Glycyrrhizic acid (GA), a triterpenoid saponin glycoside alleviates ultraviolet-B irradiation-induced photoaging in human dermal fibroblasts.
Reactive oxygen species
Human dermal fibroblast
Glycyrrhizic acid (GA), a triterpenoid saponin glycoside from the roots and rhizomes of licorice is used in traditional and modern medicine for the treatment of numerous medical conditions including skin diseases and beauty care product. In the present study, we investigated the effect of GA against ultraviolet B (UVB) irradiation-induced photoaging in human dermal fibroblasts (HDFs) and its possible mechanism of action. HDFs were subjected to photoaging by sub-toxic dose of UVB (10 mj/[cm.sub.2]) irradiation. Cell viability, matrix metalloproteinase 1 (MMP1), pro-collagen 1, cellular and nuclear morphology, cell cycle, intracellular reactive oxygen species (ROS), caspase 3 and hyaluronidase inhibition assays were performed. Western blotting was used to evaluate the expression of NF-kappa B (NF-KB) and cytochrome-C proteins. GA treatment significantly inhibited photoaging. It achieved this by reducing ROS, NF-KB, cytochrome c, caspase 3 levels and inhibiting hyaluronidase enzyme. The main mechanism seems to be, most likely by blocking MMP1 activation by modulating NF-KB signaling. These findings may be useful for development of natural and safe photoprotective agents against UVB irradiation.
[c] 2012 Elsevier GmbH. All rights reserved.
Skin is the largest human organ and is directly exposed to the harmful irradiation, toxic materials and microbial invasion and raises a physical, biochemical and immunological barrier against environmental insults (Riffle and Fisher 2002; Ding and Wang 2003). Ultraviolet (UV) irradiation has deleterious effects on human skin, including tanning, sunburn, immune suppression, cancer, and connective tissue degradation (photoaging) (Jones et al. 1999; Offord et al. 2002). Out of all subtypes of UV irradiation, only UV-B is capable of producing physiological response and is considered most hazardous environmental carcinogen (Katiyar et al. 2001; Fisher et al. 2002). Higher doses of UVB irradiation induces severe oxidative stress resulting in cell death (Morliere et al. 1995). While as sub-lethal UVB irradiation (physiological UV) induces oxidative stress and activates various intracellular signal transduction pathways leading to "mammalian UV response" (Vicentini et al. 2011). This response results in activation of nuclear factor kappa B (NF-kappa B). Upon activation, NF-kappa B-induces various genes including interleukin-1 (ILI ), tumor necrosis factor alpha (TNF alpha), and matrix metalloproteinase-1 (MMP-1). MMP-1 is the cause for extracellular matrix (ECM) degradation resulting in skin photoaging and eventually leading to cancer (Fisher et al. 1998; Jenkins 2002; Varani et al. 2002; Oh et al. 2004; Wenk et al. 2004).
Botanical antioxidants have been shown to be associated with reduced incidence of photocarcinogenesis and photoaging (F'guyer et al. 2003). Consistent with this understanding, botanical antioxidants have attracted considerable attention because of their skin photoprotective effects (Afaq et al. 2005).
Glycyrrhizic acid (GA), a triterpenoid saponin glycoside, is a major active constituent of licorice root, has been attributed with numerous pharmacologic effects, including anti-inflammatory, anti-viral, anti-tumor, hepatoprotective, and antioxidant activities (Asl and Hosseinzadeh 2008; Ni et al. 2011). Traditionally licorice has been used in various skin disorders, in cosmetics and personal care products. Licorice-derived ingredients are used in the formulation of makeup products, hair care products and skin care products.
Recently it has been suggested that GA down-regulates UVB induced signal transduction cascades in carcinogenesis and confers photoprotective effect in SKH-1 murine skin via down regulation of cell proliferation involving thymine dieter, proliferating cell nuclear antigen (PCNA), apoptosis and transcription factor NF-kappa B and inflammatory processes involving cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2) and nitric oxide (NO) (Lee et al. 2005; Cherng et al. 2011). Here we report the inhibitory effect of GA against UVB-induced photoaging in human dermal fibroblasts (HDFs) and the mechanisms involved.
Materials and methods
The normal human newborn foreskin fibroblast cell line, HS68 cell (ATCC CRL 1635), was obtained from American Type Culture Collection (Rockville, MD, USA). Glycyrrhizic acid 75% ([C.sub.42][H.sub62][O.sub.16] * [NH.sub.3], FW= 840.0), Dimethyl sulphoxide (DMSO), Dulbecco's modified eagle's media (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, trypsin-EDTA, 344,5-dimethylthiazo1-2-y1)-2,5-diphenyltetrazolium bromide (MTT), 2,7-dichlorodihydrofluorescein diacetate (DCHF-DA), propidium iodide, ribonuclease A (RNase A), hyaluronic acid (HA), hyaluronidase (ENZ) were purchased from Sigma Aldrich Chemicals Private Limited (St. Louis, MO). Pro-collagen type 1 C-peptide protein and matrix metaloproteinase-1 ELISA kits were procured from Takara, Japan; Cat.#M1001 and GE Healthcare; Code: RPN2610, respectively. NF-kappa B (p50), cytochrome C, [BETA]-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) All other biochemicals used were of high purity biochemistry grade.
HS68 cells used for the experiments were between 10 and 25 passages of their growth period. HS68 cells were plated in 175 [cm.sub.2] culture flasks and grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin streptomycin (Sigma, St. Louis, Mo, USA). The cells were pretreated with GA for 24 h, washed with phosphate buffer saline (PBS; Sigma, St. Louis, Mo, USA) and irradiated under UVB radiation. After UVB irradiation, cells were rinsed twice with PBS and incubated in fresh culture media without serum, in presence of GA for further 24 h. All UVB irradiations were performed under a thin layer of PBS.
The source of UVB radiation was a band of four UVB lamps (Daavlin, UVA/UVB Research Irradiation Unit, Bryan, OH, USA) equipped with digital controller to regulate UV dosage at a fixed distance of 24 cm from the lamps to the surface of the cell culture plates. The majority of the resulting wavelengths (>90%) were in the UVB range (280-320 nm) and UVA was less than 10%. The peak emission was recorded at 314 nm.
Cell viability was determined as described by (Moon et al. 2008), by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. For cytotoxicity experiments, cells were treated with GA at mentioned concentrations and incubated for 24 h. For cyto protection experiments, cells were pretreated with GA for 24 h, washed twice with PBS and subjected to UVB-irradiation. After irradiation, cells were again washed twice with PBS and cultured in fresh medium in presence of GA for further 24 h. After corresponding treatments the medium was removed and cell viability was evaluated by assaying the ability of functional mitochondria to catalyze the reduction of MIT (0.5 mg/ml, at 37 [degrees] C for 3 h) to form formazan salt by mitochondrial dehydrogenases, as determined by ELISA reader at 565 nm (Multiskan Spectrum; Thermo Electron Corporation, USA).
Cellular and nuclear morphology
The cellular and nuclear morphology was observed under the light microscope (Nikon Eclipse TE2000U), at 20X magnification, or under fluorescent microscopy, using Hoechst 33258 staining, as described earlier (Tasduq et al. 2008).
Pro-collagen-1 and matrix metalloproteinase-1 immunoassay
The levels of type 1 procollagen (Takara, Japan) and matrix metalloproteinase-1 proteins (GE Healthcare) in cell-free supernatants were determined by ELISA. The cell free supernatants of cultured fibroblasts after indicated treatments were collected and stored at -80 [degrees] C until used. The assays were performed according to manufacturer's instructions.
Hyaluronidase inhibition assay
Hyaluronidase (HA) was assayed as described by (Sumantran et al. 2007), based on precipitation of HA with cetyl pyridinium chloride. Enzyme (800 U/ml) and HA substrate (0.40 mg/ml) were incubated at 37 [degrees] C for 1 h. Enzyme activity was measured by monitoring the percentage of undigested HA substrate in the cetyl pyridinium chloride precipitate at absorbance 415 nm (A 415 nm) after the enzyme reaction.
Cell cycle analysis
Cell cycle was analyzed as described by Yang et al. (2007) with some modifications. Briefly, non-treated and treated skin fibroblast cells were harvested by trypsinization, centrifuged at 1500 x g for 5 min, washed with PBS, and fixed in 70% ethanol at 4 [degrees] C overnight. Fixed cells were washed twice with PBS and incubated in PBS containing 1.5 mg/l RNase A for 1 h at 37 [degrees] C, followed by staining with 5 [micro]lPI (1 mmol/l stock) for 20 min on ice. The cells were analyzed for DNA content using BD FACS Calibur cytometer using blue (488 nm) excitation from argon laser. Data were collected in list mode on 10,000 events for FL2-A vs. FL2-W.
Intracellular reactive oxygen species (ROS)
The intracellular production of ROS levels were determined as described by Heo et al. (2009) using 2',7'-dichlorofluorescein diacetate (DCFDA). Cells grown in 96-blackwell (Nunc, Denmark), after pretreatment of GA for 24 h, were washed twice with PBS and irradiated with UVB (10 mj/[cm.sup.2]) under a thin layer of PBS. Immediately after UVB irradiation, cells were again washed twice with PBS and DCFDA was added (5 [micro]M) and incubated for 30 min at 37 [degrees] C in a [CO.sub.2] incubator. DCFDA is oxidized by ROS to the highly fluorescent 2',7'-dichlorofluorescein (DCF). The fluorescence was read at ex/em of 488/525 nm with Perkin Elmer, LS 55, USA.
Caspase 3 assay
Caspase activation was measured using a caspase 3 fluorometric assay kit (BD Apoalert caspase 3 fluorescent assay kit). HDFs, treated or untreated were harvested, and centrifuged (approximately 1 mg protein) at 400 x g for 5 min. The cell pellets were re-suspended in 50 [micro]l of chilled cell lysis buffer and incubated on ice for 10 min, and the lysates were centrifuged at 15,000 x g for 10 min at 4 [degrees] C to precipitate cellular debris. A total of 50 [micro]l of cell lysates was incubated with 50 [micro]l of reaction buffer/DTT mix. DEVD-CHO was used as an inhibitor of caspase 3 in an induced sample. Five ill of 1 mmol/l caspase-3 substrate (DEVD-AFC at a final concentration of 50 [micro]mol/l) was added to each sample. The samples were incubated at 37 [degrees] C for 1 h and read on a spectrofluorometer (Perkin Elmer LS 55) with excitation wavelength 400 nm and emission wavelength of 505 nm.
Preparation of whole cell lysate for western blotting
After treatment, HDFs (HS68) were washed twice with ice-cold PBS, scraped from the dishes and transferred to microfuge tubes in cell lysis buffer (25 mM HEPES, pH 7.2, 2.5 mM Mg[Cl.sub.2], 75 mM NaCI, 0.2 mM EDTA, 0.1% Triton X-100, 0.5 mM dithiothreitol (DTT) and 20 mM b-glycerophosphate), supplemented with 10 mg/ml aprotinin, 10 mg/ml leupeptin, 10 mg/ml pepstatin A and 1 mM, phenylmethylsulfonyl fluoride (PMSF), and 1 mM sodium orthovanadate. Following 20 min of incubation at 4 [degrees] C, cell homogenates were centrifuged at 14,000 x g at 4 [degrees] C for 10 min, and supernatants were collected and used as whole cell lysates. For protein expression by western blotting, equal amounts of proteins (50 [micro]g protein per lane) were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membrane (BioRad, USA). Western blots were developed and quantified using a chemiluminescent substrate (ECF Western blot Reagents, GE Healthcare, Piscataway, NJ). Detection of chemiluminescence was performed using Hyper film (GE Healthcare, Piscataway, NJ). Densitometric analysis of the blots were performed by using image lab software version 3.0.
Protein concentration was measured with Bradford Reagent (Sigma Aldrich Chemicals Private Limited, St. Louis, MO) as per instructions of the manufacturer.
Results are expressed as mean [+ or -] SD. Data were analyzed for significant difference from the untreated control group by Student's t-test and p values <0.05 were considered statistically significant.
Effect of UVB irradiation induced cytotoxicity
In preliminary experiments, we evaluated the effect of UVB irradiation on MMP-1 and procollagen type-1 levels in cultured fibroblast over a wide range of UVB irradiation (0-100 mj/[cm.sub.2]; data not shown). A UVB dose of 10 mj/[cm.sub.2] optimally increased MMP-1 levels and decreased procollagen 1 content (markers of photoaging), without causing excessive toxicity and was chosen for subsequent experiments for evaluation of anti photoaging effect of GA. At 5 mj/[cm.sub.2], there was an increase in cell viability (11%; p < 0.001) compared to non-irradiated cells. And at 10, 15 and 20 mj/[cm.sub.2], the cell viability decreased by 11%, 28% and 30% (p <0.001) respectively compared to non irradiated cells (Fig. 1A).
Effect of GA on cell viability and cytoprotective effect of GA against UVB irradiation
Incubation of fibroblasts with various concentrations of GA (5-50 [micro]M), for 24 h, resulted in enhanced cell viability in a concentration dependent manner. GA treatment resulted in increased viability by 9-115% compared to non-treated cells (control) (p <0.001). UVB irradiation at a dose of 10 mj/[cm.sub.2] resulted in a decreased cell viability by 11% (p < 0.001) compared to non irradiated cells. Treatment with GA at 5, 10, 25 and 50 M showed a significant protective effect (8-22%, p < 0.001) against UVB irradiation induced loss of cell viability at 10 mj/[cm.sub.2].
Effect of GA on UVB induced altered cellular and nuclear morphology
Nuclei of control (non-irradiated) cells appeared prominently round in shape. After UVB irradiation caused morphological alterations in cells marked with condensation of nuclei. The prominent changes were, an increased apoptotic bodies and cellular debris. These alterations were prevented by treatment with GA (Fig. 2A and B).
Effect of GA on pro-collagen 1 and pro-matrix metalloproteinase 1 (pro-MMP-1) levels in skin fibroblasts irradiated with UVB
Irradiation of skin fibroblast with UVB irradiation (10 mj/[cm.sub.2]), resulted in 8.6-fold decrease in pro-collagen 1 levels compared to non-irradiated cells (control). This shows the intense effect of UVB irradiation on pro-collagen 1 in skin fibroblasts. This decrease in pro-collagen 1 levels was restored by GA treatment at 5, 10, 20 and 50 [micro]M in a range of 48 (p < 0.003) to 74% (p < 0.001). pro-MMP-1 levels were enhanced by 19.2-folds due to UVB irradiation (10 mj/[cm.sub.2]) compared to non irradiated cells. Treatment with GA (5-50 [micro]M) restored these levels by about 90% (p < 0.001; Fig. 3).
Inhibition of hyaluronidase by GA
Hyaluronidase enzyme (ENZ) at 800 U/ml caused 87% (p < 0.001) digestion of Hyaluronic acid (HA) (0.40 mg/ml) when incubated at 37 [degrees] C for 1 h. Incubation of ENZ and HA in presence of GA at 10, 25 and 50 [micro]M resulted in strong inhibition of digestion of HA by ENZ (above 80%, p < 0.001; Fig. 4).
Effect of UVB on cell cycle and amelioration by GA
UVB irradiation at 10 mj/[cm.sub.2], significantly caused cell cycle arrest (42% cells in sub [G.sub.0]) compared to non irradiated cells (7%). Treatment with GA at 5, 10, 25 and 50 [micro]M resulted in restoration of cell cycle arrest by 41, 30, 28 and 21% respectively (Fig. 5).
Effect of GA on UVB induced caspase 3 and intracellular reactive oxygen species (ROS) levels
UVB irradiation (10 mj/[cm.sub.2]) caused an increase of 7.7-folds in caspase 3 levels in skin fibroblasts. This increase in caspase 3 levels was effectively decreased by GA treatment at 10, 25 and 50 [micro]M by 12.7% (p < 0.002), 37% (p < 0.001) and 55% (p < 0.001), respectively. UVB irradiation (10 mj/[cm.sub.2]) caused an increase of3.5-folds in intracellular ROS levels in skin fibroblasts compared to non-irradiated cells. This increase in ROS levels was effectively quenched by GA treatment at 10, 25 and 50 [micro]M by 25% (p < 0.002), 39% (p < 0.001) and 50% (p < 0.001), respectively (Fig. 6).
Effect of GA on NF-kappa B and cytochrome C
We explored inhibition of UVB-induced expression of NF-kappa B and cytochrome C by immunoblotting, using specific antibodies. Results shows that UVB irradiation enhanced NF-kappa B by about 1.4-fold compared to non irradiated cells. GA treatment caused a decrease in the range of 1.8-to 4-folds compared to UVB irradiated cells. Cytochrome C levels were enhanced by UVB irradiation in HDFs by 4.1-folds compared to non irradiated cells. This enhancement in cytochrome C levels was decreased by GA by about 1-fold (Fig. 7).
UV-irradiation is the main environmental hazard for causing skin photo-aging. The main mechanism of skin photo-aging has been attributed to the production of pro-inflammatory cytokines, ROS and effector molecules like MMP-1. These events have been shown to be controlled by NF-kappa B, which is in turn activated as a result of mammalian UV response (Tanaka et al. 2005). NF-kappa B is an important factor in the maintenance of skin homeostasis, however its excessive activation, is involved in several skin related pathological states including psoriasis, dermatitis and skin cancer (Bel et al. 2003; Tanaka et al. 2010). In this study we have examined the association of NF-kappa B in UV-B mediated skin photo-aging, through induction of ROS and MMP-1 and inhibition of such events byGA.
Present results suggest that apart from inducing MMP-1, ROS levels were also enhanced in human skin fibroblasts irradiated with UVB (10mj/[cm.sup.2). However, there remains a difficulty in defining contribution of ROS signaling in its diverse function with respect to upstream or downstream effects within a given pathway leading to inhibition or stimulation of NF-kappa B. It has also been established that ROS stimulates NF-kappa B pathway in cytoplasm, but inhibits its activity in nucleus.This phenomenon of opposing effects of ROS with respect to NF-kappa B has been described to be cell type-specific (Morgan and Liu 2011). ROS have strongly been implicated in the activation of NF-kappa B. This hypothesis is mainly based on the findings that strong anti-oxidants like N-acetyl-L-cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC) can inhibit NF-kappa B activation in a wide variety of cell types (Schreck et al. 1991). However, both NAC and PDTC have been shown to inhibit NF-kappa B activation independent of their anti-oxidant activity and also much stronger phenolic radical scavengers like epigallocatechin gallate (EGCG) and Trolox failed to inhibit NF-kappa B activation (Hayakawa et al. 2003). Therefore, it is clear, that the ability of a phtoprotective agent, in inhibiting NF-kappa B activation is independent of its anti-oxidant activity. Our result strongly suggest an existence of a strong co-relation between ROS and NF-kappa B due to UVB-irradiation. And GA acts as a strong ROS quenching agent and an inhibitor of NF-kappa B. However, results suggest that NF-kappa B and ROS inhibitory effects of GA are independent of one another.
The photo-protective effect of GA seem to occur at several mechanistically different levels. Our results show that GA has the potential to protect HDFs from UVB irradiation-induced ROS, MMP-1 and apoptotic sunburn. One of the most important characteristic of UVB irradiation-induced photo-aging in HDFs is through apoptosis (DNA damage) and disturbed ECM (enhanced MMP-1 activity; mammalian UV response) (Fisher et al. 1998; Jenkins 2002; Varani et al. 2002; Oh et al. 2004; Wenk et al. 2004). This seems to be in collaboration with respect to present results like DNA damage (cell cycle and hoechst staining), induction of apoptosis (cytochrome c release and caspase 3 activation) and collagen degradation. MMP-1 activity was increased by about 8.6-folds in HDFs exposed to UVB-irradiation (10mj/[cm.sup.2]) leading to degradation of collagen 1 thus promoting photo-aging (Fig. 3). UVB irradiation resulting in collagen degradation is most likely via activated collagenolytic MMPs. Collagen and hyaluronic acid are the important ECMs, that provide skin with diversified structure along with elasticity, strength and resiliency. MMPs inhibition has been used as one of the strategies to prevent UVB triggered photo-damage (Bae et al. 2010). This statement is in total agreement with respect to present results showing strong inhibitory effect of GA against UVB irradiation-induced MMP-1 levels and protective effect on collagen 1 degradation.
UVB irradiation-induced collagen degradation and MMP production have been linked to ROS (Bae et al. 2008). We have previously shown that ROS mediated MMP1 induction in UVB exposed HDFs was accompanied by connective tissue breakdown. This ROS mediated photo-damage was strongly augmented by Emblica officinalis fruit extract by its strong anti-oxidant effect (Adil et al. 2010). Anti-oxidants like ascorbic acid and [alpha]-tocopherol have also been shown to prevent UVB-induced signaling cascade leading to photo-aging in HDFs (Amann et al. 1999; Shibayama et al. 2008). Accordingly, use of antioxidants, capable of scavenging and quenching ROS is an another important approach to prevent UVB induced photo-aging (Afaq et al. 2007). GA shows a strong antioxidant activity by scavenging UVB irradiation-induced intracellular ROS levels in HDFs (Fig. 6). Several other studies in agreement with this have shown that botanical compounds with an anti-oxidative activity are potential agents capable of reducing skin diseases including photo-aging (Bae et al. 2008). Therefore present results are clearly indicative of the fact that GA effectively scavenges UVB-induced cellular ROS and inhibit NF-kappa B activation. This leads to inhibition of MMP-1 levels leading to prevention of ECM degradation (collagen 1 and hyluronic acid) and also prevents apoptotic form of cell death as evident by inhibitory effect of GA on UVB-induced caspase 3 and cytochrome C release in HDF. In conclusion, we suggest that GA has a strong potential to be further evaluated and developed as a therapeutic and cosmetic product against various skin ailments including photo-aging.
We are grateful to Dr. Ram A. Vishwakarma, Director of this institute for providing constant guidance and financial support for the study (Major Lab Project No. 6005). Council of Scientific and Industrial Research (CSIR), New Delhi, India is acknowledged for Junior Research Fellowships to Adil M.D. and Rang R.A. University Grants Commission (UGC), New Delhi, India is acknowledged for Junior Research Fellowships to Afnan Q. and Nissar U.A.
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(1.) Quadri Afnan and Mushtaq Dar Adil are equal contribuwrs to the study.
Quadri Afnan (1), Mushtaq Dar Adil (1), Ashraf Nissar-Ul, Ahmad Rather Rafiq, Hussian Faridi Amir, Peerzada Kaiser, Vijay Kumar Gupta, Ram Vishwakarma, Sheikh Abdullah Tasduq *
Indian Institute of Integrative Medicine. Council of Scientific and Industrial Research (CSIR), Canal Road, Jammu Tawi, Jammu and Kashmir, India
* Corresponding author at: Experimental Toxicology Lab, PK-PD and Toxicology Division, Indian Institute of Integrative Medicine, Council of Scientific and Industrial Research (CSIR), Canal Road, Jammu Tawi, jammu and Kashmir, India. Tel.: +91 191 25690009x331; fax: +91 191 2569333; mobile: +91 9419148712.
E-mail address: email@example.com (S.A. Tasduq).
0944-7113/$--see front matter [c] 2012 Elsevier GmhH. All rights reserved.