A total of 19 patients with PDR were recruited for the present study between April 2014 and August 2014. All of the patients had previously been diagnosed with type 2 diabetes (T2D), according to the World Health Organization (WHO) criteria (20). A total of 20 patients without diabetes who presented with idiopathic macular epiretinal membranes whilst waiting for vitrectomy were recruited as control subjects. The present study was approved by the Clinical Research Ethics Committee of the First Affiliated Hospital of Chongqing Medical University (Chongqing, China) and followed the tenets of the Declaration of Helsinki. Informed consent was acquired from all participants and detailed demographics of the patients are outlined in Table I.

Vitrectomy was performed on all the participants. Fibrovascular membrane samples from patients with PDR and epiretinal membrane samples from the controls were excised during the surgery, fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5-µm sections. Briefly, 10% goat serum (Beyotime Institute of Biotechnology, Haimen, China) was used to block nonspecific binding, and the slides were incubated overnight with mouse monoclonal SIRT1 primary antibody (1:200; sc-74504; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). After washing three times with Tris-buffered saline, biotinylated secondary antibody (1:150; Santa Cruz Biotechnology, Inc.) was subsequently applied for 20 min at room temperature and the sections were visualized using a StrepABC horseradish peroxidase kit (Beyotime Institute of Biotechnology). Subsequently, goat anti-mouse biotinylated secondary antibody (1:150; sc-2039; Santa Cruz Biotechnology, Inc.) was applied for 20 min at room temperature and the sections were visualized using a StrepABC horseradish peroxidase kit (Beyotime Institute of Biotechnology). SIRT1 expression levels were semiquantitatively measured using a light microscope (magnification, ×200; BX51T-PHD-J11; Olympus Corporation, Tokyo, Japan), to generate an immunoreactive score (IRS) (21). Negative expression was defined by an IRS score of 0, low expression levels were defined by an IRS score of 1–5, whereas high expression was denoted by an IRS score of 6–12.

Circulating expression levels of IL-17 in the sera of patients with PDR and the controls were determined using enzyme-linked immunosorbent assay (ELISA; R&D Systems, Inc., Minneapolis, MN, USA), according to the manufacturer's protocol.

PBMC culture was performed as previously described (22). Briefly, fasting blood samples were harvested from all participants using Vacutainer® tubes supplemented with heparin (BD Biosciences, Franklin Lakes, NJ, USA). PBMCs were obtained using Ficoll-Hypaque™ density gradient centrifugation (GE Healthcare, Piscataway, NJ, USA). PBMCs were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (all Invitrogen, Carlsbad, CA, USA), and incubated at 37°C in an atmosphere containing 5% CO₂ for 24 h. Subsequently, 1×10⁶ PBMCs/ml were cultured on 24-well plates and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) or western blotting was used to determine the mRNA and protein expression levels of SIRT1, respectively. In order to ascertain the effects of an SIRT1 activator, resveratrol, on the expression levels of IL-17, anti-CD3 (5 µg/ml; 11-0039-41; eBioscience, Inc., San Diego, CA, USA) and anti-CD28 (1 µg/ml; 11-0289-41; eBioscience, Inc.) mouse monoclonal antibodies were added with/without 10 µM resveratrol (Sigma-Aldrich, St. Louis, MO, USA) (23). Resveratrol was stored as a powder and sterile phosphate-buffered saline solution (PBS) was added to the powder prior to use. Following 72 h incubation, the expression levels of IL-17 in the supernatants of the PBMCs were analyzed using ELISA (R&D Systems, Inc.). The mRNA and protein expression levels of SIRT1 in the PBMCs from the patients and controls were analyzed again using the methods described below. All experiments were repeated in triplicate.

An RNeasy Mini kit (Qiagen GmbH, Hilden, Germany) was used to extract the total RNA from PBMCs, according to the manufacturer's protocol. cDNA was synthesized from 1 µg total RNA using a TaqMan® Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc., Foster City, CA, USA), according to the manufacturer's protocol. RT-qPCR was subsequently performed on an ABI 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific) using SYBR® Premix Ex Taq™ II (Takara Bio, Inc., Otsu, Japan). The following primer sequences were used: β-actin, forward 5′-GGATGCAGAAGGAGATCACTG-3′ and reverse 5′-CGATCCACACGGAGTACTTG-3′; and SIRT1, forward 5′-CGGAAACATACCTCCACCTGA-3′ and reverse 5′-GAAGTCTACAGCAAGGCGAGCA-3′. The following cycling conditions were used: One cycle at 95°C for 3 min, and 40 cycles of 95°C for 3 sec and 58°C for 20 sec, followed by 1 cycle of 95°C for 15 sec, 60°C for 15 sec and 95°C for 15 sec. SIRT1 expression levels were normalized to the expression levels of a housekeeping gene, β-actin. Fold change was calculated using the 2^−ΔΔCq method (24).

PBMCs were washed twice with ice-cold PBS and NE-PER™ Nuclear Extraction reagents (Pierce Biotechnology, Inc., Rockford, IL, USA) were used to extract PBMC nuclear proteins, according to the manufacturer's protocol. Nuclear proteins were subsequently boiled for 10 min with 5% sodium dodecyl sulfate (SDS) loading buffer (4:1), separated by 8% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride (PVDF; EMD Millipore, Billerica, MA, USA) membrane. The membrane was blocked using 5% non-fat milk and rabbit monoclonal anti-SIRT1 (1:2,000; #2496; Cell Signaling Technology, Inc., Danvers, MA, USA) primary antibody was added to the membrane and incubated for 1 h at room temperature. The membrane was subsequently washed using PBS and alkaline phosphatase (ALP) buffer (Beyotime Institute of Biotechnology) containing 100 mmol/l Tris-HCl prior to incubation with ALP-conjugated secondary antibody (1:7,500; #7054; Cell Signaling Technology, Inc., Danvers, MA, USA) for 1 h at room temperature. Following this, 10 ml ALP buffer, 66 µl 5-bromo-4-chloro-3-indolyl phosphate (Beyotime Institute of Biotechnology) and 33 µl nitro blue tetrazolium chloride (Beyotime Institute of Biotechnology) were mixed, added to the membrane and incubated at 37°C. ddH₂O was added once the protein bands were clear and ImageJ software, version 1.43 (National Institutes of Health, Bethesda, MA, USA) was used to quantify the protein levels. β-actin housekeeping protein was used for normalization.

One-way analysis of variance was used to compare the expression levels of IL-17 in the supernatants of the PBMCs and the mRNA and protein expression levels of SIRT1 in PBMCs. Between-group differences were determined using Tukey's test. Student's t-test was used to compare the expression levels of IL-17 in the sera of the control and PDR groups, whereas χ² test was used to compare the differences in SIRT1 expression levels in the excised membranes from the controls and patients with PDR. Statistical tests were performed using GraphPad Prism® 5 (GraphPad Software, Inc., La Jolla, CA, USA) or SPSS software (SPSS, Inc., Chicago, IL, USA). Data are expressed as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.

The expression levels of SIRT1 in fibrovascular (n=19) and epiretinal membranes (n=20) were examined using immunohistochemical analysis (Fig. 1; Table II). A significant difference in the expression levels of SIRT1 was demonstrated between the two groups (χ²=23.85, P <0.001).

IL-17 expression levels were significantly increased in the sera from patients with PDR (21.4±5.9 pg/ml), as compared with the control group (17.3±6.2 pg/ml; P=0.038; Fig. 2). Furthermore, the expression levels of IL-17 in the supernatants of cultured PBMCs were significantly increased in patients with PDR (419.3±53.7 pg/ml), as compared with the control group (182.5±50.3 pg/ml; P<0.05; Fig. 3).

The mRNA expression levels of SIRT1 in the PBMCs of patients with PDR were significantly reduced, as compared with the control group (0.54±0.08 vs. 1.24±0.08; P<0.05; Fig. 4). The protein expression levels of SIRT1 were consistent with these mRNA results. In the PBMCs of patients with PDR that did not receive resveratrol stimulation, SIRT1 protein expression levels were significantly reduced, as compared with those from control subjects (0.11±0.01 vs. 0.19±0.03; P<0.05; Fig. 5).

In order to explore the effects of resveratrol on the expression levels of IL-17 in PBMCs, PBMCs were incubated with anti-CD3, anti-CD28 and 10 µM resveratrol for 72 h, and the mRNA and protein expression levels of SIRT1 were subsequently determined. The results demonstrated that resveratrol activated the expression of SIRT1 mRNA and protein in patients with PDR (mRNA with vs. without resveratrol, 0.80±0.10 vs. 0.54±0.08; protein with vs. without resveratrol, 0.20±0.02 vs. 0.11±0.01; both P<0.05; Figs. 4 and 5). However, SIRT1 expression levels remained lower in the PBMCs of patients with PDR following stimulation with resveratrol (P<0.05), as compared with those from the control group. SIRT1 expression levels in the controls were not significant affected by resveratrol administration (P>0.05; Figs. 4 and 5). IL-17 expression levels in the PBMC supernatants from patients with PDR were inhibited by resveratrol (with vs. without resveratrol, 368.5±62.72 vs. 419.3±53.7 pg/ml), and they were not altered in the control subjects (with vs. without resveratrol, 207.6±39.5 vs. 182.5±50.3 pg/ml; supernatant with vs. without resveratrol, 0.20±0.02 vs. 0.11±0.01; both P>0.05; Fig. 3).