Rosmarinic Acid Protects Skin Keratinocytes from Particulate Matter 2.5-Induced Apoptosis

Background: The exposure of the human skin to particulate matter 2.5 (PM2.5) results in adverse health outcomes, such as skin aging, wrinkle formation, pigment spots, and atopic dermatitis. It has previously been shown that rosmarinic acid (RA) can protect keratinocytes from ultraviolet B radiation by enhancing cellular antioxidant systems and reducing oxidative damage; however, its protective action against the adverse effects of PM2.5 on skin cells remains unclear. Therefore, in this study, we explored the mechanism underlying the protective effects of RA against PM2.5-mediated oxidative stress in HaCaT keratinocytes. Methods: HaCaT keratinocytes were pretreated with RA and exposed to PM2.5. Thereafter, reactive oxygen species (ROS) production, protein carbonylation, lipid peroxidation, DNA damage, and cellular apoptosis were investigated using various methods, including confocal microscopy, western blot analysis, and flow cytometry. Results: RA significantly inhibited PM2.5-induced lipid peroxidation, protein carbonylation, DNA damage, increases in intracellular Ca2+ level, and mitochondrial depolarization. It also significantly attenuated PM2.5-induced apoptosis by downregulating Bcl-2-associated X, cleaved caspase-9, and cleaved caspase-3 protein levels, while upregulating B-cell lymphoma 2 protein level. Further, our results indicated that PM2.5-induced apoptosis was associated with the activation of the mitogen-activated protein kinase (MAPK) signaling pathway and that MAPK inhibitors as well as RA exhibited protective effects against PM2.5-induced apoptosis. Conclusion: RA protected HaCaT cells from PM2.5-induced apoptosis by lowering oxidative stress.


Introduction
Globally, urban air pollution is a serious threat to public health.Fine particulate matter with aerodynamic diameter < 2.5 μm (PM 2.5 ) is a constituent of airborne particulate matter that is primarily derived from industrial soot [1].The components of PM 2.5 differ depending on its source, and PM 2.5 emitted by diesel exhaust predominantly contains polycyclic aromatic hydrocarbons (PAHs), black carbon, and hydrocarbons (C 14 -C 35 ) and their derivatives.PAHs in PM 2.5 have a high mutagenic potential and can easily penetrate the skin via the appendageal route and the stratum corneum, thereby causing PM 2.5 -induced skin injury [2,3].Numerous studies have demonstrated that PM 2.5 exposure increases the risk of cardiovascular and respiratory damage as well as neurotoxicity [4,5].Further, PM 2.5 has been linked to various skin disorders, including acne, atopic dermatitis, and skin aging [6,7].It has also been shown that exposure to PM 2.5 induces reactive oxygen species (ROS) generation in keratinocytes, and this excessive intracellular ROS generation induces cell damage owing to oxidative stress [3,8], which can Ivyspring International Publisher considerably harm nucleic acids, proteins, lipids, cell membranes, and organelles, such as the mitochondria, and even induce apoptosis [9].
Most phenolic compounds can function as antioxidants or free-radical scavengers.Specifically, rosmarinic acid (RA), a naturally occurring hydroxylated polyphenolic compound that is commonly found in Rosmarinus officinalis, exhibits antimicrobial, anti-inflammatory, antioxidative, antiapoptotic, and antitumor activities [10][11][12].Our previous study demonstrated that it exerts cytoprotective effects against ultraviolet B radiation by modulating cellular antioxidant systems in keratinocytes and ameliorating oxidative damage [11,12].However, the cytoprotective action of RA against PM 2.5 remains unclear.Therefore, in this study, we aimed to explore the cytoprotective effect of RA on PM 2.5 -induced skin damage.

Preparation of RA and PM 2.5
Stock solutions of RA and PM 2.5 were prepared using DMSO.Specifically, the prepared PM 2.5 stock solution (25 mg/mL) was subjected to sonication for 30 min to prevent the particles from clustering [13].

Cell culture
The human skin HaCaT cell line was procured from Cell Lines Service (Eppelheim, Germany).The cells were cultured according to standard procedures at 37 °C in an incubator with 5% CO 2 and full humidity.Further, the culture medium, DMEM, was supplemented with 10% heat-inactivated FBS and an antibiotic-antimycotic mix [14].

MTT assay
The cells were incubated with MAPK pathway-targeted chemical inhibitors (50 nM U0126; 10 µM SB203580; and 5 µM SP600125) with or without 2.5 μM RA for 30 min and then exposed to 50 μg/mL PM 2.5 for 24 h at 37 °C.Thereafter, MTT solution was added to each well after which incubation was further performed at 37 °C for 4 h.The purple precipitate thus obtained was dissolved by adding DMSO and cellular metabolic activity measurements were performed at 540 nm using a VersaMax ELISA microplate reader (Molecular Devices, Sunnyvale, CA, USA) [14].

Trypan blue assay
Cells were exposed to 30 μM Z-VAD-FMK with or without 2.5 μM RA for 30 min and then exposed to PM 2.5 at 50 μg/mL for 24 h.After this treatment, the cells were stained using 0.1% trypan blue solution and observed using a fluorescence microscope [15].

ROS measurement
Cells were treated with 2.5 μM RA or 1 mM NAC for 30 min and then exposed to 50 μg/mL PM 2.5 for 24 h.Then, for total ROS and superoxide anion detection, 25 μM H 2 DCFDA and 10 μM DHE, respectively, were added to the culture medium and fluorescent cells were analyzed using a flow cytometer (Becton Dickinson, Mountain View, CA, USA) [3,14].Further, cellular H 2 O 2 level was measured via the ROS-Glo™ H 2 O 2 assay (Promega, Madison, WI, USA) according to the manufacturer's protocols [16].

Protein carbonylation
Protein carbonylation level was assessed using the OxiSelect™ protein carbonyl ELISA kit (Cell Biolabs, San Diego, CA, USA) following the guidelines provided by the manufacturer [8].

Comet assay
Cells were collected on a slide, electrophoresed (25 V, 300 mA), stained with ethidium bromide, and observed under a fluorescence microscope equipped with image analysis software (Kinetic Imaging, Komet 5.5, UK).The percentage of total fluorescence in the comet tail and the length of the tail were measured in 50 cells/slide [17].

Intracellular Ca 2+ level
To detect intracellular Ca 2+ level, cells were stained with 10 μM Fluo-4 AM and fluorescence intensity measurements were performed via flow cytometry and confocal microscopy [3].

Western blot analysis
Extracted cell lysates were electrophoresed on SDS-polyacrylamide gel to separate proteins.Thereafter, the separated proteins were electroblotted to nitrocellulose membranes, which were then incubated with the appropriate primary antibodies (1:1,000), followed by incubation with a secondary antibody.Protein expression levels were then detected using an enhanced chemiluminescence plus western blot detection system (GE Healthcare Life Sciences, Buckinghamshire, UK) [14].

Hoechst 33342 staining
Cells were stained with a DNA-specific fluorescent dye, Hoechst 33342 and examined using a fluorescence microscope (Media Cybernetics, Rockville, MD, USA).Finally, the proportion of apoptotic cells was quantified [14].

Annexin V/PI staining assay
Apoptotic cells were quantified via flow cytometry using the Alexa Fluor TM 488 annexin V/dead cell apoptosis kit (Invitrogen, Thermo Fisher Scientific, Inc.) in accordance with the guidelines provided by the manufacturer [18].

RA protected skin cells by lowering PM 2.5 -induced increases in ROS levels
Reportedly, RA at 2.5 μM does not induce any form of cytotoxicity in HaCaT cells; it maintains cell viability at approximately > 95% [11].Therefore, 2.5 μM RA was used as the optimal RA concentration in this study.The exposure of HaCaT cells to PM 2.5 significantly reduced cell viability; however, treatment with RA significantly reversed the PM 2.5 -induced loss of cell viability (Figure 1A).Further, RA pretreatment significantly lowered the PM 2.5 -induced increase in the levels of ROS, including superoxide anion and hydrogen peroxide (Figures 1B-D).

RA attenuated PM 2.5 -induced macromolecular damage
Oxidative stress significantly increases intracellular macromolecular damage [19].In this study, we examined the protective properties of RA against macromolecular damage caused by PM 2.5 .The examination of PM 2.5 -induced lipid peroxidation via DPPP staining showed the significant enhancement of fluorescence intensity in the PM 2.5 -treated group; however, pretreatment with RA significantly decreased this fluorescence intensity (Figure 2A).Further, PM 2.5 -mediated oxidative stress resulted in a significant increase in protein carbonylation level, while RA pretreatment prevented this phenomenon (Figure 2B).Additionally, the examination of PM 2.5 -induced DNA damage via the comet assay showed that PM 2.5 -induced oxidative stress increased comet tail length and tail fluorescence percentage; however, these observations were reversed following RA pretreatment (Figure 2C).8-Oxoguanine, a major form of oxidative DNA damage, was examined using an avidin-conjugated TRITC reagent [20].The confocal microscopy images obtained thereafter revealed severe DNA lesions in the PM 2.5 -exposed cells; however, pretreatment with RA ameliorated this effect (Figure 2D).

RA decreased PM 2.5 -induced apoptotic cell death
Oxidative stress results in increased cytoplasmic Ca 2+ concentration, and such elevated Ca 2+ level can damage mitochondria and nuclei, leading to apoptosis [21].After labelling cells with Fluo-4 AM, we measured intracellular Ca 2+ level via flow cytometry and confocal imaging.Thus, we observed an increased fluorescence intensity for cells exposed to PM 2.5 , indicating increased Ca 2+ level; however, pretreatment with RA resulted in a decrease in Ca 2+ level, as depicted in Figure 3A and B. Mitochondria act as apoptosis-regulating centers.PM 2.5 -induced excessive ROS generation can induce mitochondrial oxidative damage, and reportedly, severe mitochondrial damage is associated with mitochondrial malfunction and apoptosis [22].Our results showed that PM2.5 enhanced mitochondrial depolarization, which was reversed by pretreatment with RA (Figure 3C).Previous studies have shown that PM 2.5 enhances keratinocyte apoptosis by activating the caspase signaling pathway [15,23].As shown in Figure 3D, PM 2.5 exposure upregulated Bax, cleaved caspase-9, cleaved caspase-3, and cleaved PARP protein levels, while pretreatment with RA downregulated these proteins.In contrast, the expression level of antiapoptotic protein, Bcl-2 was decreased following PM2.5 exposure; however, it was enhanced owing to RA pretreatment.To verify the effect of caspase activation on apoptosis, HaCaT cells were pretreated with a caspase inhibitor (Z-VAD-FMK) with or without RA, both of which are associated with decreases in the amounts of apoptotic bodies (Figure 3E).As was observed for Hoechst 33342-staining, the percentage of annexin V-stained cells in the PM2.5 group increased and this effect was ameliorated by Z-VAD-FMK or/with RA pretreatment (Figure 3F).Trypan blue staining further revealed that Z-VAD-FMK and RA pretreatment ameliorated PM 2.5 -induced decreases in cell viability (Figure 3G).

RA downregulated the PM 2.5 -activated MAPK signaling pathway
MAPK signaling pathways regulate various biological processes, such as apoptosis [24].To further investigate the effect of PM 2.5 on MAPK signaling pathway-mediated apoptosis, MAPK signaling cascade-associated proteins, ERK, p38, and JNK were detected via western blot analysis (Figure 4A).Thus, we observed that PM 2.5 elevated the levels of phospho-ERK, phospho-p38, and phospho-JNK in comparison with the control treatment.However, RA pretreatment reversed this effect.Cells pretreated with each ERK, p38, and JNK inhibitor (U0126, SB203580, and SP600125) exhibited decreased PM2.5-induced cytotoxicity and apoptosis, similar to cells pretreated with RA (Figure 4B-D).These results demonstrated that RA ameliorates PM 2.5 -induced apoptosis by downregulating the PM 2.5 -induced MAPK signaling pathway.

Discussion
Skin keratinocytes constitute the primary barrier against environmental stressors and exposure of keratinocytes to PM 2.5 could lead to increased ROS production [25].Recent studies have demonstrated that PM 2.5 may penetrate the skin and directly affect viable skin cells, including keratinocytes [9,25].Therefore, in this study, we aimed to explore the protective effects of RA against PM 2.5 -induced damage in HaCaT cells.Our previous study showed that at concentrations up to 2.5 μM, RA does not exert any cytotoxic effects on HaCaT and that at 2.5 μM, RA scavenges up to 60% of intracellular ROS [11].Thus, in this study, we used 2.5 μM as the optimal RA concentration.Further, based on our previous study, which showed that the optimal PM 2.5 exposure concentration required to induce excessive ROS generation in HaCaT cells is 50 μg/mL [10].In this study, we observed that RA significantly reversed PM 2.5 -induced decreases in the viability of HaCaT cells.RA also significantly reduced PM 2.5 -induced increases in ROS levels.PM 2.5 -induced excessive ROS generation can trigger oxidative macromolecular damage, leading to apoptosis [26].In the lipid peroxidation process, oxidants, such as free radicals or ROS attack several carbon-carbon double bonds in polyunsaturated fatty acids, initiating the removal of hydrogen from carbon to produce water and fatty acid radicals.The unstable fatty acid radicals thus obtained react with molecular oxygen to form lipid peroxyl radicals and hydroperoxides [27].This membrane lipid peroxidation alters the physical properties of lipid bilayers, and consequently, affects membrane permeability, lipid-lipid interactions, ion gradients, and membrane fluidity [28].Thus, lipid peroxidation negatively affects cellular functions.Additionally, oxidative stress caused by ROS overproduction results in protein carbonylation owing to protein oxidation or DNA modification [8].In this study, we observed that RA exhibited a protective effect against PM 2.5 -induced macromolecular damage by diminishing ROS.Owing to oxidative stress, Ca 2+ flows into the cytoplasm from internal cellular stores, such as the sarcoplasmic reticulum/endoplasmic reticulum and can also be imported from extracellular spaces.As the Ca 2+ concentration in the cytoplasm increases, Ca 2+ flows into the mitochondria and nuclei.The accumulation of Ca 2+ in mitochondria and nuclei as such disrupts normal cell metabolism, leading to apoptosis [21,29].In this study, we observed that RA significantly attenuated oxidative stress-mediated cellular Ca 2+ influx.Mitochondria play a major role in the onset of apoptosis.Moreover, PM 2.5 -induced oxidative stress leads to mitochondrial damage, including changes in Δψ m [15,19,30].In this study, we observed that RA regulated Δψ m and facilitated the normal functioning of mitochondria.It has been shown that mitochondrial damage is associated with Bcl-2, which maintains the integrity of the mitochondrial membrane.The loss of Δψ m induces the release of apoptotic factors into the cytosol.Further, the balance between the pro-apoptotic protein, Bax and the antiapoptotic proteins of the Bcl-2 family serves as the determining factor for the activation or inhibition of the caspase cascade.Specifically, caspase-3 initiates the intrinsic apoptotic pathway and cleaves nuclear proteins, such as PARP [31,32].It has also been shown that PM 2.5 upregulates apoptotic protein levels while downregulating the expression of antiapoptotic proteins [8,15].In this study, we observed that RA reversed these effects.
The MAPK signaling cascade plays a crucial role in mediating cellular stress response.Previous reports have shown that PM 2.5 activates MAPK pathway-associated proteins via ROS-mediated pathways [15,19].The activation of p38 and JNK in response to PM 2.5 is thought to induce apoptosis [33].In contrast, the ERK pathway plays a dual role in cell survival and death.Further, recent studies have shown that continuous ERK activation can promote apoptosis [15,34].Further exploration in this regard using ERK, p38, and JNK inhibitors revealed that RA pretreatment reduced the phosphorylation of ERK, p38, and JNK, similar to their respective inhibitors, leading to a decrease in the levels of apoptotic bodies.

Conclusion
In this study, we investigated the effects of RA on PM 2.5 -induced damage in keratinocytes.Our results revealed that PM 2.5 exacerbated skin cell damage by increasing ROS generation and activating apoptotic pathways; however, RA ameliorated these observed PM 2.5 -induced effects.Thus, it showed protective effects on skin cells against PM 2.5 -induced damage (Figure 5).

Figure 5 .
Figure 5. Summary of the protective effects of RA against PM2.5-induced skin cell damage.RA ameliorated PM2.5-induced intracellular ROS levels, hence attenuated oxidative damage to DNA, lipids, and proteins.Further, RA downregulated mitochondria-mediated caspase activation and the MAPK signaling pathway, and thus ameliorated apoptosis.