Mouse Neuro2a neuroblastoma cells obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (both Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C in a 5% CO₂ atmosphere. Then, 3×10⁵ cells/ml were seeded into a 6-well plate and medium was replaced every 1–2 days. The cells were washed with PBS prior to the experiments. The Trx inhibitor PX12 (Tocris Bioscience; Bio-Techne, Minneapolis, MN, USA) was dissolved in 1 mM dimethyl sulfoxide and stored at −20°C. The stock high-glucose (HG; 100 mM) medium (Gibco; Thermo Fisher Scientific, Inc.) was diluted with DMEM to give a final working concentration of 30 mM glucose and was stored at 4°C. GSPE was purchased from Tianjin Jianfeng Natural Product R&D, Co., Ltd. (Tianjin, China). Neuro2a cells were pretreated for 6 h with or without 10 µM PX12, and subsequently received treatment for 24 h with or without 10 µg/ml GSPE and 30 mM glucose at 37°C in a 5% CO₂ atmosphere.

Neuro2a cells that were treated with HG, HG + GSPE or HG + GSPE + PX12, and control cells, were washed with PBS three times prior to analysis. Then, 3×10⁵ cells/ml were collected by centrifugation at 112 × g for 5 min at room temperature, and then stained with annexin V (1:100)/propidium iodide (PI) (1:500) in binding buffer (BioTools, Inc., Jupiter, FL, USA) at room temperature for 15 min in the dark. Subsequently, the samples were analyzed by BD Accuri™ C6 software version 1.0.264.21 (BD Biosciences, Franklin Lakes, NJ, USA).

Total proteins were extracted from Neuro2a cells that were treated with HG, HG + GSPE or HG + GSPE + PX12, and control cells. The cells were lysed in ice-cold lysis buffer (Geno Technology, Inc., St., Louis, MO, USA) for 30 min. The lysates were centrifuged at 12,000 × g for 30 min at 4°C, and the supernatants were collected as protein samples. The concentration of protein was measured by BCA assay and equal amounts of extracted protein samples (50 µg protein/lane) were separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked in 5% non-fat milk for 1 h at room temperature and then incubated with the following primary antibodies overnight at 4°C: Anti-Trx (rabbit; 2429; 1:1,000 Cell Signaling Technology, Inc., Danvers, MA, USA), anti-78 kDa glucose-regulated protein (rabbit; GRP78; 11587-1-AP; 1:1,000; ProteinTech Group, Inc., Chicago, IL, USA), anti-Trx-interacting protein (Txnip; rabbit; 18243-1-AP; 1:1,000; ProteinTech Group, Inc.), anti-apoptosis signal-regulating kinase (ASK) 1 (rabbit; ab45178; 1:2,000; Abcam, Cambridge, UK), anti-Nrf2 (rabbit; sc-722; 1:500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and anti-β-actin (mouse; sc-4778; 1:1,000; Santa Cruz Biotechnology, Inc.). All antibodies were diluted in 5% non-fat milk. The membranes were washed three times with 1X TBS containing 0.1% Tween-20 (TBST) for 15 min and then incubated with secondary antibodies horseradish peroxidase (HRP) conjugated goat anti-rabbit immunoglobulin (Ig)G (ab6721; 1:2,000; Abcam) and HRP goat anti-mouse IgG (ab205719; 1:2,000; Abcam) for 1 h at room temperature. Following three washes with 1X TBST for 20 min, the protein bands were visualized using enhanced chemiluminescence (GE RPN2232; GE Healthcare Life Sciences) and exposed to X-ray film. Blots were semi-quantified by densitometry using Labworks software version 4.5 (Perkin Elmer, Inc., Waltham, MA, USA).

To detect cell apoptosis, 3×10⁴ cells/ml was seeding into 8-well chamber slides (Thermo Fisher Scientific, Inc.) and a TUNEL assay was performed using a FITC labeled TUNEL Cell Death Detection kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China), according to the manufacturer's protocol. Briefly, following control, HG, HG + GSPE or HG + GSPE + PX12 treatment, cells were fixed in 4% paraformaldehyde for 15 min at room temperature, washed in PBS three times for 10 min and incubated with 50 µl reaction mixture for 60 min at 37°C in a dark humidified chamber. The cells were then counterstained with PI (Vector Laboratories, Inc., Burlingame, CA, USA) diluted 1:500 for 10 min at room temperature in the dark. Apoptotic cells were observed in at least 5 fields under a fluorescence microscope (BZ 7000; Keyence Corporation, Osaka, Japan) and Image Pro Plus version 5.0 (Media Cybernetics, Inc., Rockville, MD, USA) was used to analyze data.

A total 24 male inbred BALB/c mice (age, 6 weeks; weight, 20–25 g) were supplied by Dalian Medical University (Liaoning, China) and were housed in an animal control facility for 2 weeks in a 12-h light/dark cycle with a mean illumination of 80 lx and 30–70% humidity. The animals were maintained at 22±2°C. Tap water and food pellets (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) were available ad libitum. All experimental procedures were conducted in accordance with the institutional guidelines for the care and use of laboratory animals, and experimental protocols were approved by the Institutional Animal Care and Use Committee at Dalian Medical University. A total of 6 mice were assigned to 4 groups; control, diabetic, diabetic + GSPEI and diabetic + GSPEII. The diabetic mice were fed a high fat diet (10% lard, 20% yolk, 1% cholesterol, 0.5% cholate, 20% sucrose and 48.5% standard diet) for 8 weeks and intraperitoneally injected with streptozotocin (STZ; 60 mg/kg; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) twice every 3 days to induce diabetes. When blood glucose levels reached 250 mg/dl, the model was considered to be successfully established. Subsequent experiments were conducted between 10.00 am and 2.00 pm. STZ was dissolved in cold 50 mM citric acid buffer (pH 4.5). The diabetic mice were also administered with various concentrations of GSPE (50 and 100 mg/kg) via oral gavage.

The animals were sacrificed with an overdose of carbon dioxide 2 weeks after commencement of the different treatments. The enucleated eyes were immersed in Bouin's solution for 24 h and then fixed in 70% ethanol for 24 h at room temperature. Following alcohol dehydration, the eyes were embedded in paraffin and 5-µm sagittal sections containing the entire retina, including the optic disc, were obtained. The retinal sections were stained with 0.5% hematoxylin and 1% eosin (H&E) at room temperature to observe under a light microscope. In each of the superior and inferior hemispheres, the thickness of the outer nuclear layer (ONL) was measured at 9 defined points. Each point was centered on adjacent 220-µm lengths of the retina. The first point of measurement was at a ~220 µm distance from the optic nerve head, and subsequent measurement points were located in the periphery. The average ONL thickness was determined based on measurements from 18 points in each section.

Total RNA was obtained from each retina sample using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.). RT was performed with the PrimeScript RT Reagent kit (Perfect Real Time; Takara Bio, Inc., Otsu, Japan). RT-qPCR was performed to measure Trx mRNA expression using SYBR Premix DimerEraser (Takara Bio, Inc.), with reverse-transcribed cDNA as the template. All PCR reactions were conducted in a final volume of 20 µl. The amplification was performed using an ABI Prism 7000 Sequence Detection System (Applied Biosystems; Thermo Fisher Scientific, Inc.) under the following conditions: 95°C for 30 sec followed by 40 cycles of 95°C for 3 sec, 72°C for 30 sec and 55°C for 30 sec. GAPDH was used as an internal reference gene. The primers used were as follows: Forward, 5′-GGAATGGTGAAGCAGATCGAG-3′ and reverse, 5′-ACGCTTAGACTAATTCATTAAT-3′ for Trx; forward, 5′-TGTGATGGGTGTGAACCACGAGAA-3′ and reverse, 5′-GAGCCCTTCCACAATGCCAAAGTT-3′ for GAPDH. The 2^−ΔΔCq method was used to quantify the results (15).

Data are presented as the mean + standard deviation. Each experiment was performed three times. The statistical significance of the differences between groups was assessed using one-way analysis of variance. Statistical analysis was performed using SPSS software version 17.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

In the present study, diabetes was induced in mice in vivo using an STZ injection and a high-fat diet. The morphology of the retina was observed following H&E staining and the present results demonstrated that ONL (photoreceptor cell layer) thickness was diminished in diabetic mice compared with in healthy mice. However, following treatment with various concentrations of GSPE, ONL degeneration was revealed to be prevented (Fig. 1A and B). Reverse transcription-quantitative polymerase chain reaction was used to detect the expression of Trx in tissue samples isolated from mice in the various groups. As presented in Fig. 1C, the mRNA expression of Trx was significantly downregulated in diabetic mice compared with in healthy mice. Conversely, following GSPE administration, Trx mRNA expression levels were significantly increased compared with in diabetic mice.

During the in vitro experiments used in the present study, mouse Neuro2a cells were maintained in hyperglycemic conditions (30 mM glucose) to induce an HG damage model with or without GSPE treatment. Hyperglycemia-induced apoptosis in Neuro2a cells was quantitatively analyzed using flow cytometry. As presented in Fig. 2A and B, HG treatment increased the percentage of apoptotic cells compared with the control group, whereas treatment with GSPE counteracted the proapoptotic effects of hyperglycemia.

Western blot analysis was used to detect the expression of GRP78, which is one of the markers of ER stress, Nrf2 and Trx in Neuro2a cells. The present results demonstrated that the protein expression of GRP78 was significantly upregulated in HG-exposed cells, whereas the protein expression of Nrf2 and Trx was significantly downregulated (Fig. 2C-F). However, following GSPE treatment, GRP78 expression was significantly decreased, whereas Nrf2 and Trx expression was significantly increased (Fig. 2C-F). These results indicate that HG treatment may induce ER stress in neuronal cells and downregulate the expression of Trx, thus leading to cell apoptosis; Trx may therefore be implicated in the antiapoptotic effects observed following GSPE treatment.

Neuro2a cells were pretreated for 6 h with or without the Trx system inhibitor PX12 (10 µM), following which they were treated for 24 h with or without 10 µg/ml GSPE and 30 mM glucose. Neuro2a cell apoptosis was analyzed using TUNEL staining (Fig. 3A and B) and flow cytometry (Fig. 3C and D). The present results demonstrated that GSPE inhibited Neuro2a apoptosis induced by hyperglycemia. Conversely, PX12 treatment was revealed to significantly enhance the HG-induced Neuro2a cell apoptosis compared with GSPE-treated cells (Fig. 3) and inhibit the expression of Trx (Fig. 4). These results indicate that, under hyperglycemic conditions, GSPE may exhibit neuroprotective effects in Neuro2a cells may depend on a Trx-mediated mechanism.

In order to investigate the molecular mechanisms involved in Trx-mediated neuroprotection, Neuro2a cells were treated with HG, GSPE and PX12, and the protein expression levels of ASK1, Txnip and Trx were assessed using western blot analysis. The present results demonstrated that the expression of ASK1 and Txnip was upregulated in HG-treated cells, whereas the expression of Trx was downregulated compared with control cells (Fig. 4). Compared with the HG group, GSPE treatment significantly increased the protein expression levels of Trx, and decreased the levels of ASK1 and Txnip (Fig. 4). However, in cells pretreated with PX12, the expression of ASK1 and Txnip was increased, whereas the expression of Trx was decreased compared with GSPE-treated cells (Fig. 4). These results indicate that Trx-associated pathways may be implicated in the antiapoptotic effects of GSPE in hyperglycemic Neuro2a cells.