Part 2: Anti-Inflammatory, Antioxidant, Moisturizing, And Antimelanogenesis Effects Of Quercetin 3-O-β-D-Glucuronide in Human Keratinocytes And Melanoma Cells Via Activation Of NF-κB And AP-1 Pathways

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3. Discussion

In the previously published studies, Q-3-G has been used to inhibit ear edema, peritoneal permeability, and pulmonary edema [50], acting as a potent antioxidant in blood plasma with low-density lipoprotein [42], decreasing peroxynitrite-induced oxidative modification [51], exerting an anti-inflammatory effect by inhibiting JNK and ERK signaling pathways under LPS challenge [52] and possessing anticancer properties against breast cancer cells [56]. However, unlike its widely studied parent compound, quercetin, the pharmacological benefits, and mechanisms of Q-3-G remain to be explored. To the best of our knowledge, the role of Q-3-G on skin protective effects has not been fully elucidated. In this study, using various in vitro assessments, we focused on characterizing the skin protective effects of Q-3-G against UVBor H2O2,-induced oxidative stress and inflammation, skin hydration in HaCaT (a human keratinocyte cell line), and antimelanogenesis activity in B16F10 (a murine melanoma cell line).

The viability of HaCaT, B16F10, and HEK293T cells was determined after treatment with Q-3-G over a range of non-cytotoxic concentrations. The results showed that there was no significant inhibition of cell viability in HaCaT, B16F10, and HEK293T cells over concentrations of Q-3-Gup to 20 μM （Figures 2a，3a, and 4f）. The cell viability in HaCaT， B16F10， and HEK293T cells at concentrations of O-3-G up to 20 μM was found to be lower than the concentration of quercetin when it is used to investigate the cytotoxic effects in SCC-9 and HSC-6 cells（50 μM）【57】or sperm viability （50-100μM）【58】. However， Q-3-G was shown to be much less toxic compared to its aglycone [59]. The lack of a free OH group at the three-position may contribute to the less toxic effect of Q-3-G [29].

UVB rays with a wavelength of 280-315 nm penetrate the upper layer of the skin and are more effective than UVA and UVC[60]. UVB rays are the reason for most skin cancers and cell death. Furthermore, cell damage in HaCaT cells has been shown to be induced by UVB irradiation [61]. Consequently, we investigated the cell viability of HaCaT cells after UVB irradiation compared with Q-3-G treatment. As shown in previous reports, one of the main factors that lead to damaged cells, tissues, and organs is UVB exposure [62,63]. Q-3-G restored the diminished cell viability induced by UVB irradiation (Figure 2b-d). The results are shown to be similar to the previous study on the effect and mechanism on UVB-induced cytotoxicity in HaCaT of quercetin [64].

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Several studies have described that UVB can promote severe inflammatory responses, leading to problems in the skin [1,2,4,27].UVB activates important pro-inflammatory enzymes such as COX-2 and cytokines such as TNF-α.COX-2 is an inflammation-associated enzyme that is triggered by pro-inflammatory cytokines and mediators [65], which stimulates the inflammatory response. Because COX-2 produces the inflammatory mediator PGE-2, it induces an inflammatory response [66,67], and TNF-α is the major pro-inflammatory cytokine [68] in the inflammatory responses. These inflammatory responses cause skin damage, which results in skin aging [69]. The mRNA expression levels of the inflammatory enzyme COX-2 and the pro-inflammatory cytokines TNF-α were inhibited by Q-3-G (Figure 2e,). According to a previously published result, quercetin also decreased the gene expression and production of TNF-α[70]. In regard to antioxidant properties, the DPPH and ABTS radical scavenging assays are rapid, reliable, and reproducible methods for evaluating the in vitro antioxidant activity of pure compounds and plant extracts [54] and have been used widely in common methods to investigate the scavenging activities of natural compounds or extracts [1,46,71,72]. Q-3-G was found to reduce the reactivity of radicals in both assays and flow cytometry in concentration-dependent manners. These results are in accordance with a previously reported study, in which Q-3-G significantly inhibited the formation of ROS in pheochromocytoma PC-12 cells [34]. Besides acting chemically with an antioxidant effect, we showed that Q-3-G increased the level of Nrf2, one of the important regulators of oxidative stress in cells. Several compounds or natural extracts have been reported to possess antioxidant properties through Nrf2-dependent ARE signaling [46,73,74]. Taken together, these findings indicate that Q-3-G possesses antioxidant activity(Figure 2g-j). Melanosome from melanin synthesis occurs in intracellular organelles [75-77]. Overproduction of melanin can affect skin appearances and hyperpigmentation-related skin diseases. We found that treatment with O-3-G in cα-MSH-induced B16F10 cells inhibited melanin secretion. As in previous works, intracellular melanogenesis is also controlled by quercetin metabolites and some quercetin-3-O-β-D-glucopyranosides [24,78,79].

Maintaining healthy skin requires adequate skin hydration, and NMFs and HA have an important role to play in this process [12]. FLG is a constituent of the skin barrier, so the increased level of FLG expression may be associated with moisturizing [80]. In our study, Q-3-G was found to affect skin moisture retention activity by increasing FLG and TGM-1 (Figure 3c,d). The expression levels of FLG and TGM-1 were blocked by JNK, IKKα, and IkBainhibitors (SP600125 and Bay11-7082, respectively). We focused on MAPK-related enzymes because our studies aimed to determine the molecular mechanisms by which Q-3-Gexerts its skin protective effects. JNK plays an important part in oxidative-stress-induced keratinocyte apoptosis [81]. The phosphorylation of c-Jun, TAK1, MKK4, and INK was increased by Q-3-G. Additionally, retinol did not significantly increase the phosphorylation of c-Jun, TAK1, MKK4, or JNK, suggesting that there are some differences in the inhibitory characteristics between Q-3-G and retinol in the AP-1 signaling pathway. As in previously published results, the JNK-c-Jun/AP-1 pathway is correlated with the anti-apoptotic effect of quercetin [82]. Compared to the previous study [83], by regulating NF-kB and AP-1 signaling pathways, these pathways were also found to be involved in the underlying mechanism of quercetin. In line with the mechanism of its parent compound, our results show that the NF-kB pathway was also included in the skin protective effect of Q-3-Gaby the activation of p50, p65, IKKα, IkBα, Akt, and Src after treatment with Q-3-G in HaCaT cells. In addition, Q-3-G upregulated AP-1-mediated Luc and NF-B-Luc activity (Figure 4). These findings suggest that MAPK-mediated AP-1 and NF-kB signaling contributed to Q-3-G-mediated skin protective properties.

4. Materials and Methods 

4.1. Materials

Q-3-Gwas obtained from Sigma Aldrich Co. (St.Louis, MO, USA).HaCaT, HEK293T, and B16F10 cells were purchased from the American Type Culture Collection (Rockville, MD, USA).ABTS diammonium salt, DPPH, ascorbic acid(AA), ABTS, and retinol were purchased from Sigma Chemical Co.(St.Louis, MO, USA). Fetal bovine serum(FBS), Dulbecco's Modified Eagle Medium (DMEM), and phosphate-buffered saline (PBS) were bought from Gibco(Grand Island, NY, USA).3-(4-5-Dimethylthiazol-2-yl)-25-diphenyltetra-sodium bromide (MTT)was purchased from Amresco (Brisbane, Australia). TRILzol and PCR premix were purchased from Bio-D Inc. (Seoul, Korea). The cDNA synthesis kits were bought from Thermo Fisher Scientific (Waltham, MA, USA). Forward and reverse primers for PCR (polymerase chain reaction) were synthesized by Macrogen, Inc. (Seoul, Korea). All the antibodies related to the phosphorylated or total forms of the target protein were bought from Cell Signaling Technology (Beverly, MA, USA).

4.2.Cell Cultures

HaCaT and B16F10 cells were cultured in DMEM with 10% FBS and 1% antibiotics (streptomycin and penicillin). HEK293T cells were cultured in DMEM with 5% FBS and 1% antibiotics. These cell lines were incubated in 5% CO, at 37°C.

4.3. Cell Viability Tests

B16F10 cells were seeded at 2×10° cells per well in 96-well plates for 24 h, differ-ent concentrations of Q-3-G（0-20 μM） were added to them， and then they were incubated for 48 h. HaCaT cells were plated in 96-well plates, and the cells were seeded at 6×10°cells/mL. After overnight incubation, the cells were incubated with different concentrations of Q-3-G （0-20 μM） for 24 h. HEK293T cells were seeded at 6×10°cells/mL in 96-well-plates for 24 h and then treated with Q-3-G （0-20 μM）. Incubated cells were treated with 10μL/well of MTT solution， and after 3 h were treated with 100 μL of MTT Stopping Solution. Using the conventional MTT assay for measured cell viability [84], the absorbance at 570 nm was measured using a reader(BioTek Instruments, Winooski, VT, USA).

4.4.mRNA Analysis by Semi-Quantitative RT-PCR and Quantitative Real-Time PCR

HaCaT cells were treated with Q-3-G（5-30 μM） or retinol（10ug/mL） for 12 h.RNA was precipitated using TRI reagent as reported previously [85]. A cDNA synthesis kit was used to synthesize the complementary DNA. The RT-PCR was conducted using specific reverse and forward primers. Primers for RT-PCR and real-time PCR are listed in Table 1.

4.5. DPPH Assay

A DPPH assay was used to assess the radical scavenging capacity of Q-3-G. Methanol was used to dissolve DPPHto a final concentration of 250 μM. Then，475 mL of DPPHwas

reacted with5 mL of Q-3-G（0-40 μM） or AA（500 mM）at37°C for 30 min. The positive control was AA. The determination of the DPPH scavenging effect and calculation was performed as described previously [86]. 

4.6.ABTS Assay

An ABTS assay was performed as previously reported [72]. Briefly, equal volumes of 7.4 mM ABTS solution and 2.4 mM potassium persulfate solution were mixed and kept at room temperature overnight to generate ABTS radical cations. ABTS solutions were added to each well of 96-well plates， with the addition of Q-3-G（0-40 μM） or AA（500 mM） per well. The mixtures were incubated at 37 °C for 30 min, and then their absorbance at 730 nm was measured.

4.7. Cellular ROS Assay by Flow Cytometry

To check the formation of intracellular ROS, an oxidation-sensitive dye,2',7'-dichlorodihydrofluorescein diacetate (DCFDA), was used. HaCaT cells were exposed to UV, and then the cells were harvested and resuspended with 300 ul PBS with 20 μM DCFDA for 30 min at 37 °C in the dark. The cells were then washed with PBS and the fluorescence was measured at 485/535 nm by a Beckman CytoFLEX Flow Cytometer(Beckman Coulter, Brea, CA, USA).

4.8.Western Blot Analysis

HaCaT cells were seeded at a density of 6× 10° cells/mL. After incubation overnight, different concentrations of Q-3-G（0-10 μM） or retinol （10 μg/mL） was added and further incubated for 24 h. Protein preparation and whole-cell lysates were performed as described previously [87]. Specific antibodies were used to detect the total form and phosphorylated form of Jun, JNK, MKK4, TAK1, p50, p65, IKKα, IkBα, Src, and Akt, which were visualized with chemiluminescence reagents [88].

4.9. Melanin Content and Secretion Analysis

B16F10 cells were seeded at a density of 2×10° cells/mL into 12-well plates and incubated overnight. The culture medium was changed for fresh medium supplemented with Q-3-G（10-20 μM） or arbutin （1 mM）. Melanin production was stimulated with a-MSH (100 nM) for 48 h. For the melanin secretion assay, the optical density at 475 nm was measured. Afterward, the cells were harvested to determine the intracellular melanin con-tent, using a lysis buffer to lyse the cells and pelleting them by centrifugation (12,000 rpm, 5 min). The concentrated cell pellets were dissolved in 100 mL dissolving buffer(1 M NaOH, 10%DMSO) and melted at 55°C. The absorbance was measured at 405 nm using an optical density reader [89].

4.10.UVB Irradiation

HaCaT cells were seeded at a density of 1×105 cells/mL into 6-well plates using serum-free MEM and were subjected to 24 h of starvation. HaCaT cells were pre-treated with G-3-Q for 30 min and then UVB irradiation. Then, the HaCaT cells were washed with PBS and exposed to UVB irradiation (UVBLamp BLX-312, Vilber Lourmat, France). The energy of UVB irradiation was 30 mJ/cm2²【90】.

4.11.H2O2 Treatment

HaCaT cells were seeded into 6-well plates and subjected to 24 h of starvation us-ing serum-free MEM. The cells were pretreated with different concentrations of Q-3-G（5-10 μM and after 30 min were incubated with H2O2 （500 μM） for 12 h. 

4.12. Luciferase Reporter Gene Assay

HEK293T cells were seeded at a density of 3×10°cells/mL into 24-well plates. The HEK293T cells were transferred with 2 μg of plasmids containing AP-1-Luc or NF-kB-Luc and b-galactosidase, employing polyethylenimine[91]. After 24 h of incubation, the cells were treated with Q-3-G（5-10 μM） or retinol（10 ug/mL） for a further 24 h. The luciferase assay was performed by a luminometer at the absorbance of each sample was measured at 475 nm using a Spectramax 250 microplate reader (Molecular Devices, San Jose, CA, USA), as reported previously [92].

4.13. Cell Morphology Shooting

HaCaT cells were seeded into 6-well plates at a cell density of 1× 10° cells/mL. The HaCaT cells were treated with G-3-Q for 30min and then UVB irradiation. After 24 h,4×, 10×, and 20× microscope objective photos were taken (Olympus, Tokyo, Japan).

4.14. Statistical Analysis

All data are presented as the mean ± standard deviation of at least three independent experiments. The Mann-Whitney test and Student's t-test were used to compare statistical differences between groups. A p-value<0.05 was regarded as statistically significant. All statistical tests were carried out using SPSS(Ver.25, SPSS Inc., Chicago, IL, USA).

5. Conclusions

Our research demonstrated that Q-3-Gexhibited anti-inflammatory and antioxidant activities protect against factors of oxidative stress and inflammation under UVB irradiation and HO, exposure. We also demonstrated that Q-3-G has a moisturizer-stimulating ability in HaCaT cells. In addition to the antimelanogenesis effect of Q-3-G, it was shown to inhibit melanin secretion in α-MSH-induced B16F10. Finally, the effect of Q-3-G was mediated through the activation of AP-1 and NF-kB signaling pathways. The skin-protective activities of Q-3-G in HaCaT human keratinocytes cells and B16F10 murine melanoma cells are summarized in Figure 5. This study established that Q-3-G could be effective due to its anti-inflammatory, antioxidant, moisturizing, and antimelanogenesis properties in human keratinocytes and melanoma cells via the activation of NF-kB and AP-1 pathways. In conclusion, the results clearly indicate that this conjugate metabolite has the potential to protect skin cells. Further investigations are needed to validate the effect of Q-3-G in various in vivo models, especially in relation to skin protective models.

References

1. Kim, E.; Hwang, K.; Lee, J.; Han, S.Y.; Kim, E.-M.; Park, J.; Cho, J.Y. Skin protective effect of epigallocatechin gallate. Int. J. Mol. Sci. 2018, 19, 173. [CrossRef]

2. Draelos, Z.D. New treatments for restoring impaired epidermal barrier permeability: skin barrier repair creams. Clin. Dermatol. 2012, 30, 345–348. [CrossRef] [PubMed]

3. Slominski, A.T.; Zmijewski, M.A.; Zbytek, B.; Tobin, D.J.; Theoharides, T.C.; Rivier, J. Key role of CRF in the skin stress response system. Endocr. Rev. 2013, 34, 827–884. [CrossRef] [PubMed]

4. D’Orazio, J.; Jarrett, S.; Amaro-Ortiz, A.; Scott, T. UV radiation and the skin. Int. J. Mol. Sci. 2013, 14, 12222–12248. [CrossRef]

5. Rittie, L.; Fisher, G.J. UV-light-induced signal cascades and skin aging. Ageing Res. Rev. 2002, 1, 705–720. [CrossRef]

6. Videira, I.F.; Moura, D.F.; Magina, S. Mechanisms regulating melanogenesis. An. Bras. Dermatol. 2013, 88, 76–83. [CrossRef] [PubMed]

7. Che, D.N.; Xie, G.H.; Cho, B.O.; Shin, J.Y.; Kang, H.J.; Jang, S.I. Protective effects of grape stem extract against UVB-induced damage in C57BL mice skin. J. Photochem. Photobiol. B 2017, 173, 551–559. [CrossRef]

8. Jeong, D.; Lee, J.; Jeong, S.G.; Hong, Y.H.; Yoo, S.; Han, S.Y.; Kim, J.H.; Kim, S.; Kim, J.S.; Chung, Y.S.; et al. Artemisia Asiatica ethanol extract exhibits anti-photoaging activity. J. Ethnopharmacol. 2018, 220, 57–66. [CrossRef]

9. McDaniel, D.; Farris, P.; Valacchi, G. Atmospheric skin aging-Contributors, and inhibitors. J. Cosmet. Dermatol. 2018, 17, 124–137. [CrossRef]

10. Rao, C.V.; Pal, S.; Mohammed, A.; Farooqui, M.; Doescher, M.P.; Asch, A.S.; Yamada, H.Y. Biological effects and epidemiological consequences of arsenic exposure, and reagents that can ameliorate arsenic damage in vivo. Oncotarget 2017, 8, 57605–57621.

11. Sinha, K.; Das, J.; Pal, P.B.; Sil, P.C. Oxidative stress: The mitochondria-dependent and mitochondria-independent pathways of apoptosis. Arch. Toxicol. 2013, 87, 1157–1180. [CrossRef]