Rabbit polyclonal immunoglobulin G (IgG) anti-TβRII (sc-220) and mouse monoclonal IgG_2b anti-tubulin (sc-23950) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Human skin fibroblasts (HSFs), derived from newborn skin were acquired from the Chinese Academy of Medical Science (Beijing, China). The cells were then cultured in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, Logan, UT, USA), supplemented with 10% fetal calf serum (FCS; Invitrogen Life Technologies, Victoria, Australia), 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma-Aldrich, St. Louis, MO, USA). HSFs were cultivated in 75-cm² culture flasks in an incubator at 37°C with a humidified atmosphere containing 5% carbon dioxide. When the cells reached 80–90% confluency, they were subcultivated to 60-mm culture dishes.

A total of four F36T12 ERE-VHO UV tubes were used in the present study as the UV source. A Kodacel TA401/407 filter (Kodak, Tokyo, Japan) was mounted 4 cm in front of the tubes to block UVC (wavelengths >290 nm). The irradiation intensity was monitored using a UVR radiometer equipped with a UVA sensor (Bioblock Scientific, Tournai, Belgium). Subconfluent HSFs were cultured in DMEM containing 0.1% FCS for 24 h and subsequently incubated in DMEM with various concentrations of AST (10, 20, 30, 40, 50 μml; Sigma-Aldrich) for 24 h. HSFs were then washed twice with fresh phosphate-buffered saline (PBS; Sigma-Aldrich) and exposed to UVA irradiation (10 J/cm²) in a thin layer of PBS. Following irradiation, the cells were incubated in DMEM for the indicated time.

A total of 40 μg of protein from each sample was separated by 10–12% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (EMD Millipore, Bedford, MA, USA). Following blocking with 10% instant non-fat dry milk for 1 h, membranes were incubated with specific antibodies overnight at 4°C followed by incubation with horseradish-conjugated secondary IgG antibodies (anti-rabbit, #7074 and anti-mouse, #7076; Cell Signaling Technology, Inc., Danvers, MA, USA) for 1 h. Antibody binding was detected with the enhanced chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ, USA).

Cell viability was measured using the 3-(4,5-dimethylthylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) method as described previously (12).

The ELISA Detection kit (Roche, Palo Alto, CA, USA) was used to detect MMP-1 and Smad7 in HSFs with different treatments. Briefly, following the indicated treatments, the cytoplasmic histone/DNA fragments from cells were extracted and bound to immobilized anti-histone antibody. Subsequently, the peroxidase-conjugated anti-DNA antibody was used for the detection of immobilized histone/DNA fragments The antibodies used were from the ELISA Detection kit (Roche). Following the addition of a substrate for peroxidase, the spectrophotometric absorbance of the samples was determined using the Dynatech MR5000 plate reader at 405 nm (Dynatech Laboratories, Chantilly, VA, USA).

Total RNA was isolated from human skin (Chinese Academy of Medical Science) using TRIzol reagent (Invitrogen Life Technologies, Shanghai, China) and reverse transcription was conducted on 2 μg RNA using the PrimeScript RT reagent kit (TaKaRa Bio Inc. Ohtsu, Japan) and standard RT-PCR primers for human COL1: Forward: 5′-CGC CAT CAA GGT CTA CTG C-3′ and reverse: 5′-GAA TCC ATC GGT CAT GCT CT-3′ and tubulin forward, 5′-ATCAGCAATGCCTCCTGCAC-3′ and reverse, 5′-CGTCAAAGGTGGAGGAGTGG-3′. Data were normalized to tubulin expression and the untreated group was set as one. The PCR was semi-quantitative and the cycling conditions were 50°C for 2 min, 95°C for 1 min and 40 cycles of amplification at 95°C for 15 sec, 60°C for 1 min, followed by 95°C for 15 sec, 60°C for 30 sec and 95°C for 15 sec.

The values in the figures are expressed as the mean ± standard deviation. The figures in the present study were representative of >3 different experiments. Statistical analysis of the data between the control and treated groups was performed using SPSS software version 6.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

Initially, it was assessed whether AST affects cell viability in HSFs using an MTT assay. As shown in Fig. 1, at a low concentration (20 μg/ml), AST did not affect cell viability. AST at 30 μg/ml exhibited almost a 25% increase in cell viability. When the concentration was 40 μg/ml, AST had the most significant effect (~50%) on HSFs compared with the control. Subsequently, the effect of AST on cell viability in UVA-exposed HSFs was examined. The results revealed that UVA irradiation (10 J/cm²) exhibited marked cytotoxicity. AST (40 μg/ml) enhanced cell viability in HSFs irradiated with UVA (Fig. 2).

Type I collagen is the most abundant structural protein in skin connective tissue. UV irradiation decreases type I collagen through inhibition of COL1 production. Therefore, the present study aimed to elucidate the effect of AST on UVA-induced COL1 downregulation. HSFs were pretreated with AST. Following UVA irradiation, secreted COL1 in the supernatants were harvested and identified using RT-PCR. As shown in Fig. 3, COL1 secretion was inhibited by UVA, while AST reversed this inhibitory effect. These results suggested that AST significantly prevented UV-induced reduction of COL1 mRNA expression.

It is well-established that UV irradiation damages human skin cells and causes photoaging. UVA and UVB irradiation of dermal fibroblasts induced MMP-1 expression, which is implicated in the degradation of human skin matrix proteins, including collagen and other components of the ECM. In the present study, to investigate whether AST affects the expression of MMP-1 in UVA-irradiated HSFs, cultured fibroblasts were pretreated with AST followed by UVA irradiation. MMP-1 protein levels were determined using ELISA. As expected, the results revealed that UVA irradiation significantly enhanced MMP-1 expression. The ELISA results demonstrated that pretreatment with AST markedly inhibited UVB-induced MMP-1 expression compared with the UVA-irradiated group (Fig. 4).

Smad7 is one of the negative factors in the TGF-β/Smad signaling pathway, which interacts with TβRI to prevent activation of Smad2/3, thereby inhibiting TGF-β signaling. It was subsequently investigated whether AST affects the expression of Smad7 in UVA-irradiated HSFs. Smad7 protein levels were determined using ELISA. The results demonstrated that UVA irradiation significantly enhanced Smad7 expression and the expression of Smad7 induced by UVB irradiation was significantly attenuated by AST (Fig. 5).

Significant progress has been made towards understanding the molecular mechanisms underlying the UV-induced TGF-β/Smad signaling pathway. It is reported that UV irradiation impairs TGF-β/Smad signaling through transcriptional downregulation of TβRII (7). The effect of AST on UVA-induced TβRII downregulation was further investigated. The results revealed that attenuated TβRII expression induced by UVA irradiation was significantly inhibited by AST pretreatment (Fig. 6).