Human fibroblast cells were isolated and subsequently cultured for analysis of the effects of HG treatment. All the procedures followed for purification and culture of human fibroblasts were described by Xuan et al (14). Human foreskin samples were collected from 3 patients in Department of Dermatology, the First Affiliated Hospital, Wenzhou Medical University (Wenzhou, China). This study was approved by the ethics committee of Wenzhou Medical University (Wenzhou, China) and written informed consent was obtained from all the patients involved. The fat was removed from the tissue and was cut into 3 by 2 mm strips and were incubated overnight at 4°C in 0.05% Dispase I (Sigma-Aldrich, St. Louis, MO, USA). The epidermis was removed and the dermis was placed in 25-cm² flasks pre-treated with FBS, and placed horizontally for 1 h and then vertically for 3 h in a culture chamber with 5% CO₂ at 37°C. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM; HyClone; GE Healthcare Life Sciences, Logan, UT, USA) containing 5.5 mM glucose with 10% fetal bovine serum (FBS; HyClone; GE Healthcare Life Sciences) and 1% penicillin-streptomycin (Gibco; Thermo Fisher Scientific Inc., Watham, MA, USA) the medium was changed every 3 days. When cell confluence reached 70–80% the cells were digested and passaged with 0.25% trypsin (Gibco; Thermo Fisher Scientific Inc.) Cells were cultured for 3 days in 5.5 mM glucose medium and transferred to the media containing either 5.5 mM glucose (LG) or 30 mM glucose (HG). Cells at passage 3–6 were used for the LG and HG treatment. Cells were harvested after 1 h of LG and HG treatment.

Cell proliferation was assayed using a Cell Counting Kit-8 (CCK-8) kit (Dojindo Molecular Technologies, Inc., Kumamoto, Japan). The fibroblast cell culture and measurement of cell densities under different treatments were followed as previously described (14). In summary, 50 ml of the cell resuspension solution (1×10³ cells/well) were transferred into 96-well plates following digestion with trypsin, and five parallel wells were used for each treatment. Subsequent to attachment to the culture plate, the cells were subjected to the different glucose treatments for 72 h in a 5% CO₂ incubator at 37°C. Then, 5 ml of CCK-8 was added to each well, and the cells were cultured for another 3 h. Cell density was determined by quantifying the absorbance at 450 nm using a Varioskan Flash Multimode Reader (Thermo Fisher Scientific, Inc.) using the following formula: Cell dens ity=(A_{cell+CCK8+medium}-A_CCK8+medium/A_cell+CCK8-A_CCK8+medium) × 100.

Total RNA was extracted from human foreskin fibroblasts for RNA-Seq experiments following treatment with a low (5.5 mM) or high (30 mM) concentration of glucose. RNA-Seq experiments and data analysis were performed by the NovelBio Bio-Pharm Technology Co., Ltd. (Shanghai, China). The RNA-Seq data is available upon request.

Differentially expressed genes were identified by analyzing for association with biological process gene ontology (GO) terms (15). Fisher's exact test was used to classify the GO category, and the false discovery rate (FDR) was calculated to correct the P-value (16). Enrichment of GO members among differentially expressed gene sets was identified using the one-tailed Fisher's exact test for 2×2 contingency tables (17), which measures the significance of the function that as the enrichment increases, the corresponding function is more specific, which aids the identification of GOs with a more concrete function description in the experiment. Pathway analysis was used to determine the significant pathways of the differential genes according to Kyoto Encyclopedia of Genes and Genomes (KEGG) (18), BioCarta (http://cgap.nci.nih.gov/Pathways/BioCarta_Pathways and Reactome (19). Fisher's exact test was followed by Benjamini-Hochberg multiple testing correction to select the significant pathway and the threshold of significance was defined by P-value and FDR (20).

Total RNA was extracted from stimulated or unstimulated fibroblasts treated with high-concentration glucose (30 mM), Bay11-7082 (0.5 μM; Sigma-Aldrich) or inhibitor of Wnt response (IWR) (0.5 μM; Sigma-Aldrich). The cell monolayer was rinsed with ice-cold phosphate-buffered saline once. Each sample was treated with RQ1-DNAse (Promega Corporation, Madison, WI, USA). The cells were then lysed directly in a culture dish by adding 1 ml TRIzol (Thermo Fisher Scientific, Inc.) per each 3.5 cm diameter dish, scraped with a cell scraper and then 0.2 ml chloroform was added per 1 ml TRIzol. RNA (2 μg) was reverse transcribed at 42°C for 60 min, 70°C for 5 min and following stop the reaction at 8°C using a GoScript Reverse Transcription System (Promega Corporation) following the manufacturer's protocol. A SYBR Green Master Mix (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used to perform the qPCR on an Illumina Eco 3.0 (Illumina, Inc., San Diego, CA, USA). A typical reaction consisted of an initial denaturation at 95°C for 3 min, followed by 40 cycles of denaturation for 30 sec at 95°C, annealing for 30 sec at 58°C, and extension at 72°C for 30 sec, followed by a final extension at 72°C for 5 min. The transcription levels were normalized against those of GAPDH using the 2^−ΔΔCq method (21). The gene-specific primer sequences used for RT-qPCR are described in Table I. Each experiment was repeated at least 3 times. A unreversed transcribed RNA was used as a PCR template control.

For extraction of total protein, the cells were lysed in an ice-cold lysis solution containing 7 M urea, 2 Mthiourea, 2% CHAPS detergent, 40 mM Trizma base, 40 mM dithiothreitol, 1% protease inhibitor, the lysates were centrifuged for 15 min at 15,000 × g. All reagents were sourced from Sigma-Aldrich. The supernatant from each tube was moved to a new tube. The total proteins were separated on a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel (Sigma-Aldrich) at 100 V for 2 h following extraction. Then transferred onto Immobilon-P Transfer Membranes (Merck Millipore, Tokyo, Japan). The membranes were incubated in Tris-buffered saline containing 5% skimmed milk and 0.05% Tween-20 (EMD Milipore, Billerica, MA, USA) for 1–2 h and reacted with the corresponding primary antibodies at 4°C overnight. The following primary antibodies were purchased from Abcam, all at dilution of 1:2,000 (Cambridge, MA, USA): p-IKBα (mouse monoclonal; cat no. 39A1431, reactivity - mouse, rat, cow, human), IKBα (rabbit polyclonal; cat no. ab7217; reactivity - mouse, rat, human) and GAPDH (mouse monoclonal; cat no. mAbcam 9484; reactivity - mouse, rat, rabbit, chicken, cow, dog, human, pig). The membranes were incubated for 1 h with an anti-mouse or polyclonal anti-rabbit horseradish peroxidase-linked secondary antibody (cat. no. 7074; 1:2,000; Cell Signaling Technology, Inc., Danvers, MA, USA).

Statistical calculations were performed with Prism 5 software package (GraphPad Software, Inc., La Jolla, CA, USA). Significant differences are expressed as the mean ± standard error. The comparison between two groups was analyzed by t-test. P<0.05 was considered to indicate a statistically significant difference.

To simulate diabetes, HG was utilized to study its effects on the fibroblasts (22). To analyze the effects of different concentrations of HG on fibroblasts, cell proliferation was monitored in different concentrations of glucose-containing media with 10% fetal bovine serum. HG treatment up to 30 mM did not markedly alter cell proliferation, whilst culture in 50 and 70 mM glucose significantly inhibited cell proliferation compared with culture in low-glucose (LG; 5.5 mM; P<0.01; Fig. 1A). However, this concentration of glucose is much higher than the levels recorded in patients' blood; therefore, 20 and 30 mM were used to further analyze gene expressions. Compared to 30 mM, 20 mM of glucose did not significantly affect the expression levels of caspase 3 and PAI1 (data not shown). Therefore, 30 Mm of glucose was selected for transcriptome analysis. As presented in Fig. 1A, 30 mM glucose treatment did not affect cell proliferation activity, thus, the expression levels of two apoptosis and inflammation marker genes, caspase 3 and plasminogen activator inhibitor 1 (PAI1), were further monitored at this concentration. RT-qPCR results indicated that the application of 30 mM glucose led to an increase in the expression levels of caspase 3 and PAI1 following 3-h treatment, which reached a peak at 6 h (Fig. 1B). These data indicate that HG culture damages fibroblast cells.

To identify HG-regulated genes and pathways, RNA-Seq experiments were performed using human fibroblast cells cultured in LG (5.5 mM) and HG (30 mM). Since Caspase 3 and PAI1 expression levels were highest at 6 h subsequent to HG stress, the fibroblast cells stimulated for 6 h with LG and HG were collected for RNA-Seq analysis. The RNA-Seq results demonstrated that 814 genes were differentially expressed (>1.5-fold change; P<0.05) in the HG-treated fibroblasts compared with LG-treated cells. Among them, 351 genes were downregulated, and 463 genes were upregulated (Fig. 2A), determined from statistical outcomes by analysis for association with biological process GO terms. To verify the RNA-Seq data, HG-mediated expression levels of the following four genes were assessed by RT-qPCR: Interleukin 8 (IL8), chemokine (C-C motif) ligand 13 (CCL13), frizzled class receptor 8 (FZD8) and early growth receptor 2 (EGR2). The inflammatory response genes (IL8 and CCL13) were upregulated, while the Wnt signaling gene (FZD8) and putative SUMO E3 ligase (EGR2) were repressed by HG stimulation, and the RT-qPCR results were similar to RNA-Seq data (Fig. 2B). GO analysis indicated that 31 GO terms were enriched (P<0.01; Table II). These genes were associated with multiple biological processes, including cellular triglyceride homeostasis, positive regulation of cholesterol efflux, the canonical Wnt signaling pathway and transcription (Table II). Further pathway analysis was performed with 814 genes that were altered by HG stress. The results demonstrated that these genes were divided into 20 different pathways, including NF-κB, tumor necrosis factor (TNF), Wnt, ECM/receptor interaction and hedgehog signaling pathways. Among them, certain inflammatory response pathways involving NF-κB and TNF were upregulated by HG stress, while Wnt signaling genes were downregulated (Table III). Together, these data indicate that HG regulates a large number of genes involved in various biological processes.

The NF-κB pathway was identified to be involved in HG-regulated biological processes, the effect of the inflammatory response in HG-mediated fibroblast cell damage was further examined. To further evaluate the effects of HG on NF-κB signaling, the activity of IκBα, the most characterized and studied NF-κB regulator, was examined. IκBα is phosphorylated by IκB kinases (IKK), resulting in the translocation of NF-κB to the nucleus and transcription of target genes (23). Western blot analysis indicated that HG induced IκBα phosphorylation (p-IκBα), but did not change total IκBα (t-IκBα) levels (Fig. 3A). To further analyze the effect of the inflammatory response on HG-mediated gene expression, a combination of HG stress and Bay11-7082 (0.5 μM), a representative NF-κB pathway inhibitor, was used to treat fibroblasts. RNA was extracted and RT-qPCR was performed to monitor PAI1 gene expression. The results indicated that HG induced an increase in PAI1 mRNA levels at 3 (P<0.05), 6 and 12 h (P<0.01) compared with the levels observed at 0 h, and this induction was blocked by inhibiting NF-κB with Bay11-7082 (Fig. 3B). Taken together, these results suggest that the inflammatory response is inversely correlated with HG-regulated gene expression.

Wnt signaling is known to regulate diverse aspects of numerous biological processes (24). The RNA-Seq data from the present study demonstrated repressed expression of a number of Wnt signaling genes following HG stimulation in fibroblasts. NF-κB pathway inhibition partially rescued HG-mediated fibroblast damage. Therefore, the relationship between NF-κB and Wnt signaling were investigated further. In the fibroblast cells stimulated with Bay11-7082 (0.5 μM) and HG, expression levels of the Wnt signaling gene, FZD8, were similar to the levels in the LG cells (P>0.05; Fig. 4A). To further evaluate the role of Wnt signaling in HG-mediated gene expression, IWR, a typical Wnt signaling inhibitor, was also used to treated fibroblasts alongside Bay11-7082 and HG culture, then PAI1 gene expression was measured. Notably, IWR application reduced the effect of Bay11-7082 on HG-induced PAI1 expression levels (Fig. 4B). Together, these data demonstrate that NF-κB inhibition blocked the gene expression changes induced by HG. Additionally, Wnt signaling inhibition reversed Bay11-7082-mediated PAI1 repression under HG conditions.