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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


Department of Biochemistry and Biotechnology, Annamalai University, Annamalainagar, India

Date of Submission24-Aug-2011
Date of Acceptance02-Nov-2011
Date of Web Publication23-Feb-2012

Correspondence Address:
Nagarajan Rajendra Prasad
Department of Biochemistry and Biotechnology, Annamalai University, Annamalainagar 608 002, Tamilnadu
India
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Source of Support: FAST TRACT (SR/FT/LS-016/2007) Scheme for Young Scientists. Government of India., Conflict of Interest: None


DOI: 10.4103/2231-0738.93131

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   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.

Keywords: Apoptosis, human skin dermal fibroblasts, adult, reactive oxygen species, sesamol, ultraviolet radiation


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;2:31-9

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 Jul 22];2:31-9. Available from: http://www.ijnpnd.com/text.asp?2012/2/1/31/93131


   Introduction Top


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 Top


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 Top


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: Protective effect of SM on UVB radiation-induced mitochondrial membrane potential (Δψm) alteration in HDFa. Δψm changes were observed under a fluorescence microscope after Rh-123 (bright red and green color) staining. UVB exposure increased loss of Δψm and emitted green fluorescence. SM treatment (8, 40 and 80 µM) before UVB exposure improved membrane potential loss and emitted red fluorescence. The percentage of fluorescence intensity was increased in a dose-dependent manner. values are given as means ± SD of three experiments in each group. values not sharing a common marking (a, b, c, …) differ significantly at P<0.05 (DMRT), (a) Control, (b) SM (80μM), (c) UVB-irradiation, (d) UVB + SM (8μM), (e) UVB + SM (40μM), (f) UVB + SM (80 μM)

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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: Protective effect of SM on UVB radiation-induced apoptotic cell damage in HDFa. Cellular morphological changes were observed under a fluorescence microscope after DAPI (bright blue color) staining. UVB exposure increased apoptotic cells in HDFa. SM treatment (8, 40 and 80 µM) before UVB exposure reduced the percentage of apoptotic cells in a dose-dependent manner. values are given as means ± SD of three experiments in each group. values not sharing a common marking (a, b, c, …) differ significantly at P<0.05 (DMRT), (a) Control, (b) SM (80 μM), (c) UVB + irradiation, (d) UVB + SM (8 μM), (e) UVB + SM (40 μM), (f) UVB + SM (80 μM)

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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: Flow cytometric analysis of the sub-G0/G1 peak of HDFa cells. UVB exposure prevents G1 to S phase progression and an increased in sub-G0/G1 phase was observed. SM treatment (8, 40 and 80 µM) before UVB exposure decreased apoptotic sub-G0/G1 phase in a dose-dependent manner. values are given as means ± SD of three experiments in each group. values not sharing a common marking (a, b, c, …) differ significantly at P<0.05 (DMRT)

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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: Effect of SM on UVB-induced activation of TNF-α and NF-кB in HDFa cells. HDFa cells were exposed to UVB (19.8 mJ/cm2) with or without treatment with SM (8, 40 and 80 μM) for 30 min. Cells were harvested at 4 h after UVB exposure, and cell lysates were prepared to determine the activation of TNF-α and NF-кB using Western blot analysis. SM treatment at concentrations of 8, 40 and 80 µM significantly down-regulated these protein expressions in a dose-dependent manner (compared with 0 mg/ml control group, P<0.05). The graph represents the quantification results normalized to β-actin levels. Data represent the means ± SD of three individual experiments. *Significantly different from control cells (P<0.05). **Significantly different from UVB-exposed cells (P<0.05)

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Figure 5: Effect of SM on UVB-induced activation of p53, Bax and caspase-3 and suppression of Bcl-2 in HDFa cells. HDFa cells were exposed to UVB (19.8 mJ/cm2) with or without pretreatment with SM (8, 40 and 80 μM) for 30 min. Cells were harvested at 4 h after UVB exposure, and cell lysates were prepared to determine the activation of p53, Bax and caspase-3 and suppression of Bcl-2 using Western blot analysis. The UVB plus SM treatment at concentrations of 8, 40 and 80 µM significantly down-regulated the p53, Bax and caspase-3 and up-regulated Bcl-2 in a dose-dependent manner (compared with 0 mg/ml control group, P<0.05). The graph represents the quantification results normalized to β-actin levels. Data represent the means ± SD of three individual experiments. *Significantly different from control cells (P<0.05). **Significantly different from UVB-exposed cells (P<0.05)

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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: Inhibitory effect of SM on UVB radiation-induced MMP-9 and MMP-2 activation. Cells were harvested at 24 h after UVB exposure, and cell supernatants were prepared to determine the activation of MMP-9 and MMP-2 using gelatin zymography. UVB exposure increased the activation of both MPPs in HDFa, while SM treatment at concentrations of 8, 40 and 80 µM significantly down-regulated the intracellular MMP-9 and MMP-2 activation in a dose-dependent manner (compared with 0 mg/ml control group, P<0.05). Data represent the means ± SD of three individual experiments. *Significantly different from control cells (P<0.05). **Significantly different from UVB-exposed cells (P<0.05)

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   Discussion Top


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: Schematic diagram showing inhibitory action of SM in UVB radiation-induced dermis cellular alteration and photoaging. SM blocks UVB irradiation-induced ROS-mediated MMP production and collagen degradation by interfering with TNF-α and NF-κB signaling pathways

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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 Top


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 Top


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.

 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]


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