It has been reported that AGE inhibits proliferation of ECV304 cells [5,6]. However, it is unknown whether hyperoside can inhibit these effects. A significant difference in cell viability between the AGE-treated and negative control (NC) groups was observed (p < 0.01), indicating that treatment with AGEs could inhibit cell viability in ECV304 cells. In contrast, there was a significant difference between cells treated with AGEs and hyperoside compared to those treated with AGEs alone (p < 0.05; Figure 1), indicating that hyperoside promoted cell viability.

To date, there are no data available on the effects of hyperoside in AGE-induced activation of JNK in ECV304 cells. P-JNK is the activated JNK. When JNK was activated by some kinds of stimulations, more P-JNK can be detected. As shown by Western blot analysis (Figure 2), the increase in JNK activation induced by AGEs was significantly inhibited by hyperoside in ECV304 cells (p < 0.05). This suggests that hyperoside may have an inhibitory effect on JNK activation.

In order to determine whether hyperoside affected RAGE expression induced by AGEs, quiescent ECV304 cells were treated with AGEs either with or without hyperoside. As shown by real time qPCR and Western blot analysis (Figure 3), in the absence of hyperoside AGEs significantly increased expression of RAGE relative to negative control (p < 0.05). In contrast, this effect was significantly inhibited by hyperoside (p < 0.05). These results suggest that AGEs may induce upregulation of RAGE and that this effect can be inhibited by hyperoside.

To further assess the contribution of RAGE to AGE-induced activation of JNK, quiescent ECV304 cells transfected with siRNA-RAGE were treated with AGEs either with or without hyperoside. As shown by Western blot analysis (Figure 4), activation of JNK in ECV304 cells was significantly inhibited by knockdown of RAGE relative to a control group (p < 0.05). These results suggest that RAGE may mediate JNK signaling induced by AGEs.

Human cell line ECV304, derived from urinary bladder carcinoma, showed some biomarkers expected of endothelial cell lines, namely Factor VIII, tubule formation on Matrigel, and/or Weibel-Palade bodies, and so ECV304 cells were selected to study the functions of endothelium cells.

RAGE belongs to the immunoglobulin superfamily of cell surface molecules [7]. It is expressed in multiple tissues [8] and interacts with various ligands including AGE [9,10]. The engagement of RAGE by AGEs has been reported to induce cellular oxidant stress via activation of the transcription factor nuclear factor-κB (NF-κB) [11,12], which evokes inflammatory and thrombogenic responses in various types of cells, including endothelial cells, smooth muscle cells, macrophages and renal cells, leading to the perturbation of a variety of vascular homeostatic functions. AGEs impair cell functions, such as proliferation, migration, apoptosis and adhesion. Abnormal proliferation and migration of these cells directly increase the thickness of the intima and accelerate the formation of atherosclerotic plaque. On the contrary, abnormal apoptosis of these cells leads to rupture of plaque. As such, the AGE-RAGE interaction is thought to play a central role in the development and progression of diabetic vasculopathy [4].

RAGE can engage with AGEs, thereby promoting activation of JNK (Figure 2). It is widely known that activation of JNK is an important step in the induction of cell apoptosis. Finally, a decrease in cell viability was observed (Figure 1). At the same time, in agreement with Tanaka et al. [4] and Shi L. et al. [13], our results confirmed that AGEs can induce upregulation of RAGE in ECV304 cells (Figure 3), which maybe promote cell apoptosis by the AGEs/RAGE/JNK pathway, because there is more RAGE that can engage with AGEs. In contrast, knockdown of RAGE expression with siRNA-RAGE significantly inhibited AGE-induced JNK activation in ECV304 cells (Figure 4). These results suggested that AGEs may play an important role in the genesis and progression of atherosclerosis via the RAGE/JNK signaling pathway.

The expression of vascular RAGE can be inhibited by clinical drugs and compounds. These include antihypertensive drugs (Telmisartan [14] and Candesartan [15]), vitamin A [16], selenium [17], n-3 polyunsaturated fatty acids [18], antihyperlipidemic agents (statins) [19], antidiabetic agents (thiazolidinediones) [20], peroxisome proliferator-activated receptor gamma [21] and several extracts from Chinese traditional medicines, such as ginkgo biloba extract [22] and Panax notoginseng saponins (PNS) [23]. This suggests that reduced expression of RAGE may contribute to the therapeutic value of these drugs.

Hyperoside is widely used in clinical practice to relieve pain and improve cardiovascular functions. It can protect cortical neurons from oxygen-glucose deprivation-reperfusion induced injury via the nitric oxide signaling pathway and the extracellular signal-regulated kinases (ERK), JNK and Bcl-2 family-related apoptotic signaling pathways [24]. In this study, hyperoside was found to suppress RAGE expression, resulting in a significant decrease in AGE-induced activation of JNK to promote proliferation in ECV304 cells (Figures 1–3). These results outline a new mechanism of the pleiotropic effects of hyperoside in ECV304 cell proliferation in response to AGEs.

Human cell line ECV304, derived from urinary bladder carcinoma was purchased from the Shanghai Institute of Cell Biology (SIBCB; Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China). The cells were cultured in RPMI 1640 (Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal calf serum, penicillin and streptomycin at 37 °C in a humidified atmosphere of 5% CO₂. The medium was changed every 2 days and the cells were passage by treatment with 0.05% trypsin-0.02% EDTA. Experiments were performed on ECV304 cells at the point of confluence.

AGEs were prepared in a manner similar to that described by Kim et al. [25]. Briefly, 1 mM fatty acid-free bovine serum albumin (BSA) was dissolved in phosphate buffered saline (PBS) with 0.5 M glucose and incubated under sterile condition for 8 weeks at 37 °C. Reaction mixtures were dialyzed against PBS to remove free glucose and then passed through an affinity column (Pierce, Rockford, IL, USA) to remove any endotoxins. Non-glycated BSA was subjected to the same conditions with the exception of glucose as a control. AGEs were identified by fluorescence spectrophotometry [26].

The procedures used in this experiment were similar to those described in our previous report [27]. The following RAGE small interfering RNA (siRNA-RAGE) was synthesized by Invitrogen (Carlsbad, CA, USA): sense, 5′-GACCAACUCUCUCCUGUAUTT-3′; antisense, 5′-AUACAGGAGAGAGUUGGUCTT-3′. A non-targeting siRNA duplex sequence (Invitrogen Stealth RNAi, Carlsbad, CA, USA) was used as a negative control. ECV304 cell transfection was performed according to the manufacturer’s instructions. Serum-starved ECV304 cells were subjected to AGEs and/or hyperoside for 10 min for Western blot analysis, or cultured for an additional 24 h for MTT assays.

Cell viability was determined using a CellTiter 96 Non-Radioactive Cell Proliferation Assay kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Briefly, EVC304 cells were seeded in 96-well culture plates at 3000 cells/well. After incubation at 37 °C for 24 h, the medium was replaced with fresh medium supplemented either with or without hyperoside (50 μg/mL). After incubation for a further hour, AGEs (200 μg/mL) were added to the cells for 10 min. Dye solution was added to each well 24 h after treatment and the cells were incubated at 37 °C for 2 h. Solubilization/Stop Solution was added to each well and the absorbance was measured at 570 nm using an ELISA reader. Relative cell numbers were calculated after normalizing the absorbance to untreated cells. Cell viability was calculated relative to untreated cells.

An experimental system to study of the effects of RAGE on JNK activation in response to AGEs was established by transfecting quiescent ECV304 cells with siRNA-RAGE (100 pmol/well). The cells transfected were then treated with AGEs and/or hyperoside for 10 min. Lipofectamine 2000 and non-targeting siRNA were used as controls

Western blot analysis was performed as previously described [28], with minor modifications. Treated ECV304 cells were harvested in lysis buffer using protease inhibitors. The lysate suspension was centrifuged and protein concentration was assessed by Bio-Rad protein assay. Heat-denatured proteins were resolved by SDS-PAGE and electrophoretically transferred to nitrocellulose membranes. The membranes were probed with antibodies against phosphorylated JNK (pJNK; Cell Signaling Technology, Danvers, MA, USA) and RAGE (R & D systems, Minneapolis, MN, USA) and reprobed with β-actin antibody (Cell Signaling Technology, Danvers, MA, USA). The bands were visualized using an enhanced chemiluminescence (ECL) detection system (GE Healthcare, Piscataway, NJ, USA). The intensities of the bands were quantified by densitometry.

qPCR was performed similar to those described previously[29]. In brief, it was performed with a Power SYBR Green PCR Master Mix (Takara, Dalian, China) on an ABI 7900 Real-time PCR instrument according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA, USA). The primer sequences used in this study were designed using Primer 3 (Whitehead Institute/MIT, Cambridge, MA, USA) and confirmed by BLAST in GenBank. Primers of qPCR for amplification of cDNA for RAGE and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were synthesized by TaKaRa Biotechnology (Dalian, China) as follows: for GAPDH (228 bp) F-5′-CGACCACTTTGTCAAGC TCA-3′, R-5′-AGGGGTCTACATGGCAACTG-3′, for RAGE (143bp) F-5′-AGGAGCGTGCAG AACTGA AT-3′, R-5′-GAGTTGGTCTGAGGCCAGAA-3′.

Data from three independent experiments are expressed as mean ± SD. Statistical analyses were performed by one-way analysis of variance (ANOVA). p-values <0.05 were considered significant.