Curcumi, AGEs, Triton X-100, DMSO, LY294002 and 3-methyladenine (3-MA) were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). DMEM and fetal bovine serum (FBS) were both obtained from HyClone (Thermo Fisher Scientific, Inc., Logan, UT, USA). Anti-GAPDH, anti-bax, anti-AIF, anti-caspase-3 and anti-p-AKT were obtained (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Anti-Beclin 1 and anti-LC3 were both obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). ECL kit was purchased (Pierce; Thermo Fisher Scientific, Inc.). Flow cytometer (FACSCalibur; Becton-Dickinson, Franklin Lakes, NJ, USA). All reagents used were trace element analysis grade. All water used was glass distilled.

Rat kidney tubular epithelial cell line NRK-52E was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). The cells were cultured in a 5% CO₂ atmosphere in Dulbecco's modified Eagle's medium (DMEM; low glucose), supplemented with 10% fetal bovine serum, 4 mM L-glutamine and 1% penicillin/streptomycin at a density of 6×103 cells/well in six-well culture plates.

We use 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to detect cell viability. Briefly, the sample medium were added to 10 µl MTT (500 µg/ml) and incubated for 3 h at 37°C following treatment. Subsequently, the MTT solution was removed and 100 µl dimethyl sulfoxide (DMSO) was added to dissolve the colored formazan crystals. The absorbance of each aliquot at 490 nm was measured using a Sunrise microplate reader (Tecan Group Ltd., Männedorf, Switzerland). The cell viability was determined as the ratio of the signal.

We detected apoptosis by Annexin V labeled with FITC. Propidium iodide was used to determine cell necrosis. After exposure to various experimental conditions, cells were trypsinized and labeled with fluorochromes at 37°C, and then cytofluorometric analysis was performed with a FACS can (Becton-Dickinson).

Apoptosis was also evaluated by the TUNEL method. For the TUNEL assays, cells grown on a coverslip were pretreated with various experimental conditions. The TUNEL assay kit was used to detect apoptotic cells under a fluorescence microscope (Leica TCS SPE; Leica Microsystems GmbH, Wetzlar, Germany). After treatment, the cells were washed with PBS, fixed in 4% paraformaldehyde/PBS, and permeabilized with 0.2% Triton X-100 in citrate buffer. Samples were incubated with TdT and fluorescein-labeled dUTP, counterstained with 4′,6-diamidino-2-phenylindole (DAPI), and then observed under a fluorescence microscope (Leica TCS SPE). Percentages of apoptotic cells were estimated by counting 300 cells in random fields.

NRK-52E cells were fixed with 4% paraformaldehyde at 4°C for 30 min, incubated with 0.2% Triton X-100 for 10 min. The cells were blocked at non-specific antibody binding sites by incubating with 10% goat serum in PBS containing 0.3% Triton X-100 and 0.5% bovine serum albumin (BSA) for 30 min at room temperature, followed by incubation with a mouse monoclonal antibody against Beclin 1 and LC3 (1:400 in PBS; Cell Signaling Technology, Inc.) overnight. Then TRITC and FITC-conjugated goat anti-mouse IgG (1:100 in PBS) was used to incubate for 0.5 h at room temperature. Heochst 33342 was added to the cells for 15 min. After washing three times with PBS, cells were visualized under fluorescence microscopy.

The NRK-52E cells were lysed in protein extraction reagent (Tissue Protein Extraction kit; Pierce, Rockford, IL, USA) and the lysates were extracted by centrifugation. Equal amounts of protein were loaded per sample in each experiment, separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes by electroblotting. The protein-bound membranes were blocked and washed in Tris-buffered saline (TBS)-Tween-20. The membranes were incubated overnight with primary antibodies. The primary antibodies used in the present study were as follows: anti-GAPDH, anti-bax, anti-AIF, anti-caspase-3 and anti-p-AKT (Santa Cruz Biotechnology, Inc.). Anti-Beclin 1 and anti-LC3 (Cell Signaling Technology, Inc.). After washing in TBS-0.1% Tween-20, the membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies overnight at 4°C. Finally, the blots were developed using an enhanced chemiluminescence system (Pierce). To compare the levels of proteins, the density of each signal was evaluated by image analysis software (CS Analyzer; ATTO, Tokyo, Japan).

Statistical comparison was made on the differences in mean values among multiple groups by using one-way analysis of variance with post hoc Scheffe's test. Statistical significance was achieved if P-values were <0.05.

The cell viability of NRK-52E cells under AGEs conditions were assessed by MTT. As shown in (Fig. 1A), when the cells were treated with 0–2,000 µg/ml AGEs for 24, 48 and 72 h, AGEs significantly inhibited NRK-52E cells viability in concentration-dependent manner. Similarly, when the cells were treated with 300, 700 and 1,000 µg/ml AGEs for 0–72 h, AGEs inhibited NRK-52E cell viability in time-dependent manner (Fig. 1B).

Curcumin, the active ingredient from the spice turmeric (Curcuma longa L.), has been demonstrated recently to possess anti-apoptosis effects (13). This study aimed to investigate the protective effect of curcumin on AGEs induced apoptosis and the underlying mechanism in NRK-52E cells. We then further hypothesize that curcumin may also play a role in the regulation of apoptosis under AGEs. As shown in (Fig. 2), the NRK-52E cells were treated with 700 µg/ml AGEs with or without 10 µM curcumin for 48 h. Firstly, AGEs-induced apoptosis was determined by TUNEL analysis. As can be seen from (Fig. 2A), AGEs treatment alone led to a significant increase in the apoptotic population, while curcumin cotreatment resulted in a substantial decrease of AGEs-induced NRK-52E cells apoptosis. We further evaluated the anti-apoptotic effect of curcumin by directly assessing the percentage of apoptotic cells using flow cytometry followed by Annexin V/PI double staining. Addition of curcumin reduced the percentage of AGEs-induced apoptotic cells from 46.2 to 28.2% (Fig. 2B). To further confirm the anti-apoptotic effect of curcumin, we performed western blot analysis. AGEs injury is associated with upregulation of Bax and AIF, leading to caspase cascade activation (caspase-3), However, co-incubation of NRK-52E cells with curcumin significantly reduced AGEs-induced apoptosis-related proteins (Fig. 2C-E). These results indicate that curcumin activates cytoprotective effect in AGEs induced apoptosis.

Apoptosis and autophagy are two evolutionarily conserved processes that maintain homeostasis during stress. The crosstalk between apoptosis and autophagy is complex, as autophagy can function to promote cell survival under various cellular conditions. In this text, we explored the role of autophagy in the cytoprotective effect of curcumin. As above, the NRK-52E cells were treated with 700 µg/ml AGEs with or without 10 µM curcumin for 48 h. After both curcumin and AGEs treatment, the number of LC3 and Beclin 1 fluorescent dots dramatically increased (Fig. 3A and B), suggesting that autophagic vacuolization occurs in response to curcumin treatment, compared to the AGEs alone group, From the results of fluorescence microscope. However, treatment of curcumin alone did not increase autophagy in NRK-52E cells (Fig. 3A and B). Similar changes were observed on western blot results. The expressions of Beclin 1 and the ratio of LC3II and LC3I were significantly increased in AGEs + curcumin group compared to AGEs group (P<0.01) (Fig. 3C and D). Theses results indicate that curcumin promotes autophagy in NRK-52E cells.

To investigate whether the mechanism of curcumin anti-apoptotic effects was the induction of chondrocyte autophagy, 3-Methyladenine (3-MA), a type of potent pharmacological inhibitors on autophagy, was used to suppress the autophagy. As (Fig. 4A) demonstrated that pretreatment with 2 mM 3-MA could block autophagy in NRK-52E cells without significant cytotoxicity. From the results of (Fig. 4A), curcumin significantly inhibited AGEs-induced apoptosis, however, the protective role of curcumin was greatly reduced when co-cultured with autophagy inhibitor 3-MA. These results show that autophagy is an important pathway for a protective role of curcumin. In order to further validate this conclusion, we further carried out western blot experiment. The western blot analysis offered another evidence. The upregulation of the expression of apoptosic protein AIF, Bax and cleaved caspase-3 were decreased when cells were co-treated with curcumin but this effect was attenuated by co-treatment with 3-MA (Fig. 4B and C). These results indicated that autophagy induced by curcumin serve in a protective manner, and blockage of autophagy interrupted the protection effect of curcumin in NRK-52E cells.

Given that PI3K/AKT pathway activation promotes autophagy in different cell lines (14). To investigate the involvement of PI3k/AKT pathway in curcumin-induced autophagy, 10 µM LY294002 (the inhibitor of PI3K/AKT) was added to the medium of NRK-52E cells. As the results of Fig. 5A and B, preincubation of the cells with both curcumin and AGEs activated the PI3k/AKT signaling pathway and significantly increased the expression of Beclin 1. However, the expression of Beclin 1 was greatly reduced when co-cultured with PI3k/AKT inhibitor LY294002. These results show that curcumin promotes autophagy through PI3K/AKT signaling pathway.