International Journal of Nutrition, Pharmacology, Neurological Diseases

ORIGINAL ARTICLE
Year
: 2012  |  Volume : 2  |  Issue : 1  |  Page : 31--39

Sesamol modulates ultraviolet-B-induced apoptotic and inflammatory signaling in human skin dermal fibroblasts


Samivel Ramachandran, Nagarajan Rajendra Prasad 
 Department of Biochemistry and Biotechnology, Annamalai University, Annamalainagar, India

Correspondence Address:
Nagarajan Rajendra Prasad
Department of Biochemistry and Biotechnology, Annamalai University, Annamalainagar 608 002, Tamilnadu
India

Abstract

Aim: Sesamol (SM), a dietary phenolic phytochemical, has been shown to reduce ultraviolet-B (UVB) mediated oxidative damage. The aim of the present study was to investigate the protective mechanism of SM against UVB-induced photoaging, inflammatory and apoptotic signaling in human skin dermal fibroblasts, adult (HDFa) in vitro. Materials and Methods: In this study, we examined the effect of SM on UVB radiation-induced loss of mitochondrial membrane potential (ΔΨm), DNA fragmentation, cell cycle modulation, inflammatory markers [tumor necrosis factor (TNF)-α and nuclear factor (NF)-κB] expression, pro-apoptotic (p53, Bax and caspase-3), and anti-apoptotic marker (Bcl-2) expression in HDFa. We also investigated the effect of SM and/or UVB radiation on matrix metalloproteinase (MMP-2 and MMP-9) activation by gelatin zymograpy in HDFa. Results and Conclusion: SM pretreatment prevented UVB-induced ΔΨm alteration, DNA fragmentation and down-regulated the expressions of apoptotic (p53, Bax and caspase-3) and inflammatory markers (TNF-α and NF-κB) in HDFa. SM also prevented the activation of MMP-2 and MMP-9 in a concentration-dependent manner. Our data indicated the ability of SM to block UVB-induced inflammatory and apoptotic signaling in HDFa. However, further detailed mechanistic approach warrants before claiming this compound for photoprotection.



How to cite this article:
Ramachandran S, Prasad NR. Sesamol modulates ultraviolet-B-induced apoptotic and inflammatory signaling in human skin dermal fibroblasts.Int J Nutr Pharmacol Neurol Dis 2012;2:31-39


How to cite this URL:
Ramachandran S, Prasad NR. Sesamol modulates ultraviolet-B-induced apoptotic and inflammatory signaling in human skin dermal fibroblasts. Int J Nutr Pharmacol Neurol Dis [serial online] 2012 [cited 2019 Jun 16 ];2:31-39
Available from: http://www.ijnpnd.com/text.asp?2012/2/1/31/93131


Full Text

 Introduction



Humans and animals are constantly exposed to several toxic substances in day-to-day life. [1] Changes in lifestyle over the past several decades, including much of the time spent outdoors and the use of tanning devices for cosmetic purposes by individuals, have led to an increase in the incidence of solar ultraviolet (UV) radiation-induced skin diseases, including the risk of skin cancers. A wide variety of natural phytochemicals of several fruits and vegetables have substantial anti-carcinogenic activity because of their antioxidant and anti-inflammatory properties. [2] UV radiation, particularly UVB, elicits a wide spectrum of biological effects on skin. Acute exposure to UVB radiation causes a variety of adverse skin reactions including erythema, edema, sunburn cells, hyperplasia, inflammation and immunosuppression, while chronic UVB exposure leads to skin carcinogenesis and premature skin aging. [3] Human skin keratinocytes and fibroblasts are natural target cells of UVB in epidermal and dermal potions. [4] In vitro exposure of these cells to high amounts of UVB leads to activation of many cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1α (IL-1α), IL-6, IL-10 and IL-8. [5] These cytokines are responsible for the development of UVB-induced cutaneous inflammatory response. [6] Further, acute UVB exposure results in the appearance of apoptotic cells within human epidermis in vivo[7] and in different cell types in vitro. [8] UV radiation-induced DNA damages are considered to be an important event in the activation of apoptotic signaling. It has been already established that p53 is activated during UV light-induced DNA damage. [9]

Reactive oxygen species (ROS) formed during UV exposure may act as a second messenger and modulate a variety of protein phosphorylation signal transduction pathways. [10] In addition, the death-suppressing proto-oncogene Bcl-2 plays a major role in UV-induced apoptosis. [11] Bcl-2 potently blocks Bax activation and translocation to the mitochondria, [12] preventing cytochrome-C release and caspase activation response to UVB radiation. The death-suppressing activity of Bcl-2 is regulated by Bax, which promotes cell death. The ratio of these two proteins is considered to be important when determining whether the cell undergoes apoptosis after UVB exposure. [13] Further, UVB-induced ROS activates matrix metalloproteinases (MMPs) in both epidermis and dermis, degrades collagen and other components (e.g. elastin) in the dermal extracellular matrix, [14] and eventually manifests as clinically observable macro-scars (i.e., wrinkles) termed photoaging. [15]

One approach to protecting humans from the harmful effects of UV irradiation is to use active photoprotectors. Much attention has recently been focused on naturally occurring antioxidants which provide effective protection from UV-induced damage. [16] Oral administration of a combination of the antioxidants, ascorbic acid and α-tocopherol, in humans significantly reduced both the ROS and the formation of thymidine dimers. [17] The health-promoting properties of sesame oil (Sesamum indicum) have been documented in folklore medicine. Sesame oil contains a number of phytochemicals such as sesamolin, sesamin and sesamol (SM). [18] SM is a potent phenolic antioxidant contained only in processed sesame oil. After roasting sesame oil, the sesamolin content is lost, but the content of the derivative compound, SM, is increased. [19] Previous studies showed that SM can act as a metabolic regulator and possesses chemopreventive, antimutagenic, antihepatotoxic and antiaging properties. [20],[21],[22] SM was found to induce growth arrest and apoptosis in cancer and cardiovascular cells. [23] In addition, SM was found to enhance both vascular fibrinolytic capacity through regulating gene expression of a plasminogen activator and nitric oxide (NO) release in endothelial cells. [24],[25] We have reported antioxidant and free radical scavenging property of SM [26] and its protective effect against UVB and γ-irradiation-induced cellular changes in human blood lymphocytes and human skin dermal fibroblasts (HDF). [27],[28] In this present study, to further define the anti-photodamaging mechanism of SM, we evaluated the effect SM on UVB-induced oxidative stress-mediated activation of inflammatory and apoptotic signaling pathways in HDF, adult (HDFa).

 Materials and Methods



Chemicals

HDFa-500K cells/vial were purchased from Invitrogen Bioservices, India (Catalogue No.: C0135C). Medium 106 (Catalogue No.: M-106-500), Low Serum Growth Supplement (LSGS; Catalogue No.: S-003-10), fetal bovine serum, hydrocortisone, human epidermal growth factor, basic fibroblast growth factor, heparin, trypsin/ethylenediaminetetraacetic acid (EDTA) solution (Catalogue No.: R-001-100) and trypsin neutralizer solution (Catalogue No.: R-002-100) were purchased from Casecade Biologics, Invitrogen Bioservices, India. Ursolic acid, rhodamine-123, monoclonal antibodies anti-TNF-α, anti-nuclear factor (NF)-κB, anti-p53, anti-Bax, anti-Bcl-2, anti-caspase-3 and anti-β-actin anti-mouse and goat anti-mouse IgG-HRP polyclonal antibody were purchased from Sigma chemical Co., St. Louis, MO, USA. Bovine serum albumin (BSA), radioimmunoprecipitation assay (RIPA) buffer, 4′6-diamidino-2-phenylindole (DAPI) and gelatin were purchased from Himedia, Mumbai. All other chemicals, solvents and other analytical grades were obtained from SD Fine Chemical, Mumbai, and Fisher Inorganic and Aromatic Limited, Chennai.

Culturing human skin fibroblasts

HDFa cells were cultured at 37°C in 5% CO 2 in medium-106 (Casecade Biologics, Invitrogen, India) supplemented with low serum growth supplements (LSGS) kit containing 2% v/v fetal bovine serum, 1 μg/ml hydrocortisone, 10 ng/ml human epidermal growth factor, 3 ng/ml basic fibroblast growth factor, 10 μg/ml heparin and antibiotics. The cells were allowed to grow for 7 days to reach the maximum confluence. Then, the cells were sub-cultured and used for experiments.

Study design

Cultured fibroblasts were divided into six groups as follows:

Group 1: Normal fibroblasts without any treatment;

Group 2: Normal fibroblasts with 80 μM of SM;

Group 3: UVB-irradiated fibroblasts;

Group 4: UVB-irradiated fibroblasts pretreated with 8 μM of SM;

Group 5: UVB-irradiated fibroblasts pretreated with 40 μM of SM; and

Group 6: UVB-irradiated fibroblasts pretreated with 80 μM of SM.

Treatment of the HDFa cells

Thirty minutes prior to irradiation, three test doses (8, 40 and 80 μM) of SM were added to the grouped HDFa cells. Preliminary studies were carried out to check whether these concentrations had any toxic effect by conducting trypan blue dye exclusion test. Before exposure to UV light, the cells were washed twice with phosphate buffered saline (PBS) solution. Non-irradiated HDFa showed no decrease in viability over the 30-min period of incubation.

Irradiation procedure

Cultures of HDFa were washed once with PBS and exposed to UVB radiation in a thin layer of culture medium without fetal bovine serum (FBS)The culture medium was later removed and covered with a UV-permeable membrane to prevent contamination. A battery of TL 20 W/20 fluorescent tubes (Heber Scientific, Chennai, India) served as UVB source, which had a wavelength range set at 290-320 nm, peaked at 312 nm, and with an intensity of 2.2 mW/cm 2 for 9 min. The total UVB irradiation was 19.8 mJ/cm 2 , with an average value of 1.52 × 10−3 mJ/cell. After irradiation, the HDFa cells were kept at room temperature for 4 h at 37°C in 5% CO 2 incubator. Cells were then washed twice with PBS, scraped gently and transferred to sterile centrifuge tubes for further analysis.

Changes in mitochondrial transmembrane potential (ΔΨm)

The mitochondrial membrane potential (ΔΨm) changes were measured by Rh-123 staining. After incubation with 1 μl of Rh-123 (10 mmol/l), the cells were kept in 5% CO 2 incubator for 15 min, as described in the method of Prasad et al. [29] Then, the cells were washed with PBS and viewed under fluorescence microscope using blue filter (450-490 nm). Polarized mitochondria were marked by orange-red fluorescence and depolarized mitochondria were marked by green fluorescence. The fluorescence intensity was measured at 535 nm.

Detection of apoptotic cells

The intracellular apoptotic bodies were measured by DAPI staining in UVB plus SM treated HDFa. [30] Fibroblast cultures grown in six-well culture plates were washed with PBS and stained with DAPI dye (8 μg/ml) for 5 min. The slide were examined using a fluorescence microscope with 330/380 nm excitation filter and LP 440 nm barrier filter under ×100 magnification. In each sample, a minimum of 400 cells was counted, and cells having condensed or fragmented nuclei were expressed as percentage of total cells.

Flow cytometric analysis of cell cycle

The DNA-specific fluorochrome, propidium iodide (PI), can identify a distinct hypo-diploid cell population. [31] After UVB-irradiation and/or SM treatment, the cells were kept at 37°C for 4 h. [32] 1.0 × 10 6 HDFa cells were washed with PBS and fixed in 70% ethanol, then washed twice with PBS and treated with RNase-A for 30 min at 37°C. Finally, the cells were stained with PI and incubated in the dark for 30 min. The distribution of cells in the different cell cycle phases was analyzed using Bectone Dickinson FACS Vantage flow cytometer and Cell Quest software. [33]

Western blot analysis for apoptotic and inflammatory markers expression

Immunoblot analysis was carried out for TNF-α, NF-κB, p53, Bax, Bcl-2 and caspase-3 expressions in SM plus UVB-irradiated HDFa. The results were normalized to β-actin gene expression.

Cultured cells were washed with PBS solution and detached from the culture dishes using a rapid treatment with trypsin/EDTA. Cell suspensions were centrifuged at 1000 rpm for 10 min and the pellets were lysed with an ice-cold lysis RIPA buffer [50 mM; Tris-HCl pH 7.4; 1% NP-40; 150 mM NaCl; 2 mM EDTA; 0.1% sodium dodecyl sulfate (SDS); 1 mM ethylene glycol tetraacetic acid (EGTA); 1 mM phenylmethanesulfonylfluoride (PMSF); 0.15% bME] containing a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) for 30 min. The lysate was cleared by centrifugation at 4°C for 10 min at 14,000 rpm and the supernatant was used to determine the protein concentration of the lysates using the Lowry protein assay. [34] Cell extracts containing 50 μg of proteins were fractionated on 12% SDS-PAGE gel and transferred to a nitrocellulose acetate membrane (Amersham Biosciences, Piscataway, NJ, USA) using Biorad semi-dry apparatus (Biorad, USA). Nitrocellulose membranes were blocked with 5% (w/v) nonfat milk (blocking solution) in Tris Buffer Saline Tween20 (TBST) [1.5 M NaCl, 20 mM Tris-HCl, 0.05% (v/v) Tween-20] for 6 h and then incubated overnight with primary antibodies (Sigma-Aldrich, St. Louis, MO, USA), diluted 1:1000 in blocking solution, at 37°C. The membranes were washed with TBST thrice for 10 min interval and then incubated with horseradish peroxidase conjugated secondary antibody (diluted 1:2000) in blocking solution for 2 h at 37°C. Then, the membranes were washed with TBST thrice for 10 min interval and the bands were detected using a DiaminoBenzidine (DAB) solution. The images were acquired by Image Station 2000R (Kodak, NY, USA). [35]

Gelatin zymography

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) substrate-embedded enzymography (zymography) was used to detect enzymes with gelatinase activity. Assays were carried out as previously described by Fonseca et al. [36] Briefly, the culture supernatant was collected after 24 h UVB-irradiated HDFa and centrifuged at 2000 rpm for 10 min. The supernatant was then subjected to zymography on 12% SDS-PAGE co-polymerized with 0.1% gelatin. Gel was washed in 2.5% Triton-X-100 for 30 min to remove SDS and then incubated overnight in reaction buffer (50 mM Tris-HCl pH 7, 4.5 mM CaCl 2 , 0.2 M NaCl). After incubation, the gel was stained with 0.5% Coomassie Blue in 30% methanol and 10% glacial acetic acid. The bands were visualized by destaining the gel with 30% methanol and 10% glacial acetic acid.

Statistical analysis

All the values were expressed as means of six (n=6) determinations. The data were statistically analyzed using one-way analysis of variance (ANOVA) on SPSS (statistical package for social sciences) and the group means were compared by Duncan's Multiple Range Test (DMRT). The results were considered statistically significant if the P value was less than 0.5.

 Results



SM prevents UVB-induced ΔΨm alteration in HDFa

Fluorescence microscopic images showed the accumulation of Rh-123 dye in control group and the dye accumulation had been decreased in UVB-treated cells. It has also been observed that there was decreased fluorescence indensity (86 ± 6.43) in irradiated HDFa when compared to control (123 ± 8.38). SM treatment before UVB exposure significantly prevented UVB-induced loss of ΔΨm in a dose-dependent manner [Figure 1]. There was a significant increase in fluorescence intensity (108.04 ± 6.45) in 80 μM of SM plus UVB-irradiated HDFa.{Figure 1}

Effect of SM on UVB-induced apoptotic morphological changes in HDFa

The nuclear fragmentation was evaluated by DAPI staining [Figure 2]. UVB-irradiated HDFa cells showed condensed apoptotic bodies; 52.06 ± 4.23% of apoptotic cells were observed in UVB-irradiated HDFa. The percentage of apoptotic cells was significantly decreased in SM plus UVB-treated HDFa cells (12.02 ± 1.12%). No nuclear fragmentation was detected in control cells.{Figure 2}

Effect of SM on sub-G 1 peak in HDFa cells

UVB irradiation induced a distinct sub-G 0 /G 1 peak, which represents the population of apoptotic cells [Figure 3]. Treatment with SM before UVB irradiation significantly decreased the proportion of sub-G 0 /G 1 in a dose-dependent manner.{Figure 3}

SM inhibits UVB-induced activation of TNF-α, NF-κB, Bcl-2, p53, Bax and caspase-3 in HDFa

UVB-treated cells showed increased nuclear TNF-α and total NF-κB expression 4 h after irradiation [Figure 4]. Immunoblots also indicate that treatment with SM before UVB irradiation markedly decreased the expressions of TNF-α and total NF-κB in a dose-dependent manner.

The expression pattern of p53, Bax and caspase-3 in cell lysates was significantly increased at 4 h post UVB irradiation [Figure 5]. On the other hand, Bcl-2 protein expression was significantly decreased in UVB-irradiated HDFa. SM treatment before UVB exposure down-regulated the expressions of p53, Bax and caspase-3 and up-regulated Bcl-2 in a dose-dependent manner.{Figure 4}{Figure 5}

Effect of SM on MMP-9 and MMP-2 expressions in normal, UVB-irradiated and SM-pretreated HDFa

UVB irradiation dramatically increased the activation of MMP-9 and MMP-2 activity in HDFa [Figure 6]. Treatment with SM (8, 40 and 80 μM) before UVB exposure significantly inhibited the activation of MMP-9 and MMP-2 in a dose-dependent manner.{Figure 6}

 Discussion



One approach to protecting human skin against the harmful effects of external source is to use antioxidants as photoprotectives. In recent years, naturally occurring herbal compounds such as phenolic acids, flavonoids, and high-molecular-weight polyphenols have gained considerable attention as beneficial protective agents. [37] In the previous study, UVB-induced DNA damage seemed to play an important role in apoptotic signaling cascade. [38] UVB-induced intracellular formation of ROS, mitochondrial dysfunction and cytochrome-C release were demonstrated to be additionally involved in the apoptotic program. [8] Besides MMP-2 and MMP-9 activation, proteases involved in aging process have also been found to be increased in skin dermal cells during UVB exposure. [39] Investigations on protective natural phytochemical agents which modulate UVB-induced signaling cascades appear to be an effective strategy against UV-induced pathological changes. Our previous study demonstrated UVB-induced ROS generation and its subsequent toxicity were attenuated by SM, a dietary phytochemical in cultured skin dermal fibroblasts. [28] The present study showed the modulatory role of SM on UVB-induced apoptotic, inflammatory and photoaging signaling cascades. The signaling pathways of p53 and NF-κB have a key role in the regulation of cellular senescence and photoaging. [40]

Apoptosis is a regulated form of cell death and a multifactor-related process, including gene expression and mutation. [41] In this study, we observed numerous apoptotic cells during UVB exposure in HDFa by DAPI staining. Kulms et al. in 2002 indicated that DNA damage, death receptor activation and ROS generation, all contribute to UVB-induced apoptosis in different ways. [42] UVB-induced apoptosis is a highly complex process involving the extrinsic and intrinsic pathways, but it is unclear how these pathways are interrelated. In this study, we noticed up-regulated Bax, p53 and caspase-3 and down-regulated Bcl-2 protein expression in UVB-irradiated HDFa. The balance between pro-apoptotic (e.g. bax, Bak and bid) and anti-apoptotic members of the Bcl-2 protein family (e.g. BCL-2 and BCL-XL) determines whether apoptosis is promoted or prevented. [43] A number of studies have indicated the critical role of p53 in the apoptotic process in UVB-irradiated cells. [3],[11],[43] Treatment of HDFa with SM before UVB exposure resulted in the reduction of expressions of pro-apoptotic factors (p53 and Bax) and enhancement of expression of anti-apoptotic factor (Bcl-2) in HDFa, thus protecting HDFa from UVB-induced cell death. In accordance with our findings, (−)epigallocatechin gallate hampers UVB-induced apoptotic signaling in HDFa. [44] Similarly, genistein, a soybean phytochemical, protects HDFa against UVB-induced senescence-like state in a dose-dependent manner. [45] To explore the protective impact of SM on UVB-induced apoptosis, we applied flow cytometric analysis after PI staining of the cells [Figure 3]. It has been previously reported that UVB irradiation-induced apoptosis was mediated by regulation of the cell cycle. [33] UVB-exposured HDFa decreased G1 to S phase progression and then increased sub-G0/G1 phase death progression cell was noticed (63.45 ± 4.62). Treatment with UVB plus SM showed decreased apoptotic sub-G 0 /G 1 populations (22.31 ± 2.12). Similarly, Adil et al. recently proved the effect of Emblica officinalis against UVB radiation-induced DNA damage, apoptotic cell death and its subsequent cell cycle modulation. [46]

Further, SM inhibited UVB radiation-induced activation of TNF-α and NF-κB inflammatory signaling in a dose-dependent manner [Figure 7]. TNF-α is an important mediator involved in the UVB-induced inflammatory reactions. Further, it activated NF-κB, a dominant transcription factor, responsible for inflammation. [47] Once activated, NF-κB binds to DNA and transcripts various pro-inflammatory genes, including cytokines and inducible nitric oxide synthase (iNOS). [48] It has been already proved that SM dose dependently attenuates free radical production, iNOS mRNA, NF-κB activation in lipopolysaccharide-stimulated murine BV-2 microglia. [49] Chu et al. recently established the protective effect of SM on the pulmonary TNF-α, NF-κB expression and lung injury in endotoxemic rats. [50] Another study showed that pretreatment with SM (at 50 mg/kg b.w.) significantly reduced the numbers of inflammatory cells, mitotic cells and dead cells, and blocks TNF-α activation in γ-irradiated mice spleen cells. [51] Thus, the present findings along with the previous reports indicate SM possesses protective effect against UVB-induced inflammatory signaling in HDFa.{Figure 7}

Much evidence demonstrates a close relationship between wrinkle formation and the action of MMPs. MMP-1 initiates the breakdown of collagen by unwinding the triple-helical structure and hydrolyzing the peptide bonds. [52] After its degradation, collagen is converted to denatured collagen (gelatin) and is further degenerated by gelatinases including MMP-9. [52] Inhibition of gelatinases, such as MMP-2 and MMP-9, has been shown to prevent UVB-induced photoaging. [53] In this study, UVB irradiation increased MMP-2 and MMP-9 activity in HDFa and this effect was blocked by SM treatment. This antiphotoaging capability of SM to prevent UVB-induced collagen degradation was possibly mediated via transcriptional mechanisms responsible for collagenolytic MMP production. Recently, Jung et al. showed myricetin block damage to the basement membranes by inhibiting the activity and expression of MMP-9, consequently preventing UVB-induced wrinkle formation in SKH-1 hairless mice. [54] Pretreatment of human dermal fibroblasts with epigallocatechin gallate (EGCG) also inhibited UVB-induced production of MMP-1, MMP-8 and MMP-13 in a dose-dependent manner. [44] Accumulating evidence suggests that the mitogen-activated protein kinases (MAPKs) family plays a major role in MMP up-regulation and that MMP up-regulation results in photoaged skin. [55],[56] Additionally, an siRNA study showed that MMP-9 expression is regulated by extracellular signal-regulated kinase (ERK) phosphorylation. [55] The results of our Western blot assay showed that SM inhibits the UVB-induced expression of TNF-α and NF-κB. It has been already proved in our laboratory that SM scavenges hydroxyl, lipid peroxyl and total radicals at a nanosecond time scale, and inhibits UVB radiation-induced single-strand DNA breaks. [26],[27] Recently, we have demonstrated that SM pretreatment decreased UVB-induced ROS generation and its subsequent DNA damage in HDFa. In addition, SM also restored UVB-mediated alterations of antioxidant status. [28] Hence, we hypothesized that SM-mediated redox manipulations block MMP-mediated photoaging process and this might be due to its influence on the UVB-induced MAPK signaling pathways.

 Conclusion



To conclude, we have shown that SM inhibits UVB-induced apoptotic cell death by modulating mitochondrial pathway. Further, SM prevents UVB-induced activation of MMP-2 and MMP-9; this might be due to the modulatory role of SM on TNF-alpha and NF-κB [Figure 7]. Consequently, interventions with botanical antioxidants such as SM could be promising in the design and development of new treatment strategies for UVB-induced pathological damages.

 Acknowledgments



The authors gratefully acknowledge the Department of Science and Technology (DST), Government of India, for providing financial assistance to Dr. N. Rajendra Prasad under FAST TRACT (SR/FT/LS-016/2007) Scheme for Young Scientists. Mr. S. Ramachandran is the SRF in this project.

References

1Baskaran N, Rajasekaran D, Manoharan S. Coumarin protects 7,12-dimethylbenz(a)anthracene-induced genotoxicity in the bone marrow cells of golden Syrian hamsters. Int J Nutr Pharmacol Neurol Dis 2011;1:167-73.
2Nirmala A, Mullaicharam AR, El-Khide MA. Chemistry and pharmacology of caffeine in different types of tea leaves. Int J Nutr Pharmacol Neurol Dis 2011;1:110-5.
3Afaq F, Adhami VM, Mukhtar H. Photochemoprevention of ultraviolet B signaling and photocarcinogenesis. Mutat Res 2005;571:153-73.
4Abdel-Daim M, Funasaka Y, Kamo T, Ooe M, Matsunaka H, Yanagita E, et al. Preventive effect of chemical peeling on ultraviolet induced skin tumor formation. J Dermatol Sci 2010;60:21-8.
5Xu YR, Fisher GJ. Ultraviolet (UV) light irradiation induced signal transduction in skin photoaging. J Dermatol Sci 2005;1:S1-8.
6Paz ML, Ferrari A, Weill FS, Leoni J, Gonzalez Maglio DH. Time-course evaluation and treatment of skin inflammatory immune response after ultraviolet B irradiation. Cytokine 2008;44:70-7.
7Lu YP, Lou YR, Peng QY, Xie, JG, Conney AH. Stimulatory effect of topical application of caffeine on UVB-induced apoptosis in the epidermis of p53 and Bax knockout mice. Can Res 2004;64:5020-7.
8Mantena SK, Katiyar SK. Grape seed proanthocyanidins inhibit UV-radiation-induced oxidative stress and activation of MAPK and NF-κB signaling in human epidermal keratinocytes. Free Radical Biol Med 2006;40:1603-14.
9Ichihashi M, Ueda M, Budiyanto A, Bito T, Oka M, Fukunaga M, et al. UV-induced skin damage. Toxicology 2003;189:21-39.
10Heck DE, Gerecke DR, Vetrano AM, Laskin JD. Solar ultraviolet radiation as a trigger of cell signal transduction. Toxicol Appl Pharmacol 2004;195:288-97.
11Assefa Z, Garmyn M, Vantieghem A, Declercq W, Vandenabeele P, Vandenheede JR, et al. Ultraviolet B radiation-induced apoptosis in human keratinocytes: Cytosolic activation of procaspase-8 and the role of Bcl-2. FEBS Lett 2003;540:125-32.
12Van Laethem A, Van Kelst S, Lippens S, Declercq W, Vandenabeele P, Janssens S, et al. Activation of p38 MAPK is required for Bax translocation to mitochondria, cytochrome c release and apoptosis induced by UVB irradiation in human keratinocytes. FASEB 2004;18:1946-8.
13Assefa Z, Laethem AV, Garmyn M, Agostinis P. Ultraviolet radiation-induced apoptosis in keratinocytes: On the role of cytosolic factors. Biochim Biophys Acta 2005;1755:90-106.
14Ren SW, Li J, Wang W, Guan HS. Protective effects of κ-ca3000 + CP against ultraviolet-induced damage in HaCaT and MEF cells. J Photochem Photobiol B: Biol 2010;101:22-30.
15Anggakusuma, Yanti, Hwang JK. Effects of macelignan isolated from Myristica fragrans Houtt. on UVB-induced matrix metalloproteinase-9 and cyclooxygenase-2 in HaCaT cells. J Dermatol Sci 2010;57:114-22.
16Afaq F. Natural agents: Cellular and molecular mechanisms of photoprotection. Arch Biochem Biophys 2011;508:144-51.
17Kalaivani K, Anandhan A, Mohankumar K, Preetham E, Manivasagam T. 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine is a potent neurotoxin: Gamma-tocopherol recuperate behavior, dopamine, and oxidative stress on Parkinsonic mice. Int J Nutr Pharmacol Neurol Dis 2011;1:139-45.
18Jan KC, Ho CT, Hwang LS. Bioavailability and tissue distribution of sesamol in rat. J Agric Food Chem 2008;56:7032-7.
19Fukuda Y, Nagata M, Osawa T, Namiki M. Contribution of lignan analogues to antioxidative activity of refined unroasted sesame seed oil. J Am Oil Chem Soc 1986;63:1027-31.
20Kaur IP, Saini A. Sesamol exhibits antimutagenic activity against oxygen species mediated mutagenicity. Mutat Res 2000;470:71-6.
21Kapadia GJ, Azuine MA, Tokuda H, Takasaki M, Mukainaka T, Konoshima T, et al. Chemopreventive effect of resveratrol, sesamol, sesame oil and sunflower oil in the Epstein-Barr virus early antigen activation assay and the mouse skin two-stage carcinogenesis. Pharmacol Res 2002;45:499-505.
22Sharma S, Kaur IP. Development and evaluation of sesamol as an antiaging agent. Int J Dermatol 2006;45:200-8.
23Jacklin A, Ratledge C, Welham K, Bilko D, Newton CJ. The sesame seed oil constituent, sesamol, induces growth arrest and apoptosis of cancer and cardiovascular cells. Ann NY Acad Sci 2003;1010:374-80.
24Chen PR, Lee CC, Chang H, Tsai CE. Sesamol regulates plasminogen activator gene expression in cultured endothelial cells: A potential effect on the fibrinolytic system. J Nutr Biochem 2005;16:59-64.
25Chen PR, Tsai CE, Chang H, Liu TL, Lee CC. Sesamol induces nitric oxide release from human umbilical vein endothelial cells. Lipids 2005;40:955-61.
26Kanimozhi P, Prasad NR. Antioxidant potential of sesamol and its role on radiation-induced DNA damage in whole-body irradiated Swiss albino mice. Environ Toxicol Pharmacol 2009;28:192-7.
27Prasad NR, Mahesh T, Menon VP, Jeevanram RK, Pugalendi KV. Radioprotective effect of sesamol on radiation induced DNA damage, lipid peroxidation and antioxidant levels in cultured human lymphocytes. Toxicology 2005;209:25-235.
28Ramachandran S, Prasad NR, Karthikeyan S. Sesamol inhibits UVB-induced ROS generation and subsequent oxidative damage in cultured human skin dermal fibroblasts. Arch Dermatol Res 2010;302:733-44.
29Prasad NR, Karthikeyan A, Karthikeyan S, Reddy BV. Inhibitory effect of caffeic acid on cancer cell proliferation by oxidative mechanism in human HT-1080 fibrosarcoma cell line. Mol Cell Biochem 2010;349:11-9.
30Darzynkiewiez Z, Li X, Gong J. Assay for cell viability: Discrimination of cells dying by apoptosis. Methods Cell Biol 1994;41:15-38.
31Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C. A novel assay for apoptosis flow cytometric detection of phosphatidylserine early apoptotic cells using fluorescein labelled expression on Annexin V. J Immunol Methods 1995;184:39-51.
32Li JL, Liu N, Chen XH, Sun M, Wang CB. Inhibition of UVA-induced apoptotic signaling pathway by polypeptide from Chlamys farreri in human HaCaT keratinocytes. Radiat Environ Biophys 2007;46:263-8.
33Huang JH, Huang CC, Fang JY, Yang C, Chan CM, Wu NL, et al. Protective effects of myricetin against ultraviolet-B-induced damage in human keratinocytes. Toxicol In Vitro 2010;24:21-8.
34Lowry OH, Rosebough NJ, Farr AL, Randal RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-75.
35Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Prot Nat Acad Sci USA 1979;76:4350-4.
36Fonseca YM, Catini CD, Vicentini FT, Nomizo A, Gerlach RF, Fonseca MJ. Protective effect of Calendula officinalis extract against UVB-induced oxidative stress in skin: Evaluation of reduced glutathione levels and matrix metalloproteinase secretion. J Ethnopharmacol 2010;127:596-601.
37Subash S, Subramanian P. Effect of N-phthaloyl gamma-aminobutyric acid on lipid peroxidation, antioxidants and liver markers in constant light exposed rats. Int J Nutr Pharmacol Neurol Dis 2011;1:163-6.
38Svobodova A, Zdarilova A, Vostalova J. Lonicera caerulea and Vaccinium myrtillus fruit polyphenols protect HaCaT keratinocytes against UVB-induced phototoxic stress and DNA damage. J Dermatol Sci 2009;56:196-204.
39Inomata S, Matsunaga Y, Amano S, Takada K, Kobayashi K, Tsunenag M, et al. Possible involvement of gelatinases in basement membrane damage and wrinkle formation in chronically ultraviolet B-exposed hairless mouse. J Invest Dermatol 2003;120:128-34.
40Salminen A, Kaarniranta K. Control of p53 and NF-κB signaling by WIP1 and MIF: Role in cellular senescence and organismal aging. Cell Signal 2011;23:747-52.
41Liu X, Shi S, Ye J, Liu L, Sun M, Wang C. Effect of polypeptide from Chlamys farreri on UVB-induced ROS/NF-κB/COX-2 activation and apoptosis in HaCaT cells. Photochem Photobiol B: Biol 2009;96:109-16.
42Kulms D, Zeise E, Poppelmann B, Schwarz T. DNA damage, death receptor activation and reactive oxygen species contribute to ultraviolet radiation-induced apoptosis in an essential and independent way. Oncogene 2002;21:5844-51.
43Katiyar SK, Roy AM, Baliga MS. Silymarin induces apoptosis primarily through a p53-dependent pathway involving Bcl-2/Bax, cytochrome c release and caspase activation. Mol Cancer Ther 2005;4:207-16.
44Bae JY, Choi JS, Choi YJ, Shin SY, Kang SW, Han SJ, et al. Epigallocatechin gallate hampers collagen destruction and collagenase activation in ultraviolet-B-irradiated human dermal fibroblasts: Involvement of mitogen-activated protein kinase. Food Chem Toxicol 2008;46:1298-307.
45Wang YN, Wu W, Chen HC, Fang H. Genistein protects against UVB-induced senescence-like characteristics in human dermal fibroblast by p66Shc down-regulation. J Dermatol Sci 2010;58:19-27.
46Adil MD, Kaiser P, Satti NK, Zargar AM, Vishwakarma RA, Tasduq SA. Effect of Emblica officinalis (fruit) against UVB-induced photo-aging in human skin fibroblasts. J Ethnopharmacol 2010;132:109-14.
47Cooper SJ, Bowden GT. Ultraviolet B Regulation of Transcription Factor Families Roles of Nuclear Factor-kappa B (NF-κB) and Activator Protein-1 (AP-1) in UVB-Induced Skin Carcinogenesis. Curr Cancer Drug Targets 2007;4:325-34.
48Surh YJ, Kundu JK, Na HK, Lee JS. Redox-se.nsitive transcription factors as prime targets for chemoprevention with anti-inflammatory and antioxidative phytochemicals. J Nutr 2005;135:2993S-3001S.
49Hsu DZ, Li YH, Chu PY, Chien SP, Chuang YC, Liu MY. Attenuation of endotoxin-induced oxidative stress and multiple organ injury by 3, 4-methylene dioxy phenal in rats. Shock 2006;25:300-5.
50Chu PY, Chien SP, Hsu DZ, Liu MY. Protective effect of sesamol on the pulmonary inflammatory response and lung injury in endotoxemic rats. Food Chem Toxicol 2010;48:1821-6.
51Parihar VK, Prabhakar KR, Veerapur VP, Kumar MS, Reddy YR, Joshi R, et al. Mutat Res 2006;611:9-16.
52Chung L, Dinakarpandian D, Yoshida N, Lauer-Fields JL, Fields GB, Visse R, et al. Collagenase unwinds triple-helical collagen prior to peptide bond hydrolysis. EMBO J 2004;23:3020-30.
53Fisher GJ, Datta SC, Talwar HS, Wang ZQ, Varani J, Kang S, et al. Molecular basis of sun-induced premature skin aging and retinoid antagonism. Nature 1996;379:335-9.
54Jung SK, Lee KW, Kim HY, Oh MH, Byun S, Lim SH, et al. Myricetin suppresses UVB-induced wrinkle formation and MMP-9 expression by inhibiting Raf. Biochem Pharmacol 2010;79:1455-61.
55Lin CC, Tseng HW, Lee CW, Wu CY, Cheng CY, Yang CM. Tumor necrosis factor-alpha induces MMP-9 expression via p42/p44 MAPK, JNK and nuclear factor-κB in A549 cells. Toxicol Appl Pharmacol 2008;229:386-98.
56Kim HH, Shin CM, Park CH, Kim KH, Cho KH, Eun HC, et al. Eicosapentaenoic acid inhibits UV-induced MMP-1 expression in human dermal fibroblasts. J Lipid Res 2005;46:1712-20.