Cur, with a purity of 99.8%, was purchased from Shijiazhuang spring letter Biotechnology Co., Ltd. (Shijiazhuang, China; http://www.sjzcxswkj.com) and CsA was obtained from Zhejiang Ruibang Pharmaceutical Co., Ltd. (Wenzhou, China).

Rabbit anti-human Bax primary antibody and horseradish peroxidase-conjugated goat anti-rabbit secondary antibody were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Rabbit anti-human Bcl-2 primary antibody and β-actin primary antibody were purchased from Abcam (Cambridge, UK). SOD (cat no. A001-3), malondialdehyde (MDA; cat no. A003-1), GSH-Px (cat no. A005), reactive oxygen species (ROS; cat no. E004) and catalase (CAT; cat no. A007-1-1) detection kits were all purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Other reagents were of domestic and analytical grade.

HK-2 cells were purchased from Beijing Zhongyuan Jinqiao Biotechnology Co., Ltd. (Beijing, China). The base medium for this cell line was provided by Invitrogen; Thermo Fisher Scientific, Inc. (Waltham, MA, USA) as part of a kit: Keratinocyte serum free medium (K-SFM; kit cat no. 17005-042). This kit was supplied with each of the two additives required to grow this cell line (bovine pituitary extract (BPE) and human recombinant epidermal growth factor (EGF). The following components were added the base medium: 0.05 mg/ml BPE-provided with the K-SFM kit; 5 ng/ml EGF-provided with the K-SFM kit. Atmosphere: air, 95%; carbon dioxide (CO₂), 5%, 37.0°C and humidity 70–80%.

HK-2 cells (2×10⁴ cells/well) were assigned to the following groups for 48 h at 37°C in a 5% CO₂ incubator: Control (equivalent volume of saline), 50 µM Cur, 2 µM CsA, 10 µM Cur + 2 µM CsA, 50 µM Cur + 2 µM CsA, 100 µM Cur + 2 µM CsA and 5 µM N-acetyl-L-cysteine (NAC; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Cells were washed with PBS, collected in a test tube with 1 ml normal saline and lysed with an ultrasonic crusher. After centrifugation at 12,000 × g for 10 min at 4°C, SOD activity and MDA levels in the supernatant were measured by SOD and MDA kits, a GSH-Px detection kit was used to measure GSH-Px activity, an ROS detection kit was used to measure ROS levels and a CAT detection kit was used to determine CAT activity.

HK-2 cells in logarithmic growth phase were inoculated in 96-well plates (5×10³ cells per well), added with different concentrations of Cur (0.1, 1, 10, 100 and 1,000 µM) and then co-incubated with or without (2 µM) for 24 h at 37°C. After incubating at 37°C for 4 h with 20 µl MTT (5 mg/ml), 200 µl DMSO was added. After 10 min vibration, the absorbance value of the mixture was measured at 560 nm using a microplate reader.

HK-2 cells in the control and different treatment groups were washed three times with cold PBS and lysed with RIPA lysis buffer (Beyotime Institute of Biotechnology, Haimen, China; cat. no. P0013B) on ice. Lysates were collected and centrifuged at 12,000 × g for 10 min at 4°C, the supernatants were collected and protein concentration was determined by a BCA assay. A 10% SDS-PAGE gel was prepared and samples were loaded (80 µg protein per lane), and run on the gel. After 2 h of SDS-PAGE, proteins were transferred to polyvinylidene difluoride membranes. Subsequently, blocking with 5% non-fat milk was performed for 1 h at room temperature and the membranes were incubated with diluted primary antibodies, including rabbit anti-human Bax (cat. no. 5023; 1:1,500 dilution), anti-β actin antibody (cat no. ab227387; 1:2,000 dilution) and Bcl-2 (cat. no. ab194583; 1:2,000 dilution) antibodies overnight at 4°C. The membrane was washed three times with 1X TBS 0.1% Tween-20 and then the horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (cat. no. 7074; 1:4,000) was added and incubated with the membrane at room temperature for 2 h. Grey scale and area of the protein band were analyzed by gel imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with enhanced chemiluminescent kit (SuperSignal; Pierce, Thermo Fisher Scientific, Inc.; cat no. 34094). The protein expression level was indicated by the integral grey value (D, density) using Image J software (National Institutes of Health, USA).

HK-2 cells treated with medium or different drugs for 48 h were collected and treated according to the Annexin V-FITC Apoptosis Detection kit (Vazyme Biotech; cat no. a211-01). After washing twice with PBS, cells were suspended with 1X binding buffer, added with 5 µl of fluorescein isothiocyanate labeled Annexin V and 5 µl of PI, and then mixed gently. After incubation at room temperature for 15 min, the cells were analyzed by flow cytometry. A total of 20,000 cells were analyzed each time.

Male Sprague-Dawley rats (n=60; 7 weeks of age), clean grade and weighing 200–250 g, were purchased from the Experimental Animal Center of Changzhou Cavens Laboratory Animal Co., Ltd. (Changzhou, China) and kept under controlled conditions (temperature 23±1.5°C, relative humidity 40–60%, 0.03–0.04% CO₂, 12/12 light/dark cycle)- at the Animal Center of Ningbo University (Ningbo, China). Rats were allowed free access to drinking water and food. The present study was approved by the animal ethical committee of the Medical School of Ningbo university. The rats were randomly divided into four groups according to their body weight, with n=6 per group: Control group, Cur group (30 mg/kg/day) (37)-, CsA group (20 mg/kg/day) and Cur (30 mg/kg/day) + CsA (20 mg/kg/day) group, all of which were administered for 21 days. The control group received 0.9% sodium chloride injection (4 ml/kg/day, intragastric) + vegetable oil (2 ml/kg/day, subcutaneous), the Cur group received Cur (30 mg/kg/day, intragastric) + vegetable oil (2 ml/kg/day, subcutaneous), the CsA group received 0.9% sodium chloride injection (4 ml/kg/day, intragastric) + CsA (20 mg/kg/day dissolved in vegetable oil, subcutaneous) and the CsA + Cur group received Cur (30 mg/kg/day, intragastric) + CsA (20 mg/kg/day dissolved in vegetable oil, subcutaneous).

On the 22nd day, 24 h urine of rats was collected by a metabolic cage. The rats were anesthetized with ether and 3.0–4.0 ml blood was collected to obtain serum by centrifugation at 1,400 × g for 30 min at 4°C. Serum/urine creatinine (Crea) and blood urea nitrogen (BUN) levels were measured with a Hitachi Model 7060 automatic biochemical analyzer, and creatinine clearance (Ccr) was calculated as follows: Ccr=[urine Crea concentration ×1 h urine volume (ml)]/serum Crea concentration.

On the 22nd day, rat kidneys were fixed with 5% formaldehyde at 4°C for 48 h to prepare paraffin blocks and tissue sections (5 µm) following dehydration. For histological investigation, the deparaffinized and rehydrated rat kidney tissue sections were stained with hematoxylin for 15 min and 1% eosin for 3 min at room temperature by the Hematoxylin and Eosin Staining kit (Beyotime Institute of Biotechnology; cat no. C0105). For periodic acid-Schiff (PAS) staining, the slides were immersed in periodic acid solution for 10 min, and then immersed in Schiff's solution for 30 min at 20°C after being rinsed with distilled water 4 times using a Periodic Acid Schiff (PAS) Staining kit (cat no. ab150680). After rinsing slides under hot running tap water, the slides were stained with hematoxylin for 3 min, rinsed in running tap water for 3 min and applied the bluing reagent for 30 sec. Finally, the distilled water rinsed slides were dehydrated through graded alcohols. Expression of NF-κB in kidney tissue was detected by immunohistochemistry. In brief, tissues in paraffin sections were treated with the following procedures, including dewaxing, incubation with anti-NF-kB p65 antibody (cat no. ab16502; 1:1,000 dilution) for overnight at 4°C and horse radish peroxidse-conjugated goat anti-rabbit secondary antibody (CST; cat no. 7074) for 2 h at room temperature, stained with DAB and mounted with anti-fade oil. All sections were observed at ×200 magnification on a light microscope (37).

All statistical analyses were performed with PASW Statistics 18.0 software (SPSS, Inc., Somers, USA). Data are presented as the mean ± standard deviation. One-way analysis of variance and the Tukey post-hoc test were used to analyze the significance between the groups. P<0.05 was considered to indicate a statistically significant difference.

In vitro, a concentration of 100 µM Cur improved cell viability when treated with CsA, compared with lower concentrations of Cur (Fig. 1A). The levels of ROS and MDA were significantly downregulated in a dose-dependent manner following treatment with Cur, compared with the CsA alone group, as demonstrated in Fig. 1B and C. The ROS level in the CsA alone group was 115.67±7.66 KU/g, while levels in the 100 µM Cur + CsA group were 71.67±5.35 KU/g (P<0.01; Fig. 1B). MDA levels in the CsA alone group were 39.01±1.36, while levels in the 100 µM Cur + CsA group were 23.85±1.22 nM (P<0.01; Fig. 1C). In addition, the activity of SOD was increased in the 100 µM Cur + CsA group (22.76±0.73 KU/g in the CsA alone group vs. 32.6±0.66 KU/g in the 100 µM Cur + CsA group; P<0.05; Fig. 1D) and the GSH-Px activity was also increased following treatment with 100 µM Cur (55.65±1.45 in the CsA alone group vs. 76.04±2.07 KU/g in the 100 µM Cur + CsA group; P<0.05; Fig. 1E). Furthermore, CAT activity was increased in the 100 µM Cur-treated group compared with the CsA alone group (1.54±0.07 in the CsA alone group vs. 2.31±0.12 KU/g in the 100 µM Cur + CsA group; P<0.05; Fig. 1F). CsA-induced increases in ROS and MDA, and decreases in SOD, GSH-Px and CAT activity, were dose-dependently reversed by treatment with Cur (Fig. 1B-F).

The results in Fig. 2A demonstrate that the rate of apoptosis was gradually decreased with increasing concentrations of Cur in HK-2 cells, compared with the CsA alone group, which indicated that Cur may inhibit CsA-induced cell apoptosis. The ratio of Bax/Bcl-2 is an important indicator in the balance between cell apoptosis and survival. The ratio of Bax/Bcl-2 was decreased when CsA was combined with Cur treatment, compared with the CsA alone group, as demonstrated in Fig. 2B and C, which indicates that levels of the anti-apoptotic protein Bcl-2 were higher compared with the proapoptotic protein Bax, thus indicating that Cur may improve cell survival in CsA-treated HK-2 cells.

To further evaluate the protective effect of Cur in a CsA-induced rat nephrotoxicity model, renal histopathological analysis was performed. Histopathological results are presented in Fig. 3. Hematoxylin and eosin staining demonstrated that the tissue sections of the control group exhibited normal structure. By contrast, the kidneys of rats treated with CsA exhibited marked histological changes, cellular edema, vacuolar deformation and dissolution. These changes were reduced when CsA was combined with Cur treatment. The nucleus presented blue and the glomerular basement membrane was purple. The glomerular volume was markedly increased in the CsA group, with mesangial matrix hyperplasia (positive PAS staining), glomerular swelling, an irregular morphology of certain glomeruli, lobulated capillary loops and a markedly increased renal capsular space. NF-κB-positive cell cytoplasm or nuclei were stained brown. The results demonstrated that NF-κB-positive cells, and therefore inflammation, were markedly increased in the CsA group compared with the control group, which was reduced when CsA was combined with Cur treatment.

As demonstrated in Table I, renal failure was induced by continuous administration of CsA (20 mg/kg/day) for 21 days. The serum Crea and BUN were significantly elevated and the Ccr rate was significantly reduced (P<0.05) in the CsA alone group compared with the control group. Cur (30 mg/kg/day) alone did not significantly reduce renal function in rats compared with the control group, however, Cur significantly improved CsA-induced renal failure.