Celastrol was obtained from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were obtained from Life Technologies (Grand Island, NY, USA). Penicillin, and gentamycin were purchased from Amresco, Inc. (Framingham, MA, USA). The Cell Counting Kit-8 (CCK-8) assay kit was obtained from Dojindo Laboratories (Kumamoto, Japan). The BAY-11-7082 and Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit were purchased from Beyotime Institute of Biotechnology (Shanghai, China). IL-6, IL-8, IL-10 and PGE2 ELISA Duoset kits, and recombinant human IL-1β were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Orbital fibroblasts were cultured from adipose connective tissues, which were obtained from four patients with GO (two male, two female) and severe proptosis associated with increased orbital fat volume during a process of surgical decompression. The control tissues were obtained from two patients with no history of GO or autoimmune thyroid disease, and were collected during the course of upper lid blepharoplasties from 2 individuals (one male, one female). The mean age of all subjects was 58 years. The protocol for obtaining orbital adipose connective tissue was approved by the Institutional Review Board of Longhua Hospital (Shanghai, China), and written informed consent was obtained from all patients.

GO orbital tissues samples were minced and plated directly into culture dishes. The cells were maintained in DMEM containing 10% FBS, penicillin (100 U/ml) and gentamycin (20 mg/ml), in a humidified 5% CO₂ incubator at 37°C. When the fibroblasts had grow to 80% confluence, the cell culture medium was removed and the cells were washed with phosphate-buffered saline (PBS). The fibroblasts were then passaged serially by treatment with trypsin (Sigma-Aldrich). The cell culture medium was replaced every 2 days, and cells between the third and seventh passage were used for the subsequent examinations.

Cell viability was assayed using the CCK-8 according to the manufacturer's protocol. Briefly, 100 µl cells were seeded onto 96-well plates (1×10⁴ cells/ml) for 24 h, following which the cells were treated with, or without, 1 µM celastrol for 24 h at 37°C. Subsequently, 10 µl of the CCK-8 solution was added to each well of the plate, followed by 1 h incubation at 37°C. The optical density (OD) was measured at 450 nm using a microplate reader (Multiskan MK3; Thermo Fisher Scientific GmbH., Darmstadt, Germany). The cell inhibitory rate was calculated according to the following equation: Cell inhibitory rate = [1 − (OD experiment − OD blank) / (OD control − OD blank)] × 100%. All experiments were performed in triplicate and repeated three times independently.

Apoptosis assays were performed according to the manufacturer's protocols. Briefly, the cells in the logarithmic growth phase were collected and washed with isotonic PBS, following which 1×10⁶ cells were seeded into 6-well cell culture plates. After 24 h, the cell cultures were removed, and the cells were incubated with serum-free DMEM, with or without 1 µM celastrol, for another 24 h at 37°C. The cells were then digested with trypsin and collected by centrifugation at 300 × g for 10 min at 4°C. The cells were washed with ice-cold PBS, and resuspended gently with 195 µl annexin V-FITC binding buffer and 5 µl annexin V-FITC, following which 10 µl propidium iodide (PI) solutions were added. The mixture was incubated in the dark at room temperature for 15 min. Cytometric analysis was performed using a FACS Calibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Data acquisition and analysis were performed using the WinMDI 2.9 computer program (BD Biosciences).

The cells were collected and washed with ice-cold PBS, following which the cells were centrifuged at 300 × g for 5 min at 4°C, and the supernatant was removed. The cells were lysed with radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology) at 4°C for 20 min. The lysates were centrifuged for 10 min at 12,000 × g at 4°C, and the supernatant was collected. The protein concentration was determined using a Bradford assay (BioRad Laboratories, Inc., Hercules, CA, USA). A total of 30–50 µg proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis on 10% (w/v) gels (Beyotime Institute of Biotechnology), and were then electrophoretically transferred onto a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA). Following blocking with blocking buffer (Beyotime Institute of Biotechnology) for 1 h at room temperature, the membrane was incubated with the indicated primary antibodies overnight at 4°C. This was followed by incubation in horseradish peroxidase (HRP)-conjugated corresponding secondary antibodies for 1 h at room temperature. Positive signals were visualized using ECL Advanced Solution (Bioworld Technology, Inc., St. Louis Park, MN, USA). Actin was used as a loading control. The primary antibodies used in the present study were as follows: Rabbit polyclonal ICAM-1 (1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA; cat. no. 4915), rabbit monoclonal COX-2 (1:1,000; Cell Signaling Technology, Inc.; cat. no. 12282) and rabbit monoclonal β-actin (1:1,000; Cell Signaling Technology, Inc.; cat. no. 8457).

Total RNAs were isolated from the orbital fibroblasts using TRIzol reagent (Thermo Fisher Scientific, Inc.). The total RNAs were reverse transcribed into cDNA using Reverse Transcriptase M-MLV (Takara Bio, Inc., Otsu, Japan) and were amplified using SYBR Green Master mix (Takara Bio, Inc.). The mRNA expression was analyzed using an ABI 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). Relative gene expression levels were obtained following normalization with β-actin. The thermocycling conditions used were as follows: 95°C for 20 sec; 40 cycles of 95°C for 20 sec, 60°C for 30 sec and 72°C for 30 sec. All reactions were run in triplicate. The primer sequences used were as follows: IL-6, forward 5′-ATGAACTCCTTCTCCACAAG -3′ and reverse 5′-TGTCAATTCGTTCTGAAGAG-3′ (26); IL-8, forward 5′-GTGCAGTTTTGCCAAGGAGT-3′ and reverse 5′-TAATTTCTGTGTTGGCGCAG-3′ (26); IL-10, forward 5′-CTTCGAGATCTCCGAGATGCCTTC-3′ reverse 5′-ATTCTTCACCTGCTCCACGGCCTT-3′ (27); ICAM-1, forward 5′-CTCAGTCAGTGTGACCGCAGA-3′ and reverse 5′-CCCTTCTGAGACCTCTGGCTTC-3′ (28); COX-2, forward 5′-GCTCAAACATGATGTTTGCATTG-3′ and reverse 5′-GCTGGCCCTCGCTTATGA-3′ (29); and β-actin, forward-TCACCCACACTGTGCCCAT-3′ and reverse 5′-TCCTTAATGTCACGCACGATTT-3′ (29). The 2^−ΔΔCq method was used to quantify the results (30).

The orbital fibroblasts (1×10⁶) were seeded into 6-well cell culture plates and, after 24 h, the cell culture medium was replaced with DMEM containing 1% FBS, and 10 ng/ml IL-1β was added, with or without 1 µM celastrol. Following 24 h of incubation, the supernatants from the cell cultures were collected, and the concentrations of IL-6, IL-8, IL-10 and PEG-2 were determined using an ELISA kit, according to the manufacturer's protocol. The absorbance was measured at 450 nm using a microplate reader (Molecular Devices LLC, Sunnyvale, CA, USA).

For the luciferase assays, HEK 293T cells (Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were seeded into a 24-well plate at a density of 3×10⁴ cells/well. After 24 h at 37°C, HEK 293T cells were transfected with 200 ng firefly luciferase reporter gene construct (per well) and 1 ng pRL-SV40 Renilla luciferase constructs (per well) for normalization, using cotransfection with 2.4 µl Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). At 24 h post-transfection, the cells were stimulated with 100 ng/ml lipopolysaccharide (Sigma-Aldrich), with or without 1 µM celastrol, 4 h. Cells were subsequently collected and luciferase activity was measured with the Dual-Luciferase^® Reporter (DLR™) assay system (Promega, Madison, WI, USA).

All experiments were performed at least three times and the results are presented as the mean ± standard error of the mean. Student's t-test was used to compare two independent groups using SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

In the present study, orbital fibroblasts obtained from normal controls or patients with GO were treated with different concentrations of celastrol (0, 200, 400, 600, 800, 1, 2, 3, 4 and 5 µM) for 24 h, and cell viability was examined using a CCK-8 assay. As shown in Fig. 1, exposure of the orbital fibroblasts from the GO and normal groups to celastrol at concentrations ≤1 µM for 24 h led to no significant decline in the numbers of living cells, whereas 2 µM celastrol decreased cell viability in the two groups to 85.02 and 88.94%, respectively (Fig. 1A). The results of the apoptosis assay also showed that exposure of the cells to celastrol at 1 µM for 24 h did not induce cell apoptosis (Fig. 1B). Therefore, in the subsequent experiments, the cells were treated with 1 µM celastrol for 24 h to further investigate the role of celastrol in GO.

As is already known, inflammation is critical in the pathogenesis of GO, therefore, the present study examined the expression levels of IL-6, IL-8 and IL-10 in IL-1β-induced GO cells and normal cells, which were treated with or without celastrol. As shown in Fig. 2A, following treatment with IL-1β, the mRNA expression levels of IL-6 and IL-8 were significantly increased in the GO cells, whereas no change was observed in the expression of IL-10. Celastrol was found to decrease the mRNA expression levels of IL-6 and IL-8 in the IL-1β-induced orbital fibroblasts. The results of the ELISA also showed that, following stimulation with IL-1β, the levels of IL-6 and IL-8 in the orbital fibroblast supernatant were significantly upregulated, and co-treatment of celastrol significantly attenuated the IL-1β-induced expression of IL-6 and IL-8 (Fig. 2B).

To investigate the effect of celastrol on ICAM-1 and COX-2, the GO cells were treated with 10 ng/ml IL-1β, with or without 1 µM celastrol, for 24 h, following which the cells were collected and subjected to RT-qPCR analysis. As shown in Fig. 3A, in the IL-1β-induced orbital fibroblasts, the mRNA expression levels of ICAM-1 and COX-2 were significantly increased, whereas treatment with celastrol almost completely reversed the IL-1β-induced upregulation of ICAM-1 and COX-2. In addition, following treatment with IL-1β, the protein expression levels of ICAM-1 and COX-2 were markedly enhanced, and this was also depressed by celastrol (Fig. 3B).

PGE2 is important in modulating the inflammatory process, and COX-2 is a key enzyme, which catalyzes the production of PGE2. It has been suggested that the increase in PGE2 may be attributed to the pathological inflammatory process of GO. As it was found that the IL-1β-induced expression of COX-2 was depressed by celastrol, the present study evaluated the effect of celastrol on the IL-1β-induced expression of PGE2. Following treatment of the GO orbital fibroblasts with 10 ng/ml IL-1β, with or without 1 µM celastrol for 24 h, the supernatants were analyzed using ELISA to detect the production of PGE2. As shown in Fig. 4, IL-1β significantly induced the production of PGE2 in the orbital fibroblasts, whereas co-treatment with celastrol markedly attenuated the IL-1β-induced expression of PGE2.

The NF-κB signaling pathway is important in regulating the production of several cytokines. In the cytoplasm, NF-κB is arrested by IκB, and the activation of IKK phosphorylates IκB, thereby releasing NF-κB, which translocates to the nucleus and activates the transcription of response genes (31). It has been demonstrated that celastrol is a potent inhibitor of NF-κB, therefore, the present study examined whether celastrol exerts suppressive effects on IL-1β-induced proinflammatory molecules through the inhibition of NF-κB. As shown in Fig. 5A–C, following treatment with IL-1β, the phosphorylation of IκBα was significantly upregulated, whereas cotreatment with celastrol significantly suppressed the IL-1β-induced phosphorylation of IκBα. Pretreatment with the NF-κB inhibitor, BAY-11-7082, almost completely inhibited the activation of NF-κB induced by IL-1β.

The effect of celastrol was also examined using an NF-κB luciferase system in 293T cells. As shown in Fig. 5B, celastrol significantly inhibited IL-1β-induced NF-κB activation, in a dose-dependent manner.

To further determine whether the IL-1β-induced stimulation of proinflammatory gene expression was mediated by the NF-κB-dependent pathway, the present study pretreated GO cells with BAY-11-7082 (2.5 µM) for 30 min, following which IL-1β and/or celastrol were added. Following incubation for 24 h, the cells were harvested and subjected to RT-qPCR analysis. The results showed that pre-incubation with BAY-11-7082 significantly decreased the IL-1β-induced gene expression levels of IL-6, IL-8, ICAM-1 and COX-2 (Fig. 6), which confirmed activation of the NF-κB pathway as the mechanism underlying the increased expression of these cytokines.