Part 1: 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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Abstract: Quercetin 3-O-β-D-glucuronide(Q-3-G), the glucuronide conjugate of quercetin, has been reported as having anti-inflammatory properties in the lipopolysaccharide-stimulated macrophages, as well as anticancer and antioxidant properties. Unlike quercetin, which has been extensively described to possess a wide range of pharmacological activities including skin protective effects, the pharmacological benefits and mechanisms of Q-3-G in the skin remained to be elucidated. This study focused on characterizing the skin protective properties, including anti-inflammatory and antioxidant properties, of Q-3-Against UVB-induced or H2O2-induced oxidative stress, the hydration effects, and antimelanogenesis activities using human keratinocytes (HaCaT) and melanoma (B16F10) cells. Q-3-G down-regulated the expression of the pro-inflammatory gene and cytokine such as cyclooxygenase-2 (COX-2) and tumor necrosis factor(TNF)-α in HO, or UVB-irradiated HaCaT cells. We also showed that O-3-Gexhibits have an antioxidant effect using free radical scavenging assays, flow cytometry, and an increased expression of nuclear factor erythroid 2- related factor 2 (Nrf2). Q-3-G reduced melanin production in α-melanocyte-stimulating hormone (α-MSH)-induced B16F10 cells. The hydration effects and mechanisms of Q-3-G were examined by evaluating the moisturizing factor-related genes, such as transglutaminase-1(TGM-1), filaggrin(FLG), and hyaluronic acid synthase (HAS)-1. In addition, Q-3-G increased the phosphorylation of c-Jun, Jun N-terminal kinase (JNK), Mitogen-activated protein kinase (MAPK) kinase 4(MKK4), and TAK1, involved in the MAPKs/AP-1 pathway, and the phosphorylation of IkBα, IkB kinase(IKK)-α, Akt, and Src, involved in the NF-kB pathway. Taken together, we have demonstrated that Q-3-G exerts anti-inflammatory, antioxidant, moisturizing, and antimelanogenesis properties in human keratinocytes and melanoma cells through NF-kB and AP-1 pathways.

Keywords: quercetin 3-O-β-D-glucuronide;UV protection; moisturizing; antimelanogenesis; AP-1; NF-kB

1.Introduction

The skin, the largest organ of the body, offers a barrier that plays a major role in protecting the body from the external environment and damaging factors such as ultraviolet radiation, infectious microbes, and harmful chemicals. This barrier also regulates the body's electrolyte balance and temperature and prevents moisture loss [1,2]. Human skin is composed of three layers: the epidermis, dermis with appendages, and subcutaneous tissue [3]. The epidermal layer plays an important role in maintaining body temperature.

Keratinocytes, the abundant component in the skin epidermis, tightly interact with another through desmosomes and tight junctions that allow an effective physicochemical barrier[4]. Although the skin functions as a protective barrier, ultraviolet (UV) radiation has a significant effect on most cells, including keratinocytes, the outer layer of the skin. UV radiation can cause skin aging, skin damage, wrinkles, and hyperpigmentation, which is considered to be one of the most censorious risk factors [5].UV light consists of three wavelength ranges: UVA(320-400 nm), UVB(280-320 nm), and UVC (200-280 nm). Be-sides hydrogen peroxide (H2O2), which has been known to contribute to oxidative stress, UVB can lead to the production of reactive oxygen species (ROS)through the activation of ROS-generating enzymes [6]. The uncontrolled accumulation of ROS in cells including keratinocytes has been described to cause oxidative modifications to lipids, proteins, nucleic acids, and other intracellular molecules that subsequently lead to various types of skin damage and aging-related processes such as cell death, oxidative stress, and inflammation [7,8]. In addition, the oxidative stress response is known to influence the damage of various cellular components [9]. Damaged skin cells can also lead to inflammatory responses, resulting in chronically damaged skin [10]. One type of UVB or H2O2-mediated skin damage through biological processes is the expression of inflammatory genes such as cyclooxygenase-2 (COX-2) and pro-inflammatory cytokines [11]. Therefore, the development of safer, more robust, and efficient skin protective effects is essential for preventing and blocking diseases associated with UVB or H2O2,-mediated skin damage.

Skin hydration also plays an important role in maintaining its normal function. In particular, the stratum corneum (SC)has a protective effect against water loss [12]. The water content of the stratum corneum participates in skin desquamation and proper maturation, whereas insufficient water in SC promotes the accumulation and dysfunction of corneocytes [12,13]. At the final stages of keratinocyte differentiation, its plasma membrane and cellular organelles disappear, and then calcium influx induces transglutaminase (TGM)for crosslinking with other proteins. A deficiency or loss of TGM-1 leads to defective crosslinking of the cell envelopment. Lamella ichthyosis is an example of a disease caused by a loss of TGM-1. Furthermore, filaggrin (FLG), the formed keratin matrix, plays a role in SC formation as it acts as a scaffold to bind cornified-envelope proteins and lipids[14]by catalyzing g-glutamyl lysine cross-linking reactions, ATGMs, and membrane-associated enzymes, imparting integrity to the SCs [15]. In addition, hyaluronic acid (HA) is a type of natural moisture factor (NMF) involved in moisture retention in the skin and exhibits a distinct profile in intrinsic skin aging, is a drug delivery agent, and is an important component of the extracellular matrix[16,17]. HA plays a role in increasing skin moisture by regulating the hyaluronic acid synthase (HAS)genes; besides, retinoic acid (vitamin A)can effectively regulate HA in the epidermis [17]. TGM-1, FLG, and HAS-12,3 are genes associated with NMF production [18]. Moreover, the activation of transcription factors such as activator protein-1(AP-1) and nuclear factor (NF)-kB are involved in the production of pro-inflammatory substances that optimize the skin barrier and skin hydration through the AP-1 and NF-kB pathways[19]. The upstream signaling enzymes in the AP-1 pathway include mitogen-activated protein kinases (MAPKs) such as c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38, while Src, Akt, IkBα-kinase (IKKo/β), and KBO(IkBa) belong to the NF-KB pathway [20. The skin also produces melanin in the epidermal layer, especially in the melanocytes—the major source of melanin—which contribute to the determination of skin color and melanogenesis [21]. UVB irradiation and α-melanocyte-stimulating hormone (o-MSH) can stimulate melanin secretion, thereby triggering melanogenesis [22. During melanogenesis, α-MSH activates protein kinase A (PKA), protein microphthalmia-associated transcription factor(MITF), and cAMP response element-binding (CREB)[23]. One of the functions of melanin is considered to be the prevention of skin damage by UV, including in sunlight. However, the excess production of melanin or the absence of melanin content caused by aging, melanocyte-stimulating hormone, and UV exposure promotes freckles, melasma, senile lentigines, and other hyper/hypopigmentation disorders that could have a negative impact on the appearance and skin health [24]. Thus, controlling melanogenesis is also desired to maintain both the health and cosmetic appearance of the human body.

Quercetin is a predominant allocated flavonol-type flavonoid present in vegetables, fruits, beverages, and medicinal plants [25]. Consequently, quercetin has been extensively studied to possess pharmacological benefits including anti-inflammatory, anti-atherosclerotic, antioxidant, anticancer, and skin protective properties, and is related to melanogenesis [24,26-29]. However, several works using sensitive and reliable methods have demonstrated that quercetin has not been detected in plasma [30-33] and is hardly found in the brain [34,35]. Quercetin, which generally presents as a glycoside form, is well absorbed and mostly hydrolyzed and metabolized in the small intestine, colon, liver, and kidney into conjugated metabolites [30,36-38]. Only the conjugated metabolites such as glucuronide or sulfate (methylated or unmethylated, respectively), rather than quercetin aglycone or its glycosides, are detected in the circulating blood. 3/-methyl-quercetin-3-glucuronide, quercetin-3-glucuronide, and quercetin-3'-sulfate have been identified as major plasma metabolites after 1.5 h from the consumption of fried onions[30,32]. These quercetin metabolites are formed in the small intestine and liver by biotransformation enzymes including UDP-glucuronyltransferases as a result of phase II metabolism [29,39,40]. It was also reported that conjugated metabolites of quercetin accumulate in human plasma in the concentration range of 10-'to 10-°M after the periodic ingestion of onions with meals for 1 week[41]. The half-life of quercetin metabolites is quite high, at about 11 to 28 h [29]. Since these compounds have been observed as the main forms of conjugated quercetin metabolites, studies focusing on the biological activity of flavonols using these conjugated metabolites would be essential, which prompted us to study the biological activity of one of the conjugated quercetin metabolites. One of the most abundant glucuronide metabolites found mainly in plasma and tissues including the liver, kidney, and brain is quercetin 3-O-β-D-glucuronide(Q-3-G)(Figure 1)[31,35,40,42,43], which may generate similar, stronger, or weaker activities compared with parent compounds in different models. Q-3-G is transported to target tissues via plasma to exert its biological activity [39]. O-3-G has also been shown to be an effective metabolite in rat plasma after oral administration of quercetin [43]. In other studies, Q-3-G-containing metabolite was found to be accumulated in the aorta after the administration of quercetin-rich food and has been identified as an active principle detected in human blood ranging from 0.1-10 μM【44】. Additionally， Q-3-G can be found in wine [45] and in medicinal plants such as Hypericum hirsutum, Nelumbo nucifera, Oenothera biennis, and green beans [46-49].Q-3-G has been described as an anti-inflammatory component under LPS-stimulated conditions and showed antioxidant properties in blood plasma [50-52]. However, compared with the long history of application and wide study of quercetin, the study of Q-3-G remains to be explored. Therefore, Q-3-G was chosen as a representative of quercetin metabolites as an active in vivo player of quercetin. In addition, the role of Q-3-G on skin protective effects has also not been fully elucidated yet. In this study, using various in vitro assessments, we investigated the skin protective effects of Q-3-G against UVBor H2O2-induced oxidative stress and inflammation, the skin hydration in HaCaT(a human keratinocyte cell line) cells, and antimelanogenesis activity in B16F10(a murine melanoma cell line) cells.

2. Result

2.1.Anti-Inflammatory and Antioxidant Effects of Q-3-G against UVB or H2O2-Stimulated HaCaT Cells

Skin tissue can be damaged by UV radiation. UV radiation activates several harmful factors, including oxidative stress, apoptotic cell death, inflammation, and skin aging. In order to determine the UVprotective activities of Q-3-G, initially, we evaluated whether Q-3-G has any effects on the cell viability of human keratinocytes. Using a 3-(4-5-Dimethylthiazol-2-yl)-2,5-diphenyl-243 tetrazolium bromide (MTT)assay, we investigated the cytotoxic effects of O-3-G in HaCa cells. As shown in Figure 2a, there was no significant interference on cell viability at a concentration of 2.5-20 uM by Q-3-G, which indicated that O-3-G showed no cytotoxicity up to 20 μM and did not harm the keratinocytes， the superficial layer of the skin cells. Next, we evaluated the skin-protective effects of Q-3-G in HaCaT cells exposed to UVB. After UVB irradiation at 30 mJ/cm²， Q-3-G significantly protected HaCaTcells from UVB-induced cell death in a concentration-dependent manner (Figure 2b). In agreement with MTT assay, using a camera connected to a microscope to capture the morphology of HaCaT cells, Q-3-G protected against cell death due to UVB-irradiated HaCaT cells up to a concentration of 10 μM（Figure 2c）. The number of HaCaT cells under each condition was 482 for the untreated group, 91 for the UVB 30 mJ/cm-group,276 for the UVB 30 mJ/cm²plus 5μM O-3-G group， and 320 for the UVB 30 mJ/cm² plus 10μM Q-3-G Group (Figure 2d). These results confirmed that Q-3-G inhibits UVB-induced HaCaT damage and rescues the skin-protective effect from UVB irradiation.

The occurrence and exacerbation of inflammation in the body occasionally activate macrophages and release some cytokines. Inflammation is a hallmark of oxidative stress. We investigated the anti-inflammatory effect of Q-3-G against UVB or HO2-induced inflammation. It is well known that UVB can promote DNA damage and inflammation in skin cells 【10】. We irradiated the HaCaT cells with UVB 30mJ/cm² and incubated the HaCaT cells with H2O2(500 uM). We employed a real-time PCR assay to check the mRNA expression of inflammatory enzyme COX-2 and pro-inflammatory cytokine TNF-α. As shown by the results in Figure 2e，f， a 10 μM concentration of O-3-G significantly inhibited the mRNA expression of COX-2 and TNF-α. Consequently, Q-3-G suppressed the inflammatory responses induced in HaCaT cells after exposure to UVB irradiation or H2O2.

We used 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS) and 2,2-diphenyl-lpicrylhydrazyl(DPPH) radical scavenging assays—-widely used model systems to investigate the scavenging activities in vitro of natural compounds—to assess the antioxidant property of Q-3-G at different concentrations. HaCaT cells were treated with Q-3-G in a concentration-dependent manner from 2.5-40 μM in the presence of DPPH， and the result showed a significant scavenging activity at more than 2.5 uM(Figure 2g). The ABTS assay also significantly demonstrated the effects of Q-3-G on free radical scavenging in concentration-dependent manners with an IC50 value of 1.75 μM （Figure 2h）. Ascorbic acid （AA）， as a positive control, showed the greatest antioxidant effects in both DPPH and ABTS assays. Nuclear factor erythroid 2-related factor 2 (Nrf2), one of the antioxidant response element (ARE)-dependent genes, has been reported to regulate the protective oxidoreductases and its nucleophilic substrates, thereby promoting antioxidant enzymes to remove damage and repair systems [53]. Next, we examined the expression level of Nrf2 in UVB-irradiated HaCaT cells. Strengthening the above findings, O-3-G significantly enhanced the expression level of Nrf2 upon UVB irradiation compared to the control in a concentration-dependent manner, up to 10 uM(Figure2i), which implied that Q-3-G presents an antioxidant effect both chemically and biologically with a free radical scavenging property and increases the protective effect of the Nrf2-dependent ARE signaling, respectively. Furthermore, we also performed ROS flow cytometry using dichlorodihydrofluorescein diacetate (DCFDA) in the UV condition. The antioxidant is widely known to suppress cellular stressors by inhibiting ROS production [54]. The result showed that treatment with Q-3-G at concentrations of 5 and 10 μM decreased the level of UVB-induced ROS（Figure 2j）. Taken together， these data implied that Q-3-Gexerts skin-protective properties such as anti-inflammatory and antioxidant effects against H2O, or UVB-induced oxidative stress and inflammation in HaCaT cells.

2.2.Antimelanogenesis Effect in B16F10 and Moisturizing Effect in HaCaT Cells of Q-3-G

To examine the effects of O-3-G on melanogenesis, the melanoma cells were treated with α-MSH for 48 h to secrete melanin in B16F10 cells. By employing MTT assays, we first confirmed that Q-3-G had no cytotoxicity in B16F10 cells at concentrations up to 20 μM for 48 h (Figure 3a). This result showed, for the next assessments that the effects of O-3-G in B16F10 were not due to cell death. Next, we evaluated B16F10 with Q-3-Gand α-MSH for 48 h to determine the effects of Q-3-G on melanogenesis. As shown in Figure 3b, Q-3-G significantly inhibited melanin secretion. The positive control arbutin also significantly decreased melanin secretion.

The skin moisture-related molecule is hyaluronic acid (HA). As previously mentioned, HA, distributed throughout the epidermis and dermis, is an NMF that enhances skin hydration [55]. We used a reverse transcription PCR (RT-PCR)assay to examine the transcription level of HAS-1 after 12 h with a concentration of Q-3-G of up to 30 uM. HAS promotes the accumulation of intermediate-sized HA within keratinocytes [12]. As shown in Figure 3c, the expression of HAS-1 was increased by treatment with Q-3-G up to 10 μM.In addition to the moisturizing factor， we also examined the expression of FLG and TGM-1. We used retinol as a positive control group. The transcription levels of FLG and TGM-1 increased notably after treatment with Q-3-G at concentrations up to 10 μM (Figure 3d). These findings suggest that Q-3-G has skin moisturizing activity in HaCaT cells. Moreover, using SB203580 (p38 inhibitor), SP600125(JNK inhibitor), UO126(ERK inhibitor), and Bay11-7082 (NF-kB inhibitor), we examined the involvement of MAPK and NF-kB signaling pathways in the moisturizing factor of Q-3-G-treated HaCaT cells. Based on O-3-G-induced gene expression, both the levels of TGM-1 and FLG were reduced by treatment with SP600125, a JNK inhibitor (Figure 3e), suggesting that JNK is required to increase the expression of TGM-1 and FLG in terms of the skin moisturizing effect of O-3-G. Additionally, FLG expression was also decreased by Bay11-7082, an inhibitor of IKKα and IkBα, suggesting that the pharmaceutical mechanism of Q-3-G also could belong to the NF-kB signaling pathway.

2.3.Moisturizing-Related Signaling Pathways of Q-3-G via Activation of AP-1 and NF-kB

Based on the previous mRNA results, we hypothesized that the involvement of AP-1 and NF-kB signaling on the hydration effect of Q-3-G can also occur in the protein levels. Using a Western blot assay, we examined the phosphorylation of upstream molecules of AP-1 and NF-kB. First, in the AP-1 pathway, the phosphorylation of c-Jun was increased after incubation of HaCaT cells with Q-3-G for 24 h(Figure 4a). The level of p-INK was also increased after treatment with Q-3-G at concentrations of 2.5，5 and 10 μM. The total form of JNK remained unchanged (Figure 4b). We further examined the phosphorylation of MKK4 and TAK-1, which are upstream regulators of JNK. As shown in Figure 3c, after the treatment of HaCaT cells with O-3-G, the protein levels of p-MKK4 and p-TAK1 were also upregulated.

In addition to NF-kB signaling, Q-3-G increased the phosphorylation of p50 and p65, a subunit of NF-kB(Figure 4c). Moreover, we evaluated the activation of IKKα and IkBa, which also were increased by Q-3-G up to 10 uM compared to the control group (Figure 4d). Eventually, we determined the activation of upstream molecules involved in the NF-kB signaling pathway. The phosphorylation of Src and Akt (Ser473) was enhanced by O-3-G treatment(Figure 4e). Overall, these data indicate that the skin protection effects of Q-3-G target TAK1 and Src activation by increasing the protein level, thereby upregulating the activity of AP-1 and NF-kB, respectively.

2.4.Effects of Q-3-G on the Transcriptional Activation of AP-1 and NF-kB

We investigated the cell cytotoxicity of Q-3-G in human embryonic kidney cell line HEK293T cells by an MTT assay (Figure 4f). The viability of HEK293T cells was not significantly affected after treatment with Q-3-G（2.5-20 μM） compared to untreated cells.

We employed a luciferase assay to determine whether Q-3-G could modulate the NF-kB and AP-1 promoter assays. We confirmed that O-3-G increased AP-1-mediated luciferase activity (Figure 4g)and NF-kB-mediated luciferase activity (Figure 4h) in a concentration-dependent manner. These results validate that AP-1 activation and NF-KB activation are important pharmacological targets of Q-3-G. 

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