Curcumin inhibits high glucose‑induced inflammatory injury in human retinal pigment epithelial cells through the ROS‑PI3K/AKT/mTOR signaling pathway.

Regents CUR (see Fig. 1 for chemical structure) was obtained from BioBioPha Co., Ltd. (Kunming, China). Glucose and mannitol were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). RPMI-1640 medium with glucose (5.6 mmol/l) was purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Fetal bovine serum (FBS) was purchased from HyClone (GE Healthcare, Chicago, IL, USA). Penicillin and streptomycin were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). 2′,7′-dichlorodihydrofluororescein diacetate (DCFH-DA), DAPI, LY294002, rapamycin and N-acetylcysteine (NAC) were obtained from Beyotime Institute of Biotechnology (Jiangsu, China).

Cell Counting Kit

(CCK)-8 was purchased from Invitrogen (Thermo Fisher Scientific, Inc.). AKT (catalog no. 9272), phosphorylated (p)-AKT (catalog no. 9611) and p-mTOR (catalog no. 2971) antibodies were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). mTOR (catalog no. 66888-1-Ig) and β-actin (catalog no. 60008-1-Ig) antibodies were obtained from ProteinTech Group, Inc. (Chicago, IL, USA). Horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibodies (catalog no. TA130004) and HRP-conjugated goat anti-rabbit secondary antibodies (catalog no. TA130023) were purchased from the OriGene Technologies, Inc. (Beijing, China).

Cell culture ARPE-19 human

RPECs were obtained from the Type Culture Collection of the Chinese Academy of Sciences (catalog no. CRL-4000; Shanghai, China). RPECs were cultured in RPMI-1640 medium supplemented with 10% FBS and 100 U/ml penicillin/streptomycin, and maintained at 37°C in a saturated humidified atmosphere with 5% CO 2 . Prior to experiments, RPECs were cultured in RPMI-1640 medium with 1% FBS at 37°C for 12 h, and glucose was added to the culture medium (30 mmol/l) for 0, 6, 12 and 24 h. To exclude the effects of high osmolarity on RPECs, 24.4 mmol/l mannitol was used as an equivalent osmolarity control to 30 mmol/l glucose. For specific inhibitors or antioxidant treatments, RPECs were incubated with LY294002 (1 µmol/l), rapamycin (10 µmol/l) or NAC (1 mmol/l) for 1 h at 37°C before high glucose treatment; and in CUR treatment experiments, RPECs were incubated with CUR for 1 h at 37°C prior to high glucose treatment. The concentration of LY294002 was selected based on our previous study ( 30 ), whereas the concentrations of rapamycin and NAC were determined according to previously reported methods ( 31 ).

CCK-8 assay

RPEC viability was measured using a CCK-8 method. Briefly, RPECs were seeded in 96-well plates at a density of 1–1.5×10 4 cells/ml. After culturing at 37°C for 0, 6, 12 or 24 h, 10 µl CCK-8 solution was added and the cells were incubated at 37°C for 4 h. Optical density (OD) was measured at 450 nm using a microplate reader (Molecular Devices, LLC, Sunnyvale, CA, USA). This experiment was replicated three times. ELISA RPECs were seeded in 96-well plates at a density of 1–1.5×10 4 cells/ml and incubated for 12 h at 37°C. Following incubation, the medium was collected and TNF-α (catalog no. ab181421), IL-1β (catalog no. ab100562) and IL-6 (catalog no. ab46027) content were measured by ELISA kits (Abcam, Cambridge, UK), according to the manufacturer's protocols. This experiment was replicated three times.

Detection of intracellular ROS

The intracellular ROS content of RPECs was examined following incubation with cell-permeable DCFH-DA, which is converted to fluorescent DCF in the presence of ROS. Briefly, cells were seeded in a 6-well plate at a density of 1–1.5×10 7 cells/ml. Following treatment with 30 mmol/l glucose or 24.4 mmol/l mannitol for 12 h, 10 µmol/l DCFH-DA and 1 µg/l DAPI in FBS-free RPMI-1640 were added and the plates were incubated for 20 min at 37°C. Cells were washed using RPMI-1640 with 10% FBS, and images were captured using a laser scanning confocal microscope (Olympus Corporation, Tokyo, Japan). The fluorescence intensity was measured in six random fields and analyzed using ImageJ software (version 1.4l; National Institutes of Health, Bethesda, MD, USA).

Western blot analysis

RPECs in the various treatments cultured on 6-well plates were collected with pancreatin and lysed on ice for 30 min in Western and IP Cell Lysis buffer (catalog no. P0013J; Beyotime Institute of Biotechnology) supplemented with protease inhibitor cocktail and phosphatase inhibitors (150 µl). Protein levels were quantified using a Bicinchoninic Acid Protein Assay kit (catalog no. P0009; Beyotime Institute of Biotechnology). Protein extracts (50 µg) were separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Following blocking with 10% skim milk overnight at 4°C, the membranes were incubated with primary antibodies against p-AKT (1:500), p-mTOR (1:600) or control β-actin (1:50,000) at 4°C for 12 h, washed three times with PBS, followed by incubation with HRP-conjugated secondary antibodies (1:2,000) for 2.5 h at room temperature. Each membrane was stripped and re-probed for its corresponding total protein, AKT (1:1,000) or mTOR (1:1,200). Protein bands were visualized using a chemiluminescence detection system (Pierce; Thermo Fisher Scientific, Inc.). Images were captured and analyzed using ImageJ software (version 1.41; National Institutes of Health, Bethesda, MD, USA); β-actin was used for normalization.

Statistical analysis

Statistical analysis was performed on GraphPad Prism 6.0 (GraphPad Software, Inc. La Jolla, CA, USA). All data are presented as the mean ± standard error of the mean of three independent experiments. Statistical analysis was performed by one-way analysis of variance, followed by the Tukey-Kramer post-test to determine any significant differences. P