Eckol Inhibits Particulate Matter 2.5-Induced Skin Keratinocyte Damage via MAPK Signaling Pathway

Toxicity of particulate matter (PM) towards the epidermis has been well established in many epidemiological studies. It is manifested in cancer, aging, and skin damage. In this study, we aimed to show the mechanism underlying the protective effects of eckol, a phlorotannin isolated from brown seaweed, on human HaCaT keratinocytes against PM2.5-induced cell damage. First, to elucidate the underlying mechanism of toxicity of PM2.5, we checked the reactive oxygen species (ROS) level, which contributed significantly to cell damage. Experimental data indicate that excessive ROS caused damage to lipids, proteins, and DNA and induced mitochondrial dysfunction. Furthermore, eckol (30 μM) decreased ROS generation, ensuring the stability of molecules, and maintaining a steady mitochondrial state. The western blot analysis showed that PM2.5 promoted apoptosis-related protein levels and activated MAPK signaling pathway, whereas eckol protected cells from apoptosis by inhibiting MAPK signaling pathway. This was further reinforced by detailed investigations using MAPK inhibitors. Thus, our results demonstrated that inhibition of PM2.5-induced cell apoptosis by eckol was through MAPK signaling pathway. In conclusion, eckol could protect skin HaCaT cells from PM2.5-induced apoptosis via inhibiting ROS generation.


Introduction
Natural compounds can be effective candidates for various skin diseases. Particularly, phlorotannins extracted from seaweeds have interesting properties that make them useful for cosmeceutical applications. They can whiten the skin by inhibiting melanin synthesis [1], and delay skin wrinkles by inhibiting matrix metalloproteinase [2]. Moreover, phlorotannins show antioxidant [3], anti-inflammatory [4], and hair-growth promotion activities [5]. Studies have shown that eckol, which is a kind of phlorotannin present in brown seaweeds (Phaeophyceae), decreases ultraviolet B (UVB)-induced oxidative stress in human keratinocytes at a dose of 27 µM [6], and inhibits cancer in SKH-1 mice via inhibiting UVB-induced inflammation [7], and declines matrix metalloproteinase-1 level in human dermal fibroblasts implying anti-aging effects at a dose of 10 µM [8]. Our earlier studies have proved that eckol could clear excess reactive oxygen species (ROS) and protect skin keratinocytes from apoptosis [6].
Previous studies have shown that eckol exhibited no cytotoxicity to HaCaT cells at a concentration of 30 µM [18] but showed antioxidant activity [6]. Therefore, in this study, we used 30 µM of eckol ( Figure 1a) as the optimal concentration. From Figure 1b,c, it is evident that while PM 2.5 increased the levels of ROS as indicated by 2',7'-dichlorofluorescein diacetate (DCF-DA) staining, eckol inhibited intracellular ROS generation. The results demonstrated that PM 2.5 -induced ROS could accelerate cell apoptosis and death. To confirm that eckol could help cells escape from this damage, we checked nuclei integrity, and cell viability. According to results, PM 2.5 treatment led to sub-G 1 cell population after 24 h (Figure 1d), fragmented nuclei (Figure 1e), and cell death ( Figure 1f). However, it was noted that following treatment with eckol, the apoptotic cell death was decreased, and the cell viability was also enhanced. All experiments were performed after treatment with PM2.5 for 24 h, and n = 3 for every group. * p < 0.05 and # p < 0.05 compared to control cells and PM2.5-exposed cells, respectively.

Eckol Protected Cells against PM2.5-Induced Intracellular Molecular Damage
Previous studies have shown that increment in ROS disrupted intracellular molecules involved in apoptosis [19,20]. Thus, we detected lipid peroxidation, protein carbonylation, and DNA damage. The confocal images show that PM2.5 caused generation of phosphine oxide, which is a marker of lipid peroxidation. However, this was reversed by treatment with eckol ( Figure 2a). Moreover, PM2.5 aggravated protein carbonylation level, which was decreased by eckol treatment (Figure 2b). DNA lesions and strand breaks were studied by staining the cells with avidin-tetramethylrhodamine isothiocyanate (TRITC) conjugate ( Figure 2c) and comet assay ( Figure  2d). The data show that eckol guarded DNA against PM2.5.  were reduced via treatment with eckol, determined by trypan blue assay. The arrow indicated the dead cell (stained cells by trypan blue). All experiments were performed after treatment with PM 2.5 for 24 h, and n = 3 for every group. * p < 0.05 and # p < 0.05 compared to control cells and PM 2.5 -exposed cells, respectively.

Eckol Protected Cells against PM 2.5 -Induced Intracellular Molecular Damage
Previous studies have shown that increment in ROS disrupted intracellular molecules involved in apoptosis [19,20]. Thus, we detected lipid peroxidation, protein carbonylation, and DNA damage. The confocal images show that PM 2.5 caused generation of phosphine oxide, which is a marker of lipid peroxidation. However, this was reversed by treatment with eckol ( Figure 2a). Moreover, PM 2.5 aggravated protein carbonylation level, which was decreased by eckol treatment (Figure 2b). DNA lesions and strand breaks were studied by staining the cells with avidin-tetramethylrhodamine isothiocyanate (TRITC) conjugate ( Figure 2c) and comet assay ( Figure 2d). The data show that eckol guarded DNA against PM 2.5 . All experiments were performed after treatment with PM2.5 for 24 h, and n = 3 for every group. * p < 0.05 and # p < 0.05 compared to control cells and PM2.5-exposed cells, respectively.

Eckol Protected Cells against PM2.5-Induced Intracellular Molecular Damage
Previous studies have shown that increment in ROS disrupted intracellular molecules involved in apoptosis [19,20]. Thus, we detected lipid peroxidation, protein carbonylation, and DNA damage. The confocal images show that PM2.5 caused generation of phosphine oxide, which is a marker of lipid peroxidation. However, this was reversed by treatment with eckol ( Figure 2a). Moreover, PM2.5 aggravated protein carbonylation level, which was decreased by eckol treatment (Figure 2b). DNA lesions and strand breaks were studied by staining the cells with avidin-tetramethylrhodamine isothiocyanate (TRITC) conjugate ( Figure 2c) and comet assay ( Figure  2d). The data show that eckol guarded DNA against PM2.5.  All experiments were performed after treatment with PM2.5 for 24 h, and n = 3 for every group. * p < 0.05 and # p < 0.05 compared to control cells and PM2.5-exposed cells, respectively.

Eckol Prevented PM2.5-Induced Mitochondrial Dysfunction
Mitochondria play an important role in cellular energy production, and their biogenesis is related to synthesis of molecules, such as lipids and proteins, DNA transcription, and even cell apoptosis [21]. Next, we examined mitochondrial functions. Dihydrorhodamine 123 (DHR123) staining images show that mitochondrial ROS was accumulated in PM2.5-treated group. Whereas, ROS level was decreased by pretreatment with eckol ( Figure 3a). Both flow cytometry ( Figure 3b) and confocal microscopy ( Figure 3c) data demonstrate that PM2.5 caused mitochondrial depolarization, which was arrested by treatment with eckol. Furthermore, the flux of mitochondrial calcium was increased in the PM2.5-treatment group, and it was decreased in eckol-treatment group, which was monitored using the calcium indicator, Rhod-2 acetoxymethyl ester (Rhod-2 AM), by confocal microscopy (Figure 3d  All experiments were performed after treatment with PM 2.5 for 24 h, and n = 3 for every group. * p < 0.05 and # p < 0.05 compared to control cells and PM 2.5 -exposed cells, respectively.

Eckol Prevented PM 2.5 -Induced Mitochondrial Dysfunction
Mitochondria play an important role in cellular energy production, and their biogenesis is related to synthesis of molecules, such as lipids and proteins, DNA transcription, and even cell apoptosis [21]. Next, we examined mitochondrial functions. Dihydrorhodamine 123 (DHR123) staining images show that mitochondrial ROS was accumulated in PM 2.5 -treated group. Whereas, ROS level was decreased by pretreatment with eckol ( Figure 3a). Both flow cytometry ( Figure 3b) and confocal microscopy ( Figure 3c) data demonstrate that PM 2.5 caused mitochondrial depolarization, which was arrested by treatment with eckol. Furthermore, the flux of mitochondrial calcium was increased in the PM 2.5 -treatment group, and it was decreased in eckol-treatment group, which was monitored using the calcium indicator, Rhod-2 acetoxymethyl ester (Rhod-2 AM), by confocal microscopy (Figure 3d   All experiments were performed after treatment with PM 2.5 for 24 h, and n = 3 for every group. * p < 0.05 and # p < 0.05 compared to control cells and PM 2.5 -exposed cells, respectively.

Eckol Modulated PM 2.5 -Induced Apoptotic Factors
It has been reported that urban particulate pollution penetrates the skin barrier and causes apoptosis in keratinocytes by activating caspase-3 [22]. Therefore, we evaluated the levels of the proapoptotic protein-Bax, antiapoptotic protein-Bcl-2, and cleaved caspase-3 (Figure 4a). The protein levels of Bax and activated caspase-3 were increased by PM 2.5 , but expression of Bcl-2 was decreased by treatment with PM 2.5 . However, these were reversed by eckol treatment. To investigate whether PM 2.5 could induce apoptosis, we counted apoptotic bodies via Hoechst 33342 dye staining (Figure 4b).
The number of apoptotic cells in PM 2.5 group surged four times compared to that in the control group. However, both eckol and Z-VAD-FMK (the caspase inhibitor) halted the apoptotic bodies induced by PM 2.5 .  24 h, and n = 3 for every group. * p < 0.05 and # p < 0.05 compared to control cells and PM2.5-exposed cells, respectively.

Eckol Modulated PM2.5-Induced Apoptotic Factors
It has been reported that urban particulate pollution penetrates the skin barrier and causes apoptosis in keratinocytes by activating caspase-3 [22]. Therefore, we evaluated the levels of the proapoptotic protein-Bax, antiapoptotic protein-Bcl-2, and cleaved caspase-3 (Figure 4a). The protein levels of Bax and activated caspase-3 were increased by PM2.5, but expression of Bcl-2 was decreased by treatment with PM2.5. However, these were reversed by eckol treatment. To investigate whether PM2.5 could induce apoptosis, we counted apoptotic bodies via Hoechst 33342 dye staining ( Figure  4b). The number of apoptotic cells in PM2.5 group surged four times compared to that in the control group. However, both eckol and Z-VAD-FMK (the caspase inhibitor) halted the apoptotic bodies induced by PM2.5.   All experiments were performed after treatment with PM 2.5 for 24 h, and n = 3 for every group. * p < 0.05 and # p < 0.05 compared to control cells and PM 2.5 -exposed cells, respectively.

Eckol Reduced MAPK Signaling Pathway Activated by PM 2.5
In a review, Sun et al. have pointed out that many anti-cancer therapeutics induced apoptosis by modulating the MAPK/ERK signaling pathway [23]. Thus, we checked the expression levels of MAPK-related proteins, ERK, p38, and JNK, and the results showed that PM 2.5 could stimulate ERK, p38, and JNK (Figure 5a). However, eckol inhibited the activation of ERK, p38, and JNK. Next, we examined PM 2.5 -induced apoptotic bodies by treatment with MAPK pathway inhibitors, U0126, SB203580, and SP600125 (inhibitors of ERK, p38, and JNK, respectively), and the results showed that all these three inhibitors could reduce the number of apoptotic bodies (Figure 5b). In addition, eckol enhanced the anti-apoptotic effect of MAPK-related inhibitors. In a review, Sun et al. have pointed out that many anti-cancer therapeutics induced apoptosis by modulating the MAPK/ERK signaling pathway [23]. Thus, we checked the expression levels of MAPK-related proteins, ERK, p38, and JNK, and the results showed that PM2.5 could stimulate ERK, p38, and JNK (Figure 5a). However, eckol inhibited the activation of ERK, p38, and JNK. Next, we examined PM2.5-induced apoptotic bodies by treatment with MAPK pathway inhibitors, U0126, SB203580, and SP600125 (inhibitors of ERK, p38, and JNK, respectively), and the results showed that all these three inhibitors could reduce the number of apoptotic bodies (Figure 5b). In addition, eckol enhanced the anti-apoptotic effect of MAPK-related inhibitors.

Discussion
There have been several investigations into the bioactivities of eckol, since it was first isolated from Ecklonia cava [3]. Eckol has multi-protective effects towards several cell lines, including lung fibroblast cells [24], human dermal fibroblasts [8], Chang liver cells [25], and human keratinocytes All experiments were performed after treatment with PM 2.5 for 24 h, and n = 3 for every group. * p < 0.05, # p < 0.05 and ## p < 0.05 compared to control cells, PM 2.5 -exposed cells, and both inhibitor and PM 2.5 -exposed cells respectively.

Discussion
There have been several investigations into the bioactivities of eckol, since it was first isolated from Ecklonia cava [3]. Eckol has multi-protective effects towards several cell lines, including lung fibroblast cells [24], human dermal fibroblasts [8], Chang liver cells [25], and human keratinocytes [6]. Furthermore, eckol is a compound with therapeutic potential in many areas, such as anti-oxidative stress [24], radioprotective action [26], antithrombotic and profibrinolytic activities [27], and anticancer activity [28]. Piao  and 100 µg/mL) for 24 h in HaCaT keratinocytes, and found that PM 2.5 50 µg/mL caused excessive ROS and skin dysfunction [29]. Usually, oxidative stress is caused by excessive accumulation of ROS or lack of the ability to eliminate them. PM 2.5 produces large amounts of ROS beyond the clearance ability of cells [30]. In our study, eckol showed its ability to protect cells against PM 2.5 -induced ROS, cell cycle arrest, and apoptosis, and improved cell viability.
To explore the mechanism in detail, we checked the state of intracellular molecules such as lipids, protein, and DNA, which play various important roles in the cells [31]. Furthermore, intracellular macromolecular damage can be recognized as oxidative stress [32]. Our results demonstrated that PM 2.5 indeed induced oxidation of molecules, whereas eckol relieved intracellular molecular damage. The review also points out that mitochondria-dependent ROS generation subsequently caused cell cycle arrest and apoptosis, which is ROS-mediated apoptosis via mitochondrial mechanism [33]. In addition, our previous studies showed that calcium level and mitochondrial membrane potential affect the function of mitochondria [30,34]. The data in Figure 3 show that PM 2.5 increased the calcium level and depolarized mitochondrial membrane potential as compared to the control cells, whereas eckol regulated the mitochondria and maintained a stable state. The mechanism of mitochondrial damage is related to Bcl-2 proteins, which maintains mitochondrial membrane integrity [35]. The interaction between Bcl-2 and Bax also influences antiapoptosis [36]. Bcl-2 plays an anti-apoptotic role, whereas Bax is proapoptotic [37]. There is a complex crosslink between Bcl-2 family proteins and caspase proteins in cell apoptosis, in which Bcl-2 indirectly activates the caspase cascade [38]. The caspase-3 results in apoptosis induced by both extrinsic and intrinsic stimulus [39]. The results elucidated that except for the decrease of Bcl-2, Bax and cleaved caspase-3 (activated caspase-3) were increased by PM 2.5 . However, eckol reversed these effects. Then, we treated cells with caspase inhibitor (Z-VAD-FMK) and found that upon pretreatment with caspase inhibitor, the apoptotic bodies were decreased significantly. These data prove that caspase proteins contributed to cell apoptosis induced by PM 2.5 . MAPK signaling pathway plays a role in many systems of cell proliferation, migration, and apoptosis [23]. Many drugs are used to modulate MAPK signaling pathway to induce cell apoptosis in cells, such as lung cancer [40], human colorectal cancer [41], and cervical cancer HeLa cells [42]. Finally, we checked MAPK signaling pathway-related proteins, ERK, p38, and JNK. The results show that PM 2.5 activated all three proteins, but eckol exhibited the ability to inactivate them. When we used inhibitors of ERK, p38, and JNK to treat PM 2.5 -damaged cells, the numbers of apoptotic bodies were decreased, similar to eckol. These data further prove that MAPK signaling pathway plays a vital role in the inhibition of PM 2.5 -induced apoptosis by eckol.
Although the protective effects of eckol on human keratinocytes from PM 2.5 -induced skin damage has been shown, there are limitations to this study. These results from in vitro experiments need to be validated by animal studies and clinical trials. Moreover, the concentration of air pollutants in the natural environment is different from the PM 2.5 purchased from the company, which provide certain ingredients for reference. In the future, there should be in vivo animal trials on skin protection to elucidate the protective effects and side effects of eckol under the complicated living environments.

Eckol and PM 2.5
Eckol was provided by Professor Nam Ho Lee of Jeju National University (Jeju, Korea), which belonged to Phaeophyceae. Preparation of the extract and its purification was following the reported protocol [43]. The dried brown alga Ecklonia cava was extracted with 80% methanol and the crude extract was purified by HPLC. After purification, 20 mg of pure eckol was obtained from 1 kg dry weight of the brown algae. A stock solution of eckol was prepared by dimethyl sulfoxide (DMSO). The NIST particulate matter SRM 1650b (PM 2.5 ) was bought from Sigma-Aldrich (St. Louis, MO, USA) and a stock solution in DMSO was prepared to obtain a concentration of PM 2.5 at 25 mg/mL. DMSO was as the control.

Cell Culture
The human HaCaT keratinocytes were purchased from Cell Lines Service (Heidelberg, Germany) and were grown in Dulbecco's modified Eagle's medium (Life Technologies Co., Grand Island, NY, USA) with 10% heat-inactivated fetal calf serum, streptomycin (100 µg/mL), and penicillin (100 units/mL). The cells were cultured at 37 • C in an incubator in an atmosphere containing 5% CO 2 [6].

Hoechst 33342 Staining
The apoptotic bodies were examined with Hoechst 33342 (Sigma-Aldrich), which is a nucleus-specific dye. All cells were stained with Hoechst 33342, and the images were captured under a Cool SNAP-Pro color digital camera (Media Cybernetics, Silver Spring, MD, USA) in a fluorescence microscope [44].