Anticatabolic and Anti-Inflammatory Effects of Myricetin 3-O-β-d-Galactopyranoside in UVA-Irradiated Dermal Cells via Repression of MAPK/AP-1 and Activation of TGFβ/Smad

Any potential cytotoxicity of UVA irradiation and M3G on HaCaT keratinocytes and HDFs was evaluated by MTT assay. Both cells were incubated with M3G for 24 h without UVA irradiation. Cells were also irradiated by UVA and incubated for 24 h without M3G treatment to assess the cytotoxic effects of UVA irradiation. In addition, vehicle only treatment was carried out using the same volume of 10% dimethyl sulfoxide (DMSO, in distilled water) vehicle. Effects on cell viability were analyzed by comparison with untreated control group. Treatment with M3G did not show any cytotoxicity in HaCaT keratinocytes for the doses up to 25 μM (Figure 2A). However, HDFs exhibited a 7.57% decrease in cell viability following 25 μM M3G treatment. Both cells did not show any drop in cell viability following vehicle treatment with final concentrations of 0.01%, 0.05% and 0.25% DMSO which was equivalent amount of M3G final concentrations of 1, 5 and 25 μM, respectively in wells (Figure 2B). Hantke et al. [19] reported that these concentrations did not affect the UVA-induced changes of MMP expression in both HaCaT keratinocytes and HDFs. In addition, the highest final concentration of DMSO (0.25%) as a vehicle were comparable to reported studies where similar concentrations of DMSO did not exert any effects in cultured cells against UVA-induced changes [20,21]. UVA doses up to 10 j/cm² did not show any cytotoxicity in both HaCaT and HDFs after 24 h incubation (Figure 2C). According to results, further assays used the M3G concentration of 25 μM and below, given that the concentrations higher than 25 μM in HDFs might lower the cell viability below 90% compared to untreated control.

The effect of M3G on the UVA-mediated changes of MMP-1 and type Iα1 procollagen production was determined using an ELISA to quantify the secretion levels. UVA irradiation (10 J/cm²) resulted in increased production of MMP-1 and decreased production of type Iα1 procollagen in both HaCaT keratinocytes and HDFs (Figure 3A,B). MMP-1 release was increased from 2090.0 pg/mL to 5782.7 pg/mL in HaCaT keratinocytes and from 20822.7 pg/mL to 22228.0 pg/mL in HDFs following UVA irradiation. Treatment with M3G inhibited the release of MMP-1 in UVA irradiated HaCaT keratinocytes by 66.6% (1930.7 pg/mL) at the concentration of 25 μM (Figure 3A). In HDFs, M3G (25 μM) decreased the MMP-1 release by 4.8% (21143.6 pg/mL) (Figure 3B). Pro-collagen Iα1 release was decreased from 88.0 pg/mL to 46.9 pg/mL in HaCaT keratinocytes and from 1933.9 pg/mL to 1753.3 pg/mL in HDFs following UVA irradiation. M3G (25μM) increased the type I procollagen production in UVA-irradiated HaCaT keratinocytes by 37.2% (64.4 pg/mL) (Figure 3A) and in UVA-irradiated HDFs by 9.0% (1911.5 pg/mL) (Figure 3B).

In order to investigate any possible effect of M3G on the UVA-induced reactive species production in HaCaT keratinocytes and HDFs, nitrite oxide was measured in cultured cell media as amount of nitrate. Results have showed that UVA irradiation increased the NO production in both HaCaT keratinocytes and HDFs. Following UVA irradiation, nitrate amount was increased from 10.43 to 13.27 in HaCaT keratinocytes and from 10.21 to 12.71 in HDFs. Treatment with M3G (25 μM) decreased nitrate concentration to 11.63 in HaCaT keratinocytes and to 10.92 in HDFs (Figure 4).

To determine the effect of M3G on the mRNA expression of MMP-1 and protein expression of MMP-1, MMP-9 and type I procollagen in UVA-irradiated (10 J/cm²) HaCaT keratinocytes, RT-PCR, RT-qPCR and Western blotting were utilized. Presence of M3G (1, 5, 25 μM) in UVA-irradiated HaCaT keratinocytes dose-dependently inhibited the mRNA expression of MMP-1 analyzed by both RT-PCR and RT-qPCR (Figure 5A). MMP-1 expression was increased 4.35-fold by UVA irradiation which was decreased by M3G treatment (25 μM) by 83.8%. Protein expression levels were observed to be parallel with mRNA expression where M3G treatment decreased the UVA-related increase in MMP-1 levels by 58% (Figure 5B). In addition, protein levels of MMP-9 and type I procollagen were also investigated. UVA irradiation caused upregulation (89.7%) in MMP-9 protein expression and suppression (87.9%) in type I procollagen levels both of which were reverted by M3G treatment by 29.8% and 95.0%, respectively.

Effect of M3G on the UVA-mediated inflammatory response in HaCaT keratinocytes was analyzed by investigating the protein expression levels of pro-inflammatory enzymes and cytokines (cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β) and IL6) utilizing Western blotting. Results indicated that UVA exposure (10 J/cm²) caused a notable increase in pro-inflammatory response in HaCaT keratinocytes (COX-2, 22.6%; TNF-α, 46.6%; iNOS, 86.8%; IL-1β, 35.5%; IL-6, 98.9%). M3G treatment decreased the levels of COX-2, iNOS, TNF-α., IL-1β and IL6 levels by 51.7%, 55.9%, 66.6%, 52.6% and 81.3%, respectively, at the concentration of 25 μM (Figure 6A). Results suggested that M3G was able to suppress the UVA-induced inflammation in keratinocytes via inhibiting the production of inflammatory cytokines.

Effect of M3G on the UVA-stimulated activation of MAPK signaling was investigated through phosphorylation levels of AP-1 regulating MAPKs using Western blotting. UVA irradiation (10 J/cm²) resulted in increased activation of MAPKs, shown as significantly elevated phosphorylation of p38, ERK and JNK MAPKs (Figure 6B). Following UVA irradiation, relative phosphorylation of MAPKs (normalized against their total protein levels) was increased by 82.8% for p38, 56.1% for ERK and 58.0% for JNK compared to non-irradiated group. Levels of phosphorylation were significantly lowered by 45.1% for p38, 59.2% for ERK and 62.0% for JNK in M3G treated (25 μM) HaCaT keratinocytes. Next, phosphorylation levels of c-Fos and c-Jun, building blocks of AP-1 transcription factor, were analyzed in UVA irradiated HaCaT keratinocytes (Figure 6C). UVA exposure increased the phosphorylation of c-Fos and c-Jun by 51.3% and 88.6%, respectively, compared to non-irradiated control. M3G treatment at 25 μM concentration significantly decreased the phosphorylation level of c-Jun by 13.2% but failed to affect the c-Fos phosphorylation. Results demonstrated that M3G suppressed MAPK mediated activation of AP-1 (via c-Jun) suggesting that this led to inhibition of MMP-1 production.

To investigate the effects of M3G on the mRNA and protein expressions of MMPs and collagen in UVA-irradiated dermal fibroblasts, RT-qPCR and Western blotting were utilized, respectively. UVA-irradiated HDFs expressed 4.21-fold higher MMP-1 mRNA compared to non-irradiated control group whereas type I procollagen mRNA was expressed 2.13-fold less (Figure 7A). At the concentration of 25 μM, M3G treatment suppressed MMP-1 mRNA expression by 1.90-fold and upregulated type I procollagen mRNA expression by 2.03-fold. UVA-induced increase in protein levels of MMP-1 (60.7%) and decrease in collagen (94.2%) were also ameliorated in a similar manner by M3G treatment (Figure 7B). At 25 μM, M3G treatment decreased MMP-1 protein levels by 47.5% and increased collagen levels by 39.2%. In addition, UVA-induced increase (50.4%) in MMP-3 levels were slightly decreased by 25 μM M3G treatment. However, M3G was only able to decrease the levels of MMP-9 proenzyme not the active enzyme suggesting that the effect of M3G on the suppression of MMPs is not specific to MMP-1 and also follows a common transcriptional activation of MMP-1, -3 and -9 rather than enzyme specific inhibition.

To determine the effects of M3G on the inflammation in dermal fibroblasts caused by UV exposure, HDFs were irradiated by UVA (10 J/cm²) and the protein levels of pro-inflammatory enzymes and cytokines (COX-2, iNOS, TNF-α, IL-1β and IL6) were investigated by Western blotting. Similar to its effects in HaCaT keratinocytes, UVA exposure (10 J/cm²) significantly increased the protein levels of COX-2, iNOS, TNF-α, IL-1β and IL6 which were decreased by M3G treatment, except IL6 (Figure 7C). M3G (25 μM) treated HDFs expressed 16.3%, 50.5%, 57.2%, 52.8% and 0.8% less amounts of COX-2, iNOS, TNF-α, IL-1β and IL6 proteins, respectively compared to UVA-irradiated only cells. Results suggested that M3G hindered the inflammatory response in UVA-irradiated HDFs via suppression of inflammatory cytokine production.

As a suggested mechanism for the effects of M3G against UVA induced changes in HaCaT keratinocytes, UVA mediated activation of MAPKs was also investigated in HDFs. Treatment with M3G (25 μM) inhibited the UVA-induced (10 J/cm²) increase of p38 and JNK phosphorylation by 35.3% and 12.9%, respectively (Figure 8A). However, the levels of ERK phosphorylation was further enhanced by M3G treatment compared to UVA-irradiated non-treated HDFs. At 25 μM, ERK activation was 93% higher compared to UVA-irradiated only group.

Parallel to its effects in UVA-irradiated HaCaT keratinocytes, effects of M3G on the levels of c-Fos and c-Jun phosphorylation were investigated using Western blotting. Protein levels of phosphorylated c-Fos and c-Jun in both total cell lysates and nuclear fractions of UVA-irradiated HDFs were significantly increased (Figure 8B). Presence of M3G (25 μM) decreased the phosphorylation of c-Fos and c-Jun both in cellular and nuclear fractions of HDFs suggesting that M3G inhibited MMP production via prevention of AP-1 (c-Fos and c-Jun) activation. At 25 μM treatment, phosphorylation of c-Fos and c-Jun was decreased by 41.3% and 57.8%, respectively. Nuclear fractions of M3G treated (25 μM) HDFs contained 80.3% and 87.8% less phosphorylated c-Fos and c-Jun compared to UVA-irradiated only group.

To investigate the underlying mechanism of M3G-mediated increase in collagen production of UVA-irradiated HDFs, expression and phosphorylation levels of key collagen synthesis cascade (TGFβ, Smad 2/3, Smad 7 and Smad 4) were analyzed using Western blotting. Protein levels of TGFβ and Smad 4 were significantly reduced in HDFs after UVA (10 J/cm²) exposure: 85.2% and 34.5%, respectively (Figure 9A). This was also accompanied by diminished phosphorylation of Smad 2/3 complex (79.2%). On the other hand, levels of Smad 7, negative regulator of TGFβ signaling, were observed to be increased by UVA exposure (by 119.8% of non-irradiated non-treated control). M3G treatment (25 μM) reversed the effects of UVA in type I collagen production cascade by increasing the protein levels of TGFβ (368.2%) as well as its downstream proteins Smad 4 (59.2%). In addition, phosphorylation of Smad 2/3 was significantly upregulated (338.55% of UVA-irradiated only group). Detrimental effects of UVA exposure were further showed by the decrease in phosphorylated Smad 2/3 (17.4%) and Smad 4 (37.2%) levels in the nuclear fraction of HDFs (Figure 9B). M3G treatment (25 μM) resulted in the elevation of phosphorylated Smad 2/3 levels by 13.7% and Smad 4 levels by 68.9% compared to UVA-irradiated only group.

To confirm the enhancement of ERK activation by M3G which has been suggested to be related to its ability to stimulate TGFβ activation, ERK1/2 activation was analyzed by flow cytometry. Results showed that M3G treated (25 μM) cells expressed 59.80% activated population in terms of ERK1/2 compared to 41.80% of UVA irradiated only cells (Figure 9C). Results suggested that M3G inhibited the activation of p38 and JNK MAPKs but contradictorily enhanced the ERK activation. Overall, it was demonstrated that M3G relieved the UVA-induced suppression in TGFβ signaling pathway via increasing ERK1/2 phosphorylation which potentially increased collagen synthesis.

Isolation and characterization of myricetin 3-O-β-d-galactopyranoside (M3G) has been previously reported [36]. A stock solution of 1 mM M3G in 10% DMSO (v/v, in distilled water) was prepared and stored at −20 °C until use.

HaCaT cells (300493; Cell Line Service, Eppelheim, Germany) were cultured in Dulbecco's modified Eagle medium with 10% fetal bovine serum (FBS). Cells were kept in 37 °C incubators with an atmosphere containing 5% CO₂ between the experiments.

HDF cells (C-12302; PromoCell, Heidelberg, Germany) were cultured in Fibroblast Growth Medium (C-23020, PromoCell) and kept in 37 °C incubators with an atmosphere containing 5% CO2 between the experiments.

Any possible toxic effect of M3G and DMSO in keratinocytes and dermal fibroblasts was investigated by colorimetric MTT assay as previously described [16]. Briefly, HaCaT cells and HDFs were seeded in 96-well plates (1 × 10³ cell/well) and treated with final concentrations of 1, 5, 10, 20, 25 and 50 μM M3G or equivalent amount of 10% DMSO (v/v, in distilled water). Culture medium was replaced with 100 μL MTT solution (1 mg/mL) following a 24 h incubation. The plate was then kept in dark at 37 °C for 4 h. Wells were aspired, and cells were washed with phosphate buffer saline (PBS). Ten microliters of 100% DMSO was introduced to each well to dissolve the formazan crystals and absorbance values of wells at 540 nm were measured using GENios FL microplate reader (Tecan Austria GmbH, Grodig, Austria). Same procedure was followed to investigate the effect of UVA exposure on the viability of HaCaT keratinocytes and HDFs. Cells were irradiated by UVA (0–10 J/cm²) (Section 4.3) and the cell viability assay was carried out as mentioned without sample treatment. Changes in the viability of cells were calculated as a percentage of the untreated blank group which was considered to be 100% alive and compared to each concentration of M3G treatment.

HaCaT keratinocytes and HDFs were irradiated by UVA using a Bio-Sun UV Irradiation System (Vilber Lourmat, Marine, France) fitted with a UVA source designed for microplates. Cells grown in microplates were irradiated at a sublethal dose of 10 J/cm² UVA. Briefly, cells were irradiated in phosphate-buffered saline (PBS) without the plastic lid using the Bio-Sun system illuminator with a UV peak at 365 nm. Cells were cultured in plates and incubated for 24 h prior to UVA irradiation. Irradiation was emitted from 30W T-20.L tubes (Vilber Lourmat) placed at a distance of 10 from the sample loading tray. Irradiation was carried out automatically by the programmable microprocessor which stopped the irradiation when the energy received matched the desired programmed energy (0–10 J/cm²; range of measurement, 0–20 J/cm²). Subsequently the cells were incubated with their own growth medium without FBS until analysis.

Releases of MMP-1 and type Iα1 procollagen in UVA-irradiated HaCaT keratinocytes and HDFs were analyzed by ELISA. Cells (1 × 10⁶ cell/well) were pre-incubated in 6-well plates for 24 h and washed with PBS prior to UVA (10 J/cm²) exposure. Immediately after UVA irradiation, the cells were treated with or without different doses (final concentrations of 1, 5, 25 μM) of M3G and incubated for another 24 h. Cell culture medium from each well was harvested and analyzed for its MMP-1 and type Iα1 procollagen contents per manufacturer's instructions of the ELISA kit (R&D Systems, Inc., Minneapolis, MN, USA).

UVA-induced NO generation by HaCaT keratinocytes and HDFs was quantified by measuring the nitrate amount in conditioned cell culture medium. Cell culture medium was harvested following 24 h incubation after UVA irradiation and sample treatment and centrifuged for 15 min (134× g). Supernatants were then filtered through 0.2 μM syringe filter and the nitrate amount was calculated using modified Griess reaction as previously described [37].

HaCaT keratinocytes and HDFs cultured in 6-well plates (1 × 10⁶ cell/well) were incubated for 24 h and treated with or without M3G (final concentrations of 1, 5, 25 μM) immediately after UVA (10 J/cm²) irradiation, and incubated for another 24 h. Total RNA was isolated from nonirradiated and irradiated (UVA, 10 J/cm²) HaCaT keratinocytes and HDFs using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc., Rockford, IL, USA). Total RNA (2 μg) and oligo(dT) were mixed in RNase-free water for the cDNA synthesis. Synthesis was performed in a thermo cycler (T100; Bio-Rad Laboratories, Inc., Hercules, CA, USA) with an initial denaturation of the mix at 70 °C for 5 min and cooling immediately followed by the preparation of a master mix containing 1× RT buffer, 1 mM dNTPs, 500 ng oligo(dT), 140 units M-MLV reserve transcriptase and 40 units RNase inhibitor. Next, the cDNA synthesis was carried out with two steps of 42 °C for 60 min and 72 °C for 5 min.

For RT-PCR analysis, the target cDNA was amplified using the sense and antisense primers as previously noted [16]. The amplification was carried out with 30 cycles; each cycle consisted of 95 °C for 45 s, 60 °C for 1 min and 72 °C for 45 s. The final PCR products were separated by agarose gel (1.5%) electrophoresis for 30 min at 100 V. Gels were then stained with 1 mg/mL ethidium bromide and visualized by UV light using a CAS-400SM Davinch-Chemi imager™ (Davinch-K, Seoul, Korea).

Gene expression was also measured by RT-qPCR in a Thermal Cycler Dice^® Real Time System TP800 (Takara Bio Inc., Ohtsu, Japan) following the manufacturer's protocol. Briefly 1.0 μL of cDNA sample and 10.5 μL of Luna^® Universal qPCR Mix (New England Biolabs, Ipswich, MA, USA) were mixed with forward and reverse primers in nuclease-free water. The target cDNA was amplified using following forward and reverse primers; MMP-1, forward 5'-TCT GAC GTT GAT CCC AGA GAG CAG-3′, reverse 5'-CAG GGT GAC ACC AGT GAC TGC AC-3′; type I procollagen, forward 5'-CTC GAG GTG GAC ACC ACC CT-3', reverse 5'-CAG CTG GAT GGC CAC ATC GG-3'; β-actin, forward 5′-AGA TCA AGA TCA TTG CTC CTC CTG-3′, reverse 5′-CAA GAA AGG GTG TAA CGC AAC TAA G-3′. The PCR amplification was carried out with an initial denaturation at 95 °C for 1 min, followed by 40 PCR cycles, each cycle consisting of 95 °C for 15 s and 60 °C for 30 s. Relative quantification was calculated using the 2^−(ΔΔCT) method. β-Actin was used as an internal control.

Protein levels were investigated using immunoblotting according to common Western blotting protocols. HaCaT keratinocytes and HDFs cultured in 6-well plates (1 × 10⁶ cell/well) were incubated for 24 h and were treated with or without M3G (1, 5, 25 μM) immediately after UVA (10 J/cm²) irradiation and incubated for another 24 h. Following incubation, wells were aspirated, and cells were lysed by vigorous pipetting in 1 mL of RIPA buffer (Sigma–Aldrich, St. Louis, MO, USA) at 4 °C to obtain total cell lysates. The nuclear fraction extraction was carried out using NE-PER^TM Nuclear Extraction Kit (Catalog No. #78835; Thermo Fisher Scientific) according to manufacturer's instructions. Protein content of the lysates was measured with a BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA) following kit's protocol. Same amount (20 μg) of protein from each well was loaded onto 10% SDS-polyacrylamide gel and run at 100 V. Proteins on gel were then transferred onto a polyvinylidene fluoride membrane (Amersham, GE Healthcare, Little Chalfont, UK) using a wet system run at 100 V for 1 h at 4 °C. Membranes were then incubated for 1 h at room temperature in 5% skimmed milk for blocking. Blocked membranes were washed with 1× TBST and incubated with primary antibodies of specific proteins (diluted 1:1000) in primary antibody dilution buffer containing 1× TBST with 5% bovine serum albumin overnight at 4 °C. Membranes were then incubated with horseradish-peroxidase-conjugated secondary antibodies (diluted 1:1000) specific to the primary antibody at room temperature for 1 h. β-actin and lamin B1 were used as internal housekeeping control for total cell lysate and nuclear fractions, respectively. Detection of proteins on blotted membranes was achieved using an ECL Western blot detection kit (Amersham) according to the manufacturer's instructions. Protein bands were imaged with the CAS-400SM Davinch-Chemi imager™ (Davinch-K, Seoul, Korea).

The MAPK (ERK1/2) activation levels were investigated employing flow cytometry. Cells were pre-incubated in 6-well plates (1 × 10⁶ cell/well) for 24 h and washed with PBS prior to UVA (10 J/cm²) exposure. Following UVA irradiation, the cells were treated with or without different concentrations (1, 5, 25 μM) of M3G for 24 h. Levels of ERK1/2 phosphorylation were measured with MUSE^TM MAPK Activation Dual Detection Kit (MCH200104; Merck KGaA, Darmstadt, Germany) using MUSE^TM Cell Analyzer and software (Muse Cell Soft V1.4.0.0, Merck KGaA, Darmstadt, Germany) according to the manufacturer's instructions.

All numerical data were given as the mean ± standard deviation of three separate experiments carried out in triplicates. Statistical differences between the means of the sample groups were calculated by the analysis of variance (ANOVA) followed by Duncan's multiple range test using SAS v9.1 software (SAS Institute, Cary, NC, USA). Any statistically significant difference between the groups was determined at p < 0.05 and p < 0.01 levels.