Stereoselective Skin Anti-photoaging Properties of Ginsenoside Rg3 in UV-B-Irradiated Keratinocytes

Abstract

Ginsenosides are major bioactive constituents that are responsible for the diverse pharmacological activities of ginseng. This work aimed to assess the skin anti-photoaging activities of the two stereoisomeric forms of ginsenoside Rg3, 20(S)-Rg3 and 20(R)-Rg3. When the two Rg3 stereoisomers were added to cultured human keratinocyte HaCaT cells prior to irradiation with 70 mJ/cm² UV-B, 20(S)-Rg3, but not 20(R)-Rg3, decreased the UV-B-induced intracellular reactive oxygen species (ROS) levels in a concentration-dependent manner, as detected by both fluorometric and confocal microscopic analyses. Likewise, 20(S)-Rg3, but not 20(R)-Rg3, decreased the UV-B-induced ROS levels in human dermal fibroblast cells. Both stereoisomers were unable to modulate the nitric oxide levels in HaCaT cells under UV-B irradiation, and induced no cytotoxicity in cultured keratinocytes and fibroblasts. 20(S)-Rg3 suppressed the UV-B-induced matrix metalloproteinase (MMP)-2 activities in HaCaT cells. Taken together, these results indicate that 20(S)-Rg3 possesses both ROS-scavenging and MMP-2 inhibitory activities, while 20(R)-Rg3 possesses neither activity. These findings imply that ginsenoside Rg3 stereoselectively demonstrates skin anti-photoaging activities.

Solar UV radiation is comprised of UV-A (315–400 nm), UV-B (280–315 nm), and UV-C (200–280 nm) wavelengths. Most UV-C radiation is absorbed by stratospheric ozone; very little reaches the Earth’s surface. In contrast, UV-A and UV-B reach the Earth’s surface and, subsequently, have the potential to harm humans. Although most biomolecules cannot absorb UV-A, UV-B is particularly harmful to living organisms. UV-A and UV-B contribute to detrimental effects via different molecular mechanisms, such as direct DNA damage, modulation of gene expression, and reactive oxygen species (ROS) generation.¹⁾ UV-B acts primarily on the epidermal basal cell layer of the skin. It initiates a photo-oxidation reaction that impairs the antioxidant status of the skin and increases the cellular ROS level, sequentially accelerating photoaging and the development of malignant diseases.^2–5) UV-B is one of leading causes of skin changes, such as wrinkle formation, laxity, coarseness, mottled pigmentation, epidermal thickening, degradation of matrix macromolecules, vascularization, and immunosuppression.^6,7) Keratinocytes, the major cell population in the basal layer of the skin, are the primary targets of UV-B. In keratinocytes, UV-B induces the immediate generation of superoxide radical, which is sequentially converted to other ROS species, such as hydrogen peroxide and hydroxyl radical.⁸⁾

Matrix metalloproteinases (MMPs) have been implicated in remodeling extracellular matrix (ECM) structures in wound healing,⁹⁾ dermal photoaging,¹⁰⁾ and severe pathologic states such as carcinogenesis.¹¹⁾ MMP-1 (interstitial collagenase) is produced by both dermal fibroblasts and epidermal keratinocytes. MMP-1 cleaves types I and III collagen into specific fragments that are further degraded by other MMPs, including MMP-2 (72 kDa gelatinase A/collagenase) and MMP-9 (92 kDa gelatinase B/type IV collagenase). UV radiation, including UV-B, induces the expression and secretion of MMP-1, -2, -3, -9, and/or -13 in the epidermis and dermis.^12,13) Destruction of collagen by UV irradiation, a cause of skin aging in both naturally aged and photoaged skin, is related to the enhanced induction of MMPs, which are secreted by epidermal keratinocytes and dermal fibroblasts.¹⁴⁾

Ginsenosides, also referred to as triterpenoid saponins, are the bioactive ingredients of ginseng, an herbal medication derived from the root of the Panax genus of plants that is widely used in traditional medicine. Ginsenosides are categorized into three groups on the basis of their aglycone (sapogenin) skeletons: the panaxadiol-type, including Rb1, Rb2, Rb3, Rc, Rd, Rg3, Rh2, and Rs1; the panaxatriol-type, including Re, Rf, Rg1, Rg2, and Rh1; and the oleanolic acid group, including Ro.

Ginsenoside Rg3, one of the most effective ginsenosides, exerts a wide spectrum of pharmacological actions through anti-inflammatory, anti-tumor, anti-angiogenic, anti-metastatic, and anti-diabetic activities.^15–21) Ginsenoside Rg3 exists in two stereoisomeric forms, 20(S)-Rg3 and 20(R)-Rg3 (Fig. 1), based on a chiral center at C-20 in its molecular structure. Of the two stereoisomers, 20(S)-Rg3 is the major constituent in the natural isolation process. 20(S)-Rg3 reduces amyloid β-peptide levels in cultured primary neurons and in brain tissue of a mouse model of Alzheimer’s disease.²²⁾ 20(R)-Rg3 displayed a neuroprotective effect against transient focal cerebral ischemia in rats through down-regulation of calpain I and caspase-3 and subsequent attenuation of neuronal apoptosis.²³⁾

Fig. 1. Chemical Structures of the Two Stereoisomers of Ginsenoside Rg3 (Rg3), 20(S)-Rg3 and 20(R)-Rg3

The stereospecific properties of the two Rg3 stereoisomers have been attracting interest and may help to elucidate the underlying mechanisms of Rg3 actions. Although both 20(S)-Rg3 and 20(R)-Rg3 exhibit adjuvant effects on ovalbumin-induced immune responses in mice, 20(R)-Rg3 exhibits a more potent adjuvant activity than 20(S)-Rg3, implying that Rg3 is stereospecific in stimulation of the immune response.²⁴⁾ Similarly, 20(R)-Rg3 exhibits significantly higher inhibitory effects than 20(S)-Rg3 against oxidative stress induced by cyclophosphamide in mice.²⁵⁾

Ginsenoside(s) with a protective role against skin photoaging are desirable for use in anti-photoaging cosmetics. However, the anti-photoaging activities of ginseng and its components on UV-irradiated skin have not been clearly assessed. In this work, the anti-photoaging activities of the two stereoisomers of Rg3, 20(S)-Rg3 and 20(R)-Rg3, were examined in UV-B-irradiated keratinocytes and fibroblasts.

MATERIALS AND METHODS

Chemicals

20(S)-Rg3 (purity≥98%) and 20(R)-Rg3 (purity≥98%) were purchased from Ambo Institute (Seoul, Korea). Gelatin, sodium nitrite, sodium dodecyl sulfate (SDS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2,′7′-dichlorofluorescein diacetate (DCFH-DA), and Griess reagent were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin–streptomycin, and trypsin–ethylenediaminetetraacetic acid (EDTA) were purchased from HyClone Laboratories Inc. (Logan, UT, U.S.A.). All other chemicals used were of the highest grade commercially available.

Cell Culture and UV-B Irradiation

An immortalized human keratinocyte cell line, HaCaT (ATCC, Manassas, VA, U.S.A.), and a human dermal fibroblast cell line, CCD986sk (ATCC), were cultured in DMEM containing 10% heat-inactivated FBS, 100 U/mL penicillin and 100 µg/mL streptomycin in a humidified atmosphere with 5% CO₂ at 37°C.

A UV lamp (peak, 312 nm; model VL-6M, Vilber Lourmat, Marine, France) was used as a UV-B source. The radiation intensity was monitored using a radiometer (model VLX-3W, Vilber Lourmat) with a sensor (bandwidth, 280 to 320 nm; model CX-312, Vilber Lourmat). Cultured mammalian cells were irradiated with 70 mJ/cm² UV-B for experiments.

Detection of Intracellular ROS Production

For fluorometric analysis of intracellular ROS, the redox-sensitive fluorescent probe DCFH-DA, which generates the fluorescent 2,′7′-dichlorofluorescein (DCF; λ_excitation=485 nm, λ_emission=525 nm) upon enzymatic reduction and subsequent oxidation by ROS, was used as previously described.²⁶⁾ HaCaT cells or CCD986sk cells were seeded (1×10⁵ per well) onto 24-well plates and cultured overnight, washed twice with 1 mL of phosphate buffered saline (PBS), and covered with 1 mL of FBS-free media. After incubation with 20(S)-Rg3 or 20(R)-Rg3 and 10 µM DCFH-DA for 30 min at 37°C, the cells were washed twice with 1 mL of FBS-free media. The cells were covered with 1 mL of FBS-free media and irradiated with 70 mJ/cm² UV-B. The intracellular ROS levels were immediately analyzed with a Synergy™ Mx Multi-Mode Microplate Reader (BioTek Instruments, Winooki, VT, U.S.A.).

For confocal microscopic analysis, 3×10⁵ HaCaT cells were seeded per well onto 6-well plates and cultured overnight, washed twice with 1 mL of PBS, and suspended in 1 mL of serum-free media. After incubation with 20(S)-Rg3 or 20(R)-Rg3 and 10 µM DCFH-DA for 30 min at 37°C, the cells were irradiated with 70 mJ/cm² UV-B and immediately analyzed with a Fluoview-FV300 confocal laser scanning microscope (Olympus, Tokyo, Japan). These assays were repeated at least three times.

Determination of Nitrite in Culture Supernatants

The level of accumulated nitrite (NO₂⁻), generated from cell-released nitric oxide (NO), in the culture supernatants was determined with a spectrophotometric assay based on the Griess reaction.²⁷⁾ In brief, an equal volume of Griess reagent (1% sulfanilamide–0.1% N-1-naphthyl-ethylenediamine dihydrochloride in 2.5% phosphoric acid) was incubated with culture supernatants for 10 min at room temperature. The absorbance at 550 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader (Molecular Devices, Sunnyvale, CA, U.S.A.). The calibration curve was constructed with known concentrations (0–160 µM) of sodium nitrite.

Cell Viability Assay

To assess the survival of cultured mammalian cells in the presence of 20(S)-Rg3 or 20(R)-Rg3, the cell viability was determined by an MTT assay of cellular metabolic activity.²⁸⁾ A total of 1×10⁵ cells were seeded into each well of a 24-well plate, cultured overnight at 37°C, and washed twice with 1 mL of PBS. After replacing the culture media with 1 mL of FBS-free media, the cells were treated with 20(S)-Rg3 or 20(R)-Rg3 for 30 min. The cells were washed twice with 1 mL of FBS-free media and if necessary, irradiated with 70 mJ/cm² UV-B. After the media was removed by suction, the cells were incubated with 5 µg/mL MTT solution for 4 h. The cells were then lysed with dimethyl sulfoxide. The amount of formazan, produced from the reduction of MTT by the mitochondria of living cells, was determined by absorbance measurements at 540 nm.

Gelatin Zymography

The gelatinolytic activities of MMP-2 and MMP-1 in culture supernatants were determined by zymographic analysis, as previously described.²⁹⁾ HaCaT cells (1×10⁵ per well) were seeded onto 24-well plates, cultured overnight at 37°C, and washed twice with 1 mL of PBS. Cells were re-suspended in 1 mL of FBS-free media, incubated for 24 h at 37°C, and washed twice with 1 mL of PBS. Cells in 1 mL of FBS-free media were incubated with 20(S)-Rg3 or 20(R)-Rg3 for 30 min and irradiated with 70 mJ/cm² UV-B.

Culture supernatants obtained from the incubation of irradiated cells for 24 h at 37°C were fractionated on a 10% (w/v) SDS-polyacrylamide gel electrophoresis (PAGE) gel impregnated with 1 mg/mL gelatin under non-reducing conditions. Proteins in the gel were renatured by two consecutive 30-min periods of shaking with 2.5% Triton X-100 at room temperature. The gel was stored in incubation buffer (50 mM Tris buffer, pH 7.8, 5 mM CaCl₂, 0.15 M NaCl, 1% Triton X-100) for 24 h. After the gel was stained with a solution of 0.1% Coomassie Brilliant Blue R-250, gelatin-degrading activities were observed as clear zones against a blue background. MMP-1 and MMP-2 activity bands were identified in accordance with their molecular weights, which were estimated by molecular mass markers.

Statistical Analysis

The results were expressed in terms of the mean±S.D. Statistical comparisons between experimental groups were performed using the Kruskal–Wallis test, followed by Dunn’s post-hoc test for pairwise individual comparison. A p-value of less than 0.05 was considered statistically significant.

RESULTS

ROS-Scavenging Activity

At physiological concentrations, ROS are closely linked with various cellular functions, including intracellular signaling and redox regulation. Excessive ROS produced during abnormal metabolic reactions lead to a variety of damages to macromolecules, resulting in genetic mutation, physiological dysfunction, and, ultimately, cell death.³⁰⁾ The intracellular ROS level is also elevated in the presence of exogenous oxidative stress-inducing agents. When the defense systems of living cells cannot thoroughly cope with stress-inducing agents, the cells experience oxidative stress, which adversely affect many different cellular processes and functions. Because the intracellular ROS level increases under a variety of stresses, particularly oxidative stress, it is one cellular marker that is closely linked with the internal stress level of cells.

In the present work, cultured HaCaT cells were pretreated with varying concentrations of the two Rg3 stereoisomers, 20(S)-Rg3 and 20(R)-Rg3, prior to irradiation with 70 mJ/cm² UV-B. As shown in Figs. 2A and 3A, the UV-B irradiation, in the absence of prior 20(S)-Rg3 treatment, induced a 5.2-fold increase in the ROS level over that in the non-irradiated control HaCaT cells. 20(S)-Rg3 attenuated the irradiation-induced ROS increase in a concentration-dependent manner (Fig. 2A). Treatment with 20(S)-Rg3 at concentrations of 5, 12, and 30 µM blocked the irradiation-induced ROS increases by 21.5%, 54.8%, and 56.8%, respectively (Fig. 2A). However, 20(R)-Rg3, used at the same concentrations, was unable to block the increase in ROS level induced by UV-B irradiation (Fig. 3A). This discrepancy in the ROS-scavenging activities of the two Rg3 stereoisomers was also ascertained using confocal microscopic analysis. The microscopic patterns were weakened in a concentration-dependent manner by treatment with 20(S)-Rg3 (Fig. 4A), whereas no changes were observed in the microscopic patterns obtained with the treatment of 20(R)-Rg3 (Fig. 4B).

Fig. 2. Effects of 20(S)-Rg3 on Reactive Oxygen Species (ROS, (A) and Nitric Oxide (NO, (B) Levels and Cellular Viability (C) of Human HaCaT Keratinocytes Subjected to Irradiation with 70 mJ/cm² UV-B

Mammalian cells in fresh media were treated with the indicated concentrations (0, 5, 12 or 30 µM) of 20(S)-Rg3 for 30 min before irradiation. In (A), the ROS levels were determined using DCFH-DA in a microplate fluorometer. The ROS level is represented as DCF fluorescence, an arbitrary unit. In (B), accumulated nitrite, an index of NO, in the culture supernatants was determined by the Griess reaction. In (C), the cell viability, represented as relative percentage, was determined by an MTT assay. Each bar shows the mean±S.D. of the three independent experiments repeated in triplicate. * p<0.05; ** p<0.01; *** p<0.001 versus the non-treated control (irradiation only).

Fig. 3. Effects of 20(R)-Rg3 on the Reactive Oxygen Species (ROS, (A) and Nitric Oxide (NO, (B) Levels and Cellular Viability (C) of Human HaCaT Keratinocytes Subjected to Irradiation with 70 mJ/cm² UV-B

Mammalian cells in fresh media were treated with the indicated concentrations (0, 5, 12 or 30 µM) of 20(R)-Rg3 for 30 min before irradiation. In (A), the ROS levels were determined using DCFH-DA in a microplate fluorometer. The ROS level is represented as DCF fluorescence, an arbitrary unit. In (B), accumulated nitrite, an index of NO, in the culture supernatants was determined by the Griess reaction. In (C), the cell viability, represented as relative percentage, was determined by an MTT assay. Each bar shows the mean±S.D. of the three independent experiments repeated in triplicate.

Fig. 4. Confocal Microscopic Analysis of the Effects of 20(S)-Rg3 (A) and 20(R)-Rg3 (B) on the Reactive Oxygen Species (ROS) Level in Human HaCaT Keratinocytes Subjected to Irradiation with 70 mJ/cm² UV-B

Mammalian cells were irradiated with 70 mJ/cm² UV-B in the presence of 20(S)-Rg3 and 20(R)-Rg3. ROS levels were determined using DCFH-DA followed by confocal laser scanning microscopy. Representatives of three independent experiments are shown. Scale bar, 20 µm.

The stereospecific ROS-scavenging activity of 20(S)-Rg3 was also examined in fibroblasts. When 20(S)-Rg3 was administered at concentrations of 5, 12, and 30 µM to cultured human fibroblast CCD986sk cells, the ROS levels dropped to 69.3%, 46.1%, and 33.7%, respectively, of that observed in cells treated with UV-B irradiation only (Fig. 5A). 20(R)-Rg3 exhibited no ROS-scavenging activity in the UV-B-irradiated fibroblasts (Fig. 5B). Taken together, the data indicate that the two Rg3 stereoisomers act differently in scavenging the ROS levels of keratinocytes and fibroblasts under UV-B irradiation, and only the S-type stereoisomer of Rg3, 20(S)-Rg3, is capable of reducing the ROS level enhanced by UV-B irradiation.

Fig. 5. Effects of 20(S)-Rg3 (A), (C) and 20(R)-Rg3 (B), (D) on the Reactive Oxygen Species (ROS, A, B) Levels and Cellular Viabilities (C, D) of Human Dermal Fibroblast CCD986sk Cells

Mammalian cells were subjected to fresh media with the indicated concentrations (0, 5, 12 or 30 µM) of 20(S)-Rg3 and 20(R)-Rg3 for 30 min before irradiation with 70 mJ/cm² UV-B. In (A) and (B), ROS levels were determined using DCFH-DA in a microplate fluorometer. The ROS level is represented as DCF fluorescence, an arbitrary unit. In (C) and (D), an MTT assay was performed using non-irradiated fibroblast cells incubated in the presence of 20(S)-Rg3 and 20(R)-Rg3, and the cell viabilities were represented as relative percentages. Each bar shows the mean±S.D. of three independent experiments repeated in triplicate. ** p<0.01; *** p<0.001 versus the non-treated control cells (irradiation only).

NO-Scavenging Activity

Nitric oxide (NO^·; NO) possesses physiological effects when produced in minute quantities by constitutive nitric oxide synthases (cNOSs). NO is involved in various pathologic phenomena when produced in excessive quantities by inducible nitric oxide synthase (iNOSs) in response to a variety of stress-inducing agents, such as nitrogen starvation. The direct effects of NO occur from direct reaction with its biological target. Its indirect effects are mediated by formation of reactive nitrogen species (RNS), which undergo subsequent reactions with their respective biological targets, such as proteins, lipids, and DNA. RNS play crucial roles in cellular signaling, but when produced at high concentrations, they induce cellular nitrosative stress, which may ultimately lead to cell death.

To examine whether NO is involved in the response against UV-B irradiation, NO levels were measured in UV-B-irradiated HaCaT cells with or without pretreatment with 20(S)-Rg3 and 20(R)-Rg3. As shown in Fig. 2B, NO levels in UV-B-irradiated cells were very close to those in non-irradiated control cells, and remained unchanged even in the presence of 20(S)-Rg3. Similarly, 20(R)-Rg3 was unable to modulate the NO level in HaCaT cells subjected to UV-B irradiation (Fig. 3B). Collectively, these data show that NO is not linked with the response against UV-B irradiation, at least at the current experimental intensity, and is not modulated by either 20(S)-Rg3 or 20(R)-Rg3 in HaCaT cells.

Cell Viability

To test the cytotoxicity of the current experimental concentrations of 20(S)-Rg3 and 20(R)-Rg3 in cultured mammalian cells, their effects on the viabilities of HaCaT and CCD986sk cells were determined by an MTT assay. UV-B irradiation exhibited no cytotoxicity on HaCaT cells. 20(S)-Rg3, up to 30 µM, was unable to modulate the viability of HaCaT cells subjected to UV-B radiation (Fig. 2C). A similar finding was observed with 20(R)-Rg3 (Fig. 3C). Likewise, both 20(S)-Rg3 and 20(R)-Rg3 exhibited no cytotoxicity on cultured CCD986sk cells (Figs. 5C, D). In brief, both 20(S)-Rg3 and 20(R)-Rg3, at the current experimental concentrations, are not harmful to cultured keratinocytes or fibroblasts.

Matrix Metalloproteinases

MMPs are a family of Zn²⁺- and Ca²⁺-dependent endopeptidases that are capable of degrading almost all proteins of the ECM. MMPs are synthesized as proenzymes that are activated by proteolytic removal of the propeptide. MMP-9 and -2 preferentially digest denatured collagen (gelatin) and type IV and V collagens, whereas MMP-1 is primarily responsible for degradation of ECM. Inflammation and subsequent accumulation of ROS, generated by UV, also play a crucial role in skin aging.^31,32) Scavenging and quenching of ROS by antioxidants and inhibition of MMP activity and expression are regarded as efficient strategies to prevent and reduce skin photoaging.

The gelatinolytic activities of MMPs were detected in supernatant obtained from the UV-B-irradiated HaCaT cell culture. The gelatinolytic activity of MMP-2 was significantly enhanced, whereas that of MMP-1 remained at control levels (Fig. 6). 20(S)-Rg3, at 5, 12, and 30 µM, was capable of attenuating the UV-B-induced MMP-2 activity in a concentration-dependent manner, and failed to modulate the MMP-1 activity further (Fig. 6A). At the same concentrations, 20(R)-Rg3 insignificantly decreased the UV-B-induced MMP-2 activity and had no effects on MMP-1 activity (Fig. 6B). These data collectively suggest that only the S-form of the two Rg3 stereoisomers is capable of suppressing UV-B-induced MMP-2 activity.

Fig. 6. Effects of 20(S)-Rg3 (A) and 20(R)-Rg3 (B) on Matrix Metalloproteinase-2 and -1 (MMP-2 and -1) Activities in Human HaCaT Keratinocytes Subjected to Irradiation with 70 mJ/cm² UV-B

Mammalian cells were treated with the indicated concentrations (0, 5, 12 or 30 µM) of 20(S)-Rg3 and 20(R)-Rg3 for 30 min before irradiation, and returned to the incubator for a further 24 h. The gelatinolytic activities of MMP-2 and -1 in the culture supernatants were detected using gelatin zymography. Representatives of three independent experiments are shown.

DISCUSSION

UV-B can harm epidermal keratinocytes via induction of inflammatory cytokines, chemokines, and prostaglandins, inflammatory mediators that are responsible for cutaneous reactions, such as inflammation, hyperpigmentation, erythema, hyperproliferation, and carcinogenesis.³³⁾ As expected, when keratinocytes and fibroblasts were irradiated with 70 mJ/cm², their intracellular ROS levels increased significantly. Prior treatment with the two Rg3 stereoisomers led to different cellular ROS-scavenging activities. However, only the S-type stereoisomer appeared to possess ROS-scavenging activity in both cell types. This stereospecificity may be partly supported by previous findings. The heat processing-induced hydroxyl radical scavenging activities of two protopanaxadiol-type ginsenosides, Rb1 and Rb2, were closely related to the generation of 20(S)-Rg3.^34,35) In another report, 20(S)-Rg3 decreased the generation of ROS in lipopolysaccharide (LPS)-stimulated macrophages and decreased the ROS level in non-stimulated HaCaT cells.³⁶⁾ These observations may support the ROS-scavenging activity of 20(S)-Rg3 in UV-B-irradiated keratinocytes and fibroblasts.

The apoptotic cell death of HaCaT cells peaked 48 h after UV-B irradiation. ROS and superoxide radical levels were highest 6 and 12 h after irradiation, respectively.³⁷⁾ However, the NO levels of HaCaT cells remain unchanged at these periods,³⁷⁾ an observation that is similar to the finding obtained with this work. Therefore, the cellular damage caused by UV-B irradiation seems to be mediated by the intracellular ROS level, but not by the NO level. However, the mechanism(s) responsible for the stereospecific ROS-scavenging effect of Rg3 remain unknown. As a possibility, 20(S)-Rg3 may inhibit the enzymatic activities responsible for ROS generation in a stereospecific manner. Another possibility is that only 20(S)-Rg3 directly reacts with ROS, which is hardly understood when considering the stereospecific effect of 20(S)-Rg3, a relatively small molecule. As a third possibility, 20(S)-Rg3 may only activate or induce antioxidant proteins that are able to cope with ROS.

UV radiation activates and induces one or more MMPs and, subsequently, causes damage to the skin, resulting in skin photoaging. For example, UV-A activates MMP-1 via a condition of oxidative stress, under which excessive generation of ROS or depletion of antioxidant functions in the skin cells, including keratinocytes and fibroblasts, occurs.³⁸⁾ 20(S)-Rg3, which is capable of scavenging intracellular ROS, down-regulated the UV-B-enhanced MMP-2 activity, implying a skin anti-photoaging activity for this stereoisomer. 20(R)-Rg3 also suppressed the UV-B-enhanced MMP-2 activity, although its suppressive activity was much weaker than that of 20(S)-Rg3. These results suggest that 20(S)-Rg3 elicits its skin anti-photoaging effects through the down-regulation of UV-B-induced MMP-2 activity. 20(S)-Rg3 may suppress ROS-induced MMP-2 activity by an ROS-dependent mechanism, whereas 20(R)-Rg3 may marginally suppress MMP-2 activity by an ROS-independent mechanism. Elucidation of precise mechanism(s) on these effects of Rg3 stereoisomers will require further experimental approaches.

Other findings on UV-B-induced MMP-2 have been obtained. Increased production of MMP-2, but not MMP-9, was found in human corneal fibroblasts in response to UV-B, especially in the acute phase after irradiation.³⁹⁾ A daily-use cream containing a photostable combination of UV-B and UV-A absorbers reduced MMP-2 mRNA expression in the buttock skin biopsies of healthy volunteers exposed to solar-simulated radiation.⁴⁰⁾ Although pyruvate does not inhibit UV-B-induced increases in intracellular ROS in HaCaT cells, it was able to improve the survival rate of the UV-B-irradiated cells through suppression of UV-B-induced mRNA expression of inflammatory mediators.⁴¹⁾

The pharmacological efficacies of 20(S)-Rg3 and 20(R)-Rg3 have been individually assessed, and a few findings on stereoselective differences in the pharmacological actions of Rg3 have been documented. For instance, 20(S)-Rg3 enhanced glucose-stimulated insulin secretion, whereas 20(R)-Rg3 showed no such effect.⁴²⁾ In C2C12 myotubes, both 20(S)-Rg3 and 20(R)-Rg3 phosphorylated AMP-activated protein kinase and acetyl-CoA carboxylase, although 20(R)-Rg3 had slightly less effect.⁴²⁾ 20(S)-Rg3 and 20(R)-Rg3 were capable of inhibiting lung metastasis of tumor cells; the anti-metastatic mechanism was based on inhibition of the adhesion and invasion of tumor cells, as well as anti-angiogenic activity.⁴³⁾ 20(S)-Rg3 and 20(R)-Rg3 showed stereospecific relaxation effects on swine coronary artery contractions caused by high K⁺ and 5-hydroxytryptamine receptor activation.⁴⁴⁾

Various pure and mixture components have been shown to contain MMP inhibitory activities in diverse mechanisms. Macelignan, a natural compound belonging to a group of lignans from Myristica fragrans HOUTT. (nutmeg), protected HaCaT cells from UV-B-induced damage and inhibited MMP-9 and cyclooxygenase-2 expression by attenuating the activation of mitogen-activated protein kinases (MAPKs) and phosphatidylinositol 3-kinase (PI3K)/Akt.⁴⁵⁾ Sargachromenol, isolated from Sargassum horneri, a brown alga growing on the coastal sea of Korea and Japan, helped to down-regulate the UV-B-increased MMP-2 mRNA level in skin dermal fibroblasts.⁴⁶⁾ An ethanol extract of the red alga Bonnemaisonia hamifera protected HaCaT cells against UV-B-induced oxidative damage by scavenging ROS and absorbing UV-B photons, thereby diminishing injury to cellular components.⁴⁷⁾ An aqueous extract of Labisia pumila, known as the “queen of plants” of all Malaysian herbs, restored UV-B-suppressed collagen synthesis of human fibroblasts to normal levels and down-regulated UV-B-induced MMP-9 expression in HaCaT cells.⁴⁸⁾ An ethanol extract of Gynura procumbens (LOUR.) MERR., a decumbent perennial herb belonging to the family of Asteraceae and widely distributed in Southeast Asian countries, suppressed UV-B-induced MMP-1 expression, MMP-9 activity, and ROS production in human primary dermal fibroblasts.⁴⁹⁾ A hydroethanolic extract of Butea monosperma (LAM.) TAUB. flowers, a common Indian plant widely used for inflammatory diseases, decreased MMP-1, -2, -9, and -10 secretion, in addition to pro-inflammatory cytokine and prostaglandin E2 production, in UV-B-treated normal human epidermal keratinocytes.⁵⁰⁾ Although retinoids are well-known anti-aging ingredients, their application can cause photosensitive responses, such as skin irritation, which prevent their daytime usage.⁵¹⁾

In conclusion, ginsenoside Rg3 exhibits a skin anti-photoaging activity by down-regulating MMP-2 activity, but its stereoisomers, 20(S)-Rg3 and 20(R)-Rg3, act differently to decrease UV-B-induced MMP-2 activity, most likely in a stereoselective manner.

Acknowledgments

This study was supported by a Grant of the Korea Healthcare Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (Grant no. A103017). This study was also supported by 2014 Research Grant from Kangwon National University (No. 120140161). The authors are grateful to Ms. Hannah Jo and Ms. Suyeon Lee for their technical assistances.

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