Cur (99% purity) was obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany) and dissolved in dimethyl sulfoxide (DMSO) as a 10 mmol/l stock solution and stored at −20°C. MTT and DMSO were purchased from Sigma-Aldrich (Merck KGaA). Dulbecco's modified Eagle medium (DMEM), fetal bovine serum (FBS), penicillin and streptomycin were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The Annexin V-Fluorescein isothiocyanate (FITC) Apoptosis Detection kit and was purchased from BD Biosciences (San Jose, CA, USA). Primary antibodies against transforming growth factor-β (TGF-β; rabbit; cat. no. ab50716; 1:400), β-actin (rabbit; cat. no. ab8227; 1:5,000), NF-κB p65 (rabbit; cat. no. ab16502; 1:2,000), MMP-3 (rabbit; cat. no. ab52915; 1:2,000), Bcl-2 (rabbit; cat. no. ab59348; 1:1,000), caspase-3 (rabbit; cat. no. ab4051; 1:500) and anti-glucose-regulated protein 78 (GRP78; rabbit; catalog no. ab21685; 1:1,000) were purchased from Abcam (Cambridge, MA, USA). The antibody against MMP-1 (human, cat. no. AF901; 1:1,000) was purchased from Bio-Techne (Minneapolis, MN, USA). The antibody against Smad2/3 (rabbit; cat. no. 3102; 1:1,000) was purchased from Cell Signaling Technology, Inc., (Danvers, MA, USA). Smad7 polyclonal antibody (rabbit; cat. no. PA1-41506; 1:200 and C/EBP-homologous protein (CHOP) monoclonal antibody (9C8; mouse; cat. no. MA1-250; 1:1,000) were purchased from Thermo Fisher Scientific, Inc.. Horseradish peroxidase-conjugated anti-rabbit IgG (cat. no. sc-2357; 1:5,000) and anti-mouse IgG (cat. no. sc-516102; 1:5,000) secondary antibodies were purchased from Santa Cruz Biotechnology, Inc., (Dallas, TX, USA). N-acetyl cysteine (NAC) and 4-phenylbutyric acid (4-PBA) were obtained from Sigma-Aldrich (Merck KGaA) and were dissolved in deionized water as a 50 mmol/l stock solution. The stock solutions (50 mm) were diluted with PBS to the desired final concentration prior to use and subsequently added to the cell culture medium.

HDFs were isolated from the foreskin of a 5-year-old old boy undergoing circumcision at The Third Affiliated Hospital of Soochow University (Changzhou, China). The study was approved by the ethics committee of Soochow University and written informed consent was obtained from the parent of the child. The foreskin was digested with dispase solution (5 mg/ml) at 4°C. After 16 h of incubation, the epidermal layer was removed and the dermis was cut into small pieces (~2 mm³). Dermal explants were allowed to adhere to 25 cm² culture flasks containing complete Dulbecco's modified Eagle's medium (DMEM), which consisted of DMEM supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin and 50 µg/ml amphotericin solution (all from Invitrogen; Thermo Fisher Scientific, Inc.), for 30 min at 37°C in an incubator. Following incubation of cells in a CO₂-regulated incubator at 37°C under 5% CO₂ with 95% relative humidity for 2 weeks, fibroblasts reached confluence and were detached with trypsin-EDTA solution and passaged. The cells were observed under the inverted microscope (Olympus IX51; Olympus Inc., Tokyo, Japan) and demonstrated spindle-shape morphology with bipolar projections and were refractile. The cultured cells were further identified by immunocytochemistry. The cover-slips carrying cultured cells were washed two times with ice-cold PBST (PBS containing 1% Triton X-100) and then fixed with 4% paraformaldehyde in ice-cold PBS for 10 min at room temperature. The cells were then blocked with 4% bovine serum albumin (BSA) dissolved in PBST for 30 min at room temperature and stained with anti-viemtin (rabbit, cat. no. ab137321; 1:200; Abcam) or anti-cytokeratin 10 (CK10) antibodies (rabbit, cat. no. ab76318; 1:200; Abcam) at 4°C overnight. After washed three times with PBS, incubated with secondary antibodies horseradish peroxidase-conjugated anti-rabbit IgG (cat. no. sc-2357; 1:5,000; Santa Cruz Biotechnology, Inc.) and anti-mouse IgG (cat. no. sc-516102; 1:5,000; Santa Cruz Biotechnology, Inc.) in dark at room temperature for 30 min. Washed with PBS three times, the DAB staining was performed at temperature according to the DAB kit (cat. no. AR 0611, 20 x, Beijing Dingguo Changsheng Biotechnology Co., Ltd., Beijing, China). Following 5 min, the cells washed with double distilled water to end the DAB staining, then stained with HE. With inverted microscope (magnification ×400, Olympus IX51; Olympus Inc., Tokyo, Japan), the immunocytochemistry demonstrated that the cells were positive for vimentin (dark brown) and negative for cytokeratin 10 (blue). The cells were confirmed as fibroblasts. Fibroblasts in the log phase of growth were used in all experiments in the present study.

Fibroblasts were seeded at a density of 3×10⁵ cells in 6-well plates and were incubated in DMEM complete medium for 24 h at 37°C, followed by incubation in serum-free DMEM containing a final concentration of 0, 2.5, 5 or 10 µM Cur in DMSO (final DMSO concentration, 0.1%) at 37°C. After 2 h, the cells were washed twice with PBS and covered with a thin layer of PBS. Cells were subsequently exposed to various intensities of UVA irradiation (5 J/cm² for 17 min, 10 J/cm² for 33 min or 15 J/cm² for 51 min). During exposure, the plate was placed on ice in order to reduce the effect of heat. The UVA source used in the experiment was a UV phototherapy instrument (SS-04A; Shanghai Sigma High-tech Co., Ltd., Shanghai, China) equipped with a 15-W ozone-free UVA lamp (CEL015 W; Philips, Groningen, The Netherlands) with a peak emission at 350 nm. UVA radiation was uniformly exposed to samples at a distance of 15 cm from the cell cultures and the intensity of UVA was calibrated with a digital radiometer (Shanghai Sigma High-Tech Co., Ltd.). The cells were divided into the following four groups: Control group, which was cultured in regular medium and without any treatment; Cur group, which was only treated with Cur (5 µM for 2 h); UVA irradiation group, which was treated with UVA alone (10 J/cm² for 33 min); and Cur + UVA irradiation group, which was pre-incubated with Cur (5 µM for 2 h) and subsequently exposed to UVA (10 J/cm² for 33 min). For the positive control groups, the cells were pre-incubated either with NAC (5 mM for 2 h) or 4-PBA (5 mM for 2 h) and then exposed to UVA (10 J/cm² for 33 min). After UVA irradiation, the MTT, transmission electron microscopy (TEM), ROS, malondiadehyde (MDA), catalase (CAT) and glutathione (GSH) assays were performed immediately. For the apoptosis and western blotting analysis, cells were further incubated in complete DMEM for 24 h under standard conditions without rinsing following the irradiation.

Cell viability was examined using MTT assays. Briefly, cells were seeded in 96-well plates at a density of 1×10⁴ cells/well and incubated at 37°C overnight. Treatments were performed as described above and the cells were immediately incubated with 100 µl serum-free DMEM containing 10 µl MTT (5 mg/ml) at 37°C for 2 h. Subsequently, the medium was replaced with 100 µl DMSO. Following incubation for 20 min at room temperature, the absorbance was read by measuring the optical density (OD) at 490 nm in a microplate reader (Molecular Devices, LLC, Sunnyvale, CA, USA). The 50% inhibitory concentration (IC₅₀) was calculated as follows: Inhibition rate (%)=(OD490_Cur-OD490_blank)/(OD490_control-OD490_blank) ×100. Inhibition curves were drawn and the IC₅₀ value of Cur was calculated. The experiment was repeated three times.

HDFs in control, Cur, UVA and Cur + UVA groups were harvested and fixed with 2.5% (v/v) glutaraldehyde for 24 h at 4°C, collected by centrifugation (200 × g, 4°C for 5 min) and washed twice with cold PBS. All samples were post-fixed in 1% osmium tetroxide in 0.1 M phosphate buffer (pH 7.2) at 4°C for 1 h, dehydrated through a graded ethanol series and embedded in Epon 812 at 60°C for 48 h. Ultrathin sections (70-nm) were cut and stained with 0.5% uranyl acetate for 15 min and 3% lead citrate for 5 min at room temperature and examined under a transmission electron microscope (Tecnai G2; Thermo Fisher Scientific, Inc.).

Cell apoptosis was analyzed by an Annexin V-FITC Apoptosis Detection kit (BD Biosciences) according to the manufacturer's protocol. HDF cells (5×10⁵) were seeded in 60-mm dishes and grew for 24 h, then pre-incubated with or without Cur (5 µM) for 2 h. After UVA irradiation, cells were collected by trypsinization and washed twice in cold phosphate-buffered saline (PBS). Cells (1.0×10⁶/ml) were added to 1X combination buffer (100 µl). A total of 5 µl Annexin V and 10 µl propidium iodide were then added. The mixture was vortex-mixed and incubated in the dark for 15 min at room temperature. Loading buffer (300 µl) was then added. Flow cytometry was then employed using a FACS Calibur flow cytometer (BD Biosciences). A minimum of 10,000 cells per sample was required and analyzed. All experiments were conducted 3 times. The data was analyzed by the BD FACStation software (2007; BD Biosciences).

Intracellular ROS levels were measured using the fluorogenic compound, 2′,7′-dichlorofluorescin diacetate (H₂DCFDA; EMD Millipore, Billerica, MA, USA). The 1.5×10⁴ cells were seeded in 96 well plates and incubated for 30 min at 37°C with H₂DCFDA at a final concentration of 40 µM in the dark. When the non-fluorescent ester H₂DCFDA penetrates into cells and undergoes deacetylation to dichloro-dihydro-fluorescein (DCFH) by cellular esterases, the DCFH probe is rapidly oxidized to the highly fluorescent compound 2′,7′-dichlorofluorescin by ROS. The fluorescence intensity was measured using a microplate fluorescence reader (excitation wavelength 488 nm and emission wavelength 521 nm; SpectraMax M3; Molecular Devices, LLC).

The activities of SOD, CAT, MDA and GSH were measured by SOD assay kit (WST-1 method; cat. no. A001-3), CAT assay kit (Ultraviolet method; cat. no. A007-2), Cell MDA assay kit (colorimetric method; cat. no. A003-4) and reduced GSH assay kit (colorimetric method; cat. no. A006-2), respectively. All assays were conducted according to the manufacturer's protocols (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Western blot analysis was performed to investigate protein expression. Cells cultured in 6-well plates were washed with PBS twice and harvested on the floor of the plate. Cells were lysed using radioimmunoprecipitation assay lysis buffer (Sigma-Aldrich; Merck KGaA) supplemented with a protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA). Cell lysates were centrifuged at 13,000 × g for 15 min at 4°C and protein concentration was determined with a BCA assay. Samples (10 µg protein) were loaded onto 10% SDS-PAGE gels. Gel proteins were electrophoretically transferred to polyvinylidene difluoride membranes (PVDF; Thermo Fisher Scientific, Inc.). Membranes were blocked with 3% non-fat dried milk in PBS at 4°C overnight. Subsequently, PVDF membranes were incubated with specific primary antibody solutions against antigens for 24 h at 4°C. Detection of the primary antibodies was performed using secondary, horseradish peroxidase-conjugated antibodies (Santa Cruz Biotechnology, Inc.). Details of the antibodies used have been provided earlier in the manuscript. Finally, the antigen-antibody complexes were visualized by a Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, Inc.; cat. no: 32106) and quantitated using the Versa DOC system and Quantity One software (#1709600; Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Statistical analysis was performed with SPSS version 18.0 (SPSS, Inc., Chicago, IL, USA). Data are presented as the mean ± standard deviation of three independent experiments. The data were analyzed with one-way analysis of variance, followed by Dunnett's test and Tukey's test. For all tests, P<0.05 was considered to indicate a statistically significant difference.

Pre-experimental results demonstrated that the cell viability and cell morphology were almost unchanged when treated with the range of experimental concentrations of Cur, regardless of the incubation time, indicating that Cur did not affect the growth of HDFs (data not shown). Subsequently, the cell viability of HDFs following exposure to UVA irradiation at 5, 10 and 15 J/cm² intensities was determined. As demonstrated in Fig. 1A, at the 10 J/cm² dose of irradiation, cells underwent marked morphologic alterations observed under inverted microscope, including the development of a round cell shape, shrinkage and the presence of blebs, which is an established marker of cellular damage, on the surface of the cells. At the 15 J/cm² intensities, the morphological alterations of the cell became more severe. MTT assays demonstrated that the cell viability decreased in a dosage-dependent manner in response to UVA irradiation (Fig. 1B). Compared with the control group (0 J/cm²), the cell viability of 10 J/cm² group decreased by ~46% (P<0.01; Fig. 1B). However, 5 µM Cur pretreatment for 2 h prior to exposure to 10 J/cm² UVA irradiation prevented a UVA-induced reduction in cell viability (Fig. 1C) and no evident alterations in cell morphology were observed (data not shown), indicating that Cur may protect against photodamage. Therefore, 5 µM Cur pretreatment for 2 h combined with 10 J/cm² UVA irradiation were employed in the following experiments due to the optimal protective efficiency of this concentration of Cur in HDFs.

As UVA radiation induces phototoxicity by generating ROS, the present study investigated whether Cur may inhibit the accumulation of UVA-induced ROS. Cells with pretreatment of 5 mM NAC were employed as a positive control for ROS protection. The results demonstrated that UVA irradiation alone led to a marked increase in the levels of ROS compared with the control group (P<0.01; Fig. 1D), confirming that UVA induced the generation of ROS. However, compared with the control group, no marked alterations in the fluorescent signals were observed in cells treated with Cur alone, Cur + UVA irradiation or the NAC positive control (Fig. 1D). These results indicated that Cur inhibited the accumulation of UVA-induced ROS.

As cells have natural antioxidative systems as a defense against high levels of ROS, the present study also measured the levels of MDA, an indicator of ROS, and the activity/levels of members of the cellular antioxidant defense system, including SOD, CAT and GSH, in the cells with or without Cur pretreatment. NAC was employed as the positive control described. The results demonstrated that the levels of MDA were increased, and the activities of SOD and CAT, and GSH levels, were significantly reduced, in the UVA irradiation alone group compared with the control group (P<0.01; Fig. 1E-G). However, these effects of UVA on MDA, SOD, CAT and GSH were partially reversed in the Cur + UVA irradiation and NAC positive control groups compared with the UVA irradiation alone group (P<0.01; Fig. 1E-G), indicating that Cur was able to restore the antioxidant capacities of HDFs subjected to UVA irradiation.

As the accumulation of UVA-induced ROS often leads to ER stress, the present study further investigated whether the ER ultrastructure was altered under transmission electron microscopy. The results demonstrated that the morphological hallmarks of ER stress were represented by the expansion of the ER compartment, the shift from membrane-bound ribosomes to free forms and an increase in the large polysomes in the group treated with UVA irradiation alone, and such ultrastructural changes of the ER were not observed in the control, Cur alone or NAC positive control groups (Fig. 2A).

Furthermore, ER stress is closely associated with the activation of the unfolded protein response (UPR), which includes proteins such as GRP78 and CHOP (19). Therefore, the expression of these two proteins were determined by western blotting. 4-PBA, an ER stress inhibitor, was employed as the positive control. The results of western blotting revealed that the protein expression of GRP78 and CHOP was significantly increased in the group treated with UVA irradiation alone, compared with the control group (P<0.01; Fig. 2B and C). However, the levels of these two proteins were markedly decreased in the Cur + UVA irradiation group and 4-PBA positive control group, compared with the UVA irradiation alone group (P<0.01; Fig. 2B and C). These data indicated that Cur may prevent ER stress in HDFs upon exposure to UVA irradiation.

The results of annexin V-FITC/propidium iodide staining followed by flow cytometry demonstrated that the total apoptosis rate in the group treated with UVA irradiation alone increased compared with the control group to ~66.4%; however, the total values were reduced to ~38.2 and 48.2% in the Cur + UVA irradiation and 4-PBA positive control groups, respectively, with the Cur + UVA irradiation group exhibiting a significantly lower total apoptosis value compared with the UVA irradiation alone group (P<0.01; Fig. 3A and B). Furthermore, the expression of proteins associated with apoptosis was determined by western blotting. The results demonstrated that the cleaved caspase-3 protein expression was upregulated and the Bcl-2 expression downregulated in the group treated with UVA irradiation alone compared with the control group, while the levels of caspase-3 were decreased and Bcl-2 increased in the Cur + UVA irradiation and 4-PBA positive control groups, compared with the UVA irradiation alone group (Fig. 3C and D). These data confirmed that Cur may attenuate apoptosis induced by UVA, as demonstrated by increases in the expression of the antiapoptotic protein, Bcl-2, and reduced caspase-3 levels.

It is established that UVA radiation results in inflammatory processes and the degradation of the collagen in the skin. Therefore, the present study also investigated the effects of Cur on the protein expression of NF-κB, MMP-1 and MMP-3, which are proteins implicated in inflammation and collagen metabolism in UVA-irradiated cells (20–22). NAC, which is also an inhibitor of the NF-κB signaling pathway, was employed as a positive control. The results of western blot analysis demonstrated that UVA irradiation alone promoted NF-κB, MMP-1 and MMP-3 expression compared with the control group (P<0.01; Fig. 4A and B). However, combined treatment with Cur + UVA irradiation led to a moderate inhibitory effect on the induction of the expression of these three proteins, compared with the UVA irradiation alone group (P<0.05; Fig. 4A and B), indicating that Cur may inhibit inflammation and restore normal collagen metabolism in HDFs exposed to UVA.

As UVA irradiation damages cells, we hypothesized that Cur may exert its protective effects on UVA-irradiated cells by promoting fibrogenesis. Increasing evidence has demonstrated that TGF-β signaling is a key pathway in the process of wound repair, particularly for fibroblasts, and Smads are the regulators of the TGF-β signaling pathway (23–25). Therefore, the present study further investigated the protein expression of TGF-β, Smad2/3 and Smad7 in the various treatment groups. Western blotting results demonstrated that the levels of TGF-β and Smad2/3 in the UVA irradiation group were lower compared with the control group (P<0.01), while the levels of Smad7 were increased compared with the control group (P<0.01; Fig. 4C and D). However, the levels of Smad2/3 were upregulated, and Smad7 levels were downregulated, in the Cur + UVA irradiation group compared with the UVA irradiation alone group (P<0.01; Fig. 4C and D). Interestingly, the level of TGF-β expression in Cur+UVA group were not restored to the same degree as that of the control and Cur group alone, indicating that increase of Smad2 and Smad3 expression might be independent from TGF-β but through another signaling pathway, for example the mitogen-activated protein kinase 1 pathway (26,27). The detailed mechanism requires further studies. These data indicated that Cur may stimulate the repair of UVA-induced damage in HDFs through regulating the TGF-β pathway and the Smads that regulate the TGF-β signaling pathway upon exposure to UVA irradiation.