Tart Cherry Extract Containing Chlorogenic Acid, Quercetin, and Kaempferol Inhibits the Mitochondrial Apoptotic Cell Death Elicited by Airborne PM10 in Human Epidermal Keratinocytes

Tart cherry (Prunus cerasus L.), a medicinal food containing high concentrations of phytochemicals, has a variety of antioxidant activities and health benefits. Here, we investigate the functional effect of tart cherry during apoptotic cell death elicited by airborne particulate matter with a diameter of <10 μm (PM10) in human epidermal keratinocyte HaCaT cells. The PM10 particles significantly induced cytotoxicity in the HaCaT cells. The decrease in cell viability was restored upon treatment with tart cherry extract (200 μg/mL) containing chlorogenic acid, quercetin, and kaempferol. Tart cherry inhibited the intracellular reactive oxygen species (ROS) responsible for the distinctive activations of the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) in PM10-treated HaCaT cells. Interestingly, tart cherry significantly inhibited the expression of apoptosis-related genes (B-Cell Lymphoma 2 (Bcl-2), Bcl-2 associated X protein (Bax), and caspase-3) as regulated by the activation of transcription factor nuclear factor-kappa B (NF-κB). These results demonstrate that tart cherry is a medicinal food that blocks the mitochondrial pathway of apoptosis induced by PM10 in human epidermal keratinocytes.


Introduction
Fine particulate matter (PM 10 ), referring to particles with an aerodynamic diameter of less than 10 µm, mainly consists of air pollutants in the form of a fine dust (FD) that can contain metals, organic toxic compounds, and biological materials depending on the natural and the anthropogenic dust source [1]. PM is primarily formed from the chemical reactions triggered by exhaust gases from internal combustion engines, smoke from factory chimneys, and fuel combustion in the atmosphere [2]. This fine dust can have various adverse effects on humans' health and on the ecosystem [1]. For instance, PMs can cause numerous health problems that are associated with respiratory, cardiovascular, and skin diseases [3].
Skin, the primary tissue exposed to FD, plays an important role as an interface between air pollutants and the body. However, it was proven that PM is able to be absorbed by the skin barrier and causes many inflammatory and allergic reactions and delays the skin wound healing process induced by topical exposure [4]. Keratinocytes are a major cell type in the epidermis, which is the outermost layer of the skin and which serves mainly to protect against microbial, viral, fungal, and ambient forms of air pollution as a major skin barrier [5]. Recent studies suggest that keratinocytes, when exposed to PM, induce the mitochondrial apoptosis pathway by producing reactive oxygen species (ROS) to interact extract (1 mg) was mixed with 10 mL of 30% methanol and extracted using an ultrasonic microwave extractor (Powersonic 505; Hwashin Tech, Daegu, Korea) for 1 h. The test liquid was filtered using a membrane filter with a diameter of <0.2 µm. On the other hand, proper amounts of internal standards (chlorogenic acid, quercetin, and kaempferol) were measured accurately and dissolved in methanol to obtain stock solutions at a concentration of 1 mg/mL. Each solution was diluted in methanol to obtain working solutions with standard at concentrations of 12.5, 25, 50, and 100 µg/mL. The analysis conditions are presented in Table 1. The standard curve determination coefficient (R 2 ) value of all standard materials exceeded 0.999. The injection volume was 2 µL and the flow rate was kept constant at 0.4 mL/min. The mobile phase consisted of a 13-min gradient system combining water and acetonitrile containing 0.1% aqueous formic acid (FA). Data were processed using the Empower 3 (Waters ® ) software (Waters, Prague, Czech Republic). The chromatograms detected by UPLC-PDA (Waters, Prague, Czech Republic) were recorded at a wavelength of 330 nm for chlorogenic acid and quercetin and at 380 nm for the kaempferol. Peaks were identified by comparing retention times and were quantitated by reference standards.

Culture of Human Skin Keratinocyte and Inhibitor Treatments
Human epidermal keratinocyte HaCaT cells were obtained from the American Type Culture Collection (ATCC ® , Manassas, VA, USA) and cultured at 37 • C in an incubator supplied with 5% CO 2 in Dulbecco's Modified Eagle's medium (DMEM) cell culture medium (Invitrogen Co., Carlsbad, CA, USA) treated with FBS (10%), streptomycin (100 mg/mL), and penicillin (100 U/mL). The medium was replaced twice a week. All the cells were serum-starved for 24 h before treatment with tart cherry and PM 10 in DMEM without FBS. To determine the relevance of signaling molecules in the apoptotic cell death pathway induced by PM 10 , HaCaT cells were pretreated with N-acetylcysteine (NAC, 10 µM), SB203580 (10 µM), PD98059 (10 µM), and Bay 11-7082 (10 µM) for the inhibition of ROS, p38 mitogen-activated protein kinase (MAPK), ERK, and NF-κB for 30 min prior exposure to PM 10 , respectively.

Detection of Intracellular Reactive Oxygen Species (ROS)
Cells were treated with 10 mM of CM-H 2 DCFDA for 30 min to quantify the level of intracellular ROS. After two washes with PBS, cells were scraped and loaded into a black 96-well plate. The fluorescence, which corresponds to the amount of intracellular ROS, was determined using a microplate reader designed for the detection of fluorescent and luminescent signals (SPARK, Seestrasse, Männedorf, Switzerland) at excitation and emission wavelengths of 485 and 535 nm.

Western Blot Analysis
The expression and phosphorylation of proteins related to the intracellular signaling pathway were determined by Western blot analysis and performed as previously described [12]. The protein bands transferred to a polyvinylidene fluoride membrane were detected and quantified using the Chemi Doc™ XRS + System (Bio-Rad, Hercules, CA, USA).

Live/Dead Assay and Immunofluorescence Analysis
HaCaT cells treated with PM 10 and tart cherry were labeled with a live/dead cell assay kit (Abcam, Cambridge, MA, USA) according to the manufacturer's protocol. Live cells with esterase activity (green) and dead cells compromising plasma membranes (red) were directly counted per random microscopic field and the numbers were converted to a percentage by multiplying by 100. Ten random fields per coverslip were counted. Nuclear translocalization of NF-κB was determined by immunofluorescence staining as previously described [12]. Cells incubated with phospho-NF-κB antibody were labeled with goat anti-rabbit IgG/IgM (H + L) (Invitrogen Co., Carlsbad, CA, USA) and counterstained with 4 ,6-diamidino-2-phenylindole (DAPI) in 5% normal goat serum. The immunofluorescence signals were detected using an Olympus FluoView™ 300 confocal microscope (Center Valley, PA, USA) with 400× objective.

Cell Viability Assay
The viability of HaCaT cells treated with PM 10 and tart cherry was determined by using the EZ-CYTOX kit (Dail-Lab Service, Seoul, Korea) as previously described [12]. Cells were treated with EZ-CYTOX master mix for 30 min. Cell viability was directly analyzed using a microplate reader (SPARK, Seestrasse, Männedorf, Switzerland) at 450 nm.

Annexin V Apoptosis Assay
The apoptosis of HaCaT cells was determined by using an Annexin V-fluorescein isothiocyanate (FITC) kit (Invitrogen, Carlsbad, CA, USA) to detect phosphatidylserine on the exterior surface of the cellular membrane as previously described [13]. The proportion of healthy, early apoptotic, late apoptotic, and necrotic cells was analyzed using the NucleoCounter ® advanced image cytometer (ChemoMetec, Gydevang, Allerod, Denmark) and NucleoView NC-3000 software (ChemoMetec, Gydevang, Allerod, Denmark, version 1.4.).

Statistical Analysis
Data are expressed as mean value± standard errors (S.E.). Statistical significance was determined by ANOVA, followed, in some cases, by a comparison of treatment means with a control using the Bonferroni-Dunn test in SPSS 16 software (IBM Corp, Armonk, NY, USA). p < 0.05 is considered significant.

Tart Cherry Inhibits Keratinocyte Apoptosis Induced by PM 10
To confirm that fine particulate matter 10 (PM 10 ), which has an aerodynamic diameter of less than 10 µm, induces cytotoxicity in human epidermal keratinocytes, HaCaT cells were treated with PM 10 at various concentrations (0-200 µg/mL) for 24 h. PM 10 significantly induced cytotoxicity in HaCaT cells at concentrations ranging from 100 to 200 µg/mL compared to untreated cells ( Figure 1A). A decrease in cell viability was observed after incubation with 100 µg/mL of PM 10 for 24 h ( Figure 1B). To investigate the protective effect of tart cherry on the cytotoxicity of fine dust, cells were treated with 100 µg/mL of PM 10 for 24 h prior to exposure to tart cherry at various concentrations (50-200 µg/mL) for 30 min. A pretreatment of 200 µg/mL of tart cherry significantly reversed the reduced cell viability caused by PM 10 ( Figure 1C). We also determined the functional role of tart cherry to block cell death induced by PM 10 by means of a LIVE/DEAD viability/cytotoxicity assay for simultaneous determination of live and dead cells over a period of 24 h ( Figure 1D). PM 10 significantly induced a number of dead cells, whereas for the live cells, a significant cytotoxic effect was noted. However, tart cherry significantly prevents cytotoxicity induced Antioxidants 2021, 10, 443 5 of 13 by PM 10 . These results indicate that the protective effect of tart cherry against fine dust exposure is related to the blocking of the death of keratinocytes caused by PM 10 . effect of tart cherry on the cytotoxicity of fine dust, cells were treated with 100 μg/mL of PM10 for 24 h prior to exposure to tart cherry at various concentrations (50-200 μg/mL) for 30 min. A pretreatment of 200 μg/mL of tart cherry significantly reversed the reduced cell viability caused by PM10 ( Figure 1C). We also determined the functional role of tart cherry to block cell death induced by PM10 by means of a LIVE/DEAD viability/cytotoxicity assay for simultaneous determination of live and dead cells over a period of 24 h ( Figure 1D). PM10 significantly induced a number of dead cells, whereas for the live cells, a significant cytotoxic effect was noted. However, tart cherry significantly prevents cytotoxicity induced by PM10. These results indicate that the protective effect of tart cherry against fine dust exposure is related to the blocking of the death of keratinocytes caused by PM10.

Antioxidative Effect of Tart Cherry Containing Chlorogenic Acid, Quercetin, and Kaempferol in HaCaT Cells Treated with PM10
Fine dust has been shown to facilitate the production of reactive oxygen species (ROS), which results in the prominent amplification of apoptotic signals. A significant increase in the ROS level appeared between 5 and 30 min after incubation with 100 μg/mL of PM10 (Figure 2A), which was blocked by tart cherry treatment ( Figure 2B). The inhibitory effects of tart cherry on ROS production were further visualized by staining HaCaT cells with a fluorescent dye, 2′,7′-dichlorofluorescein diacetate (CM-H2DCFDA) (Figure

Antioxidative Effect of Tart Cherry Containing Chlorogenic Acid, Quercetin, and Kaempferol in HaCaT Cells Treated with PM 10
Fine dust has been shown to facilitate the production of reactive oxygen species (ROS), which results in the prominent amplification of apoptotic signals. A significant increase in the ROS level appeared between 5 and 30 min after incubation with 100 µg/mL of PM 10 ( Figure 2A), which was blocked by tart cherry treatment ( Figure 2B). The inhibitory effects of tart cherry on ROS production were further visualized by staining HaCaT cells with a fluorescent dye, 2 ,7 -dichlorofluorescein diacetate (CM-H 2 DCFDA) ( Figure 2C). Tart cherry extracts have substantial amounts of flavonols and non-flavonoid polyphenolic compounds responsible for their antioxidant properties. To know the underlying cause of the antioxidant effects, we further investigated the contents of chlorogenic acid as a non-flavonoid polyphenol and quercetin and kaempferol as flavonols in tart cherry by mean of a UPLC analysis. The resulting chromatograms are shown in Figure 2. The concentrations of chlorogenic acid, quercetin, and kaempferol in tart cherry as determined by the calibration curves of standard compounds were 20.90 ± 0.18 mg/L at area 27,304 ± 117.5 mV × sec and height 4446 ± 19.1 mm, 8.20 ± 0.74 mg/L at area 37,875 ± 1708.5 mV × sec and height 5906 ± 266.4 mm, and 4.57.3 ± 0.12 mg/L at area 21,373 ± 280 mV × sec and height 3406 ± 44.7 mm, respectively (Table 2 and Figure 2D,E). The validation method confirmed the stability and reliability of the results, showing the consecutive separation of the three major compounds in tart cherry. On the other hand, the decrease in cell viability that occurred due to PM 10 was significantly restored by treatment with N-acetylcysteine (NAC) as an antioxidant ( Figure 2F). These results demonstrate that the pharmacological effect of tart cherry containing chlorogenic acid, quercetin, and kaempferol on cytotoxicity in human epidermal keratinocytes is mediated by its antioxidative potential against PM 10 . of the three major compounds in tart cherry. On the other hand, the decrease in cell viability that occurred due to PM10 was significantly restored by treatment with N-acetylcysteine (NAC) as an antioxidant ( Figure 2F). These results demonstrate that the pharmacological effect of tart cherry containing chlorogenic acid, quercetin, and kaempferol on cytotoxicity in human epidermal keratinocytes is mediated by its antioxidative potential against PM10.    PM 10 induced the phosphorylation of p38 MAPK and ERK as interesting downstream candidates for ROS production but did not affect the activation of JNK ( Figure 3A), and its effect at 30 min was inhibited by treatment with tart cherry (Figure 3B), as was ROS scavenging after treatment with an antioxidant, N-acetylcysteine (NAC) ( Figure 3C). The blockage of ERK and p38 MAPK upon treatment with PD98059 and SB203580 also significantly restored cell viability affected by PM 10 ( Figure 3D). These data indicate that the phosphorylation of ERK and p38 MAPK triggered by ROS is the critical step in the cell death signaling pathway initiated by PM 10 and that the fine dust signaling pathway can be significantly blocked by treatment with tart cherry. its effect at 30 min was inhibited by treatment with tart cherry (Figure 3B), as was ROS scavenging after treatment with an antioxidant, N-acetylcysteine (NAC) ( Figure 3C). The blockage of ERK and p38 MAPK upon treatment with PD98059 and SB203580 also significantly restored cell viability affected by PM10 ( Figure 3D). These data indicate that the phosphorylation of ERK and p38 MAPK triggered by ROS is the critical step in the cell death signaling pathway initiated by PM10 and that the fine dust signaling pathway can be significantly blocked by treatment with tart cherry.

Tart Cherry Regulates the Activation of NF-κB Responsible for the Keratinocyte Apoptosis Caused by PM10
We subsequently examined the effect of tart cherry on the activation of NF-κB mediated by the phosphorylation/degradation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα) responsible for the transcriptional activation of apoptosis-related genes in keratinocytes treated with fine dust. Significant increases in the phosphorylation outcomes of IκBα and NF-κB were revealed from 30 min after treatment with 100 μg/mL of PM10 ( Figure 4A), though the increase could be suppressed by 200 μg/mL of tart cherry ( Figure 4B), the ERK inhibitor PD98059 (Figure 4C), and by the p38 MAPK inhibitor SB203580 ( Figure 4D). The regulatory effect of tart cherry on the nucleic translocation of p-NF-κB induced by PM10 was also assessed by immunofluorescence staining ( Figure 4E). The decrease in cell viability that occurred due to PM10 was significantly restored after incubation with the NF-κB inhibitor Bay 11-7082 ( Figure  4F). Thus, these data demonstrate that tart cherry negatively regulates the activation of

Tart Cherry Regulates the Activation of NF-κB Responsible for the Keratinocyte Apoptosis Caused by PM 10
We subsequently examined the effect of tart cherry on the activation of NF-κB mediated by the phosphorylation/degradation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα) responsible for the transcriptional activation of apoptosis-related genes in keratinocytes treated with fine dust. Significant increases in the phosphorylation outcomes of IκBα and NF-κB were revealed from 30 min after treatment with 100 µg/mL of PM 10 ( Figure 4A), though the increase could be suppressed by 200 µg/mL of tart cherry ( Figure 4B), the ERK inhibitor PD98059 ( Figure 4C), and by the p38 MAPK inhibitor SB203580 ( Figure 4D). The regulatory effect of tart cherry on the nucleic translocation of p-NF-κB induced by PM 10 was also assessed by immunofluorescence staining ( Figure 4E). The decrease in cell viability that occurred due to PM 10 was significantly restored after incubation with the NF-κB inhibitor Bay 11-7082 ( Figure 4F). Thus, these data demonstrate that tart cherry negatively regulates the activation of NF-κB potentiated by ERK and p38 MAPK, which is necessary for the apoptotic pathway triggered by PM 10 during the promotion of keratinocytes' cell death. NF-κB potentiated by ERK and p38 MAPK, which is necessary for the apoptotic pathway triggered by PM10 during the promotion of keratinocytes' cell death.

The Pharmacological Effect of Tart Cherry on Keratinocytes Apoptosis Induced by PM10
Having demonstrated the necessity of NF-κB in the regulation of keratinocyte apoptosis by PM10, we questioned how activated NF-κB actually coordinates with apoptosisrelated proteins. PM10 augmented the expression of the mitochondrial pro-apoptotic regulator Bax but decreased the expression of Bcl-2 which has the anti-apoptotic function ( Figure 5A), indicating that PM10 evokes apoptotic cell death mediated by the mitochondrial process. Moreover, PM10 also augmented the cleavage of caspase-3, known as an executioner caspase in apoptosis ( Figure 5A). However, the increased level of the apoptosisrelated proteins elicited by PM10 was significantly abrogated by treatment with tart cherry ( Figure 5B) and NF-κB inhibitor ( Figure 5C). To confirm the pharmacological effect of tart cherry in preventing the activation of the keratinocyte apoptotic signaling pathway, we subsequently undertook flow cytometric analyses over a period of 24 h by means of an Annexin V/Propidium Iodide (PI) assay for an accurate assessment of cell death outcomes ( Figure 5D). PM10 significantly induced keratinocyte apoptosis, whereas for necrotic cell death, a marginal effect was noted. However, tart cherry significantly prevented apoptotic cell death induced by PM10. These results indicate that the protective effect of tart cherry on cytotoxicity in human epidermal keratinocytes is related to the blocking of apoptosis caused by PM10.

The Pharmacological Effect of Tart Cherry on Keratinocytes Apoptosis Induced by PM 10
Having demonstrated the necessity of NF-κB in the regulation of keratinocyte apoptosis by PM 10 , we questioned how activated NF-κB actually coordinates with apoptosisrelated proteins. PM 10 augmented the expression of the mitochondrial pro-apoptotic regulator Bax but decreased the expression of Bcl-2 which has the anti-apoptotic function ( Figure 5A), indicating that PM 10 evokes apoptotic cell death mediated by the mitochondrial process. Moreover, PM 10 also augmented the cleavage of caspase-3, known as an executioner caspase in apoptosis ( Figure 5A). However, the increased level of the apoptosisrelated proteins elicited by PM 10 was significantly abrogated by treatment with tart cherry ( Figure 5B) and NF-κB inhibitor ( Figure 5C). To confirm the pharmacological effect of tart cherry in preventing the activation of the keratinocyte apoptotic signaling pathway, we subsequently undertook flow cytometric analyses over a period of 24 h by means of an Annexin V/Propidium Iodide (PI) assay for an accurate assessment of cell death outcomes ( Figure 5D). PM 10 significantly induced keratinocyte apoptosis, whereas for necrotic cell death, a marginal effect was noted. However, tart cherry significantly prevented apoptotic cell death induced by PM 10 . These results indicate that the protective effect of tart cherry on cytotoxicity in human epidermal keratinocytes is related to the blocking of apoptosis caused by PM 10 . xidants 2021, 10, 443 9 of 13

Discussion
In this study, we demonstrated that tart cherry can block the apoptotic signaling pathways triggered by fine particulate matter (PM) through the inhibition of MAPKs/NF-κB, a process that occurs due to ROS production in human epidermal keratinocytes (Figure 5E). Despite the wealth of evidence indicating that tart cherry has diverse biological/pharmacological effects, the anti-apoptotic mechanism of tart cherry against stressful stimuli induced by PM10 remained less well understood. PM10 is a critical air pollutant that may promote apoptotic cell death through the destruction of cellular organelles such as endoplasmic reticulum (ER), mitochondria, and lysosomes [1]. The destruction of mitochondria in skin cells causes significant pathogenesis or the progression of various diseases via ROS production, resulting in mitochondrial dysfunction and, often, in cell death [1]. In the present study, we found that PM10 amplifies keratinocyte apoptotic signals by producing ROS. These results are also supported by our previous findings showing that

Discussion
In this study, we demonstrated that tart cherry can block the apoptotic signaling pathways triggered by fine particulate matter (PM) through the inhibition of MAPKs/NF-κB, a process that occurs due to ROS production in human epidermal keratinocytes ( Figure 5E). Despite the wealth of evidence indicating that tart cherry has diverse biological/pharmacological effects, the anti-apoptotic mechanism of tart cherry against stressful stimuli induced by PM 10 remained less well understood. PM 10 is a critical air pollutant that may promote apoptotic cell death through the destruction of cellular organelles such as endoplasmic reticulum (ER), mitochondria, and lysosomes [1]. The destruction of mitochondria in skin cells causes significant pathogenesis or the progression of various diseases via ROS production, resulting in mitochondrial dysfunction and, often, in cell death [1]. In the present study, we found that PM 10 amplifies keratinocyte apoptotic signals by producing ROS. These results are also supported by our previous findings showing that fine dust together with phthalates, a ubiquitous environmental contaminant, significantly induces programmed cell death and inflammation of epidermal keratinocytes through ROS production [14,15]. Given that keratinocytes in the skin basal layer are in close contact with extracellular matrix (ECM) components and considering the associated growth factors through integrins and receptors [16], it is important to find pharmacological substances that regulate keratinocyte apoptosis as induced by PM 10 , leading to the degradation of macromolecules in the ECM and, thus, premature skin aging [6] and inflammatory diseases [7]. Although the efficacy of tart cherry or its constituents against PM 10 in epidermal keratinocyte cells has not been reported, many scientists insist that tart cherry contains various pharmacological substances, including polyphenol-rich contents, with numerous beneficial biological activities in the skin. In the present study, we have proven that tart cherry, which contains chlorogenic acid, quercetin, and kaempferol, has the ability to inhibit the oxidative keratinocyte apoptosis induced by PM 10 . Although identification of all of the chromatograms detected by UPLC-PDA remains a major challenge to us in our further research, tart cherry extract might be included in many functional phytochemicals related to phenolics, anthocyanins, flavonols, phenolic acids (non-flavonoid polyphenolic compounds), carotenoids, and other compounds as previously reported [17]. These data are supported by previous results showing that ROS production and cytotoxicity caused by airborne particles are attenuated by treatment with epigallocatechin gallate, a phenolic antioxidant found in a number of plants such as green and black teas [18]. These findings also, therefore, suggest that polyphenol-rich tart cherries are potential antioxidant prophylactic agents that can prevent oxidative stress-related diseases that affect human epidermal keratinocytes when they are exposed to fine dust.
Mitogen-activated protein kinases (MAPKs) involving ERKs, p38 MAPK, and JNKs are interesting candidates as downstream mediators of ROS. Given that ROS constitute the major regulators of apoptosis via regulation of MAPK signaling pathways [19], our data demonstrate that tart cherry abrogates the MAPK phosphorylation elicited by ROS to suppress the apoptotic signaling pathway mediated by the mitochondria in PM 10 -treated HaCaT cells. Our findings are supported further by the authors who revealed that MAPK is critical for the cell viability triggered by intracellular free radicals during exposure to air pollutants such as benzo[a]pyrene, cadmium, and acrolein [20][21][22]. Thus, our results suggest that the functional effect of tart cherry on the phosphorylation of ERK and p38 MAPK plays a key role in blocking the keratinocyte apoptotic signaling pathway elicited by PM 10 . Indeed, it was reported that PM exposure results in the phosphorylation of ERK and p38 MAPK in bronchial epithelial cells [23]. Hence, ERK and p38 MAPK inactivation by food phytochemicals may effectively prevent cell death as caused by various air pollutants, suggesting that ERK and p38 MAPK are major modulators and potential targets for the keratinocyte cytotoxicity caused by fine dust. These results collectively demonstrate that tart cherry has the ability to protect against apoptotic cell death by blocking the phosphorylation of the ERK and p38 MAPK pathways induced by PM 10 .
Next, we tried to understand the mechanism by which ROS and the ERK/p38MAPK pathway are associated with other molecular events closely linked to keratinocyte apoptosis induced by PM 10 . NF-κB, a redox-sensitive transcription factor, plays an important role in the gene transcription of the apoptotic signaling pathway in many epithelial cells when during exposure to various environmental contaminants, including fluoride, insecticides, and PM [24][25][26]. Indeed, it has been reported that NF-κB transcriptionally mediates the expression levels of many pro-inflammatory regulators and apoptotic genes in skin inflammation and damage [27]. The present results indicate that PM 10 induces the phosphorylation of NF-κB as mediated by IκBα degradation during the promotion of keratinocyte apoptosis. In contrast, we found that tart cherry has an inhibitory effect on NF-κB mediated by ERK and p38 MAPK, which is necessary for the signaling events triggered by PM 10 during the augmentation of apoptosis. Regarding the role of ERK and p38 MAPK on the phosphorylation of NF-κB, it was previously reported that the ERK and p38 MAPK pathways stimulated by ROS potentiate the transcriptional activities of NF-κB to enhance apoptotic genes' expression [28,29]. Hence, these results imply that ROS initiated by PM 10 have a significant role in enhancing the NF-κB pathway via the activation of ERK and p38 MAPK. Together, these observations demonstrate that ROS induced by PM 10 can activate ERK, p38 MAPK, and NF-κB, whereas tart cherry blocks this activation of apoptosis-related signaling events in human epidermal keratinocytes.
Finally, we have shown that tart cherry significantly restores the levels of Bax and Bcl-2 affected by PM 10 . Having shown that fine dust induces oxidative stress and, thus, stimulates apoptotic cell death mediated by mitochondria via Bax oligomerization, Bax has been proven as a major determining factor for apoptotic susceptibility [30][31][32]. Moreover, earlier work indicated that increased NF-κB activity induces the transcription of many apoptotic genes, including Bax and Bcl-2, in response to butyric acid [33]. The mitochondrial translocation of Bax induces oligomer formation and mitochondrial membrane permeabilization, augmenting the mitochondrial cytochrome c release and caspase-9 activation that are required for caspase-3 activation [34,35]. On the other hand, Bcl-2 is localized to the mitochondrial outer membrane, where it plays a functional role in enhancing the survival and abrogating the activation of pro-apoptotic proteins. Consistently, we showed that tart cherry normalizes the levels of Bax and Bcl-2 triggered by PM 10 , representing unique downstream events of NF-κB activation in the mitochondrial apoptotic pathway related to mitochondrial ROS and caspase-3 activation.

Conclusions
The results of this study imply that tart cherry has the unique function of blocking the cellular mechanism caused by PM 10 , thus preventing the activation of the mitochondrial apoptotic pathway in human epidermal keratinocytes. These findings, therefore, highlight the relevance of a novel action of polyphenol-rich tart cherries in preventing the skin oxidative stress that leads to senescence and inflammatory diseases caused by FD. Moreover, our results provide an important insight into the potential for the development of therapeutic strategies and agents for improving the skin barrier functions injured by the natural and the anthropogenic dust sources. Data Availability Statement: Data available on request due to restrictions eg privacy or ethical. The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.