2 Effects of rutin on T-AOC, T-SOD, CAT, GSH-Px and MDA in the renal cortex (mean ± SD). Group
n
CAT (U/mgprot)
T-AOC (U/mgprot)
T-SOD (U/mgprot)
GSH-PX (U/mgprot)
MDA (nmol/ml)
NS DN RL RM RH CAP
10 8 8 9 9 9
55.44 ± 16.15 18.08 ± 5.83# 26.59 ± 3.99⁎ 42.09 ± 8.15⁎⁎ 43.19 ± 7.56⁎⁎ 25.70 ± 4.23⁎
7.30 ± 1.77 1.10 ± 0.30# 2.35 ± 0.76⁎ 3.92 ± 1.22⁎⁎ 4.11 ± 0.44⁎⁎ 2.76 ± 0.65⁎⁎
329.54 ± 78.00 220.78 ± 38.37# 263.22 ± 24.15⁎ 267.40 ± 21.62⁎⁎ 277.40 ± 27.08⁎⁎ 253.90 ± 39.96
2169.90 ± 170.71 1037.50 ± 153.14# 1323.10 ± 138.82⁎ 1444.70 ± 306.52⁎⁎ 1785.30 ± 317.74⁎⁎ 1265.40 ± 89.59⁎
4.45 ± 1.49 12.71 ± 2.93# 10.77 ± 1.48⁎ 8.05 ± 2.20⁎⁎ 6.49 ± 1.11⁎⁎ 10.95 ± 1.22⁎
Mean ± SD. NS: normal standard group. DN: diabetic nephropathy. RL, RM, RH: rutin 10, 30, 90 mg/kg. CAP: captopril 10 mg/kg. # P b 0.01 vs NS group. ⁎ P b 0.05 vs DN group. ⁎⁎ P b 0.01 vs DN group.
Table 3 Effects of rutin on AGE, collagen IV and laminin in the renal cortex (Mean ± SD). Group
n
AGE (pg/mgprot)
Collagen IV (ng/mgprot)
Laminin (ng/mgprot)
NS DN RL RM RH CAP
10 8 8 9 9 9
24.88 ± 4.13 76.64 ± 13.70# 64.80 ± 8.21⁎ 50.82 ± 9.85⁎⁎ 36.09 ± 9.36⁎⁎ 58.94 ± 9.79⁎⁎
0.98 ± 0.19 4.38 ± 1.68# 3.41 ± 0.44⁎ 2.17 ± 0.72⁎⁎ 1.65 ± 0.66⁎⁎ 3.35 ± 0.77⁎
0.48 ± 0.10 3.14 ± 0.88# 2.75 ± 0.12 1.99 ± 0.45⁎⁎ 0.94 ± 0.29⁎⁎ 2.03 ± 0.78⁎⁎
Mean ± SD. NS: normal standard group. DN: diabetic nephropathy. RL, RM, RH: rutin 10, 30, 90 mg/kg. CAP: captopril 10 mg/kg. # P b 0.01 vs NS group. ⁎ P b 0.05 vs DN group. ⁎⁎ P b 0.01 vs DN group.
that the early DN model was successful. When comparing with the DN group, low, moderate and high doses of rutin significantly reduced blood glucose, urine protein, Cr, BUN, and kidney index levels in the DN rats (P b 0.05 or P b 0.01). The administration of CAP did not alter plasma glucose levels, but the urine protein, Cr, BUN, and kidney index levels were significantly reduced compared with the DN group (P b 0.01) (Table 1). These results suggest that rutin can effectively decrease blood glucose and improve renal function of DN rats.
Effects of rutin on levels of AGEs, collagen IV and laminin in the renal cortex The levels of AGEs, collagen IV and laminin of the DN group were increased significantly, when comparing with those of the NS group (P b 0.01). Compared with the DN group, there was a significant decrease in the laminin levels of the RM, RH and CAP groups (P b 0.01), whereas the RL group had no significant difference on the laminin (P > 0.05). Collagen IV and AGE levels of the RL, RM, RH, and CAP groups were strikingly lower than those of the DN group (P b 0.05 or P b 0.01) (Table 3).
Effect of rutin on the expression of CTGF in the renal cortex CTGF mainly expresses in tubular epithelial cells and some interstitial cells. With the increase of interstitial lesions, the expression of CTGF is increased. The results of immunohistochemistry stained CTGF was brown yellow (Fig. 1A–F) and the intensity of positive staining was used to quantify CTGF (Fig. 1G). Compared with the NS group, brown yellow granules increased on the mesangial cells, vascular endothelial cells and epithelial cells of renal tubules, which suggested that the CTGF levels of the DN group was significantly increased (P b 0.01). The levels of CTGF in the RL, RM, RH and CAP groups were significantly lower than that of DN group (P b 0.05 or P b 0.01). These results indicated that treatment of DN rats with rutin significantly reduced the expression of CTGF in the renal cortex.
Effects of rutin on oxidative stress Effect of rutin on p-Smad 2/3 and p-Smad 7 in the renal cortex As listed in Table 2, renal MDA levels, index of lipid peroxidation, were higher in the DN group rats than those of the NS group (P b 0.01) and markedly decreased by rutin. The T-AOC and the activities of the CAT, GSH-Px, and T-SOD in the DN group were markedly decreased (P b 0.01), which suggested that the oxidative stress was exhibited in the DN group. When comparing with the DN group, low, moderate, and high doses of rutin increased the T-AOC and the activities of antioxidase (P b 0.05 or P b 0.01). These results indicated that rutin could ameliorate the oxidative stress state of DN rats. CAP also significantly decreased MDA levels and increased T-AOC and the activities of CAT and GSH-Px (P b 0.05 or P b 0.01), but it had no evident effect on T-SOD (P > 0.05) (Table 2).
The protein levels of p-Smad 2/3 and p-Smad 7 in rat renal cortex were separated into distinct bands using Western blotting. As shown in Fig. 2A and B, the level of p-Smad 2/3 was strikingly higher and p-Smad 7 level was significantly lower in the DN group than those of the NS group (P b 0.01). Compared with the DN group, the expression of p-Smad 2/3 in the RL, RM, RH and CAP groups was significantly decreased (P b 0.05 or P b 0.01), whereas the level of p-Smad 7 in the RL, RM, RH and CAP groups was significantly increased (P b 0.05 or P b 0.01). These results strongly revealed that Smad 2/3 activation in DN rat kidney was significantly inhibited by rutin whereas the activation of Smad 7 increased obviously.
Fig. 1. Immunohistochemical micrographs of CTGF in the renal cortex. CTGF was stained brown yellow (A–F, magnification ×400). Compared with the NS group (A), the expression of CTGF in the kidney of the DN group (B) was increased. Rutin 90 mg/kg (D), 30 mg/kg (E), and 10 mg/kg (F) treatments decreased the overexpression of CTGF. Captopril 10 mg/kg (C) also obviously decreased the CTGF level. The intensity of positive staining of CTGF (G) was decreased by rutin. n=6, mean±SD, #Pb 0.01 vs NS group; ⁎Pb 0.05, ⁎⁎Pb 0.01 vs DN group.
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Fig. 2. Effects of rutin on the relative levels of p-Smad 2/3 (A) and p-Smad 7 (B). Protein extracts from the renal cortex were probed via Western blotting using anti-(p-Smad 2/3), anti-(total Smad 2/3), anti-(p-Smad 7) and anti-(total Smad 7) antibodies. The density of each band was determined with Image J analysis software version 1.34s. The NS group and DN group were treated with 1% CMC solution. RL, RM, and RH were treated with rutin 10, 30, and 90 mg/kg; CAP group was treated with 10 mg/kg of captopril. n=6, mean±SD. # Pb 0.01 vs NS group; ⁎Pb 0.05, ⁎⁎Pb 0.01 vs DN group.
Effect of rutin on the expression of TGF-β1 in the renal cortex In this study, we examined whether rutin was capable of reducing TGF-β1 expression in STZ-induced DN rats. As shown in Fig. 3A, the relative quantity of TGF-β1 mRNA in the kidney tissue of DN rats increased significantly when comparing with that of the NS rats (P b 0.01). Western blot analyses also showed that expression of TGF-β1 protein was markedly upregulated in DN group (P b 0.01) (Fig. 3B). In comparison with the DN group, the levels of TGF-β1 mRNA and TGF-β1 protein expressions in the RL, RM, RH and CAP groups were significantly decreased (P b 0.05 or P b 0.01). These findings suggest that rutin can significantly inhibit the overexpression of TGF-β1 in the renal cortex of DN rats. Effects of rutin on morphological change in kidneys An electronic microscopy was used to look into the ultrastructural changes in the kidneys. As shown in Fig. 4, when comparing with the
Fig. 3. RT-PCR and Western blotting for the determination of the expression of TGF-β1 in renal cortex. (A) Agarose electrophoresis of RT-PCR products amplified from the total RNA extracts of the renal cortex, beta-actin was used as the internal standard in each sample. The data for relative quantity of TGF-β1 mRNA was performed by densitometric analysis. NS group and DN group were treated with 1% CMC solution. RL, RM, and RH were treated with rutin 10, 30, and 90 mg/kg; CAP group was treated with 10 mg/kg of captopril. n=6, mean±SD. #Pb 0.01 vs NS group; ⁎Pb 0.05, ⁎⁎Pb 0.01 vs DN group. (B) Western blotting analysis TGF-β1 protein using anti-TGF-β1 and anti-GAPDH antibodies. The density of each band was determined with Image J analysis software version 1.34s. NS group and DN group were treated with 1% CMC solution. RL, RM, and RH treated with rutin 10, 30, and 90 mg/kg; CAP group was treated with 10 mg/kg of captopril. n=6, mean±SD. # Pb 0.01 vs NS group; ⁎Pb 0.05, ⁎⁎Pb 0.01 vs DN group.
NS group, significant thickening of the GBM, mild mesangial expansion, and local foot process effacement were found in the DN group. Compared with the DN group, these phenomena in the RL, RM, RH and CAP groups were ameliorated.
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Discussion DN is the leading cause of end-stage renal disease and approximately 30% of type 1 diabetic patients suffer from DN (Krolewski et al., 1996). STZ effectively induces renal injury in a type 1 diabetic animal model
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and the STZ diabetic rat model has been widely used to study early DN. Typical morphological changes in the diabetic kidney are represented by diffuse GBM thickening, mesangial expansion, hyalinosis of the mesangium and arteriolar walls, broadening and effacement of podocyte foot processes, reduction in podocyte number, glomerulosclerosis and
Fig. 4. Effects of rutin on the ultrastructural changes in kidneys were shown by the transmission electron microscopy (×10,000). NS group: the thickness of the glomerulus basement membrane was normal; there were no mesangial cell proliferation and extracellular matrix deposition. DN group: the glomerular basement membrane was thickened partly and foot process effacement was obvious. RL group (rutin 10 mg/kg): a part of visceral epithelial cell foot showed microvillous transformation. RM and RH groups (rutin 30 and 90 mg/kg) and CAP (captopril 10 mg/kg) group: there were no significant mesangial cell proliferation and mesangial matrix deposition; the glomerular basement membrane was normal. Among them, the apoptosis of mesangial cell was shown obviously in the RH group.
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tubulointerstitial fibrosis (Fioretto and Mauer, 2007). Several decades of extensive research has elucidated various pathways implicated in the development of diabetic kidney disease. It has been reported that high glucose can enhance the level of reactive oxygen species (ROS) and stimulate the expression of TGF-β1 (Fukami et al., 2004). TGF-β1 can induce the accumulation of ECM mediated by signals such as CTGF, Smads, protein kinase C (PKC), mitogen-activated protein kinase (MAPK), Ca2+, etc., and these signals interact with each other in a complicated network that aggravates DN. The hyperglycemic condition and predisposition to oxidative stress are well-documented conditions underlying the DN condition (Tan et al., 2007), and formation of ROS is a direct consequence of hyperglycemia. Several pathways have been related to an increased production of oxidative stress in the context of DN. Moreover, the intensity and durability of oxidative stress facilitate the formation of AGEs. At the same time, the interactions between AGEs and RAGE induce the activation of oxidative stress and stimulate the production and release of cytokines, which amplify tissue damage (Suzuki et al., 2006; Yuan et al., 2011). Thus, oxidative stress and AGEs interact mutually and upregulate each other, which can lead to ECM accumulation and mesangial cell hypertrophy. In our study, after the DN rats were treated with rutin, it was found that the T-AOC and activities of T-SOD, CAT, and GSH-Px all were increased significantly, and MDA levels were decreased obviously in the renal cortex. The AGE levels of rutin groups were also significantly reduced. These results strongly suggest that rutin has the characteristics of antioxidant and anti-AGEs in vivo, and this could be of benefit for the prevention of DN. DN is characterized by increased urinary protein and loss of renal functions (Ziyadeh and Wolf, 2008). In this study, the DN rats showed the overexcretion of urinary protein, decrease of creatinine clearance and glomerular enlargement. Albuminuria in diabetes is considered to have both homodynamic (glomerular capillary hypertension and hyperfiltration) and structural/cellular basis (changes in GBM, mesangial cell matrix, and podocyte function) (Gruden et al., 2005; Fowler, 2008). In our study, albuminuria of DN group was significantly higher than that of the NS group, but rutin treatment groups were found to attenuate microalbuminuria. Interestingly, we also found that significant thickening of the GBM, mild mesangial expansion, and local foot process effacement phenomena were alleviated when DN rats were treated with rutin using electronic microscopy. Therefore, we suggest that the amount of proteinuria may be related with the degree of renal ultrastructural damage. TGF-β1 plays a critical role in renal fibrosis and the accumulation of mesangial ECM. The Smad protein following TGF-β1 is thought to be one of the most important factors in the process of ECM accumulation (Wolf, 2003). TGF-β1 signals through a heteromeric receptor complex of the type I and type II receptors to activate the downstream intracellular mediators Smad 2 and Smad 3 by phosphorylation. The p-Smad 2/3 will then activate TGF-β1‐responsive genes in the process of tissue fibrosis. Furthermore, increased p-Smad 2/3 is thought to be some of the most important factors in the process of ECM accumulation (Mulay et al., 2010), whereas, Smad 7, as one of the inhibitory Smads, blocks TGF-β signaling pathway by inhibiting Smad 2/3 phosphorylation and thereby exerting its anti-fibrotic effect (Henique and Tharaux, 2012). In our experiments, the expression of type IV collagen and laminin of the DN group strikingly increased. We also observed an increase in the expression of p-Smad 2/3 and the relative TGF-β1 levels, whereas a decrease in the inhibitory signal, p-Smad 7. These findings indicate that it is the high glucose that initially induces the increased expression in TGF-β1, ultimately resulting in the accumulation of type IV collagen and laminin. In other words, it is the activated TGF-β1/Smad signaling pathway that induces the accumulation of ECM. Therefore, TGF-β1/Smad/ECM is obviously a linked response. Our results are consistent with the conclusions of other researchers (Li et al., 2003; Yu et al., 2004; Gagliardini and Benigni, 2006). In our study, after diabetic rats induced by STZ were treated with rutin, the
expressions of TGF-β1 and p-Smad 2/3 were lowered significantly, and p-Smad 7 was raised greatly, suggesting that rutin can suppress the TGF-β1/Smad/ECM response and prevent the development of DN. Those findings are consistent with our previous report in vitro about the protective effects of rutin on rat glomerular mesangial cells cultured by high glucose (Tang et al., 2011). CTGF, one of the most recent identified growth factors with a role in DN, has been considered as downstream mediator of TGF-β1 and a potent inducer of ECM in the fibrotic process (Nguyen et al., 2008). Some scholars believe that the TGF-β1/Smad signaling should be greatly responsible for CTGF expression. However, there is also evidence of a TGF-β-independent regulation of CTGF, which is related to a direct activation of its synthesis by hyperglycemia, AGEs and static pressure (Wahab et al., 2001). It is becoming clear that the coordinated expression of TGF-β1 and CTGF is crucial for the induction of ECM proteins and thus, for the development of DN. Numerous studies indicate that hyperglycemia induces an increase in TGF-β1 expression at both the mRNA and protein levels in experimental and human diabetes, as well as in cultured mesangial cells, and that increased signaling by TGF-β1 is also markedly influenced by CTGF. However, the expression of some ECM proteins, such as fibronectin, is CTGF-dependent, and its promoter region does not contain any Smad-binding elements. Thus, CTGF may mediate the induction of the ECM protein expression both directly and indirectly by potentiating the TGF-β1/Smad signaling pathway. In other words, CTGF is a crucial mediator for the TGF-β1-stimulated matrix protein expression. In our experiments, the expression of CTGF in the DN group increased clearly and the accumulation of type IV collagen and laminin had the same trend. After DN rats were treated with rutin, those parameters were ameliorated significantly. In this study, CTGF and TGF-β1 had the same trend, this finding may indicate that there might be a TGF-β1/CTGF/ECM linked response. The interaction of ROS, AGEs, CTGF and TGF-β1/Smads has become a focus in research of DN. It has been found that AGEs-RAGE-mediated ROS generation activates TGF-β1-Smad signals and subsequently induces mesangial cell hypertrophy and ECM accumulation (Fukami et al., 2004). It was concluded that ROS and TGF-β1-dependent signals mutually interact and upregulate each other in the pathogenesis of DN. In our present study, rutin, an anti-oxidant bioflavonoid, can attenuate the symptom of DN and downregulate TGF-β1/Smad and CTGF expressions in rats. This means that rutin may exert a protective effect on the early development of DN in STZ-induced diabetic rats. Conclusion Rutin can significantly decrease the levels of fasting blood glucose, Cr, BUN, urine protein and the thickness of GBM, and also can ameliorate oxidative stress, inhibit the accumulation of type IV collagen and laminin, decrease AGEs, TGF-β1, p-Smad 2/3 and CTGF, and increase p-Smad 7 expression in the renal cortex of DN rats. All these results indicated that rutin may postpone renal damage and have protective effects on STZ-induced DN rats. Rutin may be a potential drug for the prevention of early DN. Conflict of interest statement None.
Acknowledgments This work was supported by the Project of the National Natural Science Foundation of China (No. 81173104), the Natural Science Research Funds of Jiangsu Province (No. BK2011211), a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, President Special Grant of Xuzhou Medical College (09KJZ22 and 2010KJZ25) and the Natural Science Foundation of Xuzhou City (No. XF10C074).
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Preventive effects of rutin on the development of experimental diabetic nephropathy in rats Hui-hui Hao a, Zhu-min Shao b, Dao-quan Tang a, c,⁎, Qian Lu a, Xu Chen a, c, Xiao-xing Yin a,⁎⁎, Jing Wu a, c, Hui Chen a, c a b c
Key Laboratory of New Drug and Clinical Application, Xuzhou Medical College, Xuzhou 221004, China Department of Pharmacy, Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, China Department of Pharmaceutical Analysis, School of Pharmacy, Xuzhou Medical College, Xuzhou 221004, China
a r t i c l e
i n f o
Article history: Received 29 March 2012 Accepted 12 September 2012 Keywords: Rutin Diabetic nephropathy CTGF Smads TGF-β1 Oxidative stress
a b s t r a c t Aims: Diabetic nephropathy (DN) is an important microvascular complication and one of the main causes of end-stage renal disease. In this study, the preventive effect and mechanism of rutin on the development of DN in streptozotocin (STZ)-induced diabetic rats were investigated. Main methods: After an early DN model was induced by STZ, rats were orally administered rutin at 3 doses for 10 weeks. Fasting blood glucose, creatinine (Cr), blood urea nitrogen (BUN), urine protein, kidney index, antioxidase, advanced glycosylation end products (AGEs), extracellular matrix (ECM) including collagen IV and laminin, connective tissue growth factor (CTGF), phosphorylated Smad 2/3 (p-Smad 2/3) and Smad 7 (p-Smad 7), and transforming growth factor-β1 (TGF-β1) were determined by different methods, respectively. The ultrastructural morphology was observed by a transmission electron microscope. Key findings: Compared with the DN group, rutin decreased the levels of fasting blood glucose, Cr, BUN, urine protein, the intensity of oxidative stress and p-Smad 7 significantly. The expression of AGEs, collagen IV and laminin, TGF-β1, p-Smad 2/3 and CTGF was inhibited by rutin significantly. Moreover, rutin was observed to inhibit proliferation of mesangial cells and decrease thickness of glomerular basement membrane (GBM) by electron microscopy. Significance: The preventive effect of rutin on the development of DN is closely related to oxidative stress and the TGF-β1/Smad/ECM and TGF-β1/CTGF/ECM signaling pathways. Those results suggest that rutin can prevent the development of experimental DN in rats. © 2012 Elsevier Inc. All rights reserved.
Introduction The early stage of diabetic nephropathy (DN) is characterized by renal hypertrophy, glomerular hypertrophy, glomerular hyperfiltration, and microalbuminuria (Wolf and Ziyadeh, 1999; Makino et al., 1996; Raptis and Viberti, 2001). These changes are related to the subsequent development of glomerular morphological abnormalities and the prognosis of DN. One of the characteristic pathological changes in DN is the accumulation of extracellular matrix (ECM) components including collagens, fibronectin and laminin in the glomeruli and the interstitium of kidney (Kanwar et al., 2008). Hyperglycemia is usually considered as the main determinant factor of the initiation and progression of DN which increases the formation of advanced glycosylation end products (AGEs), oxidative stress and transforming growth factor-β1 (TGF-β1) ⁎ Correspondence to: D.Q. Tang, Key Laboratory of New Drug and Clinical Application, Xuzhou Medical College, Xuzhou 221004, China. Tel./fax: +86 516 83262136. ⁎⁎ Corresponding author. Tel./fax: +86 516 83262009. E-mail addresses: [email protected] (D. Tang), [email protected] (X. Yin). 0024-3205/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.lfs.2012.09.003
mRNA expressions. TGF-β1 is the most potent growth factor contributing to ECM accumulation, which it does by stimulating ECM production and suppressing its degradation (Border et al., 1996). Smad proteins are transcription factors that mediate TGF-β/activin signaling by forming complexes with each other. Smads 2 and 3 are specific for TGF-β signaling, and they are phosphorylated by TGF-β receptor type 1 after binding with TGF-β (Wang et al., 2005; Gagliardini and Benigni, 2006). Either Smad 2 or 3 forms a heterodimer with Smad 4, and the complex immediately translocates into the nucleus and induces transcription of various genes by combining specific binding sites of genomic DNA. Smad 7 can be an inhibitory Smad protein that blocks the phosphorylation of Smad 2/3, counterbalancing the TGF-β signaling (Stopa et al., 2000). Connective tissue growth factor (CTGF) has been described as a growth factor that acts downstream of TGF-β1 and is a potent inducer of ECM in the fibrotic process (Hishikawa et al., 2001). TGF-β1/Smad/ ECM or TGF-β1/CTGF/ECM seems to be a linked response in the pathogenesis of DN. Rutin is found in many foods and many traditional Chinese medicines, whose treatment on diabetic mellitus has been proved by the Stanely Mainzen Prince group. Their innovative achievements have
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published that rutin can improve glucose homeostasis of diabetic rats by decreasing fasting plasma glucose and increasing insulin levels, increasing the content of glycogen in the liver and muscle whereas decreasing that in the kidney, increasing the activity of hexokinase and decreasing the activities of glucose-6-phosphatase and fructose1,6-bisphosphatase in the tissues (Stanley Mainzen Prince and Kamalakkannan, 2006). This research group also proved that rutin can decrease the levels of lipids, low density and very low density lipoprotein cholesterol in plasma, increase the levels of plasma high density lipoprotein cholesterol and the activity of plasma lipoprotein lipase and lecithin cholesterol acyltransferase and decrease the levels of lipids and glycoproteins and the activity of 3-hydroxy 3-methylglutaryl coenzyme A reductase in the liver and in the kidney in diabetic rats basing on its antioxidant property (Stanely Mainzen Prince and Kannan, 2006). Moreover, Stanely Mainzen Prince group also reported that rutin can protect the diabetic rats kidney by decreasing the content of hydroxyproline and collagen and the levels of tissue inhibitors of metalloproteinases, increasing the activity of matrix metalloproteinases in the kidney (Kamalakkannan and Stanely Mainzen Prince, 2006). Other scholars also proved that rutin can inhibit the formation of AGEs (Cervantes-Laurean et al., 2006) in STZ-induced rats. All of these offer us a potential pharmacological foundation of rutin for DN therapy. We previously reported that rutin can decrease the accumulation of ECM mediated by TGF-β1/Smads in high glucose cultured-rat mesangial cells (Tang et al., 2011). Although Kamalakkannan and Prince have reported that rutin can decrease fasting plasma glucose, glycosylated hemoglobin thiobarbituric acid reactive substances and lipid hydroperoxides, increase insulin, C-peptide, hemoglobin, protein levels and non-enzymic antioxidants in diabetic rats induced by streptozotocin (STZ), their work haven't focused on the creatinine (Cr), blood urea nitrogen (BUN), and urine protein in DN rats (Kamalakkannan and Prince, 2006). Moreover, the protective effects and mechanism of rutin on early experiment DN in vivo have not been studied by Stanley Mainzen Prince group and other scholars to date, which may restrict the clinical application of rutin. Thus, the present study was designed to investigate the effect of rutin on the kidney of STZ-induced type 1 diabetic rats by observing the changes of renal morphology and some signaling including TGF-β1, phosphorylated Smad 2/3 (p-Smad 2/3), phosphorylated Smad 7 (p-Smad 7), CTGF, collagen IV, and laminin. The blood glucose, Cr, BUN, urine protein, kidney index, antioxidases, and AGEs in DN rats were simultaneously observed. Captopril (CAP) was used as a positive control drug in this study. Materials and methods Animals Adult male Sprague–Dawley rats (Certificate No. SYXK 2010-0011), weighing 180 g–220 g, procured from the Laboratory Animal Center of Xuzhou Medical College were used in this study. The rats were maintained in a controlled environment (12 h light/dark cycle) and temperature (21 ±2 °C). All animals were fed on a standard pellet diet and water was freely available. The animals were acclimatized to the laboratory conditions before starting the experiment. All the experiments were approved by Institutional Animal Ethics Committee and followed the Guiding Principles for Care and Use of Laboratory Animals of Xuzhou Medical College. Chemicals Rutin and STZ were purchased from Sigma Chemicals Company (St. Louis, MO, USA). Captopril (CAP, Lot No. 050050), serving as a positive control drug, was kindly provided by Changzhou Pharmaceutical Factory (Changzhou, China).
Induction and assessment of diabetes STZ was freshly dissolved in citrate buffer (0.01 mol/L, pH 4.4) and maintained on ice before use. The overnight fasted rats were made diabetic with a single intraperitoneal injection of STZ (60 mg/kg). Age-matched control normal standard rats (NS group, n = 10) received an equivalent amount of citrate buffer. Diabetes was confirmed in the STZ-injected rats by measuring the fasting plasma glucose levels 72 h post injection. After an overnight fast, rats with blood glucose levels above 13.89 mmol/L (250 mg/dL) were considered as diabetic and were used in this study. Treatment with rutin was started on the third day after STZ-injection. Experimental design Diabetic rats were randomly allotted into 5 groups as follows: diabetic rats were treated with 1% sodium carboxymethyl cellulose (CMC) solution (DN group, n =8); diabetic rats with 10, 30, and 90 mg/kg rutin for the RL group (low dose, n=8), the RM group (moderate dose, n= 9), and the RH group (high dose, n =9), respectively; the diabetic rats were treated with 10 mg/kg of CAP (CAP group, n=9). The low dose of rutin was calculated according to its content in the moderate effective dose of Ginkgo biloba extract for therapy of DN published by our laboratory (Ginkgo biloba extract employed in that experiment (Lu et al., 2007) contains 11.4% rutin (Tang et al., 2010)). Drugs were suspended in 1% CMC solution and orally administered to rats using an intragastric tube for a period of 10 weeks. The same volume of CMC solution was administered to the NS group (n= 10). Normal and diabetic rats were housed 5 per cage in the barrier environment referred to as breed specific pathogen-free grade animals. At the end of the experiments, after a 24 h urine sample collection, the blood samples of the rats were withdrawn from the abdominal aorta. Serum was collected to assess the renal function by measuring plasma levels of Cr and urea nitrogen. Kidneys were removed, after being weighed, parts of fresh kidney cortexes were stored in 10% formaldehyde solution for immunohistochemical assays, and 1 mm× 1 mm× 1 mm cubes of renal cortices were fixed in 2.5% glutaraldehyde for electron microscopic measurement. The rest of the kidneys were stored at −80 °C for the later analysis. Measurement of blood glucose and renal function Blood glucose was measured by blood glucose meter (OneTouch UltraEasy, USA). The 24 h urinary albumin was measured by ELISA kit purchased from R&D Systems Company (R&D, USA). BUN and Cr were determined by the automatic biochemistry analyzer (Olympus2000, Tokyo, Japan). The kidney index was expressed as 1000 × kidney weight/body weight. Assessment of renal oxidative stress and determination of AGEs, collagen IV and laminin The kidney tissue was minced and a homogenate was prepared with 10% (w/v) phosphate buffered saline (0.1 mol/L, pH 7.4) using a homogenizer. The kidney homogenate was centrifuged at 15,000 rpm for 15 min at 4 °C. The supernatant was collected to determine the content of protein by bicinchoninic acid (BCA) protein assay kit purchased from Beyotim Institute of Biotechnology (Nantong, China). The total antioxidative capability (T-AOC), catalase (CAT), total superoxidase dismutase (T-SOD) and glutathione-per-oxidase (GSH-Px) in the renal cortex and the level of malonaldehyde (MDA) were measured by using the commercially available kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). AGEs play a critical role in DN by stimulating type IV collagen and laminin, the main component of ECM. The quantity of type IV collagen,
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laminin and AGEs in the renal cortex was determined by ELISA kit purchased from R&D Systems Company (R&D, USA). Immunohistochemical measurements of CTGF Kidney cortexes, fixed in 10% formaldehyde previously, were embedded in paraffin and 4 μm thick sections were prepared for immunohistochemistry analysis of CTGF. The renal tissue sections were incubated with goat polyclonal anti-CTGF (Lot No. B1030, Zhongshan Golden Bridge Biological Technology, Beijing, China) at a dilution of 1:100 at 37 °C for 2 h. After washing in PBS, the sections were incubated with rabbit anti-goat IgG horseradish peroxidase (Lot K115214D, Beijing, China) for 30 min at room temperature. After washing, to visualize CTGF, the renal tissue sections were stained with 3,3′-diaminobenzidine (DAB) for 10 min and then washed in tap water for 10 min. After the nuclei were stained with hematoxylin for 20 s, the sections were evaluated by a light microscope (×400) and were photographed. Photomicrographs of at least 20 fields in each rat were quantified for the intensity of positive staining in the renal tissue with IPLab software (Scanalytics, Fairfax, VA). Western blot analysis for Smads and TGF-β1 in the renal cortex For detection of p-Smad 2/3, p-Smad 7 and TGF-β1 in the renal cortex, gel electrophoreses and subsequent western blotting was performed. Firstly, renal cortex tissues were homogenized in icecold RIPA buffer containing protease inhibitors. The homogenates were centrifuged at 15,000 rpm for 15 min at 4 °C and an aliquot of the supernatant was kept for protein determination. The content of protein in the supernatant was determined by BCA protein assay kit. All samples were boiled for 5 min in Laemmli sample buffer before loading. A 60 μg sample of total protein was separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to nitrocellulose membranes (Pall Corporation, USA). After being blocked with 5% nonfat dry milk in TBS-Tween (0.1%) for 2 h at room temperature, the membranes were incubated separately with the following antibodies: rabbit anti-phospho-Smad 2/3 (Santa Cruz Biotechnology, USA), rabbit anti-total-Smad 2/3 (Bioworld Technology, USA), rabbit anti-phospho-Smad 7 (Santa Cruz Biotechnology, USA), rabbit anti-total-Smad 7 (Abcam, UK), rabbit anti-TGF-β1 (Santa Cruz Biotechnology, USA) and rabbit anti-GAPDH (Beyotim Institute of Biotechnology, China) antibodies. The membranes were probed with the primary antibodies overnight at 4 °C and then washed three times in TBS-Tween for 5 min, followed by incubation with the appropriate secondary horseradish peroxidase conjugate antibody for 1 h at room temperature. After extensive washing, the signals were visualized by the enhanced chemiluminescence system (ECL; Santa Cruz Biotechnology, USA) and the density of each band was determined with Image J analysis software version 1.34s (Wayne Rasband National Institutes of Health, USA).
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RT-PCR for the determination of relative quantities of TGF-β1 mRNA A reverse transcription polymerase chain reaction (RT-PCR) procedure was performed to determine the relative quantities of TGF-β1 mRNA in the renal cortex, while β-actin mRNA, a housekeeping gene, being used as an internal control. The total RNA was extracted from the renal cortex with RNA simple Total RNA Kit (Lot No. J9117, TIANGEN Biotechnology Company, Beijing, China). The upstream and downstream primers for rat TGF-β1 mRNA were: 5′-CCCGCATC CCAGGAC CTCTCT-3′ and 5′-CGGGGGACTGGCGAGCCTTAG-3′, yielding a 519 bp product; whereas those for β-actin were: 5′-GCTGCGT GTGGCCCCTGAG-3′ and 5′-ACGCA-GGATGGCATGAGGGA-3′, yielding a 252 bp product. Equal amounts (1 μg) of each total RNA sample were added in a 50 μL reaction mixture exerting one-step amplification with Quant One Step RT-PCR Kit (Lot No. J9104, TIANGEN Biotechnology Company, Beijing, China). RT-PCR conditions were set to reverse transcription for 30 min at 50 °C, initial denaturation for 2 min at 94 °C, 40 cycles at 94 °C for 30 s, 55 °C for 45 s, 72 °C for 1 min, and final elongation at 72 °C for 10 min. The RT-PCR products were separated by 1.5% agarose gel electrophoresis, and the band densities were analyzed using Image J analysis software version 1.34s. The relative quantities of TGF-β1 mRNA in the renal cortex were represented by the ratio of band density of TGF-β1 versus that of β-actin. Electronmicroscopy for morphological observation Renal tissues, fixed in glutaraldehyde, were postfixed with 1% OsO4 for 2 h and dehydrated with graded ethanol. Samples were embedded with epoxy resin and polymerized at 37 °C for 24 h, 45 °C for 24 h, and 60 °C for 24 h. The renal samples were cut into ultrathin sections and then stained with plumbum citrate for ultrastructural observation under a transmission electron microscope (H600A-2, Hitachi, Japan). The images were amplified 10,000 and the photos were scanned into an image analysis system (Leica Qwin Standard V2.6, Leica Microsystems, Germany). Statistical analysis All the grouped data were analyzed by ANOVA and Dunnett's t-test (2-side) for different groups using SPSS 16.0 software. The values are expressed as mean ± SD. P value b 0.05 was considered significant and included in the study. Results Effect of rutin on blood glucose, kidney index and the renal function of rats In our experiment, the fasting blood glucose levels, urine protein, Cr, BUN, and the kidney index in rats of the DN group were significantly higher than those of the NS group (P b 0.01), which suggested
Table 1 Effects of rutin on blood glucose, urine protein, Cr, BUN and kidney index of rats (mean ± SD). Group
n
Fasting blood glucose (mmol/L)
24 h urine protein (μm/24 h)
Kidney index ×1000
BUN (mmol/L)
Cr (μmol/L)
NS DN RL RM RH CAP
10 8 8 9 9 9
6.16 ± 2.16 20.91 ± 2.02# 17.83 ± 3.14⁎ 13.92 ± 2.64⁎⁎ 12.54 ± 1.75⁎⁎
3.42 ± 0.24 38.71 ± 3.73# 27.15 ± 2.46⁎ 20.18 ± 1.96⁎⁎ 15.14 ± 1.69⁎⁎ 14.50 ± 2.63⁎⁎
7.88 ± 0.69 14.96 ± 1.89# 13.26 ± 0.87⁎ 12.84 ± 1.10⁎⁎ 11.57 ± 1.32⁎⁎ 11.34 ± 1.32⁎⁎
7.53 ± 1.27 26.69 ± 5.90# 22.72 ± 2.56⁎ 18.78 ± 1.97⁎⁎ 16.74 ± 1.92⁎⁎ 17.44 ± 3.48⁎⁎
37.17 ± 5.17 67.44 ± 9.21# 60.42 ± 5.91⁎ 50.13 ± 7.12⁎⁎ 46.14 ± 6.52⁎⁎ 44.63 ± 4.50⁎⁎
18.54 ± 4.47
Mean ± SD. NS: normal standard group. DN: diabetic nephropathy. RL, RM, RH: rutin 10, 30, 90 mg/kg. CAP: captopril 10 mg/kg. # P b 0.01 vs NS group. ⁎ P b 0.05 vs DN group. ⁎⁎ P b 0.01 vs DN group.
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Table 2 Effects of rutin on T-AOC, T-SOD, CAT, GSH-Px and MDA in the renal cortex (mean ± SD). Group
n
CAT (U/mgprot)
T-AOC (U/mgprot)
T-SOD (U/mgprot)
GSH-PX (U/mgprot)
MDA (nmol/ml)
NS DN RL RM RH CAP
10 8 8 9 9 9
55.44 ± 16.15 18.08 ± 5.83# 26.59 ± 3.99⁎ 42.09 ± 8.15⁎⁎ 43.19 ± 7.56⁎⁎ 25.70 ± 4.23⁎
7.30 ± 1.77 1.10 ± 0.30# 2.35 ± 0.76⁎ 3.92 ± 1.22⁎⁎ 4.11 ± 0.44⁎⁎ 2.76 ± 0.65⁎⁎
329.54 ± 78.00 220.78 ± 38.37# 263.22 ± 24.15⁎ 267.40 ± 21.62⁎⁎ 277.40 ± 27.08⁎⁎ 253.90 ± 39.96
2169.90 ± 170.71 1037.50 ± 153.14# 1323.10 ± 138.82⁎ 1444.70 ± 306.52⁎⁎ 1785.30 ± 317.74⁎⁎ 1265.40 ± 89.59⁎
4.45 ± 1.49 12.71 ± 2.93# 10.77 ± 1.48⁎ 8.05 ± 2.20⁎⁎ 6.49 ± 1.11⁎⁎ 10.95 ± 1.22⁎
Mean ± SD. NS: normal standard group. DN: diabetic nephropathy. RL, RM, RH: rutin 10, 30, 90 mg/kg. CAP: captopril 10 mg/kg. # P b 0.01 vs NS group. ⁎ P b 0.05 vs DN group. ⁎⁎ P b 0.01 vs DN group.
Table 3 Effects of rutin on AGE, collagen IV and laminin in the renal cortex (Mean ± SD). Group
n
AGE (pg/mgprot)
Collagen IV (ng/mgprot)
Laminin (ng/mgprot)
NS DN RL RM RH CAP
10 8 8 9 9 9
24.88 ± 4.13 76.64 ± 13.70# 64.80 ± 8.21⁎ 50.82 ± 9.85⁎⁎ 36.09 ± 9.36⁎⁎ 58.94 ± 9.79⁎⁎
0.98 ± 0.19 4.38 ± 1.68# 3.41 ± 0.44⁎ 2.17 ± 0.72⁎⁎ 1.65 ± 0.66⁎⁎ 3.35 ± 0.77⁎
0.48 ± 0.10 3.14 ± 0.88# 2.75 ± 0.12 1.99 ± 0.45⁎⁎ 0.94 ± 0.29⁎⁎ 2.03 ± 0.78⁎⁎
Mean ± SD. NS: normal standard group. DN: diabetic nephropathy. RL, RM, RH: rutin 10, 30, 90 mg/kg. CAP: captopril 10 mg/kg. # P b 0.01 vs NS group. ⁎ P b 0.05 vs DN group. ⁎⁎ P b 0.01 vs DN group.
that the early DN model was successful. When comparing with the DN group, low, moderate and high doses of rutin significantly reduced blood glucose, urine protein, Cr, BUN, and kidney index levels in the DN rats (P b 0.05 or P b 0.01). The administration of CAP did not alter plasma glucose levels, but the urine protein, Cr, BUN, and kidney index levels were significantly reduced compared with the DN group (P b 0.01) (Table 1). These results suggest that rutin can effectively decrease blood glucose and improve renal function of DN rats.
Effects of rutin on levels of AGEs, collagen IV and laminin in the renal cortex The levels of AGEs, collagen IV and laminin of the DN group were increased significantly, when comparing with those of the NS group (P b 0.01). Compared with the DN group, there was a significant decrease in the laminin levels of the RM, RH and CAP groups (P b 0.01), whereas the RL group had no significant difference on the laminin (P > 0.05). Collagen IV and AGE levels of the RL, RM, RH, and CAP groups were strikingly lower than those of the DN group (P b 0.05 or P b 0.01) (Table 3).
Effect of rutin on the expression of CTGF in the renal cortex CTGF mainly expresses in tubular epithelial cells and some interstitial cells. With the increase of interstitial lesions, the expression of CTGF is increased. The results of immunohistochemistry stained CTGF was brown yellow (Fig. 1A–F) and the intensity of positive staining was used to quantify CTGF (Fig. 1G). Compared with the NS group, brown yellow granules increased on the mesangial cells, vascular endothelial cells and epithelial cells of renal tubules, which suggested that the CTGF levels of the DN group was significantly increased (P b 0.01). The levels of CTGF in the RL, RM, RH and CAP groups were significantly lower than that of DN group (P b 0.05 or P b 0.01). These results indicated that treatment of DN rats with rutin significantly reduced the expression of CTGF in the renal cortex.
Effects of rutin on oxidative stress Effect of rutin on p-Smad 2/3 and p-Smad 7 in the renal cortex As listed in Table 2, renal MDA levels, index of lipid peroxidation, were higher in the DN group rats than those of the NS group (P b 0.01) and markedly decreased by rutin. The T-AOC and the activities of the CAT, GSH-Px, and T-SOD in the DN group were markedly decreased (P b 0.01), which suggested that the oxidative stress was exhibited in the DN group. When comparing with the DN group, low, moderate, and high doses of rutin increased the T-AOC and the activities of antioxidase (P b 0.05 or P b 0.01). These results indicated that rutin could ameliorate the oxidative stress state of DN rats. CAP also significantly decreased MDA levels and increased T-AOC and the activities of CAT and GSH-Px (P b 0.05 or P b 0.01), but it had no evident effect on T-SOD (P > 0.05) (Table 2).
The protein levels of p-Smad 2/3 and p-Smad 7 in rat renal cortex were separated into distinct bands using Western blotting. As shown in Fig. 2A and B, the level of p-Smad 2/3 was strikingly higher and p-Smad 7 level was significantly lower in the DN group than those of the NS group (P b 0.01). Compared with the DN group, the expression of p-Smad 2/3 in the RL, RM, RH and CAP groups was significantly decreased (P b 0.05 or P b 0.01), whereas the level of p-Smad 7 in the RL, RM, RH and CAP groups was significantly increased (P b 0.05 or P b 0.01). These results strongly revealed that Smad 2/3 activation in DN rat kidney was significantly inhibited by rutin whereas the activation of Smad 7 increased obviously.
Fig. 1. Immunohistochemical micrographs of CTGF in the renal cortex. CTGF was stained brown yellow (A–F, magnification ×400). Compared with the NS group (A), the expression of CTGF in the kidney of the DN group (B) was increased. Rutin 90 mg/kg (D), 30 mg/kg (E), and 10 mg/kg (F) treatments decreased the overexpression of CTGF. Captopril 10 mg/kg (C) also obviously decreased the CTGF level. The intensity of positive staining of CTGF (G) was decreased by rutin. n=6, mean±SD, #Pb 0.01 vs NS group; ⁎Pb 0.05, ⁎⁎Pb 0.01 vs DN group.
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Fig. 2. Effects of rutin on the relative levels of p-Smad 2/3 (A) and p-Smad 7 (B). Protein extracts from the renal cortex were probed via Western blotting using anti-(p-Smad 2/3), anti-(total Smad 2/3), anti-(p-Smad 7) and anti-(total Smad 7) antibodies. The density of each band was determined with Image J analysis software version 1.34s. The NS group and DN group were treated with 1% CMC solution. RL, RM, and RH were treated with rutin 10, 30, and 90 mg/kg; CAP group was treated with 10 mg/kg of captopril. n=6, mean±SD. # Pb 0.01 vs NS group; ⁎Pb 0.05, ⁎⁎Pb 0.01 vs DN group.
Effect of rutin on the expression of TGF-β1 in the renal cortex In this study, we examined whether rutin was capable of reducing TGF-β1 expression in STZ-induced DN rats. As shown in Fig. 3A, the relative quantity of TGF-β1 mRNA in the kidney tissue of DN rats increased significantly when comparing with that of the NS rats (P b 0.01). Western blot analyses also showed that expression of TGF-β1 protein was markedly upregulated in DN group (P b 0.01) (Fig. 3B). In comparison with the DN group, the levels of TGF-β1 mRNA and TGF-β1 protein expressions in the RL, RM, RH and CAP groups were significantly decreased (P b 0.05 or P b 0.01). These findings suggest that rutin can significantly inhibit the overexpression of TGF-β1 in the renal cortex of DN rats. Effects of rutin on morphological change in kidneys An electronic microscopy was used to look into the ultrastructural changes in the kidneys. As shown in Fig. 4, when comparing with the
Fig. 3. RT-PCR and Western blotting for the determination of the expression of TGF-β1 in renal cortex. (A) Agarose electrophoresis of RT-PCR products amplified from the total RNA extracts of the renal cortex, beta-actin was used as the internal standard in each sample. The data for relative quantity of TGF-β1 mRNA was performed by densitometric analysis. NS group and DN group were treated with 1% CMC solution. RL, RM, and RH were treated with rutin 10, 30, and 90 mg/kg; CAP group was treated with 10 mg/kg of captopril. n=6, mean±SD. #Pb 0.01 vs NS group; ⁎Pb 0.05, ⁎⁎Pb 0.01 vs DN group. (B) Western blotting analysis TGF-β1 protein using anti-TGF-β1 and anti-GAPDH antibodies. The density of each band was determined with Image J analysis software version 1.34s. NS group and DN group were treated with 1% CMC solution. RL, RM, and RH treated with rutin 10, 30, and 90 mg/kg; CAP group was treated with 10 mg/kg of captopril. n=6, mean±SD. # Pb 0.01 vs NS group; ⁎Pb 0.05, ⁎⁎Pb 0.01 vs DN group.
NS group, significant thickening of the GBM, mild mesangial expansion, and local foot process effacement were found in the DN group. Compared with the DN group, these phenomena in the RL, RM, RH and CAP groups were ameliorated.
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Discussion DN is the leading cause of end-stage renal disease and approximately 30% of type 1 diabetic patients suffer from DN (Krolewski et al., 1996). STZ effectively induces renal injury in a type 1 diabetic animal model
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and the STZ diabetic rat model has been widely used to study early DN. Typical morphological changes in the diabetic kidney are represented by diffuse GBM thickening, mesangial expansion, hyalinosis of the mesangium and arteriolar walls, broadening and effacement of podocyte foot processes, reduction in podocyte number, glomerulosclerosis and
Fig. 4. Effects of rutin on the ultrastructural changes in kidneys were shown by the transmission electron microscopy (×10,000). NS group: the thickness of the glomerulus basement membrane was normal; there were no mesangial cell proliferation and extracellular matrix deposition. DN group: the glomerular basement membrane was thickened partly and foot process effacement was obvious. RL group (rutin 10 mg/kg): a part of visceral epithelial cell foot showed microvillous transformation. RM and RH groups (rutin 30 and 90 mg/kg) and CAP (captopril 10 mg/kg) group: there were no significant mesangial cell proliferation and mesangial matrix deposition; the glomerular basement membrane was normal. Among them, the apoptosis of mesangial cell was shown obviously in the RH group.
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tubulointerstitial fibrosis (Fioretto and Mauer, 2007). Several decades of extensive research has elucidated various pathways implicated in the development of diabetic kidney disease. It has been reported that high glucose can enhance the level of reactive oxygen species (ROS) and stimulate the expression of TGF-β1 (Fukami et al., 2004). TGF-β1 can induce the accumulation of ECM mediated by signals such as CTGF, Smads, protein kinase C (PKC), mitogen-activated protein kinase (MAPK), Ca2+, etc., and these signals interact with each other in a complicated network that aggravates DN. The hyperglycemic condition and predisposition to oxidative stress are well-documented conditions underlying the DN condition (Tan et al., 2007), and formation of ROS is a direct consequence of hyperglycemia. Several pathways have been related to an increased production of oxidative stress in the context of DN. Moreover, the intensity and durability of oxidative stress facilitate the formation of AGEs. At the same time, the interactions between AGEs and RAGE induce the activation of oxidative stress and stimulate the production and release of cytokines, which amplify tissue damage (Suzuki et al., 2006; Yuan et al., 2011). Thus, oxidative stress and AGEs interact mutually and upregulate each other, which can lead to ECM accumulation and mesangial cell hypertrophy. In our study, after the DN rats were treated with rutin, it was found that the T-AOC and activities of T-SOD, CAT, and GSH-Px all were increased significantly, and MDA levels were decreased obviously in the renal cortex. The AGE levels of rutin groups were also significantly reduced. These results strongly suggest that rutin has the characteristics of antioxidant and anti-AGEs in vivo, and this could be of benefit for the prevention of DN. DN is characterized by increased urinary protein and loss of renal functions (Ziyadeh and Wolf, 2008). In this study, the DN rats showed the overexcretion of urinary protein, decrease of creatinine clearance and glomerular enlargement. Albuminuria in diabetes is considered to have both homodynamic (glomerular capillary hypertension and hyperfiltration) and structural/cellular basis (changes in GBM, mesangial cell matrix, and podocyte function) (Gruden et al., 2005; Fowler, 2008). In our study, albuminuria of DN group was significantly higher than that of the NS group, but rutin treatment groups were found to attenuate microalbuminuria. Interestingly, we also found that significant thickening of the GBM, mild mesangial expansion, and local foot process effacement phenomena were alleviated when DN rats were treated with rutin using electronic microscopy. Therefore, we suggest that the amount of proteinuria may be related with the degree of renal ultrastructural damage. TGF-β1 plays a critical role in renal fibrosis and the accumulation of mesangial ECM. The Smad protein following TGF-β1 is thought to be one of the most important factors in the process of ECM accumulation (Wolf, 2003). TGF-β1 signals through a heteromeric receptor complex of the type I and type II receptors to activate the downstream intracellular mediators Smad 2 and Smad 3 by phosphorylation. The p-Smad 2/3 will then activate TGF-β1‐responsive genes in the process of tissue fibrosis. Furthermore, increased p-Smad 2/3 is thought to be some of the most important factors in the process of ECM accumulation (Mulay et al., 2010), whereas, Smad 7, as one of the inhibitory Smads, blocks TGF-β signaling pathway by inhibiting Smad 2/3 phosphorylation and thereby exerting its anti-fibrotic effect (Henique and Tharaux, 2012). In our experiments, the expression of type IV collagen and laminin of the DN group strikingly increased. We also observed an increase in the expression of p-Smad 2/3 and the relative TGF-β1 levels, whereas a decrease in the inhibitory signal, p-Smad 7. These findings indicate that it is the high glucose that initially induces the increased expression in TGF-β1, ultimately resulting in the accumulation of type IV collagen and laminin. In other words, it is the activated TGF-β1/Smad signaling pathway that induces the accumulation of ECM. Therefore, TGF-β1/Smad/ECM is obviously a linked response. Our results are consistent with the conclusions of other researchers (Li et al., 2003; Yu et al., 2004; Gagliardini and Benigni, 2006). In our study, after diabetic rats induced by STZ were treated with rutin, the
expressions of TGF-β1 and p-Smad 2/3 were lowered significantly, and p-Smad 7 was raised greatly, suggesting that rutin can suppress the TGF-β1/Smad/ECM response and prevent the development of DN. Those findings are consistent with our previous report in vitro about the protective effects of rutin on rat glomerular mesangial cells cultured by high glucose (Tang et al., 2011). CTGF, one of the most recent identified growth factors with a role in DN, has been considered as downstream mediator of TGF-β1 and a potent inducer of ECM in the fibrotic process (Nguyen et al., 2008). Some scholars believe that the TGF-β1/Smad signaling should be greatly responsible for CTGF expression. However, there is also evidence of a TGF-β-independent regulation of CTGF, which is related to a direct activation of its synthesis by hyperglycemia, AGEs and static pressure (Wahab et al., 2001). It is becoming clear that the coordinated expression of TGF-β1 and CTGF is crucial for the induction of ECM proteins and thus, for the development of DN. Numerous studies indicate that hyperglycemia induces an increase in TGF-β1 expression at both the mRNA and protein levels in experimental and human diabetes, as well as in cultured mesangial cells, and that increased signaling by TGF-β1 is also markedly influenced by CTGF. However, the expression of some ECM proteins, such as fibronectin, is CTGF-dependent, and its promoter region does not contain any Smad-binding elements. Thus, CTGF may mediate the induction of the ECM protein expression both directly and indirectly by potentiating the TGF-β1/Smad signaling pathway. In other words, CTGF is a crucial mediator for the TGF-β1-stimulated matrix protein expression. In our experiments, the expression of CTGF in the DN group increased clearly and the accumulation of type IV collagen and laminin had the same trend. After DN rats were treated with rutin, those parameters were ameliorated significantly. In this study, CTGF and TGF-β1 had the same trend, this finding may indicate that there might be a TGF-β1/CTGF/ECM linked response. The interaction of ROS, AGEs, CTGF and TGF-β1/Smads has become a focus in research of DN. It has been found that AGEs-RAGE-mediated ROS generation activates TGF-β1-Smad signals and subsequently induces mesangial cell hypertrophy and ECM accumulation (Fukami et al., 2004). It was concluded that ROS and TGF-β1-dependent signals mutually interact and upregulate each other in the pathogenesis of DN. In our present study, rutin, an anti-oxidant bioflavonoid, can attenuate the symptom of DN and downregulate TGF-β1/Smad and CTGF expressions in rats. This means that rutin may exert a protective effect on the early development of DN in STZ-induced diabetic rats. Conclusion Rutin can significantly decrease the levels of fasting blood glucose, Cr, BUN, urine protein and the thickness of GBM, and also can ameliorate oxidative stress, inhibit the accumulation of type IV collagen and laminin, decrease AGEs, TGF-β1, p-Smad 2/3 and CTGF, and increase p-Smad 7 expression in the renal cortex of DN rats. All these results indicated that rutin may postpone renal damage and have protective effects on STZ-induced DN rats. Rutin may be a potential drug for the prevention of early DN. Conflict of interest statement None.
Acknowledgments This work was supported by the Project of the National Natural Science Foundation of China (No. 81173104), the Natural Science Research Funds of Jiangsu Province (No. BK2011211), a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, President Special Grant of Xuzhou Medical College (09KJZ22 and 2010KJZ25) and the Natural Science Foundation of Xuzhou City (No. XF10C074).
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