To demonstrate the importance of Nrf2 activity, we developed a Nrf2 gene knock-down
model in HaCaT cells by siRNA transfection. Knock-down of the Nrf2 gene was confirmed by western blot analysis, which showed that the transfection of siNrf2 (100 pM) led to reduction of the Nrf2 protein level. As shown in Fig. 7A, significant increases in Nrf2, HO-1, NQO-HO-1, and -GCLC protein levels were observed in lucidone (4 M) treated cells, whereas lucidone-induced increase of Nrf2, HO-1, NQO-1, and -GCLC expression was barely observed in Nrf2 knock-down cells. This result is evidence that the induction of antioxidant genes by lucidone occurs due to the activation of Nrf2. Transfection of scrambled siRNA (control) did not affect the levels of Nrf2, HO-1, NQO-1, and -GCLC in HaCaT cells.
To further confirm our hypothesis that lucidone protects keratinocytes from UVA-induced cell death through the up-regulation of Nrf2, HaCaT cells were transfected with siNrf2 and pre-incubated with lucidone (4 M) for 24 h, then exposed to UVA (15 J/cm2). Cell viability was measured by the MTT assay. Compared to UVA-treated cells, lucidone pre-treatment significantly increased the amount of viable cells in the control siRNA system (Fig. 7B;
upper panel), whereas the percentage of viable cells in the Nrf2 knock-down system was further decreased by UVA. Lucidone, therefore, failed to protect keratinocytes from UVA-induced cell death in the Nrf2 knock-down system, as shown by the reduction of cell viability (Fig. 7B; lower panel). In addition, the UVA-induced intracellular ROS generation was significantly inhibited by lucidone in a control siRNA system, whereas lucidone failed to inhibit ROS generation in siRNA-transfected cells, which suggests that the lucidone-mediated inhibition of intracellular ROS generation requires Nrf2 activation (Fig. 7C).
Furthermore, the UVA-induced GSH depletion in HaCaT cells was significantly inhibited by lucidone in control siRNA-transfected cells, whereas lucidone pre-treatment failed to protect against UVA-induced GSH depletion in HaCaT cells (Fig. 7D). These data clearly indicate that the cyto-protective effect of lucidone is mediated by Nrf2.
4. Discussion
The chronic exposure of UVA radiation can cause serious problem, including premature aging of skin, suppression of immune system, damage to eyes, and skin cancer (Kulms &
Schwarz, 2000). Although sunscreens have been in used for many years, they are relatively ineffective in protecting the UVA-induced photo-aging and skin cancers (Haywood, Wardman, Sanders, & Linge, 2003). In the present study, we demonstrated that when HaCaT cells were exposed to UVA, the cell viability was decreased, while intracellular ROS generation was increased. However, pre-treatment with lucidone enhanced cell viability through the inhibition of excessive ROS generation and protected cells from UVA-induced oxidative stress. The obtained results are consistent with previous studies that UVA-induced apoptosis in human keratinocytes occurs through the aberrant generation of intracellular ROS (Han, Hanawa, Saberi, & Kaplowitz, 2006; Hseu et al., 2012). The inhibitory effects of ROS generation by lucidone were also observed in human hepatic cells and keratinocytes (Kumar et al., 2013; Kumar, Liao, Xiao, Gokila Vani, & Wang, 2012). Therefore, with substantial evidences, our findings are much more enlightening and revealed that lucidone is effective in the elimination of ROS in UVA-irradiated skin cells and prevent the skin damage.
Many sun-protecting phytochemicals have been consumed by humans for centuries as a part of plant-rich diet and presumed to be of low toxicity without overdose effects of naturally occurring antioxidant. The fruit extracts of Lindera erythrocarpa used in this study have been reported as inflammatory substance by our team. We identified four anti-inflammatory cyclopentenediones by bioactivity-guided fractionation, with lucidone being the strongest inhibitor of nitric oxide production (Wang et al., 2008). We further demonstrated that lucidone inhibits LPS-induced inflammation through the down-regulation of the NF-κB and AP-1 signaling pathways in vitro and in vivo (Kumar, Yang, Chu, Chang,
& Wang, 2010; Senthil & Wang, 2009). Moreover, lucidone inhibits the tyrosinase enzyme
activity and MITF-mediated tyrosinase protein expression in melanoma cells, which are involved in the melanogenesis (Kumar, Yang, Chu, Chang, & Wang, 2010). Another study showed the heptato-protective properties of lucidone against alcohol-induced oxidative damage through the up-regulation of the Nrf2 signaling pathway (Kumar, Liao, Xiao, Gokila Vani, & Wang, 2012). A recent study reported that lucidone protects human skin keratinocytes against 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH)-induced oxidative damage and inflammation through the up-regulation of HO-1/Nrf2 antioxidant genes and down-regulation of NF-κB signaling pathways (Kumar et al., 2013). Based on the above evidences, lucidone dermato-protective properties and molecular mechanism behind the beneficial effects were emphasized in this study.
A growing body of evidence suggests that UVA radiation generates ROS in the form of free radicals, such as O2● — and •OH, as well as non-radical intermediates such as H2O2 and
1O2 (Gniadecki, Thorn, Vicanova, Petersen, & Wulf, 2000; Miyachi, 1995). A previous study demonstrated that H2O2 produced by UVA irradiation is responsible for UVA-induced oxidative stress and cell death in HaCaT cells (Petersen, Gniadecki, Vicanova, Thorn, &
Wulf, 2000). In the present study, we also observed the H2O2-induced ROS generation and cell death in skin keratinocytes, but this damage was significantly inhibited by lucidone pre-treatment. Free radicals can cause irreversible damage to lipids and DNA, and resulted byproducts can be used as biomarkers of oxidative stress. The lipid peroxidation of polyunsaturated fatty acids gives rise to a vast range of aldehydes, which diffuse rapidly in aqueous media, whereas other lipophilic compounds remain in the lipid phase. Remarkably, the UVA-induced accumulation of another aldehyde (TBARS) in keratinocytes was significantly reduced by lucidone pre-treatment. Similar UVA-induced lipid peroxidation in human keratinocytes was reported by Tebbe et al. (1997). These findings further support our assumption that lucidone directly interacts with ROS.
A number of studies show that many epidermal skin cells, including keratinocytes, undergo apoptosis following UVA exposure as a result of DNA strand breaks because DNA is a major target for photochemical modifications (Ichihashi et al., 2003). To examine DNA damage, we used the comet assay, a sensitive method for detecting single-strand DNA breaks in individual cells. Our findings also showed that UVA exposure caused apoptosis, as evidenced by single-strand DNA breaks in human keratinocytes. However, pre-treatment with lucidone effectively eliminated the UVA-induced single-strand DNA breaks, the levels of which were close to those of non-irradiated cells. This effect was concomitant with our previous observation that AAPH-induced DNA damage in HaCaT cells was prevented by lucidone pre-treatment (Kumar et al., 2013).
Apoptosis is a complex process of deliberate suicide by a cell in a multi-cellular organism. It is one of the main types of programmed cell death and involves an orchestrated series of biochemical events leading to a characteristic cell morphology and death (Kulms &
Schwarz, 2000). UVA exposure has been reported to induce apoptosis in human keratinocytes and to be dependent on the DNA fragmentation caused by DNase activity during apoptotic processes, which can be directly measured using the TUNEL assay (Morley et al., 2006). In this study, we also found that UVA exposure markedly increased DNA fragmentation in HaCaT cells, which was significantly prevented by lucidone. Multiple signaling cascades including mitochondrial-, death receptor-, ER stress-, and ROS-mediated apoptosis appear to be involved in the apoptosis of keratinocytes following UVA exposure (Hseu et al., 2012). In particular, a number of studies demonstrate the involvement of mitochondrial-dependent (intrinsic) apoptotic pathways in UVA-induced cell-death. Bcl-2 family proteins such as the pro-apoptotic proteins (e.g., Bax, Bak, and Bid) and anti-apoptotic proteins (Bcl-2 and Bcl-xL) play an important role in UVA-induced apoptosis in keratinocytes (Kulms & Schwarz, 2000). Indeed, the balance between pro- and
anti-apoptotic proteins determines whether apoptosis is promoted or prevented (Kulms &
Schwarz, 2000). Our results demonstrate that UVA-induced apoptosis is mainly mediated by the activation of mitochondrial signaling pathways. Thus, supplementation with lucidone may be therapeutically effective by restoring Bcl-2 levels and preventing aberrant apoptosis in dermal keratinocytes.
On the other hand, the induction of Bax by UVA exposure promotes mitochondrial membrane damage and releases pro-apoptotic molecules such as cytochrome c. MitoTracker Green is commonly used to determine mitochondrial injury. MitoTracker Green accumulates in the mitochondria regardless of mitochondrial membrane potential and is therefore considered to be a suitable stain for mitochondria because it remains stable after cell death or aldehyde fixation, whereas other short-chain carbocyanine dyes are not retained after cell death or fixation (Buckman et al., 2001). Fluorescence imaging of the UVA-treated cells stained with MitoTracker Green showed a significant decrease in fluorescence intensity, which confirmed that UVA treatment affected the mitochondrial membrane potential, whereas lucidone pre-treatment significantly protected against UVA-induced mitochondrial damage in human keratinocytes.
The major endogenous antioxidants, such as HO-1, SOD, NQO-1, GSTA2, γ-GCLC and γ-GCLM, are regulated by various transcription factors, including Nrf2 (Surh, 2003). These enzymes are vital for protecting cells from oxidative stress and electrophilic toxicity as well as preventing carcinogenesis. In particular, Nrf2 has been shown to be involved in the induction of various detoxifying (phase II) enzymes and antioxidants by chemical or UV inducers in human keratinocytes (Zhong, Edwards, Raval, Li, & Tyrrell, 2010).
Investigations have also shown that plant-derived polyphenols enhance Nrf2-dependent ARE activity and induce HO-1, NQO-1, and γ-GCLC expression in a variety of cells including keratinocytes (Kleszczynski, Ernst, & Wagner, 2013; Tao, Justiniano, Zhang, &
Wondrak, 2013). Not surprisingly, lucidone up-regulates HO-1, NQO-1, γ-GCLC, and Nrf2 antioxidant genes in human keratinocytes. Thus, lucidone protects keratinocytes from UVA damage mainly by elevating intracellular antioxidative enzyme levels via the enhanced accumulation of a transcription factor for antioxidant genes, Nrf2, and dramatically induces the expression of the antioxidant genes HO-1, NQO-1, and γ-GCLC following UVA irradiation.
The discovery of RNA interference has revolutionized the approach to studying specific gene functions and pathways (Narayanan, Narayanan, Davis, & Nargi, 2006). Importantly, siRNA-directed transcriptional silencing is conserved in mammalian cells and thus provides a means to inhibit specific mammalian gene functions (Narayanan, Narayanan, Davis, &
Nargi, 2006). Our data show that the siRNA-mediated knock-down of Nrf2 expression leads to enhanced HaCaT cell death. Lucidone failed to protect the Nrf2-knock-down keratinocytes from UVA-induced oxidative stress. These data demonstrate that the protective effect of lucidone is mainly mediated by Nrf2 activation.
The Nrf2/ARE signaling pathway is a common molecular target for natural products (Surh, 2003). Under normal physiological conditions, Nrf2 forms a covalent complex with Keap-1 via intermolecular disulfide bonds. When specific cysteine residues of the Keap-1 protein are oxidized or modified by electrophile inducers, Nrf2 is no longer sequestered by Keap-1 and is subsequently translocated into the cell nucleus, where it binds to the promoter and activates the transcription of several phase-II detoxifying enzymes. Notably, chemical compounds with high electrophilic activity may attack the reactive cysteine residues in the Keap-1-binding region, resulting in a conformational change in the associated motif of Nrf2-Keap-1 and the dissociation of Nrf2 from Nrf2-Keap-1 (Dinkova-Kostova, Holtzclaw, & Kensler, 2005; Magesh, Chen, & Hu, 2012). Nakazato et al. reported that EGCG, a polyphenol from green tea, induced ROS production in malignant human B cells, which suggests that
polyphenols may have a potent ability to modify the SH-residue in Keap-1 through ROS production (Nakazato, Ito, Ikeda, & Kizaki, 2005). However, we did not detect ROS signals from DCFH2-DA in HaCaT cells during lucidone treatment. This result implies that a ROS-independent pathway is involved in the activation of Nrf2 by lucidone. In another study, the well-known electrophile quercetin exerted a protective effect against UVA-induced oxidative stress in human keratinocytes through the up-regulation of Nrf2 (Kimura et al., 2009). Therefore, this study mainly aimed to characterize the induction of Nrf2 and its target antioxidant genes. Structural activity relationship studies revealed that phyto-compounds with , -unsaturated ketone moieties could act as Michael reaction acceptors that are able to modify the cysteine thiol residues in Keap-1 (Surh, 2003). Certain dietary phyto-compounds, such as curcumin, caffeic acid pheneythl ester (CAPE), and sulforaphane, contain ,-unsaturated ketone moiety; therefore, these compounds could serve as potent antioxidants in biological systems (Surh, 2003). Interestingly, lucidone also contains an ,-unsaturated ketone moiety. Therefore, we believe that the highly conserved ,-,-unsaturated ketone moiety may be responsible for the antioxidant potential of lucidone; further structural activity relationship studies may confirm this hypothesis.
Our findings demonstrated that lucidone extracted from the dried fruits of Lindera erythrocarpa has potential dermato-protective properties. This study revealed the molecular mechanism by which lucidone protects human keratinocytes from UVA-induced oxidative stress; the activation of the antioxidant system through the up-regulation and stabilization of Nrf2 is critical for the protection of skin cells from UV-induced skin damage. Suppressed oxidative stress by lucidone may be useful in the treatment of UVA-induced skin damage, photo-aging, and skin cancers. Thus our findings suggest that lucidone could be an effective component in food supplements for skin protection. We further believe that lucidone can be used in the preparation of skin care products, including dermatological creams or lotions, to
support the repair and regeneration of UVA-irradiated skin.
Conflict of interest statement
The authors have no conflicts of interest to declare.
Acknowledgments
This work was supported by the grants NSC-101-2320-B-039-050-MY3, NSC-103-2622-B-039-001-CC2, 101-ASIA-04, and CMU 101-ASIA-12 from the National Science Council, Asia University, and China Medical University, Taiwan. We thank Mallikarjuna Korivi and K.J. Senthil Kumar revised the manuscript.
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