Protective Effects of Timosaponin AIII against UVB-Radiation Induced Inflammation and DNA Injury in Human Epidermal Keratinocytes

Abstract

UVB radiation changes several photoaging pathway in the body, thereby prompting skin injury. Besides, chronic UVB radiation leads to photoaging, sustained immunosuppression, and photocarcinogenesis. We investigated the protective effect of Timosaponin AIII (TA-III), a naturally occurring steroidal saponin separated from Anemarrhena asphodeloides, against UVB-induced invasive properties of human epidermal keratinocytes (HEKs) and human dermal fibroblasts (HDF). No cytotoxicity was observed up to 50 nM concentration of TA-III. Similarly, TA-III inhibited UVB-induced cyclooxygenase-2 (COX-2), matrix metalloproteinase-9 (MMP-9) transcription level and protein expression in a dose-dependent manner at non-cytotoxic dose. Further, TA-III decreased UVB-induced invasion in primary skin cells. Additionally, TA-III suppressed UVB-stimulates mitogen-activated protein kinase (MAPK) signaling, activator protein-1 (AP-1) and nuclear factor kappa B (NF-κB) activation, thereby preventing the overexpression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and COX-2 in human epidermal keratinocytes cells. Furthermore, TA-III prevented UVB-mediated formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) and activation of DNA repair enzymes and, cell cycle arrest genes like as proliferating cell nuclear antigen (PCNA), structural maintenance of chromosomes protein 1 (SMC1). This results support that understanding into the molecular action of TA-III, which can be useful for developing photoprotective agents.

INTRODUCTION

The Skin is major human organ and, functions as a physiological hurdle to support an organism opposite environmental UV irradiation, biochemical contaminants and corporeal body damage.^1,2) UVB radiation is one of the greatest hazardous damage to the skin by causing, skin senescence, erythema, sunburn, and photo-damage. There are close association with the UVB radiation exposure dose and skin injury a higher dose sources further irreparable injury. UVB radiation stimulates the transcription element nuclear factor (NF)-κB,³⁾ which plays a significant performed in regulating the inflammatory response through causing of multifunctional cytokines, as well as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) by straight binding to their promoter region.⁴⁾ Stimulation of NF-κB more prompts the expression of matrix metalloproteinases (MMPs), which are important pathophysiological elements in photo-aging. Constantly UVB the exposure induces expression of positive members of the MMP family, which degrade collagen by stimulating of collagen breakdown and inhibiting procollagen biosynthesis.⁵⁾

Prolonged UVB radiation leads to photo-aging, sustained immunosuppression and photo carcinogenesis. UVB radiation induces a signaling response through two altered pathways: one dependent on and the other independent of DNA injury. Cellular DNA, undergoes damage generate 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG), an oxidative altered DNA base, is reflected as an suitable biomarker for evaluating oxidative DNA damage.⁶⁾ Additionally, DNA injury motivates the tumor suppressor p53, which prompts cell-cycle arrest and the concurrent procedures of DNA repair and apoptotic cell death.^7,8) We previously showed that application of Timosaponin AIII, (TA-III), a major steroidal saponin constituents isolated from Anemarrhena asphodeloides BUNGE, decreases melanoma cell migration by suppressing cyclooxygenase (COX-2) and cancer metastasis.⁹⁾ It has been shown that TA-III has capable pharmacological action in improving leaning, memory, and antineoplastic activity.^10,11) However, the precise mechanism of skin cell invasion by TA-III is not well understood. It is also unclear whether TA-III decreases UVB radiation-induced inflammatory mediator in skin cells. Therefore, we hypothesized that prevention of the inflammation response by TA-III is intervened through repair of UVB-induced inflammatory cytokines in human epidermal keratinocytes (HEKs). Here, we found that TA-III inhibits UVB-stimulated MMP-9 and COX-2 expression by inhibiting NF-κB, activator protein (AP-1) activation, and mitogen-activated protein kinase (MAPK) signaling. Additionally, we revealed that TA-III prevents UVB-induced DNA injury by motivating of DNA repair enzymes.

MATERIALS AND METHODS

Plant Material and Chemicals

The Anemarrhena asphodeloides was acquired from Kwangmyungdang Medicinal herbs (Ulsan, Korea). The origin of component was identified by Dr. G. Choi (Korea Institute of Oriental Medicine). A voucher specimen (Anemarrhena asphodeloides, 2-13-0040) has been deposited at the Herbal Medicine Resources Research Center (Korea Institute of Oriental Medicine). Antibodies against MMP-9, COX-2, MMP-2, TIMP-1, TIMP-2, IL-6, c-JUN, c-FOS, TBP, 8-oxo-dG, and β-actin were obtained from Cell Signaling Technology (Beverly, MA, U.S.A). NF-κB-luciferase assay kit, AP-1-luciferase assay kit were obtained from Promega Corporation (Madison, WI, U.S.A). Phosphorylation antibodies AKT (p-AKT), p38 (p-p38), extracellular-signal-regulated kinase (ERK) (p-ERK), c-Jun N-terminal kinase (JNK) (p-JNK) were from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A). DRAQ5 fluorescent dye, proliferating cell nuclear antigen (PCNA), SMC1 antibodies were acquired from Cell Signaling Technology.

Extraction and Isolation

The TA-III was isolated from Anemarrhena asphodeloides as previously described.¹⁰⁾ The Anemarrhena asphodeloides rhizomes (3 kg) were extracted with 70% ethanol three times under reflux for 3 h. This 70% ethanol extract (1.1 kg) was then suspended in distilled water and continuously fractionated with n-hexane, ethylacetate, and n-butanol. The n-butanol fraction (9.1 g) was subjected to diaion HP-20 resin chromatography and eluted with a gradient solvent system of H₂O : methanol (100 : 0 to 70 : 30), to afford 5 fractions (A1–A5). Fraction A4 was chromatographed over LiChroprep^® RP-18 with H₂O : methanol (100 : 0 to 50 : 50) to afford Timosaponin AIII (TA-III).

Cell Culture and Cell Viability

Human dermal fibroblasts (HDFs) and HEKs cells culture were determined as previous described.¹²⁾ HDF cells were propagated in fibroblast medium supplemented with 5% fetal bovine serum (FBS), 1% fibroblast growth supplement, and 1% penicillin and streptomycin (P/S), HEKs were purchased from Lonza (Walkersville, MD, U.S.A.) and maintained in KGM-Gold SingleQuots™ medium containing supplements and growth factors. Both cell cultures were maintained in a humidified 5% CO₂ incubator at 37°C. Viabilities of HDF and HEK were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as previously described.¹³⁾

UVB Radiation

Cells were pre-incubated with the indicated concentrations of TA-III for 12 h, washed with phosphate buffer saline (PBS) and exposed to UVB in a UV crosslinker (302 nm UV, 5 × 8 W UV dual bipin discharge type CL-1000 M, UVP, Ultra-Violet Products Ltd., Cambridge, U.K.), The UVB intensity of 20 mJ/cm² for 15 s. And the cells were additional incubated for 24 h, and subjected to different biochemical analyses.

Real Time Quantitative PCR

Total cellular RNA was extracted using Trizol reagent according to the manufacturer’s instructions (Thermo Fisher Scientific Company, Waltham, MA, U.S.A.). To quantify MMP-9 and, COX-2 gene expression in HEK cells, TaqMan Primers (Hs01110250_ml) were used, RT-qPCR was completed with TaqMan^®Universal PCR Master Mix (Applied Biosystems, Foster City, CA, U.S.A.) using an ABI QuantStudio™ 6 Flex Real-Time PCR system (Applied Biosystems). The relative mRNA expression levels of MMP-9 and, COX-2 were normalized to that of β-actin. Relative mRNA expression levels were estimated using the ΔΔCt method.

Western Blotting

Western blotting assay was determined as previously described.¹²⁾ Briefly, Cell lysates were prepared from HDF and HEK (5 × 10⁵) in 1× Laemmli lysis buffer (2.4 M glycerol, 0.14 M Tris–HCl (pH 6.8), 0.21 M sodium dodecyl sulfate, and 0.3 mM bromophenol blue), and boiled for 10 min. The protein content was measured using BCA Protein Assay Reagent (Pierce, Rockford, IL, U.S.A.). Protein samples (20 µg) were diluted with 1× lysis buffer, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred onto polyvinylidene fluoride membranes. The antibodies include MMP-9 (1 : 1000), COX-2 (1 : 1000), TIMP1 (1 : 1000), TIMP2 (1 : 1000), c-Jun (1 : 1000), c-fos (1 : 1000), AKT (1 : 1000), p-38 (1 : 1000), and β-actin (1 : 1000), and detected using an enhanced chemiluminescence detection system from Amersham Bioscience (Buckinghamshire, U.K.).

Luciferase Assay

Luciferase assay was conducted as previously described.¹⁴⁾ HEK cells were seeded at a density of 1 × 10⁵ cells/well in 6-well plates and grown to 60–70% confluence. Cells were then transfected with 100 ng of the NF-κB and AP-1 luciferase reporter construct using Lipofectamine™ 2000 transfection reagent according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA, U.S.A.). Renilla-CMV-Renilla luciferase was used to correct for variations in transfection efficiency. At 24 h afterward transfection, the cells were treated with TA-III for 12 h. After incubation, the cells were with reporter lysis buffer. Cell lysates were treated with a luciferase substrate (Promega, Madison, WI, U.S.A.), and luciferase activity was determined using a Tristar LB 941 multimode microplate reader (Tristar, Berthold, Wildbad, Germany).

Migration Assay

Chemotactic motility assayed was performed as previous described.⁹⁾ HDFs and HDKs were assayed using Transwell chambers (Corning Costar, Corning, NY, U.S.A.) with 6.5-mm diameter polycarbonate filters (8-µm pore size). The lower surface of each filter was coated with 10 µg of gelatin. Fresh Dulbecco’s modified Eagle’s medium (DMEM) medium (with 0.5% FBS) was added to the lower wells. Cells were trypsinized and suspended at a final concentration of 1 × 10⁵ cells/mL in DMEM containing 0.5% FBS, followed by treatment with the indicated concentrations of TA-III at room temperature for 30 min prior to seeding. The cell suspension (200 µL per well) was loaded into the upper wells and the chambers were incubated for 12 h at 37°C, after which the cells were fixed and stained with crystal violet. Non-migrating cells on the upper surface of each filter were removed with a cotton swab. Chemotaxis was calculated by counting the cells that had migrated to the lower side of the filter with an optical microscope (100 × magnification). Five fields were counted in each assay.

Detection of 8-oxo-7,8-Dihydro-2′-deoxyguanosine (8-oxo-dG) Positive Cells

UVB-induced DNA injury in the form 8-oxo-dG-positive cells was detected as previously described.¹⁵⁾ A primary anti-8-oxo-dG antibody was used at a 1 : 100 dilution and incubated with the cells overnight at 4°C. A goat anti-rabbit immunoglobulin G (IgG) H&L (Alexa Fluor^® 647) secondary antibody (Abcam, Cambridge, U.K.) was used at a 1 : 100 dilution and incubated with the cells for 45 min at room temperature. Nuclei were stained with DRAQ5 (Cell Signaling Technology, Danvers, MA, U.S.A.) at a 1 : 1000 dilution. Fixed and immunofluorescence labeled cells were imaged using a confocal microscope (Olympus, FV10i, Tokyo, Japan).

Statistical Analysis

All data were expressed as the mean ± standard deviation of at least three separate experiments. Comparisons among multiple comparison test were made used by the Dunnett’s test with Graph Pad Software (GraphPad, Inc., San Diego, CA, U.S.A.); significance was established at * p < 0.05, ** p < 0.01, *** p < 0.001.

RESULTS

Effect of TA-III on Cytotoxicity

The molecular structure of TA-III is shown in Fig. 1A. We first observed the cytotoxic effects of UVB radiation and TA-III using the MTT assay. After treating the HEK for 24 h TA-III showed no cytotoxic effects at concentrations of up to 50 nM, additionally, Time-dependent and the concentration of TA-III treated with TA-III did not alter cell viability (Figs. 1B, C). Therefore, we used a TA-III concentration of 50 nM in subsequent experiments. We also found that UVB stimulated, even at the lowest dose 20 mJ/cm² reduced cell viability by up to 80%, TA-III appeared to produce a modest recovery of cell viability, but it did not restore cells to initial viability (Fig. 1D). These results suggest that TA-III (1–50 nM) was not toxic does and not reflect the effect of UVB in HEK cells.

Fig. 1. Effect of TA-III and UVB on HEK Cell Viability

(A) Chemical structure of timosaponin AIII. (B) The cytotoxicity of various concentrations of TA-III on HEK cells was determined by MTT assay. (C) HEK cells were treated with TA-III (50 nM) for the indicated times followed by MTT assay. (D) The protective effects of TA-III on HEK cell viability. HEK cells were treated with different doses of TA-III for 12 h and then exposed to UVB (20 mJ/cm²) radiation for 15 s, cell viability was assessed by the MTT assay. Data were obtained from three independent experiments and expressed as the means ± standard deviation (S.D.), * p < 0.05 versus control groups ** p < 0.01 indicates significant difference versus control groups.

TA-III Inhibits COX-2 and MMP-9 Levels in HEKs

COX-2 and MMP-9 play significant functions in cell migration and invasion.¹⁶⁾ We observed the effect of TA-III on the expression of COX-2 and MMP-9. HEK were stimulated to various doses of non-toxic concentrations of TA-III. As shown in Figs. 2A and B, TA-III considerably reduced COX-2, MMP-9 expression at the mRNA levels by real-time PCR. Moreover, expression of the endogenous inhibitors TIMP-1 and TIMP-2 was increased by TA-III in a concentration-dependent manner following exposure to UVB (Fig. 2C). These results indicated that MMP-2, MMP-9 also COX-2 expression was reserved by TA-III in UVB-stimulated HEKs.

Fig. 2. Effects of TA-III on MMP-9, MMP-2 COX-2, TIMP-1, and TIMP-2 in HEK Cells

(A and B) Total RNAs extracted from HEK cells were treated with different doses of TA-III for 12 h and then exposed to UVB (20 mJ/cm²) radiation. MMP-9, COX-2 relative expression was determined by real-time qPCR relative to GAPDH mRNA expression. (C) HEK cells were treated with TA-III for 12 h and then exposed to UVB (20 mJ/cm²) radiation. MMP-9, MMP-2, COX-2, TIMP-1, and TIMP-2 were evaluated by Western blotting. Data were obtained from three independent experiments and expressed as the means ± S.D., ** p < 0.01 versus control groups. *** p < 0.001 indicates significant difference versus UVB radiation.

TA-III Inhibits UVB-Induced Migration in Keratinocytes and Fibroblast Cells

Pro-inflammatory cytokines and chemokines are conventional regulators of cell migration. We next examined whether inhibition of TA-III affects cell migration of HDF and HEK, in a migration assay. As shown in Figs. 3A and B, treatment of HDF and HEK with TA-III dose-dependently inhibited UVB-induced cell migration. Thus, we demonstrated that TA-III may be effective for preventing cell migration.

Fig. 3. Inhibitory Effects of TA-III on the Migration of Human Skin Cells

(A) Treatment of HDF cells with TA-III (5, 10, 20 nM) inhibited UVB-enhanced cell migration. (B) Treatment of HEK cells with TA-III (5, 10, 20 nM) inhibited UVB-enhanced cell migration. Data were obtained from three independent experiments and expressed as the means ± S.D., *** p < 0.001 versus control groups. ^# p < 0.05 indicates significant difference versus UVB radiation. ** p < 0.01 indicates significant difference versus UVB radiation.

TA-III Suppresses COX-2 and IL-6 Levels through the NF-κB or AP-1 Activity

IL-6 and COX-2 expression is measured by NF-κB and AP-1. It has been described that homo or hetero dimers control several pro-inflammatory genes involved in inflammation, and various stress responses.¹⁷⁾ Thus, we checked whether the inhibitory effect of TA-III on COX-2, IL-6, and TNF-α expression is facilitated by the NF-κB and AP-1. UVB stimulation led to phosphorylation of NF-κB (p65), pretreatment with TA-III effectively reduced phosphorylation of p65 in a concentration dependent manner. Likewise, the levels of the AP-1 subunits c-Jun also c-Fos were considerably decreased by TA-III (Fig. 4A). We added confirmed the effect of TA-III on luciferase reporter assay of NF-κB and AP-1. As shown in Fig. 4B, TA-III dose-dependently decreased NF-κB and AP-1 luciferase reporter gene activity. Next, we examined the COX-2, IL-6, and TNF-α expression levels by Western blot analysis, showed that TA-III treatment significantly augmented inflammatory cytokines COX-2, IL,-6 and TNF-α expression in HEKs (Fig. 4C). These results demonstrate that TA-III suppresses COX-2, IL-6 and TNF-α expression by suppressing of NF-κB and AP-1 activities.

Fig. 4. NF-κB and AP-1 Are Involved in TA-III Mediated Downregulation of COX-2, TNF-α and IL-6 Expression

(A) HEK cells were treated with different does of TA-III for 12 h and then exposed to UVB (20 mJ/cm²) radiation. p-NF-κB, c-JUN, c-Fos, and TBP were evaluated by Western blotting. (B) Cells were transfected with NF-κB and AP-1 luciferase reporter construct. After 24 h, the cells were incubated with different doses of TA-III for 8 h and then exposed to UVB (20 mJ/cm²) radiation. At 12 h after UVB radiation, equal amounts of cells extracts were assayed for dual-luciferase activity. (C) HEK cells were treated with TA-III for 12 h and then exposed to UVB (20 mJ/cm²) radiation. The protein levels of COX-2, IL-6, and TNF-α were evaluated by Western blotting. Data were obtained from three independent experiments and expressed as the means ± S.D., * p < 0.05 versus the respective UVB radiation. ** p < 0.01 indicates significant difference versus UVB radiation.

TA-III Attenuates UVB-Induced Akt and MAPK Activation in HEKs

UVB radiation well-known to activate downstream MAPK signaling pathways,¹⁸⁾ and Akt.¹⁹⁾ We investigated the UVB-induced phosphorylation of Akt and MAPK family proteins. After UVB radiation the phosphorylation of Akt was increased compared to in the control, this effect was significantly decreased by 20 nM TA-III. Additionally, TA-III after UVB radiation affected activation of the MAPK pathway. TA-III markedly decreased the phosphorylation of p38, and JNK, but not ERK (Fig. 5). Our results suggested that TA-III inhibited UVB-exposed MMP-9 and COX-2 likely by inhibiting Akt and p38, as well as JNK activation in HEKs.

Fig. 5. Effect of TA-III on UVB-Induced Activation of Akt and MAPK Signaling Pathways

(A) HEK cells were treated with TA-III for 12 h and then exposed to UVB (20 mJ/cm²) radiation. The phosphorylation of Akt, p38, ERK, and JNK were evaluated by Western blotting. Data were obtained from three independent experiments and expressed as the means ± S.D., ^# p < 0.05 versus control groups. ** p < 0.01 indicates significant difference versus UVB radiation.

TA-III Prevents UVB-Mediated Formation of 8-oxo-dG and DNA Repair Enzymes

UVB radiation induces oxidative damage in DNA, resulting in the formation of the adduct 8-oxo-dG.²⁰⁾ We evaluated the effect of TA-III on UVB-induced DNA damage by examining 8-oxo-dG formation. UVB radiation exposed cells showed extensive staining in the nuclei. However, TA-III treatment of the cells prior to UVB radiation resulted in significantly decreased formation of 8-oxo-dG (Fig. 6A). We further evaluated the levels of PCNA and SMC1, which active nuclear proteins involved in DNA repair and the cell cycle. UVB stimulation led to increased levels of PCNA, and pretreatment with TA-III, effectively reduced. PCNA levels. Similarly, the levels of SMC1 were significantly attenuated by TA-III (Fig. 6B).

Fig. 6. Inhibitory Effects of TA-III on UVB-Induced Formation of 8-oxo-dG in HEK

(A) HEKs were exposed to UVB (20 mJ/cm²) radiation and pretreated with TA-III (50 nM) as described in Materials and Methods. Immunofluorescence staining for 8-oxo-dG was performed using a specific antibody. (B) HEK cells were treated with TA-III for 12 h and then exposed to UVB (20 mJ/cm²) radiation. The protein levels of PCNA and, SMC1 were evaluated by Western blotting. Data were obtained from three independent experiments and expressed as the means ± S.D., *** p < 0.001 versus control groups. ** p < 0.01 indicates significant difference versus UVB radiation.

DISCUSSION

UV radiation is an environmental mutagen that has concerns increased focus. The level of UV radiation has amplified because of depletion of the stratospheric ozone layer. Specifically, several properties of UV radiation injury on skin physiology have been observed with respect to skin protection. Particularly, UVB radiation can cause photoaging, inflammation, cellular/tissue senescence and cell damage, and has been associated with skin photocarcinogenesis progression. Keratinocytes, which play important roles in the epidermis, have established extensive protective measures to regulate cellular injury in term of UVB irradiation.²¹⁾ Recent studies revealed that TA-III induces antiplatelet and antithrombotic activity,²²⁾ as well as antineoplastic activity in cancer cells.¹¹⁾ On the other hand, the specific cellular molecular mechanisms underlying the anti-inflammation effects and photoprotective effects remain generally unidentified. Our study was conducted to validate the claim that TA-III has anti-inflammation effects and photoprotective effects. Our result indicated that the percentage of viable HEK cells decreased upon exposure to UVB irradiation compared to normal cells. Consequently, we used 20 mJ/cm² in this study because at this dose cell viability remains high, marking it possible to conducts further studies (Fig. 1). We found that the mRNAs level and MMP-2, COX-2 and MMP-9 were considerably decreased by treatment with TA-III (Fig. 2). Furthermore, a non-toxic concentration of TA-III suppressed, cell migration of HDFs and HEKs (Fig. 3). Accordingly, we suggest that TA-III exerts its inhibitory effect on UVB-stimulated cells migration in correlation with decreased of MMP-9 expression through the AP-1 and NF-κB. Some transcription elements, containing NF-κB and AP-1 are related in regulating the expression of COX-2 and MMP-9.^23,24) These results shown that the anti-invasive effects of TA-III are related with inhibition of AP-1 and NF-κB. TA-III significantly inhibited AP-1, and NF-κB activation (Figs. 4A, B). Also, we examined the upstream molecular pathways regulating UVB-induced COX-2 and MMP-2 protein levels. MAPKs and Akt affect the expression of COX-2 and MMP-2. We observed whether TA-III regulates the activity of MAPKs and confirmation that TA-III relatively decreased Akt, p-38 and p-JNK phosphorylation, but did not stimulate ERK (Fig. 5). These results indicated that TA-III diminished MMP-2 and, COX-2 expression and activity inhibiting NF-κB, AP-1, Akt, p-38 and JNK in HEK. We also observed notably decreased 8-oxo-dG formation and expression of DNA repair enzymes such as PCNA and, SMC1 throughout UVB radiation exposure resulting in increased DNA injury (Fig. 6). Recent studies announced that the transcriptional initiation and down regulation of precise DNA repair genes are controlled by inflammation responses.^25–27) 7-Hydroxycoumarin (7-OHC) pretreatment considerably elevated the expression of repair enzymes and downregulated inflammatory cytokines expression.²⁸⁾ These results reveal the direct associated with between improved DNA repair enzyme expression and downregulated pro-inflammation indicators. Additionally, our results of the mechanism revealed reduced DNA damage by TA-III treatment, as well as reduced cell cycle and induction of DNA repair enzymes. Collectively, our results demonstrate that TA-III inhibits UVB-stimulated COX-2 and MMP-9 expression by inhibiting NF-κB, AP-1 activation and MAPK signaling.

CONCLUSION

Our results demonstrated that TA-III protects skin cells from UVB-induced inflammatory response and DNA injury via inhibiting the inflammatory factors and suppresses DNA damage in HEK.

Acknowledgments

This work was supported by the R&D Program for Forest Science Technology [FTIS: 2017055A00-1819-AB02] provided by Korea Forest Service (Korea Forestry Promotion Institute) and Korea Institute of Oriental Medicine (NAN1713041)

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

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