Polydatin ameliorates experimental diabetes-induced fibronectin through inhibiting the activation of NF-κB signaling pathway in rat glomerular mesangial cells

Molecular and Cellular Endocrinology 362 (2012) 183–193

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Polydatin ameliorates experimental diabetes-induced fibronectin through inhibiting the activation of NF-jB signaling pathway in rat glomerular mesangial cells Xi Xie a,1, Jing Peng b,1, Kaipeng Huang a, Juan Huang a, Xiaoyan Shen a, Peiqing Liu a, Heqing Huang a,⇑ a b

Laboratory of Pharmacology & Toxicology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, China Yue Bei people’s Hospital, Shaoguan, China

a r t i c l e

i n f o

Article history: Received 25 February 2012 Received in revised form 17 May 2012 Accepted 15 June 2012 Available online 22 June 2012 Keywords: Diabetic nephropathy Fibronectin NF-jB Polydatin Glomerular mesangial cells

a b s t r a c t A number of studies have recently demonstrated the involvement of nuclear factor-kappa B (NF-jB) activation and the subsequent coordinated inflammatory responses in the pathogenesis of diabetic nephropathy (DN). Polydatin has been shown to have the ability of anti-adhesive inflammation. However, the possible protective and beneficial effects of polydatin on DN via suppressing inflammatory damage and extracellular matrix (ECM) accumulation are not fully elucidated. We found that the polydatin could inhibit the induction and activity of NF-jB, and meanwhile ameliorating ECM accumulation in streptozotocin-diabetic rats. We aimed to investigate the effect of polydatin on fibronectin (FN) protein expression, and to elucidate its potential mechanism involving the NF-jB inflammatory signaling pathway in rat glomerular mesangial cells (GMCs) cultured under high glucose. The results revealed that polydatin significantly suppressed high glucose-induced FN production, inhibited NF-jB nuclear translocation, reduced the DNA-binding activity of NF-jB, as well as decreased the protein expression of ICAM-1 and TGF-b in GMCs. These findings suggested that polydatin significantly represses high glucose-induced FN expression in rat GMCs, which may be closely related to its inhibition of the NF-jB signaling pathway. Hence, we elucidated the potential mechanisms of the anti-inflammatory effects and ECM accumulation alleviation of polydatin in GMCs of DN in vitro. Ó 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Diabetic nephropathy (DN) is a morbid microvascular complication associated with diabetes, and is the most common cause of end-stage renal disease. In DN, the accumulation of extracellular matrix (ECM) components in the glomerular mesangium and tubulointerstitium causes early glomerular hypertrophy, as well as later glomerulosclerosis and tubulointerstitial fibrosis (Mauer et al., 1984; Adler, 1994). Glomerular mesangial cells (GMCs) play important roles in the physiological and pathological processes of kidneys. GMC has been postulated to be a key contributor to glomerulosclerotic lesions in diabetic patients (Young et al., 1995). GMC proliferation and hypertrophy, ECM accumulation, as well as con-

Abbreviations: NF-jB, nuclear factor-kappa B; DN, diabetic nephropathy; ECM, extracellular matrix; FN, fibronectin; GMCs, glomerular mesangial cells; ICAM-1, intercellular adhesionmolecule-1; TGF-b1, transforming growth factor-beta; STZ, Streptozocin; PDTC, pyrrolidinedithiocarbamate. ⇑ Corresponding author. Address: Lab of Pharmacology & Toxicology, School of Pharmaceutical Sciences, Sun Yat-sen University, WaiHuanDong Road 132, Guangzhou Higher Education Mega Center, Guangzhou 510006, China. Tel.: +86 2039943028; fax: +86 2039943110. E-mail address: [email protected] (H. Huang). 1 These authors contributed equally to this study. 0303-7207/$ - see front matter Ó 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mce.2012.06.008

sequent renal fibrosis have been recognized as major pathogenic events in the progression of renal failure in DN (Schena and Gesualdo, 2005; Ichinose et al., 2007; Raptis and Viberti, 2001; Tack et al., 2002; Chow et al., 2004). As one of the important ingredients of ECM, fibronectin (FN) is often used as an index to evaluate the extent of matrix accumulation. Inhibiting FN production is regarded as an effective strategy to ameliorate DN. However, the pathogenesis of DN has not been fully elucidated. DN has been recently believed as a kind of chronic inflammation (Chow et al., 2004; Mora and Navarro, 2005; Navarro and Mora, 2005; Nelson et al., 2005; Saraheimo et al., 2003). An increasing number of studies have demonstrated that the activation of nuclear factor-kappa B (NF-jB) and the subsequent coordinated expression of gene products may play important roles in the pathogenesis of DN. In diabetes, activated NF-jB translocates into the nucleus and triggers the expression of target genes, including intercellular adhesionmolecule-1 (ICAM-1) and transforming growth factorbeta 1 (TGF-b1). Consequently, persistent and enhanced inflammation as well as excessive FN production is induced. The root and rhizome of Polygonum cuspidatum have long been commonly used in traditional Chinese herbal medicine as an analgesic, antipyretic, diuretic, and expectorant. The major active component of P. cuspidatum is polydatin, also known as piceid, is a glucoside of resveratrol whose glucoside group bonded in the C-3

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position substitutes a hydroxyl group. Polydatin is more resistant to enzymatic oxidation than resveratrol and is soluble in water. Unlike resveratrol, which passively penetrates cells, polydatin enters cells via an active mechanism using glucose carriers (Fabris et al., 2008; Mikulski and Molski, 2010). These properties endow polydatin molecules with a greater bioavailability than resveratrol. Polydatin could obviously ameliorate inflammatory damage-induced intercellular adhesion and reduce the expression of adhesion molecules (Wang et al., 2002; Shu et al., 2004). These pharmacological activities of polydatin convey its influence on the field of cardio-cerebrovascular diseases. Polydatin has been shown to ameliorate inflammatory damageinduced adhesion. However, the underlying mechanisms of this protective effect on suppressing inflammatory damage leading to excessive FN production as well as ECM accumulation, and later, glomerulosclerosis, are not fully elucidated in GMCs in DN. We firstly explored the expression of NF-jB and FN in kidney tissues of Streptozocin (STZ)-induced diabetic rats treated with polydatin. Furthermore, to reveal the effects of polydatin on FN production and the activation of NF-jB signaling pathways in high glucose-induced rat GMCs model in vitro, we used the NF-jB specific inhibitor ammonium pyrrolidinedithiocarbamate (PDTC) as a positive control. We finally elucidated the protective effects and potential mechanisms of polydatin in GMCs of DN to a certain extent, providing further mechanistic insight into the nephroprotective action of polydatin. 2. Materials and methods 2.1. Chemicals and reagents Streptozocin (STZ) was produced by Sigma–Aldrich Corporation (St. Louis, MO, USA). Polydatin used for animal (purity = 95.1%, HPLC) treatment was purchased from Shanxi Scidoor Hi-tech Biology Co., Ltd. (Batch No.: 20050220; Xi’An, China). Polydatin used for cell experiments, ammonium PDTC, and dimethyl sulfoxide were obtained from Sigma–Aldrich (St. Louis, MO, USA). Trypsin was from Hyclone Thermo Scientific (Rockford, USA). Penicillin/ streptomycin solution and Dulbecco’s modified Eagle’s medium (DMEM) were from Gibco Invitrogen Corporation (Carlsbad, CA, USA). Newborn calf serum was from PAA Cell Culture Company (Pasching, Austria). A nuclear extraction kit was from Active Motif (CA, USA). An enhanced chemiluminescence (ECL) substrate for the detection of horseradish peroxidase (HRP) was obtained from Thermo Scientific (Rockford, USA). Antibodies against NF-jB p65, IjB-a, ICAM-1, and FN were supplied by Santa Cruz Biotechnology Co. (Santa Cruz, CA, USA). TGF-b rabbit monoclonal antibody was from Cell Signaling Technology, Inc. (Danvers, MA, USA). HRP-conjugated secondary antibodies were from Promega Corporation (Madison, USA). a-Tubulin antibody was obtained from Sigma–Aldrich (St. Louis, MO, USA). A polyvinylidene difluoride (PVDF) Ò membrane was purchased from immobilon -PSQ (Millipore, CA, Ò USA). Alexa Fluor 488 Goat Anti-Mouse IgG (H + L) was from InvitÒ rogen Molecular Probes, Inc. (Eugene, OR, USA). A LightShift Chemiluminescent electrophoretic mobility shift assay (EMSA) kit was obtained from Pierce Thermo Scientific (Rockford, USA). 2.2. Animal experiment Male Sprague–Dawley (SD) rats (n = 30, bodyweight: 200 ± 10 g) from Laboratory Animal Center, Sun Yat-sen University, Guangzhou, China. All animal procedures conformed to the China Animal Welfare Legislation and were reviewed and approved by the Sun Yat-sen University Committee on Ethics in the Care and Use of Laboratory Animals. (Animal quality certificate No.: 0005201). All

animals were housed under standard conditions with free access to regular food and water. After fed with regular diet for 1 week, they were randomly assigned to an Streptozocin-induced diabetic group (n = 22), which were given a single tail-vein injection of freshly prepared STZ (60 mg/kg), and a normal control group (n = 8), which were injected with equal volume of citrate buffer alone. Diabetic rats were confirmed by the levels of fasting blood glucose measurement P16.7 mmol/l after 72 h injection, which were randomized (8/group) to receive polydatin (150 mg/kg, i.g. daily) or orally given equal volumes of physiological saline. Control rats were also fed equal volumes of physiological saline. Treatment was continued for 12 weeks, at which time rats were sacrificed. The rats were housed in individual metabolic cages for collection of urine on the day before the end of the experiment. Blood sample was collected by drainage from the retroorbital venous plexus. Serum was obtained by centrifuge at 3000g for 15 min and stored at 80 °C. Meanwhile, kidney samples were rapidly excised, weighed and frozen in liquid nitrogen and stored at 80 °C or fixed in 10% neutral-buffered formalin. 2.3. Biochemical analysis and morphological studies Blood glucose was determined by glucose oxidase method. Blood urea nitrogen and serum creatinine were measured by oxidase and phosphoglycerol oxidase dynamical enzyme method, respectively. Urine protein was detected by sulfosalicylic acid–sodium sulfate turbidity method (available kits from Beijing Chemclin Biotech Co., Ltd., Beijing, China). For morphometric studies, the kidneys were fixed in 10% neutral buffered formalin and subsequently embedded in paraffin. The 4-lm sections of paraffinembedded tissues were stained with periodic acid-Schiff (PAS). The cross section yielding the maximum diameter of the glomerulus was photographed and converted into a digital image by an examiner, blinded to the source of the tissue, using light microscopy equipped with camera (Olympus BX-50; Olympus Optical, Tokyo, Japan). Glomerular tuft areas were measured with image analysis software Image Pro. Plus (Media Cybernetics, Inc., Bethesda, MD, USA). Twenty glomeruli were chosen at random form 3 slides in each animal as previous report (Peng et al., 2008). 2.4. Immunohistochemistry Sections (4-lm thick) of kidney were processed using a standard immunostaining protocol. After deparaffinization, hydration and blockage of endogenous peroxidase routinely, sections were pretreated by microwave for 20 min in 10 mmol/L sodium citrate buffer (pH 6.0) for antigen retrieval, followed by incubation sequentially with blocking agent, mouse anti-NFjB p65 antibody (1:1000, Santa Cruz, CA, USA) and secondary antibody (1:50). Slides were counterstained with hematoxylin after 3 min of diaminobenzidine reaction, and cover slipped using Vectashield (Vector Labs, Burlingame, CA, USA), then photographed and converted to a digital image using light microscopy equipped with camera. Negative control was carried out by omitting the primary antibody. Positive staining (dark brown) for NFjB p65 in each glomerulus was quantified by two investigators in a blinded manner at a magnification of 400 using image analysis software Image Pro. Plus, and expressed as the ratio of the mean of normal SD rats. Twenty glomeruli were chosen at random form 3 slides in each animal as previous report (Peng et al., 2008) (total 160 glomeruli for each group). 2.5. Cell culture Rat GMCs were separated from the glomeruli of SD rats by our research group and identified with a specific assay as previously

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described (Geoffroy et al., 2004; Gennero et al., 2002). All animal procedures were conducted in accordance with the China Animal Welfare Legislation, and were approved by the Ethics Committee on the Care and Use of Laboratory Animals of Sun Yat-sen University (Guangzhou, China). Five-week-old male SD rats (180–200 g) were purchased from the Center of Experimental Animals in Sun Yat-sen University (permit No.: 20100805003). GMCs were cultured in DMEM (normal glucose, 5.6 mM) supplemented with 10% fetal bovine serum at 37 °C under an atmosphere of 5% CO2, and were subcultured by 0.25% trypsin every 2 or 3 days. The cultured cells were used at confluence between the 5th and 8th passages. At subconfluence, the GMCs were serum-starved for 24 h and then divided into the following six groups: (a) control, where cells were kept in DMEM without fetal bovine serum (control group); (b) high glucose treatment group, in which cells were cultured in DMEM without fetal bovine serum containing 30 mM high glucose (model group); (c) low-dose polydatin-treated group (10 lM); (d) medium-dose polydatin-treated group (20 lM); (e) high-dose polydatin-treated group (40 lM); and (f) PDTC treatment group (100 lM), a specific inhibitor of NF-jB (Ha et al., 2002). 2.6. Processing methods and index detection The GMCs were preincubated in the presence or absence of polydatin (10, 20, and 40 lM) for 40 min or PDTC (100 lM) for 30 min, and then stimulated with or without 30 mM high glucose for 30 min. The cells were collected for nuclear and cytoplasmic protein extraction by a commercially available assay kit (NEPERÒ Nuclear and Cytoplasmic Extraction Reagents, Pierce Thermo Scientific, USA) for the Western blot assay of NF-jB p65 and IjB-a. Otherwise, adherent cells were fixed with 4% paraformaldehyde for the immunofluorescence detection of NF-jB nuclear translocation by a laser scanning confocal microscopy (LSCM) system (LSM710, Carl Zeiss, Germany). After preincubation with polydatin (40 lM) or PDTC (100 lM) for 30 min, the cells were incubated with 30 mM high glucose for 2 h for nuclear protein extraction for use in the EMSA. After preincubation in the presence or absence of different concentrations of polydatin for 12 h or PDTC for 30 min, the cells were incubated with 30 mM high glucose for 24 h for total protein extraction for use in the Western blot assay of FN, ICAM1, and TGF-b1. The adherent cells were fixed with 4% paraformaldehyde for the immunofluorescence detection of FN expression by an LSCM system (LSM710, Carl Zeiss, Germany). 2.7. Western blotting assay Kidney tissues were lysed and protein was extracted as published (Liu, Zhang, Liu et al., 2010; Jiang et al., 2011). The supernatants of kidney lysates were collected. The nuclear and cytoplasmic proteins of GMCs were extracted by a commercially available assay kit and the total proteins were extracted as published (Jiang et al., 2011). The protein concentration was determined using a BCATM Protein Assay kit (Pierce, USA) according to the manufacturer’s instructions. Supernatant including from kidney tissues and GMCs were separated on 10% SDS–PAGE gels, respectively, and Western blotting was performed as described (Jiang et al., 2011). The signals were visualized by GE ImageQuant LAS4000mini, quantified by densitometry using Gel Doc XR System (Bio-Rad Laboratories, Hercules, CA, USA), and analyzed using the Quantity One Protein Analysis Software (Bio-Rad Laboratories, Hercules, CA, USA). Antibodies included mouse monoclonal antibodies against p65 and FN (1:1000), rabbit polyclonal antibody against IjB-a (1:1000), goat polyclonal anti-body against ICAM-1 (1:1000), rabbit monoclonal antibody against TGF-b1 (1:1000), and mouse monoclonal antibody against a-tubulin (1:10,000).

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2.8. LSCM Different groups of adherent cells were washed three times with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde (in PBS) for 20 min, and permeabilized with 1% TritonX-100 for 5 min at room temperature. After further washing, cells were blocked with 10% goat serum for 30 min at room temperature. The cells were then incubated with a mouse polyclonal antibody directed against p65 (1:200) for 2 h at room temperature or overnight at 4 °C. Cells were washed again and then incubated in the dark at room temperature for 1 h with secondary antibody (Alexa FluorÒ 488-conjugated goat anti-mouse IgG, 1:1000, in 10% goat serum). After washing with PBS in the dark, a Hoechst33342 solution (5 g/ml in PBS) was used to counterstain the nucleus for 10 min (in the dark at room temperature). Cells were subsequently washed with PBS and placed under a laser scanning confocal microscope (LSM710, Carl Zeiss, Germany) for observation and image acquisition. 2.9. EMSA For the EMSA, nuclear extracts were prepared from the control and treated cells using a kit (Pierce Biotechnology) according to the manufacturer’s instructions. The nuclear extracts (2–3 lg of protein) were incubated with biotin-labeled oligonucleotide probes for NF-jB p65 containing a consensus NF-jB sequence (50 -AGT TGA GGG GAC TTT CCC AGG-30 ; Beyotime, Jiangsu, China) in a reaction mixture containing 3.5 ll ddH2O, 1 ll 10 binding buffer, 5% glycerol, 100 mM MgCl2, 0.5 ll of poly(deoxyinosinic–deoxycytidylic) acid, and 1% NP-40 for 30 min at room temperature in a final volume of 12.5 ll. To verify the specificity of the shifted bands, competition analyses in each experiment were performed using a 50-fold excess of unlabeled oligonucleotide (NF-jB) that was coincubated with the nuclear extracts for 10 min at room temperature before the addition of the biotin-labeled oligonucleotide probe. The reaction mixtures were then separated by 7% non-denaturing PAGE at 100 V for 70 min to 80 min, transferred onto a nitrocellulose membrane by electroblotting, and cross-linked for 5 min to 10 min. After being blocked in blocking buffer for 1 h at room temperature and incubation with strepto-avidin conjugated to HRP (1:300) for 15 min, the mixtures were subsequently washed with washing buffer four times for 10 min each time. Peroxidase activity was detected using an ECL substrate system. Images were captured and quantified using the GE ImageQuant LAS4000mini. 2.10. Statistical analysis All experiments were performed at least in triplicate. The data were assessed by SPSS 11.5. All values were expressed as mean ± SD. Statistical analyses of data were performed by oneway ANOVA using post hoc multiple comparisons. P < 0.05 was considered to be statistically significant. 3. Results 3.1. Effect of polydatin on metabolic and renal parameters in STZinduced diabetic rats Several metabolic and renal parameters of polydatin-treated STZ-induced diabetic rats at the end of the experimental period are presented in Table 1. The kidney weight and Kidney hypertrophy index (KW/BW) were significantly increased, while the body weight was significantly decreased in STZ-induced diabetic rats compared with those in the control group (P < 0.05). KW/BW was obviously ameliorated in polydatin-treated diabetic rats

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Table 1 Body and kidney weight, fasting blood glucose, and renal function parameters of STZinduced diabetic rats after 12 weeks of polydatin (PD) treatment. Parameters

Control (n = 8)

Diabetic (n = 8)

Diabetic + PD (n = 8)

Body weight (g) Kidney weight (g) Kidney/body weight (%) Blood glucose (mmol/L) Blood urea nitrogen (mmol/L) Serum creatinine (lmol/ L) Albuminuria ((g/L/24 h)

490 ± 8.12 2.34 ± 0.17 0.67 ± 0.05 5.60 ± 0.64 6.13 ± 1.27

362.14 ± 18.14a 3.30 ± 0.69a 0.96 ± 0.07a 22.53 ± 3.81a 13.53 ± 2.12a

417.57 ± 17.26a,b 2.89 ± 0.34b 0.73 ± 0.06b 18.03 ± 1.46a 8.37 ± 1.19b

25.38 ± 5.56

48.43 ± 10.08a

30.00 ± 4.21b

14.40 ± 3.56

100.74 ± 12.87a

54.18 ± 9.89a,b

Data are mean ± SD. a P < 0.01 vs. normal. b P < 0.05 vs. model by ANOVA.

(P < 0.05). Fasting blood glucose, blood urea nitrogen, serum creatinine and urine protein over 24 h were significantly enhanced compared with the control rats (Table 1, P < 0.05). After 12 weeks of therapy with Polydatin, the diabetic rats exhibited a significant reduction in those parameters except fasting blood glucose (P < 0.05). 3.2. Effect of polydatin on extracellular matrix proteins in STZ-induced diabetic rats The representative glomerular histology of PAS-stained sections is shown in Fig. 1. Compared with age-matched control rats, the glomerular accumulation of PAS-positive matrix was prominent

in STZ-induced diabetic rats. In polydatin-treated group, matrix expansion was significantly less than that in STZ-induced diabetic rats. The accumulation of ECM represented by increased FN expression in kidney was observed in the diabetic group, while it was significantly reduced by polydatin-treated (Fig. 1, P < 0.05). Collectively, these results confirmed the renal injury as characterized by renal hypertrophy, ECM accumulation, glomerulosclerosis, and renal dysfunction in STZ-induced diabetic rats as expected. Polydatin could ameliorate the renal injury and down regulate the FN expression in STZ-induced diabetic rats.

3.3. Effect of polydatin on NF-jB activation in STZ-induced diabetic rats To investigate whether NF-jB pathway is involved in diabetic nephropathy, we determined the expression and activity of NF-jB in diabetic kidney. Immunostaining for NF-jB demonstrated its glomerular localization in kidney sections from normal control and diabetic rats (Fig. 2A). In the control rats, NF-jB staining was predominantly located in cytoplasm, while nuclei remained relatively unstained. Increased expression of NF-jB in both cytoplasm and nuclei was observed in diabetes group (2.5-folds increment compared with the control group, P < 0.05, Fig. 2A, n = 20 glomerulus). NF-jB activity in diabetic kidney was substantially increased relative to the controls, while it was partially reduced by polydatin treatment (38% decrement compared with the diabetes group, P < 0.05). There was no visible staining in the negative group. In addition, the protein levels of IjB-a in kidney from the diabetic group were decreased compared with that from the control group (Fig. 2B, P < 0.05), while the renal ICAM-1 and TGF-b protein in

Fig. 1. Glomerular injury in STZ-induced diabetic kidney. (A and B) Glomerular histopathology analysis by Periodic Acid-Schiff (PAS) staining. The pictures display representative glomeruli of PAS-stained sections in control, diabetic and polydatin group at an original magnification of 400. The mesangial matrix index represented the ratio of mesangial matrix area divided by tuft area. ⁄P < 0.01 vs. control. #P < 0.05 vs. diabetes. (C) The protein expression levels of FN in the rat kidneys were detected by Western blot analysis. Data are means ± SD, n = 8. ⁄P < 0.01 vs. normal, #P < 0.05 vs. diabetes by ANOVA.

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Fig. 2. Effect of polydatin on NF-jB localization and expression in diabetic kidney by immunohistochemistry and western blotting. (A) Staining without NF-jB p65 antibody was used as a negative control. Immunostaining showed NF-jB (dark brown) was predominantly located in cytoplasmin normal control rats. Increased expression of NF-jB in both cytoplasm and nuclei was observed at 12 weeks after STZ induction in the diabetes group and was partially reduced by the polydatin treatment. (B–D) The protein expression levels of IjB-a, ICAM-1 and TGF-b in the rat kidneys were detected by Western blotting analysis. Data were means ± SD, n = 8. ⁄P < 0.01 vs. diabetic by ANOVA. Original magnification 400. PD, Polydatin.

the diabetic group was increased compared with that in the control group (Fig. 2C and D, P < 0.05). After the polydatin treatment, the down regulated IjB-a level was partially restored (Fig. 2B, P < 0.05). Meanwhile, ICAM-1 and TGF-b protein expression in the polydatin-treated diabetic group were significantly reduced compared with that of the diabetic group (Fig. 2C and D, P < 0.05). These findings further demonstrate that the NF-jB pathway is activated in diabetic kidney and suggest polydatin could potentially suppress the activation of NF-jB pathway in diabetic renal injury.

by Western blotting. This increment was significantly decreased by 20 and 40 lM polydatin as well as PDTC in high glucose-treated GMCs (Fig. 3B, P < 0.05). Polydatin had no effect on the FN expression in GMCs cultured in normal glucose (Fig. 3C, P < 0.05). Mannitol, served as a hyperosmotic control, has no effect on the production of protein FN (Fig. 3D). In the immunoflurescence staining by LSCM, we also found that the increment in FN expression induced by high glucose was significantly decreased by 40 lM polydatin and PDTC in GMCs (Fig. 3E).

3.4. Effect of polydatin on the protein expression of FN in high glucoseinduced GMCs

3.5. Polydatin inhibited the high glucose-induced nuclear translocation of NF-jB p65 protein in GMCs

High glucose stimulation for 24 h significantly increased the protein level of FN compared with the control (Fig. 3A), as detected

To explore the role of NF-jB in the anti-inflammatory effects of polydatin, we measured the protein expression of NF-jB p65 in the

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Fig. 3. Polydatin suppressed 30 mM glucose-induced increment of FN. (A) GMCs were exposed to HG as the indicated time periods measured by western blotting. Treatment of GMC with HG led to a significant increase in FN expression and that the maximal effect occurred at 24 h of the treatment. (B) Polydatin reduced the protein expression of FN in 30 mM glucose-induced GMCs. (C) Polydatin had no effect on the FN expression in GMCs cultured in normal glucose. (D) Mannitol, served as a hyperosmotic control, had no effect on the production of protein FN. (E) Immunofluorescence images of FN distribution under the laser scanning confocal microscopy were show (magnification 400). Green fluorescence indicates localization of FN. (a) normal glucose (5.5 mM) stimulation FN was hardly found in the cytoplasm, (b) while 30 mM glucose stimulation, FN was remarkably over expressed. (c) 40 lM Polydatin had proper inhibiting effect on high glucose-induced FN expression. (d) The addition of 100 lM PDTC clearly inhibited the high glucose-induced FN expression. Scale bar represents 20 lM. Experiments were performed at least three times with similar results. ⁄P < 0.05 vs. control group, #P < 0.05 vs. 30 mM glucose-treated group. PD, polydatin.

nucleus and cytoplasm by Western blotting. The results demonstrated that high glucose stimulation resulted in an significant increment in p65 contents in the nucleus, and a decrement in p65 contents in the cytoplasm in GMCs compared with the control group and PDTC as well as 20 and 40 lM polydatin exerted potent inhibitory effects on the increasing expression of p65 in the nucleus and the decrease in the cytoplasm compared with the high glucose treatment group (Fig. 4A and B; P < 0.05). Polydatin could inhibit NF-jB p65 nuclear translocation induced by IL-1 and TNF-a in normal glucose (Fig. 4C, P < 0.05). Polydatin could inhibit NF-jB p65 nuclear translocation induced by IL-1 and TNF-a under HG stimulation (Fig. 4D, P < 0.05). Polydatin had no effect on NF-jB p65 nuclear translocation in GMCs cultured in normal glucose (Fig. 4E, P < 0.05). Mannitol, served as a hyperosmotic control, has no effect on the translocation of protein p65 (Fig. 4F). This finding indicated that polydatin inhibited the NF-jB nuclear translocation induced by high glucose in a concentration-dependent manner.

We also performed immunofluorescence staining by LSCM to investigate the distribution of NF-jB p65 in GMCs. As shown in Fig. 5A, NF-jB p65 was predominately found in the cytoplasm of the control cells. After high glucose treatment for 30 min, pronounced NF-jB p65 staining was observed in the nucleus (Fig. 5B). Polydatin (40 lM) markedly decreased the distribution of NF-jB p65 staining in the nucleus, suggesting the inhibition of NF-jB p65 translocation to the nucleus by polydatin (Fig. 5C). No intensified staining in the nucleus was found in cells treated by 100 lM PDTC (Fig. 5D). The results were consistent with the changes in NF-jB protein revealed by Western blotting. 3.6. Polydatin inhibited the high glucose-induced protein degradation of IjB-a in GMCs To confirm further the inhibitory effect of polydatin on NF-jB activation in high glucose-induced GMCs, the protein expression

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Fig. 4. Effects of Polydatin on protein expression of NF-jB p65 in HG-induced GMCs measured by Western blotting. (A) Polydatin inhibited 30 mM glucose-induced p65 expression in nucleus. (B) Polydatin increased 30 mM glucose-induced p65 expression in cytoplasm. (C) Polydatin could inhibit NF-jB p65 nuclear translocation induced by IL-1 and TNF-a in normal glucose. (D) Polydatin could inhibit NF-jB p65 nuclear translocation induced by IL-1 and TNF-a under HG stimulation. (E) Polydatin had no effect on NF-jB p65 nuclear translocation in GMCs cultured in normal glucose. (F) Mannitol, served as a hyperosmotic control, has no effect on the translocation of protein p65. Experiments were performed at least three times with similar results. ⁄P < 0.05 vs. control group, #P < 0.05 vs. 30 mM glucose -treated group. ⁄⁄P < 0.05 vs. IL-1a-treated group. ##P < 0.05 vs. TNF-a-treated group. PD, polydatin.

of IjB-a in cytoplasm was detected by Western blotting. The protein level of IjB-a in cytoplasm treated by high glucose significantly decreased compared with that in the control group (Fig. 6, P < 0.05). This decrement was significantly reversed by PDTC and 40 lM polydatin treatment (P < 0.05). These results were in accordance with the changes in p65 protein expression in the nucleus and cytoplasm, implying that the inhibitory effect of polydatin on NF-jB activation induced by high glucose may have resulted from the inhibition of IjB-a degradation.

3.7. Polydatin attenuated high glucose-induced NF-jB DNA binding activity in GMCs NF-jB activation involves increased DNA binding activity. The DNA binding activity of NF-jB was measured by EMSA in high glucose-stimulated GMCs. We found that high glucose increased the band intensity of the NF-jB-DNA oligonucleotide complex, suggesting increased NF-jB binding to consensus DNA sequences upon high glucose stimulation. The maximal effect occurred after

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Fig. 5. Immunocytochemical images of NF-jB p65 nuclear translocation under the laser scanning confocal microscopy were show. Green fluorescence indicates localization of NF-jB p65. Polydatin suppressed 30 mM glucose-induced nuclear translocation and activation of NF-jB: (A) without 30 mM glucose stimulation NF-jB is predominately found in the cytoplasm, (B) while 30 mM glucose stimulation, NF-jB is translocated into the nucleus. (C) 40 lM Polydatin have proper inhibiting effect on NF-jB translocation, as there are proper p65 staining in the nuclei. (D) The addition of 100 lM PDTC clearly inhibits the 30 mM glucose induced NF-jB translocation, as there is no nuclear p65 staining found. Magnification 400. Scale bar represents 20 lM. Representative images of three independent experiments were shown.

2 h of treatment (Fig. 7A). The addition of polydatin (40 lM) or PDTC (100 lM) along with high glucose decreased the intensity of NF-jB bands to the baseline level, as seen in the control after 2 h of treatment. This finding suggested the attenuation of the high glucose-induced NF-jB DNA binding activity by polydatin (Fig. 7B, P < 0.05).

3.8. Polydatin inhibited the high glucose-induced protein expression of ICAM-1 and TGF-b in GMCs As shown in Fig. 8A, high glucose treatment for 24 h markedly increased the ICAM-1 and TGF-b protein level compared with the control group (P < 0.05). Polydatin (40 lM) and PDTC significantly reduced the ICAM-1 and TGF-b protein level in high glucose-treated cells (Fig. 8B, P < 0.05). Polydatin (40 lM) showed a similar inhibitory effect on ICAM-1and TGF-b expression with 100 lM

PDTC. Polydatin had no effect on the expression of ICAM-1 and TGF-b in GMCs cultured in normal glucose (Fig. 8C).

4. Discussion DN is characterized by mesangial matrix expansion resulting from the accumulation of ECM components mainly secreted from the proliferation of mesangial cells (Raptis and Viberti, 2001). FN is an important ingredient of ECM, and its excessive synthesis contributes to glomerular basement membrane thickening as well as ECM deposition in the mesangium (Young et al., 1995; Poljakovic et al., 2003). Therefore, suppressing the protein synthesis of FN effectively inhibits both the extent of glomerular basement membrane thickening and ECM accumulation, thereby preventing or delaying DN progression. To explore the effect of polydatin in vivo, STZ-induced diabetic rats were administrated with polyd-

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Fig. 6. Polydatin inhibited 30 mM glucose-induced IjB-a degradation in cytoplasm measured by Western blotting. a-Tubulin was set as control normalization. Experiments were performed at least three times with similar results. ⁄P < 0.05 vs. control group, #P < 0.05 vs. 30 mM glucose-treated group. PD, polydatin.

Fig. 7. Polydatin inhibited 30 mM glucose-induced NF-jB DNA binding activity in GMCs. (A) GMCs were exposed to HG as the indicated time periods. Treatment of GMC with HG led to a significant increase in DNA binding activity of NF-jB and that the maximal effect occurred at 2 h of the treatment (Fig. 7(A)). (B) GMCs were incubated in the presence (lanes 2–4) or absence (lane1) of the indicated concentrations of HG for 2 h, together with PD (40 lM, lane3) or PDTC (100 lM, lane4) Nuclear extracts from these cells were analyzed by EMSA with a biotinlabeled probe (⁄P < 0.05 vs. control group, #P < 0.05 vs. 30 mM glucose-treated group). EMSA, electrophoretic mobility shift assay. PD, Polydatin.

atin. Morphologically, the diabetic rats developed glomerular sclerosis with increased periodic acid-Schiff-positive extracellular matrix synthesis and increased mesangial matrix area in the glomeruli, however, polydatin was able to ameliorate the morphological deterioration. Furthermore, fibronectin, a major extracellular matrix protein, was induced in diabetic rats but normalized by polydatin. Consistently, in vitro study, polydatin effectively ameliorates FN production in high glucose-induced mesangial cells.

Fig. 8. Effect of Polydatin on the protein expression of ICAM-1 and TGF-b in HGinduced GMCs measured by Western blotting. (A) GMCs were exposed to HG as the indicated time periods measured by Western blotting. Treatment of GMC with HG led to a significant increase in ICAM-1 and TGF-b expression and that the maximal effect occurred at 24 h of the treatment. (B) Polydatin decreased the protein production of ICAM-1 and TGF-b in HG-induced GMCs. (C) Polydatin had no effect on the expression of ICAM-1 and TGF-b in GMCs cultured in normal glucose. aTubulin was set as control normalization. Experiments were performed at least three times with similar results. ⁄P < 0.05 vs. control group, #P < 0.05 vs. 30 mM glucose-treated group. PD, Polydatin.

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NF-jB widely exists in all kinds of cells in the kidney. After NFjB activation accompanied with the phosphorylation and degradation of IjB, the major subunit p65 of NF-jB translocates into the nucleus and binds to a specific DNA sequence on a target gene promoter. Finally, the transcription of target genes is triggered, leading to inflammatory injury (Poljakovic et al., 2003). Such physiological and biochemical changes promote GMC proliferation and mononuclear macrophage infiltration, and finally quicken glomerular sclerosis in a diabetic kidney (Mezzano et al., 2004; Park et al., 2000). Recent clinical and experimental studies have shown that inflammatory cytokines play important roles in the pathogenesis of DN. Prominent inflammatory markers have reportedly appeared in the kidney of DN patients, and 54 genes out of 138 known NF-jB targets have been upregulated. A specific NF-jB promoter module, NF-jB-IRFF-01, has also been identified. It has been activated under the inflammatory stress response of progressive DN (Mora and Navarro, 2005). In accordance with the above studies, our previous study has demonstrated that NF-jB in the kidney is markedly activated in alloxan-induced diabetic mice (Liu et al., 2010). All these findings signify that inflammatory processes contribute to DN progression, and that the activation of NF-jB may play a pivotal role in renal injury caused by the inflammatory factors of diabetes. Immunostaining manifested that NF-jB expressed slightly in cytoplasm and nuclei in normal glomeruli, and intensively in diabetic glomeruli, suggesting the activation of NF-jB pathway in renal tissue of diabetic rats. After polydatin treatment for 12 weeks, the NF-jB nucleus translocation and NF-jB protein production were significantly inhibited, implying the therapeutic effect of polydatin on diabetic rats with renal injury may result from the inhibition of NF-jB activation. And in diabetic kidney, the protein levels of IjB-a were decreased, while polydatin treatment could up-regulated IjB-a level and decreased the expression of inflammatory cytokines regulated by NF-jB, such as ICAM-1 and TGFb1, which increased in diabetic rats. These data suggest that polydatin may protect against renal injury in diabetic nephropathy by preventing activation of NF-jB. To confirm our conclusion, We further explored the activation of NF-jB and NF-jB nuclear translocation in high glucose-induced GMCs to verify whether the inhibitory effect of polydatin on FN was associated with the NF-jB-mediated inflammatory pathway. In vitro study showed high glucose decreased the protein level of IjB-a in the cytoplasm, and simultaneously increased NF-jB p65 expression in nuclei. High glucose also increased nuclear NF-jB DNA binding activity in GMCs. All these findings suggested that the activation of the NF-jB pathway in GMCs is stimulated by high glucose. However, polydatin enhanced the IjB-a protein level in the cytoplasm and significantly inhibited NF-jB nuclear translocation in a concentration-dependent manner in GMCs exposed to high glucose. Polydatin also suppressed the upregulation of nuclear NF-jB DNA binding activity induced by high glucose in GMCs, implying that polydatin resists the activation of NF-jB induced by high glucose, which may contribute to the amelioration renal injury progression caused by the inflammatory factors of DN. In DN, the activation of NF-jB increases the transcription and protein synthesis of many inflammatory mediators, such as ICAM-1 and TGF-b1. These mediators not only accelerate ECM accumulation, but also activate NF-jB in monocytes and GMCs. The results are basement membrane thickening, glomerulosclerosis, and tubulointerstitial fribrosis (Chen et al., 2008). Hence, we further investigated the effects of polydatin on the protein expression of target genes of NF-jB including ICAM-1 and TGF-b1 in high glucose-treated GMCs. ICAM-1 is a known important downstream inflammatory factor whose overexpression promotes inflammatory cells, including mononuclear macrophage infiltration into glomeruli and renal

interstitium, as well as accelerates glomerular sclerosis in diabetes (Miyatake et al., 1998). The ICAM-1 gene contains a NF-jB binding site in the promoter region. Once NF-jB binds to this site, the transcription of ICAM-1 gene encoding protein is triggered (Kumar et al., 2004). High glucose could promote ICAM-1 protein and mRNA production in rat mesangial cells through the protein kinase C-NF-jB pathways (Park et al., 2000). Recent studies have indicated that both ICAM-1 gene deficiency (Jiang et al., 2011; Okada et al., 2003; Chow et al., 2005) and anti-ICAM-1monoclonal antibody (Sugimoto et al., 1997) blockage obviously inhibit the infiltration of monocytes/macrophages into the glomerulus and alleviate the extent of renal injury. In the present study, ICAM-1 protein expression was significantly enhanced in high glucose-induced rat GMCs, which was consistent with the result of animal experiments. Polydatin treatment significantly inhibited the increased ICAM-1 protein level induced by high glucose in a concentrationdependent manner. This result implied that the nephroprotective effect of polydatin may involve the inhibition of ICAM-1 expression in mesangial cells. TGF-b1 is recognized as another important factor in the pathogenesis of DN by mediating inflammatory response, which aggravates ECM including FN and collagen accumulation, as well as accelerates glomerularbrosis in diabetes (Sharma et al., 1996; Murphy et al., 2008). High glucose has been confirmed to induce the excessive synthesis of the TGF-b1 gene and protein in both diabetic animal models and patients, contributing to ECM deposition, and ultimately, to fibrosis (Nakamura et al., 1992; Evindar et al., 2009; Kim et al., 2003; Wang et al., 2007). TGF-b1 synthesis increases in the mesangial cells of DN patients and mediates early glomerular hypertrophy (Shankland et al., 1994). In the metabolism of ECM, the main function of TGF-b is to increase ECM synthesis and inhibit its degradation. NF-jB is reportedly involved in the oxidized low-density lipoprotein-mediated increment of TGF-b1 transcription. The TGF-b1 promoter has been confirmed to contain a sequence located at 715 to 707 bp (AGGGACTT) where NF-jB binds to target TGF-b1 gene expression (Lan et al., 2004). Hence, the overexpression of TGF-b1 leading to the hyperplasia of the mesangial matrix may be the main course of glomerulosclerosis and tubulointerstitial fibrosis. In other words, the inhibition of TGF-b1 expression benefits the treatment of diabetic kidney disease by alleviating matrix accumulation. In the current research, high glucose treatment for 24 h markedly increased the TGF-b1 protein level in rat GMCs. However, polydatin treatment significantly inhibited the increased TGF-b1 protein level in a concentration-dependent manner, together with the animal data, implying that polydatin ameliorates the production of TGF-b1 under DN. In summary, the present study demonstrated that polydatin significantly inhibited NF-jB activation as well as reduced the protein expression of ICAM-1, TGF-b1, and FN in STZ-induced diabetic rats and high glucose-treated GMCs. This inhibition suggested that the anti-inflammatory and antifibrosis effects of polydatin on DN was closely associated with suppressing the activation of the NFjB-mediated inflammatory pathway. As a major complication of diabetes, diabetic nephropathy often leads to end-stage renal failure and high mortality, and finding effective treatment and prevention for diabetic nephropathy has been a major challenge facing modern medicine. These in vitro and in vivo studies provide a basis for further exploring the therapeutic potentials of polydatin in the intervention and prevention of diabetic nephropathy in the future. Considering the renoprotective effect of polydatin observed in our research, we next plan to investigate the effect of polydatin alone or together with other drugs on diabetic nephropathy patients, cooperating with some clinical institutions, which will supply clinical basis considering polydatin as a drug for the treatment of diabetic nephropathy.

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