Effects of baicalin against UVA-induced photoaging in skin fibroblasts.

The American Journal of Chinese Medicine, Vol. 42, No. 3, 709–727 © 2014 World Scientific Publishing Company Institute for Advanced Research in Asian Science and Medicine DOI: 10.1142/S0192415X14500463

Am. J. Chin. Med. 2014.42:709-727. Downloaded from www.worldscientific.com by NANYANG TECHNOLOGICAL UNIVERSITY on 06/05/14. For personal use only.

The Effects of Baicalin Against UVA-Induced Photoaging in Skin Fibroblasts Wei Min,*,†,a Xin Liu,‡,a Qihong Qian,*,a Bingjiang Lin,† Di Wu,† Miaomiao Wang,* Israr Ahmad,§ Nabiha Yusuf § and Dan Luo† *Department

of Dermatology, The First Affiliated Hospital of Soochow University, Suzhou, China

†

Department of Dermatology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China ‡

Department of Dermatology, Jiangsu Official Hospital Nanjing, China

§Department

of Dermatology and Skin Diseases Research Center University of Alabama at Birmingham, AL, USA

Abstract: Ultraviolet A (UVA) radiation contributes to skin photoaging. Baicalin, a plantderived flavonoid, effectively absorbs UV rays and has been shown to have anti-oxidant and anti-inflammatory properties that may delay the photoaging process. In the current study, cultured human skin fibroblasts were incubated with 50 g/ml baicalin 24 hours prior to 10 J/ cm2 UVA irradiation. In order to examine the efficacy of baicalin treatment in delaying UVA-induced photoaging, we investigated aging-related markers, cell cycle changes, antioxidant activity, telomere length, and DNA damage markers. UVA radiation caused an increased proportion of β-Gal positive cells and reduced telomere length in human skin fibroblasts. In addition, UVA radiation inhibited TGF-β1 secretion, induced G1 phase arrest, reduced SOD and GSH-Px levels, increased MDA levels, enhanced the expression of MMP-1, TIMP-1, p66, p53, and p16 mRNA, reduced c-myc mRNA expression, elevated p53 and p16 protein expression, and reduced c-myc protein expression. Baicalin treatment effectively protected human fibroblasts from these UVA radiation-induced aging responses, suggesting that the underlying mechanism involves the inhibition of oxidative damage and regulation of the expression of senescence-related genes, including those encoding for p53, p66 Shc and p16. Keywords: UVA; Fibroblast; Baicalin; Photoaging. Correspondence to: Dr. Dan Luo, Department of Dermatology, The First Affiliated Hospital of Nanjing Medical University, Hanzhong Road 140#, Nanjing, 210029, China. Tel: (þ86) 25-8371-4511, E-mail: daniluo2005@163. com or Dr. Nabiha Yusuf, Department of Dermatology, University of Alabama at Birmingham, 1670 University Boulevard, VH 566A, PO Box 202, Birmingham, Alabama 35294, USA. Tel: (þ1) 205-934-7432, E-mail: [email protected] a These authors contributed equally to this work.

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Introduction Skin aging is a complex biological process that is a consequence of both intrinsic, or genetically programmed aging that occurs with time, and extrinsic aging, caused by environmental factors such as ultraviolet (UV) radiation-induced cell aging, or photoaging, being the main contributor to the latter process (Grunebaum and Heffelfinger, 2011; Rock and Fischer, 2011). Ultraviolet A (UVA, wavelength of 320–400 nm) rays can reach the dermal layer of the skin and are one of the most important environmental factors that induce cutaneous photodamage and aging (Debac-Chainiaux et al., 2012; Polefka et al., 2012). Photoaging is not only characterized by changes in epidermal thickness, like atrophy or hyperplasia, but is also complicated by alterations in the dermal cells and matrix. With continuous improvements in cosmetic health and quality of life, the development of safe and highly effective sunscreens and anti-aging products has been given a high priority. The development of novel photo-protective products from natural plants and traditional herbs has gradually become a hot topic of research worldwide (Chanchal and Swarnlata, 2008; Nichols and Katiyar, 2010). Traditional Chinese medicine (TCM) has played a critical role in the prevention and treatment of diseases for thousands of years in China and other Asian countries. Baicalin (7-glucuronic acid 5, 6-dihydroxyflavone) is one of the most active constituents isolated from the dried root of Scutellaria baicalensis Georgi, which is well-known as one of the most widely used traditional medicinal herbs and is utilized as a key ingredient in many TCM formulations. Traditionally, baicalin was used for heat-clearing and detoxifying, as a diuretic, and to protect the gallbladder, among other actions. Recent studies have shown that baicalin elicits a wide range of pharmacological properties, such as anti-oxidant, antiinflammatory, and antitumor activities, as well as free radical scavenging ability. In addition, baicalin absorbs UV rays with high efficiency (Guo et al., 2011; Li et al., 2012; Chen et al., 2013). Our previous study showed that baicalin effectively inhibits UVBinduced damage to both human skin cells (including keratinocytes and fibroblasts) and mouse skin, by reducing inflammatory cytokine secretion and photoproduct production and accelerating the scavenging of cyclobutane pyrimidine dimers (CPDs) (Min et al., 2008; Zhou et al., 2011; Xu et al., 2012; Zhou et al., 2012). The present study was designed to examine the efficacy of baicalin on UVA radiation-induced changes in cultured fibroblasts, focusing on photoaging, and to explore the underlying mechanisms.

Materials and Methods Materials The following materials were used in the current study: SUV-100 solar simulator and radiant emittance monitor (Sigma Com. ShangHai); Dulbecco’s modified eagle medium (DMEM, Gibco/BRL, USA); β-Galactosidase kit (Mirus Bio Co, USA); Human TGF-β1 ELISA Kit (R&D Com, USA); 128C ELIASA (Clinibio Com, Austria); SOD GSH-Px and MDA assay kits (Nanjing Jiancheng Bioengineering Institute, China); ABI 7300 RealTime

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PCR System (Applied biotechnology company, USA); Genomic DNA Extracted Kit (Beyotime, China); reverse transcription kit and real-time quantitative PCR kit (TaKaRa Com., DaLian); TeloTAGGG Telomerase PCR ELISA kit (Roche, USA); rabbit monoclonal antibody against p53, p-16 and c-myc (Abcam, USA); baicalin (Sigma, USA, purity>95%).


Cell Culture and Subgroups The CCC-ESF-1 human embryonic skin fibroblast cell line was purchased from Union Medical University Cell Center and cultured in DMEM supplemented with 2 mM glutamine and 10% fetal bovine serum at 37 C in 5% CO2. Cells (1 10 6 /ml density) were plated into 6-well plates or 100-mm dishes for the follow-up experiments. When the fibroblasts grew to sub-confluence, they were then divided into the following subgroups: control group, baicalin group, UVA irradiation group, and UVAþbaicalin group. According to our previous study, baicalin was added into culture medium for 24 h before UVA irradiation at a 50 g/mL concentration (Min et al., 2008; Zhou et al., 2012). UVA Irradiation The UVA source consisted of a bank of 9 fluorescent bulbs (TL12, Philips) that emitted most of their energy within the UVA range (320–400 nm). The intensity of irradiation was measured with an IL443 phototherapy radiometer and an SED240/UV/W photodetector. Before UV irradiation, cells were washed and covered with phosphate-buffered saline (PBS). Monolayers of fibroblasts in a thin layer of PBS were irradiated with 10 J/cm2 UVA and then harvested as described in the next section. The experiment was repeated three times. Senescence-Associated ß-Galactosidase (SA-ß-Gal) Staining Fibroblasts for β-Galactosidase staining were irradiated by 10 J/cm2/day UVA for 2 weeks and collected. SA-β-gal staining was performed according to kit instruction. Briefly, cells were fixed in 2% formaldehyde/0.2% glutaraldehyde, washed with PBS and incubated at 37 C with fresh SA-β-Gal stain solution. Quantitative analysis of SA-β-gal signal intensity was performed using the AxioVision 4.7.1 software (Zeiss, Oberkochen, Germany). The senescence rate (%) ¼ the number of blue colored cells/the total cells 100%. Real-Time PCR for Telomere Length Telomere length of fibroblasts was measured by the quantitative PCR (qPCR) technique. The cells were harvested after 15 days of 10 J/cm2/day UVA irradiation. Genomic DNA was isolated using a Genomic DNA extraction kit according to the protocol provided. Experiments were performed as previously described (Gil and Coetzer, 2004). The


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telomere specific primer pair sequences were (5 0 to 3 0 ): tel1 5 0 -CGGTTTGTTTGGG TTTGGGTTTGGGTTTGGGTTTGGGTT-3 0 , tel2 5 0 -GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT-3 0 . 36B4 was used as the single copy gene primers: forward 5 0 -CAGCAAGTGGGAAGGTGTAATCC-3 0 , reverse 5 0 -CCCATTCTATCATCAACGG GTACAA-3 0 . The 20 L PCR reaction system was used, including SYBR Premix EX Taq 10 L, ROX Reference dye 0.4 L, each primer 0.4 L, DNA template 1 L, add DNAasefree water to 20 L. All PCRs were run on the ABI 7300 RealTime PCR System. The thermal cycling profile for both amplifications began with the 94 C incubation for 10 min to activate the Taq DNA polymerase followed by cycling for 5 s at 95 C and 31 s at 52 C for 40 cycles for telomere, or 5 s at 95 C and 31 s at 54 C for 40 cycles for 36B4. The PCR data were analyzed with ABI’s SDS v.1.7 software as the Ct i.e. the fractional cycle number at which the well’s accumulating fluorescence crossed a set threshold that is several standard deviations above baseline fluorescence. During this analysis, the amplification efficiency was calculated for each sample along with the mean efficiency of the run, which is used in calculating the relative concentration of each sample relative to the calibrator sample. This calculation coupled with the use of the same calibrator samples on all runs corrects for any inter-run variation. This process was done for both telomere (T) and single-copy (S) gene reactions, and telomere length was expressed as a ratio of C ðteloÞ C ð36B4Þ 1 the two, using the mean data from duplicate runs. T=S ratio ¼ ½2 t =2 t ¼ 2 ½Ct ðteloÞCt ð36B4Þ ¼ 2 Ct . To determine the relative telomere length, we used the Ct value of UVA group as the control. The relative T/S ratio is 2 ðCt1 Ct2 Þ ¼ 2 Ct . TRAP-ELISA for Telomerase Telomerase activity was detected by the TeloTAGGG Telomerase PCR ELISA kit according to the manufacturer’s protocol. Briefly, 2 10 5 cells per single reaction were centrifuged for 5 min at 4 C. The supernatant was removed, the cells were resuspended in PBS and centrifugation was repeated. Cells were resuspended in 100 ml lysis reagent for 30 min, and the lysate was centrifuged at 16,000 rpm for 20 min at 4 C. The supernatant was removed, and 1.25 ml (250 cell equivalents) was transferred into a new tube containing 48.75 ml lysis reagent; the cell extract aliquots were frozen in liquid nitrogen and stored at 80 C. Telomeric Repeat Amplification Protocol (TRAP) reactions were carried out on each sample. Briefly, 25 ml of reaction mixture and 5 ml of the internal standard were transferred into a tube. Cell extract (2 ml) corresponding to 10 cell equivalents was added. Nuclease-free water was added to a total volume of 50 ml. Tubes were transferred to the PCR Cycler to primer elongation/amplification reaction. Finally, the absorbance of each well was measured at a wavelength of 450 nm (reference wavelength, 690 nm) in 30 min, A ¼ A450 nm A690 nm . Samples are to be considered as telomerase-positive if the difference in absorbance (A ¼ AS-AS, 0) is higher than the 2-fold background activity (background activity 5 value of negative control or heat-treated sample). The relative telomerase of each sample was obtained using the formula proposed by the manufacturer, RTA ¼ [(AS-ASO)/AS,IS]/[(ATS8ATS8,0)/ATS8,IS] 100.


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TGF-ß 1 ELISA Twenty-four hours after UVA irradiation, the supernatant was collected and stored at 80 C in refrigerator until analysis. TGF-β1 total release was measured using a Human immunoassay ELISA kit according to manufacturer’s instructions.


SOD, GSH-Px and MDA Activity Assay To determine the enzymatic activity of SOD, GSH-Px and MDA in fibroblasts, cell suspensions were collected at 24 h after UVA irradiation and the enzymatic activity was measured according to the manufacturer’s instructions from the assay kits. Cell Cycle Analysis Fibroblasts were harvested at 24, 48, and 72 hours after UVA irradiation. The cells were then subjected to flow cytometric analysis according to the manufacturer’s recommendation. Briefly, the cells were fixed with ice cold 80% ethanol and kept at 20 C for 2 hours. The cells were washed three times with PBS, dissolved, and stained in 1 ml PI/Triton X-100 staining solution (0.1% Triton X-100 and RNase 20 mg/ml in PBS). The cells were then incubated for 30 min at 4 C and analyzed with a flow cytometer (BD FACSCalibur, USA). Quantitative Real-Time PCR Fibroblasts were harvested at 24 h after UVA irradiation and total RNA was isolated using 1 mL of Trizol reagent according to the manufacturer’s instructions. cDNA was synthesized in a 10 L reaction volume containing MgCl2 (25 mM), 10 RTBuffer, RNaseFree dH2O, dNTP Mixture (10 mM), RNase Inhibitor (40 U/L), AMV Reverse Transcriptase (5 U/L), Oligo dT-Adaptor Primer (2.5 pmol/L), and RNA. The reaction mixture was incubated for 1 h at 55 C followed by incubation at 95 C for 10 min. The cDNA product was stored at 80 C. The primers used for MMP-1, TIMP-1, p66 Shc , p53, p16 and c-myc were described as follows: MMP-1 for 5 0 -CTGCTTACGAATTTGCCGAC-3 0 , MMP-1 rev 5 0 -GCAGCATCGATATGCTTCAC-3 0 ; TIMP-1 for 5 0 -GATGGACTCTTGCACATCACT-3 0 , TIMP-1 rev 5 0 -TGGATAAACAGGGAAACACTG-3 0 ; p66 Shc for 5 0 -ACTACCCTGTGTTCCTTCTT TC-3 0 , p66 Shc rev 5 0 -TCGGTGGATTCCTGAGATACTGT-3 0 ; p53 for 5 0 -TGCCCAACAACACCAGCTC-3 0 , p53 rev 5 0 -CCAAGGCCTCATTCAGCTCTC-3 0 ; p16 for 5 0 CTGCTTACGAATTTGCCGAC-3 0 , p16 rev 5 0 -GCAGCATCGATATGCTTCAC-3 0 ; c-myc for 5 0 -AGGCTATTCTGCCCATTT-3 0 , c-myc rev 5 0 -TCGTAGTCGAGGTCATAGTTC-3 0 . The reaction mixture was prepared as follows: QuantiTect RT Mix, F and R primers (10 mol/L), 2 QuantiTect SYBR Green I RT-PCR Master Mix, cDNA and DNAase-free water. The PCR amplification was performed as follows: 10 min incubation at 95 C; 45 cycles for 15 s at 94 C, 20 s at 59 C, and 30 s at 72 C.

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Western Blotting Fibroblasts were harvested at 24 h after UVA irradiation and total proteins were purified using protein extraction reagents according to the manufacturer’s instructions. Different samples with an equal amount of protein (50 g) were separated on 12% SDS polyacrylamide gels, transferred to nitrocellulose membranes and blocked in 5% non-fat dry milk buffer. The membranes were incubated overnight at 4 C with the rabbit monoclonal antibody against p53 (1:1000), p16 (1:1000) and c-myc (1:10,000), followed by incubation with horseradish-peroxidase-conjugated secondary antibodies. Protein expression was detected with an ECL detection system and exposed on an X-ray film. Tubulin was used as a loading control. The optical densities of protein bands on the X-ray film were quantitatively analyzed with Quantity One software (Bio-Rad). Statistical Analysis Experimental data were presented as mean SD. Statistical analyses were performed using ANOVA followed by LSD test for individual comparisons between group means. p < 0:05 was considered statistically significant. All analyses were performed using SPSS 11.0 software. Results Baicalin Decreases the Proportion of ß-Galactosidase (ß-Gal) Positive Cells After fibroblasts were incubated with 50 g/ml baicalin and/or 10 J/cm2 UVA irradiation for 2 weeks, senescence cells were examined using SA-β-Gal cytochemical staining. The results showed that the rate of senescence cells in the baicalin group and the control group are both low. There was no statistical significance between the baicalin treated and untreated UVA irradiated fibroblasts (p > 0:05). In contrast, UVA irradiation induced an increased expression of SA-β-Gal-positive cells to 71.36%. Incubation with baicalin significantly decreased the number of SA-β-Gal-positive cells to approximately 30.62% compared to the UVA group, suggesting that baicalin could slow the photoaging process ( p < 0:05, Fig. 1). Effect of Baicalin on Telomere Length and Telomerase Activity A real-time qPCR assay was performed to detect changes in telomere length of the fibroblasts after exposure to UVA radiation. The results showed that baicalin treatment alone did not cause significant changes in telomere length in fibroblasts (p > 0:05). The telomere length of fibroblasts was significantly reduced following UVA radiation at a dose of 10 J/cm2 per day for 2 successive weeks. When the telomere length in the UVAirradiated cells was used as the baseline control, the telomere length of normal fibroblasts was 3.21 0.23, which was significantly longer than that in the UVA-treated cells (p < 0:05); and the telomere length of fibroblasts in the baicalin-treated cells was 2.99 0.15, which was not significantly different from that in the control cells (p > 0:05).


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(B) Figure 1. Effect of baicalin on UVA-induced photoaging of human dermal fibroblasts by using cytochemistry staining of SA-β-Gal (original magnification 100). Cells were incubated with baicalin and/or UVA irradiated. The number of SA-β-Gal positive stained cells showed an increasing trend in the UVA group compared to the control and baicalin only treated group. Treatment of fibroblasts with baicalin prior to UVA exposure caused a further increase in the senescence rate. The percentage of SAβ-Gal positive cells are presented as mean SD of three independent experiments, # p < 0:01 relative to control group at 14d post-irradiation.

In cells that were both irradiated with UVA and also treated with baicalin, the telomere length of the fibroblasts recovered to 63.9% of that of normal fibroblasts, which was also significantly longer than that in the cells exposed to UVA (p < 0:05, Fig. 2). The telomerase activity in human skin fibroblasts was determined using the TRAP-ELISA Telomerase Detection Kit. The results showed that UVA radiation and baicalin treatment induced changes in relative telomerase activity (RTA). But the A value of all groups was lower than two times of background activity, which indicated that the telomerase activity of fibroblasts was negative in all treatment groups. Thus, UVA radiation and/or baicalin treatment did not significantly promote the expression of telomerase activity. Effect of Baicalin on TGF-ß1 Secretion in the Supernatant of Human Skin Fibroblasts Following Exposure to UVA Radiation Cultured human fibroblasts exposed to UVA radiation were treated with baicalin, or treated with vehicle alone as the negative control. Baicalin treatment increased the TGF-β1 levels in the culture supernatant of human skin fibroblasts from 1343.58 45.58 pg/ml (in the control cells) to 2096.43 49.51 pg/ml (after drug treatment). Irradiation with 10 J/cm2

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(B) Figure 2. Effect of baicalin on changes in telomere length and telomerase activity. Cells were incubated with baicalin and/or 10 J/cm2 UVA irradiation for 2 weeks. Relative telomere length was calculated as the ratio of the amount of telomere DNA versus single copy DNA (T/S ratio). Data are expressed as mean SD of three individual experiments. # p < 0:05 relative to control group at 14d post-irradiation. For telomerase activity, A values of all groups were lower than two times of background activity, which indicated that the telomerase activity of fibroblasts was negative in all treatment groups.

UVA reduced the TGF-β1 level to 818.97 37.99 pg/ml, while the TGF-β1 secretion was increased to 1848.32 74.45 pg/ml following baicalin treatment. There were significant differences between the control and baicalin-treated groups (p < 0:05, Fig. 3). Effect of Baicalin on Changes in Superoxide Dismutase (SOD), Malondialdehyde (MDA), and Glutathione peroxidase (GSH-Px) Activities After 24 h of irradiation with 10 J/cm2 UVA, the activities of SOD and GSH-Px were significantly reduced in fibroblasts in comparison with those in the control cells (p < 0:05), while the MDA level was significantly increased (p < 0:05), indicating that UVA



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Figure 3. Effect of baicalin on TGF-β1 secretion in the supernatant of human skin fibroblasts following exposure to UVA radiation. The concentration of TGF-β1 are presented as mean SD. # p < 0:01 relative to control group at 24 h post-irradiation.

decreases the capacity for cellular anti-oxidant defense and causes oxidative damage. The SOD and GSH-Px activities were significantly increased in the baicalin-treated cells ( p < 0:05), compared to the activities in the control cells and in the UVA-irradiated cells. The MDA level was significantly reduced in the baicalin-treated cells, compared to the activities in the control cells and in the UVA-irradiated cells (p < 0:05). In addition,

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(C) Figure 4. Effect of baicalin on changes of SOD, MDA and GSH-Px activities. The enzymatic activity of OD, MDA and GSH-Px were assayed and presented as mean SD. # p < 0:01 relative to control group at 24 h post-irradiation.

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(B) Figure 5. Effect of baicalin on UVA induced G1 phase arrest. Cells were incubated with baicalin for 24 h before UVA irradiation. After 24, 48, and 72 h, the cell cycle was analyzed using flow cytometry. (A) Representative cell cycle distribution in human dermal fibroblasts. (B) G1 phase ratios are presented as mean SD of three individual experiments. # p < 0:05 relative to control group.

baicalin treatment significantly inhibited UVA-induced MDA production (4.85 0.6) in baicalin pre-treated cells, compared with UVA-irradiated cells not treated with baicalin (9.84 0.7) ( p < 0:05, Fig. 4). Effect of Baicalin on Changes in the Cell Cycle G1 phase arrest of the cell cycle is an important mechanism for the response to UV radiation-induced damage in human cells; however, a prolonged G1 phase arrest may also cause cell aging. After 24 h of irradiation with 10 J/cm2 UVA, G1 phase arrest was easily detected in fibroblasts, and the proportion of fibroblasts in G1 increased from 59.94% in the control cells to 81.04% in UVA-treated cells. With the prolongation of incubation time, the proportion of fibroblasts in G1 increased slightly, and rose to 89.09% after 72 h of radiation. With baicalin treatment, the proportion of fibroblasts in G1 was reduced to 65.55% at 24 h after radiation, indicating that baicalin significantly alleviated UVA-induced cell cycle arrest ( p < 0:05, Fig. 5). Effect of Baicalin on Changes in Matrix Metalloproteinase (MMP)-1, Tissue Inhibitor of Metalloproteinase (TIMP)-1, p66 Shc , p53, p16, and c-myc mRNA Levels The effects of UVA radiation and baicalin treatment on the expression of genes involved in dermal photoaging and the cellular response to DNA damage were assessed using qPCR.



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Figure 6. Effect of baicalin on changes in MMP-1, TIMP-1, p53, p16, and c-myc mRNA levels. Quantitative real-time PCR was used to analyze mRNA level. Data are expressed as mean SD of three individual experiments. # p < 0:05 relative to control group at 24 h post-irradiation.

The results showed that baicalin treatment alone did not cause significant changes in the expression of MMP-1, p66 Shc , p53, or p16 mRNA, compared to the mRNA levels in the control cells (p > 0:05). Further, TIMP-1 mRNA expression was slightly elevated, but not significantly different (p > 0:05), in comparison with the control group. Irradiation with 10 J/cm2 UVA caused the significantly elevated the expression of MMP-1, TIMP-1, p66 Shc , p53, and p16 mRNA (p < 0:05). Baicalin treatment prior to UVA radiation significantly reduced the expression of MMP-1, p66 Shc , p53, and p16 mRNA in comparison with the levels in the UVA-treated cells (p < 0:05), but the mRNA levels were still higher

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(B) Figure 7. Effect of baicalin on the expression of p53, p16, and c-myc proteins. Representative protein expression bands of p16 and c-myc in human dermal fibroblasts were detected by western blot (A), and the data were summarized in (B). Data are expressed as mean SD of three individual experiments. # p < 0:05 relative to control group at 24 h post-irradiation.

than those in the control cells ( p < 0:05). Following baicalin treatment and UVA radiation, TIMP-1 mRNA expression did not exhibit a decrease and, in fact, was significantly higher than that observed in the control cells (p < 0:05). However, TIMP-1 expression was not significantly different from the level in the UVA-treated cells ( p > 0:05). In addition, baicalin treatment decreased c-myc mRNA expression, while UVA radiation further reduced c-myc mRNA expression (p < 0:05). Treatment with baicalin after UVA radiation caused a slight recovery of c-myc mRNA expression, but this recovered level was not significantly different from that in the UVA-irradiated cells (p < 0:05, Fig. 6). Effect of Baicalin on Expression of p16 and c-myc Proteins Baicalin treatment alone did not cause significant changes in p53 or p16 protein expression in comparison with control cells ( p > 0:05). On the other hand, c-myc protein expression


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was significantly reduced in the baicalin-treated cells, compared with the control cells ( p < 0:05). Irradiation with 10 J/cm2 UVA caused a significant elevation in p53 and p16 protein expression and a further reduction in c-myc protein expression (p < 0:05). Baicalin treatment after UVA radiation caused a significant reduction in p53 and p16 protein expression, compared with that in the UVA-irradiated cells (p < 0:05), while no significant changes in c-myc protein expression were detected (p < 0:05, Fig. 7).


Discussion Skin aging is a complex biological phenomenon. Unlike intrinsic aging, photoaging can be prevented and alleviated. Fibroblasts are the major target site of UVA radiation, and they play an important role in modulating the changes in aging-specific biological characteristics of skin. SA-β-Gal serves as an aging-related biologic marker, and the proportion of β-Gal expression can partly demonstrate the degree of aging in the tested cells. In this study, an obvious aging phenomenon was observed in primary cultured human skin fibroblasts after they were successively irradiated with UVA at a dose of 10 J/cm2 per day for two weeks. This irradiation-induced aging was characterized by increases in β-Galpositive cell counts by 442%, in comparison with that in the non-irradiated cells. In addition, the β-Gal-positive cell counts in the baicalin-treated cells were reduced by 66.8% compared to that in the irradiated cells, indicating that baicalin slows the UVA radiationinduced cell aging process. Telomeres are regions of repetitive DNA sequences at the ends of eukaryotic chromosomes, and their length is continuously shortened with increasing cycles of cell division. When telomeres are shortened beyond a certain length, cell replication ceases, and aging or even death, may result. The clinical pathology and pathogenesis of cutaneous photoaging is different from that of natural aging of the skin, but both have the same end effector pathway — telomere shortening. UV radiation causes ROS production and DNA damage in skin cells, which promotes telomere shortening (Gilchrest et al., 2009; Stout and Blasco, 2013). Considering that high proportions of TT and G bases in the telomere structure are the target sites of UV radiation-induced DNA damage, UV radiation causes more severe damage to telomeric DNA in comparison with other sites on the chromosomes. Telomerase is directly involved in the maintenance of the telomeric regions and based on the template supplied by telomeric RNA; telomerase synthesizes telomeric DNA to maintain telomere length and slows cell aging. Telomerase reactivation is considered as a major mechanism of escape from cellular senescence. Recent studies have indicated that some kinds of traditional Chinese medicines can mediate the elongation of telomere length and enhance telomerase activity to slow the cellular aging process (Chan et al., 2010; Yung et al., 2012). Telomere shortening or damage leads to the disruption of the mitotic cycle, thus inducing cell cycle arrest, which is a major event and key characteristic of the cellular aging process. In this study, UVA radiation clearly induced G1 phase arrest in fibroblasts. Baicalin treatment significantly inhibited the occurrence of G1 phase arrest, and our results showed that this effect may be associated with the reduction of cellular DNA damage. In addition, changes in fibroblast telomere length were also observed. Our results showed that baicalin


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treatment alone did not elongate the telomere length of fibroblasts, indicating that baicalin has no effect on the natural aging process of fibroblasts. Following successive UVA radiation baicalin treatment significantly restored the telomere length of fibroblasts, which can perhaps be explained by one or both of the following mechanisms: (1) Baicalin reduces telomere length shortening by alleviating UVA radiation-induced DNA damage; (2) Baicalin is directly involved in promoting the formation of telomeric DNA, by activating telomerase to help maintain telomere length. In order to clarify the specific mechanism involved in baicalin-induced telomere maintenance, we measured the telomerase activity of fibroblasts that were or were not exposed to UVA radiation, in the presence or absence of baicalin treatment. The results showed that baicalin treatment attenuates telomere length shortening and slows the photoaging process, which is likely to be associated with the biological activities of baicalin, such as anti-oxidant properties and the ability to absorb UV radiation, but probably does not involve direct effects on the telomerase activity. To further explore the mechanism of baicalin-mediated protection against photoaging, the changes in TGF-β1 secretion in the supernatant of cultured human skin fibroblasts were detected. TGF-β1, a multifunctional cytokine, is a positive growth factor of the dermis, which also promotes synthesis of extracellular matrix (ECM) and inhibits collagenase production (Lang et al., 2011; Cho et al., 2012). Previous studies showed that autocrine secretion of TGF-β1 by fibroblasts is reduced in aging skin, and the cellular responses to TGF-β1 stimulation are also attenuated (Huh et al., 2007). The results of the present study showed that baicalin treatment alone significantly elevated the production of TGF-β1 in cultured fibroblasts. Following UVA radiation, the production of TGF-β1 was significantly reduced. Baicalin treatment before UVA irradiation resulted in the recovery of TGF-β1 levels. These results suggest that elevated TGF-β1 secretion may be one mechanism involved in baicalin-mediated protection against photoaging. One of the histological changes in photoaging skin is collagen degradation (Hwang et al., 2011; Cherng et al., 2012). It has been shown that MMPs play an important role in the collagen degradation process (Rijken and Bruijnzeel, 2009). UV radiation induces the expression of MMPs through the activation of the mitogen-activated protein kinase (MAPK) signal transduction pathway and by increasing the expression of transcription factor activator protein 1 (AP-1) (Dickinson et al., 2011; Lope-Camarillo et al., 2012; Hwang et al., 2013). Moreover, studies have confirmed that the reduction of collagen in naturally aging skin is mainly associated with a decrease in collagen synthesis in fibroblasts, but that it is also associated with an increase in secretion of MMPs in photoaging skin. MMP-1 is an important collagenase which mainly hydrolyzes collagen type I and induces the hydrolysis, destruction, and restructuring of the ECM, leading to the clinical manifestations of skin aging (Moon et al., 2009; Kim et al., 2010). Matrix metalloproteinase inhibitors, including TIMP-1, TIMP-2, TIMP-3, and TIMP-4, have the ability to inhibit MMP activity (Bonnema et al., 2007). TIMP-1 mainly inhibits the activity of MMPs 1, 3, and 9. The balance between TIMP and MMP activities determines whether the protease is activated or inhibited (Chirco et al., 2006; Vo et al., 2013). In this study, we observed that the expression of MMP-1 mRNA increased significantly after UVA irradiation, and that baicalin treatment inhibited UVA radiation-induced MMP-1 expression. At



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the same time, baicalin treatment alone induced a modest increase in TIMP-1 mRNA expression. A previous report showed that UV irradiation of 1–2 minimal erythemic doses (MED) did not affect TIMP-1 mRNA expression in human skin fibroblasts (Naru et al., 2005). However, we found that UVA irradiation increased TIMP-1 mRNA levels in fibroblasts, and baicalin intervention further promoted TIMP-1 expression, compared to untreated cells, after UVA irradiation. These results suggest that baicalin can reduce collagen degradation and alleviate the UVA-induced changes in photoaging skin performance by inhibiting MMP-1 expression and enhancing TIMP-1 expression. In skin cells, UVA induces the production of high concentrations of reactive oxygen species (ROS), which cause oxidative damage to many biological macromolecules, such as those in the cell membrane, proteins, and nucleic acids (Bossi et al., 2008; Svobodova et al., 2008). ROS play an important role in the initiation and progression of skin aging. MDA is one of the end products of lipid peroxidation, and its level reflects the severity of free radical attack on cells (Hseu et al., 2012). Therefore, MDA is regarded as a common indicator for assessing lipid peroxidation. Normal cells have a defense system against ROS, including anti-oxidant enzymes like SOD and GSH-Px (Ali et al., 2010; Song et al., 2012). The results of this study showed that UVA radiation significantly reduced the activities of SOD and GSH-Px and significantly elevated MDA levels in fibroblasts, indicating that UVA decreases the ability of human dermal tissues to scavenge ROS and causes oxidative damage. Baicalin treatment by itself significantly increased SOD and GSH-Px activities and reduced MDA levels in fibroblasts. Baicalin treatment also significantly inhibited the changes in the activities of SOD, GSH-Px and MDA levels induced by UVA radiation. These results demonstrated that baicalin has the ability to protect fibroblasts from UVAinduced oxidative damage and that it effectively increases the anti-oxidant activity of fibroblasts enabling the cellular systems to scavenge oxygen free radicals. To further explore the underlying anti-oxidative mechanism invoked by baicalin, the expression of p66 Shc mRNA in fibroblasts was determined. P66 Shc is a product of a protooncogene and is an isoform of the Src homology 2-domain-containing (SHC) transforming protein. The p66 Shc gene has been shown to be involved in the regulation of oxidative stress, cell senescence, and related signaling pathways (Wang et al., 2010; Suski et al., 2011). It was reported that p66 Shc knockout causes a low oxidative stress in mice (Giorgio et al., 2012). It has been found that UV radiation induces p66 Shc activation, which stimulates ROS production and regulates a series of signaling pathways involving oxidative stress responses (Gertz et al., 2009). The results of the present study showed that baicalin treatment alone had no significant effect on p66 Shc mRNA expression, while UVA radiation significantly elevated p66 Shc mRNA expression in fibroblasts. Moreover, baicalin treatment inhibited the UVA radiation-induced up-regulation of p66 Shc , which suggested that the anti-oxidant effect of baicalin may be associated with the inhibition of p66 Shc mRNA expression. Further studies need be carried out to investigate the signaling pathways involved in this process. In the present study, the changes in the expression of p53, p16, and c-myc genes were also determined. Telomere loop (T-loop) ruptures have been shown to occur when telomeres are shortened to a certain length. Then, the single-stranded-DNA overhang at the end


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of the T-loop is exposed, which leads to the activation of the tumor suppressor p53-based signal pathway and induces cellular senescence or apoptosis (Gilchrest, 2011). Changes in expression and function of the p16 gene have also been shown to be an important cause of G1 phase arrest of cell cycle after the induction of DNA damage (Munday et al., 2011). Inhibition of p16 expression can reduce telomere shortening and slow cell aging in the absence of telomerase activation (Shao et al., 2008). The present study showed that UVA radiation induced a significant elevation of the levels of p53 and p16 mRNA, as well as protein levels, while baicalin treatment inhibited UVA radiation-induced p53 and p16 expression, which may be the molecular mechanism of baicalin-mediated protection against photoaging. In addition, the proto-oncogene c-myc has a major role in regulating the cell cycle, and the overexpression of c-myc leads to shortening of the cell cycle and acceleration of cell division (Calado et al., 2012). The results of the present study showed that UVA radiation down-regulates c-myc mRNA and protein expression. Treatment with baicalin alone inhibited c-myc expression, but had no effects on the changes in c-myc expression induced by UVA radiation. Recently, baicalin was shown to exhibit antitumor activity by inhibiting tumor cell proliferation and promoting apoptosis of tumor cells (Kumagai et al., 2007; Parajuli et al., 2009). Therefore, the inhibition of c-myc expression may be one of the antitumor activities of baicalin. In summary, UVA radiation causes oxidative damage in skin fibroblasts and leads to the occurrence of stress-induced senescence. Baicalin effectively protects human skin fibroblasts from oxidative damage and slows the photoaging process. Baicalin-mediated antiphotoaging mechanisms may be associated with slowing of telomere shortening and regulation of aging-related genes such as p66 Shc , p53, p16, c-myc, MMP-1, and TIMP-1.

Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (30876232), science project from the Traditional Chinese Medicine Bureau of Jiangsu Province (LZ11095) and Jiangsu National Natural Science Foundation (BK2012170). References Ali, D., R.S. Ray and R.K. Hans. UVA-induced cyototoxicity and DNA damaging potential of benz (e) acephenanthrylene. Toxicol. Lett. 199: 193–200, 2010. Bonnema, D.D., C.S. Webb, W.R. Pennington, R.E. Stroud, A.E. Leonardi, L.L. Clark, C.D. McClure, L. Finklea, F.G. Spinale and M.R. Zile. Effects of age on plasma matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs). J. Card. Fail. 13: 530–540, 2007. Bossi, O., M. Gartsbein, M. Leitges, T. Kuroki, S. Grossman et al. UV irradiation increases ROS production via PKCdelta signaling in primary murine fibroblasts. J. Cell Biochem. 105: 194– 207, 2008. Calado, D.P., Y. Sasaki, S.A. Godinho, A. Pellerin, K. Kochert, B.P. Sleckman, I.M. de Alboran, M. Janz, S. Rodig and K. Rajewsky. The cell-cycle regulator c-Myc is essential for the formation and maintenance of germinal centers. Nat. Immunol. 13: 1092–1100, 2012.



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Effect of Microalgal Extracts of Tetraselmis suecica against UVB-Induced Photoaging in Human Skin Fibroblasts.

Molecular mechanisms of green tea polyphenols with protective effects against skin photoaging.

The protective effect of baicalin against UVB irradiation induced photoaging: an in vitro and in vivo study.

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Pinus densiflora extract protects human skin fibroblasts against UVB-induced photoaging by inhibiting the expression of MMPs and increasing type I procollagen expression.

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New insights in photoaging, UVA induced damage and skin types.

Photoaging.

Ethanol extract of Dalbergia odorifera protects skin keratinocytes against ultraviolet B-induced photoaging by suppressing production of reactive oxygen species.

Beneficial effects of dried pomegranate juice concentrated powder on ultraviolet B-induced skin photoaging in hairless mice.

Plant extracts and natural compounds used against UVB-induced photoaging.

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Skin photosensitizing agents and the role of reactive oxygen species in photoaging.

Polycomb group proteins: Novel molecules associated with ultraviolet A-induced photoaging of human skin.

Effect of photoaging on skin test response to histamine independent of chronologic age.

The protective effect of Kaempferia parviflora extract on UVB-induced skin photoaging in hairless mice.