Chlorogenic Acid, a Component of Oenanthe javanica (Blume) DC., Attenuates Oxidative Damage and Prostaglandin E 2 Production Due to Particulate Matter 10 in HaCaT Keratinocytes

: Oenanthe javanica (OJ) is a perennial herb that grows wildly or is cultivated in Asia, and it is used as food or in traditional medicine. The antioxidant and anti-inﬂammatory effects of OJ-derived materials have been extensively explored previously, but their effects on the cytotoxicity of air pollution are currently unknown. Therefore, the present study aimed to evaluate the effect of the hot water extract of OJ on atmospheric particulate matter 10 (PM 10 )-induced cytotoxicity and oxidative damage in human HaCaT keratinocytes, and to identify its active ingredient and mechanism of action. When the hot water extract of OJ was divided into methylene chloride, ethyl acetate (EA), n-butanol (BA), and water fractions, caffeic acid was enriched in the EA fraction and chlorogenic acid was enriched in the BA fraction. PM 10 increased reactive oxygen species (ROS) production, lipid peroxidation, protein carbonylation, and inﬂammatory prostaglandin (PG) E 2 production in cells. The BA fraction reduced the PM 10 -induced ROS production in cells more effectively than the total extract and other solvent fractions. Chlorogenic acid was more effective in reducing ROS levels than caffeic acid and N-acetyl cysteine (NAC). Chlorogenic acid attenuated the increase in lipid peroxidation and the PG E 2 production of cells due to PM 10 exposure. Of the genes involved in PG E 2 production, phospholipase A2 group IVA ( PLA2G4A ), Prostaglandin-endoperoxide synthase 1 ( PTGS1 ), and 2 ( PTGS2 ) were transcriptionally up-regulated by PM 10 , whereas phospholipase A2 group IIA ( PLA2G2A ) was down-regulated and prostaglandin E synthetase 1 ( PTGES1 ) and 2 ( PTGES2 ) were a little altered. The PM 10 -induced increase in PLA2G4A mRNA was alleviated by chlorogenic acid and NAC. Accordingly, PM 10 increased the expression levels of cytosolic phospholipase A2 (cPLA2) protein and its phosphorylated form, which were attenuated by chlorogenic acid and NAC. Thus, chlorogenic acid may attenuate the PM 10 -induced PG E 2 production through the suppression of PLA2G4A mRNA and cPLA2 protein expressions. This study suggests that chlorogenic acid contained in OJ extract may help alleviate the oxidative damage to and inﬂammatory responses of the skin cells due to exposure to air pollutants.


Introduction
Air pollution has a detrimental effect on skin health.The particulate matter (PM) that is suspended in the atmosphere with an approximate diameter of less than 10 and 2.5 µm is called PM 10 and PM 2.5 , respectively [1].PM is a mixture of various organic compounds, heavy metals, and biological constituents [1,2].PM is capable of penetrating the skin through pores and weak skin barriers [3,4].PM can exacerbate various skin diseases, such as atopic dermatitis, acne, and psoriasis [5,6].PM can also cause premature skin aging [7] and hyperpigmentation [8].Combined exposure to PM and ultraviolet (UV) rays exerts a synergistic harmful effect, accelerating skin photo-aging and cancer development [9,10].
PM exposure stimulates Ca 2+ signaling and increases the production of reactive oxygen species (ROS), such as superoxide radical (O 2 •− ), hydrogen peroxide (H 2 O 2 ), and hydroxyl radical ( • OH), which mediate the oxidative damage to and inflammatory responses of cells [11,12].ROS can be generated in the aryl hydrocarbon receptor-mediated metabolism of organic components of PM and the chemical reactions catalyzed by its transition metal components [13,14].PM induces the activation of the NADPH oxidase family, including dual oxidase 2, which plays a critical role in the production of ROS and the maintenance of redox balance in cells [12,15].
PM-derived ROS can cause the activation of the mitogen-activated protein kinase (MAPK) family, including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 kinase, and the stimulation of the nuclear factor-kappa B (NF-κB) signaling pathway, leading to the activation of redox-sensitive transcription factors [16].This increases the gene expression and secretion of the inflammatory eicosanoid mediators, such as prostaglandin (PG) E 2 [17,18], and the inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-8 [19].PM stimulates the expression of matrix metalloproteinases (MMPs), causing the loss of the extracellular matrix, including collagen [20], and decreases the expression of filaggrin, causing the loss of the skin barrier function [21,22].Thus, there is a need for antioxidants that alleviate the adverse effects of PM.
Oenanthe javanica (Blume) DC, abbreviated to OJ herein, is generally called water dropwort, water parsley, water celery, or 'minari' in Korean.It is a dicotyledonous perennial plant that grows in wetlands or watersides and is cultivated for food or medicinal purposes [27,28].OJ contains a substantial amount of phenolic compounds, with chlorogenic acid being in the highest concentration, followed by caffeic acid [29][30][31].The extract of OJ alleviates the hepatic steatosis and oxidative damage induced by ethanol poisoning [32] via acting as an antioxidant with free radical scavenging capabilities and reducing power [29], enhancing cellular antioxidant enzymes, such as superoxide dismutase (SOD)-1, SOD-2, catalase, and glutathione peroxidase [33], or accelerating the metabolic removal of ethanol [34].Its anti-inflammatory effects have also been demonstrated in RAW 264.7 macrophages, which were stimulated by lipopolysaccharide [35,36] in the skin of mice irradiated with UV rays [37], and in mice with sodium dextran sulfate-induced colitis [31].
Although the antioxidant and anti-inflammatory effects of OJ-derived materials have been explored in various experimental models, no previous studies have reported their protective effects against the cytotoxicity of air pollution.Therefore, the present study aimed to evaluate the effect of the hot water extract of OJ on PM 10 -induced cytotoxicity and oxidative damage in human HaCaT keratinocytes, and to identify its active ingredient and mechanism of action.The results of this study demonstrated that chlorogenic acid, a main component of OJ, attenuated ROS production, lipid peroxidation, and protein carbonylation in PM 10 -stimulated cells.It also attenuated PM 10 -induced PG E 2 production in cells, which was associated with the suppressed expression of cytosolic phospholipase A2 (cPLA2).Thus, the chlorogenic acid contained in OJ extract is suggested to help alleviate the oxidative damage to and inflammatory responses of the skin cells caused by exposure to air pollutants.

Preparation of the Hot Water Extract and Fractions of OJ
Dried Leaves and stems of OJ (Oenanthe javanica (Blume) DC.) purchased from Cheongmyeongyakcho (Chungbuk, Republic of Korea) (http://www.good1075.com,accessed on 31 March 2023) were used for extraction.The plant material (100 g) was extracted with water (600 mL) at 90 • C for 2 h.After filtering, the filtrate was concentrated to dryness using a rotary evaporator (Eyela, Bohemia, NY, USA) under reduced pressure, and the total extract of OJ (3.702 g) was obtained.The total extract was suspended in water (100 mL) and transferred to a separating funnel where the aqueous suspension was partitioned with an equal volume of methylene chloride (MC), ethyl acetate (EA), and n-butyl alcohol (BA), sequentially [34].The evaporation of each solvent fraction under reduced pressure yielded an MC fraction (0.168 g), EA fraction (0.181 g), BA fraction (0.555 g), water (WT) fraction (13.781 g), and insoluble materials (8.096 g).Each fraction was re-dissolved in 30% (v/v) aqueous ethanol at a 10% concentration and kept at −20 • C until use.

High-Performance Liquid Chromatography with Photodiode Array Detection (HPLC-DAD)
The HPLC-DAD profiles of the total extract of OJ and its solvent fractions were compared using a Waters Alliance HPLC system (Waters, Milford, MA, USA) equipped with an e2695 separation module and a 2996 photodiode array detector [30].Chromatographic separation was performed using a Hector-M C 18 column (4.6 mm × 250 mm, 5 µm) (RS Tech Co. Daejeon, Republic of Korea) as the stationary phase, and a mixture of 0.1% phosphoric acid (solvent A) and acetonitrile (solvent B) as the mobile phase.The mobile phase composition was changed as follows: 0-30 min, a linear gradient from 0 to 100% B; 30-40 min, 100% B; 40-45 min, a linear gradient from 100 to 0% B. The flow rate of the mobile phase was set at 0.6 mL min −1 .Samples were diluted with water, filtered, and injected into HPLC in 10 µL aliquots.

Cell Culture and Treatments
An immortalized human keratinocyte HaCaT cell line (CLS Cell Lines Service GmbH, Eppelheim, Germany) was cultured as previously described [26].The growth medium consisted of DMEM/F-12 medium (GIBCO-BRL, Grand Island, NY, USA), 10% fetal bovine serum, antibiotics (100 U mL −1 penicillin, 100 µg mL −1 streptomycin, 0.25 µg mL −1 amphotericin B), and 10 µg mL −1 of hydrocortisone.In each experiment, cells were cultured in 96-well, 12-well, or 6-well culture plates (SPL Life Sciences, Pocheon, Republic of Korea) for 24 h and then subjected to various treatments.PM 10 was suspended in phosphatebuffered saline (PBS) at 100 times the final treatment concentrations.Other test materials were diluted or dissolved in 30% (v/v) aqueous ethanol at 100 times the final treatment concentrations.Cells were treated with the total extract of OJ, its fractions, compounds, and PM 10 individually, in combination with each other at the specified concentrations, or with vehicles for the indicated time.

Cell Viability Assay
Cell viability was assessed by measuring the reduction of MTT to water-insoluble formazan in the viable cells [38,39].Cells were seeded on 96-well culture plates (4 × 10 3 cells/well) and cultured in a growth medium (200 µL) for 24 h.After various treatments with the total extract of OJ, its fractions, compounds, and PM 10 for 48 h, the cells were washed with PBS and incubated in 100 µL of growth medium containing 1 mg mL −1 of MTT at 37 • C for 2 h.After discarding the medium, cells were washed with PBS, and the formazan dye that had accumulated inside the cells was extracted with 100 µL of dimethyl sulfoxide per well.Then, the absorbance was measured at 570 nm using a Spectrostar Nano microplate reader (BMG LABTECH GmbH, Ortenberg, Germany).

Cellular Reactive Oxygen Species (ROS) Production Assay
Cellular ROS production was assessed by measuring the oxidation of DCFH-DA to a fluorescent compound inside the cells [40].The cells were seeded on 12-well culture plates (1.4 × 10 5 cells/well) and cultured in a growth medium (1 mL) for 24 h.Cells were treated with 10 µM DCFH-DA for 30 min.The spent medium was replaced with the growth medium containing various test materials, which was followed by the incubation of cells for 60 min.Cells were then washed with PBS and the fluorescing cells were observed under a LEICA DMI3000 B microscope (Leica Microsystems GmbH, Wetzlar, Germany).For a quantitative analysis of the fluorescing compound formed inside the cells, the washed cells were extracted with 150 µL of the cell lysis buffer (1% sodium dodecyl sulfate, 20 mM of Tris-Cl, 2.5 mM of EDTA, pH of 7.5), followed by centrifugation at 14,500× g for 15 min.After 200 µL aliquots of the supernatants were transferred to black 96-well plates (SPL Life Sciences, Pocheon, Republic of Korea), the fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 538 nm with a Gemini EM fluorescence microplate reader (Molecular Devices, Sunnyvale, CA, USA).

Cellular Lipid Peroxidation Assay
Cellular lipid peroxidation was assessed by measuring the production of malondialdehyde (MDA) [41].Cells were seeded on 6-well culture plates (2 × 10 5 cells per well) and cultured in the growth medium (2 mL) for 24 h.Cells were then treated with various test materials, such as the total extract of OJ, its fractions, compounds, and PM 10, for 48 h.Then, cells were washed with PBS and harvested using 150 µL of the cell lysis buffer (1% sodium dodecyl sulfate, 20 mM of Tris-Cl, 2.5 mM of EDTA, pH of 7.5).The reaction mixture (500 µL) consisting of 100 µL of cell lysate (ca.200 µg protein), 50 µL of 1.0% m-phosphoric acid, and 350 µL of 0.9% 2-thiobarbituric acid was heated at 95 • C in a water bath for 45 min.The reaction was also run with 100 to 400 nM of TMP as a donor of MDA to construct a standard curve.After the reaction mixture was cooled to room temperature, 500 µL of BA was added to the reaction mixture, mixed vigorously, and centrifuged at 14,500× g for 15 min to separate the mixture into a BA layer and an aqueous layer.After 200 µL of the BA layer was transferred to black 96-well plates, the fluorescence intensity was measured at an excitation wavelength of 544 nm and an emission wavelength of 590 nm using a Gemini EM fluorescence microplate reader.Data are presented as MDA levels normalized to protein content.

Protein Carbonylation Assay
Protein carbonylation was measured using a fluorometric assay kit (ab235631) from Abcam (Cambridge, MA, USA).The whole cell lysate (75 µg of protein in 50 µL) was mixed with 50 µL of 0.2 mM fluorescein-5-thiosemicarbazide (FTC) fluorophore in an assay buffer in microcentrifuge tubes, followed by incubation overnight at 25 • C in the dark.Then, proteins were precipitated by adding 200 µL of ice-cold 20% trichloroacetic acid solution, keeping the tubes on ice for 10 min.The tubes were centrifuged at 14,500× g for 15 min and the supernatant was removed by suction aspiration.The pellet was washed with 200 µL of ice-cold isopropanol 3 times and then dissolved in 50 µL of 6 M guanidine solution by heating it at 50 • C for 1 h.The tubes were cooled to 50 • C and the samples were diluted with 70 µL of sample dilution buffer.Aliquots of the diluted samples (100 µL) were transferred to a 96-well plate and the fluorescence was measured at a 485 nm excitation and at a 535 nm emission using a Gemini EM fluorescence microplate reader.Protein carbonyl contents were estimated by comparing a standard curve that was prepared using FTC fluorophore.The protein carbonyl contents were normalized to the protein content.
2.9.Enzyme-Linked Immunosorbent Assay (ELISA) for PG E 2 The secreted level of PG E 2 protein in the culture medium was measured using a PG E 2 ELISA kit (Enzo Life Sciences, Inc., Farmingdale, NY, USA).In this colorimetric competitive enzyme immunoassay, a fixed amount of PG E 2 -phosphatase conjugate is used as a PG E 2 tracer, whose binding to the PG E 2 monoclonal antibody is inversely proportional to the amount of PG E 2 derived from the sample.Cells were seeded on 6-well culture plates (8 × 10 4 cells per well) and cultured in a growth medium (1 mL) for 24 h.Cells were then treated with various test materials for another 48 h.The conditioned culture medium was centrifuged at 14,500× g for 15 min, and the supernatants were used as samples in the assay.Briefly, 100 µL samples of the culture medium or a standard PG E 2 solution were transferred to microtiter plates coated with a goat antibody specific to mouse IgG.To each well, 50 µL of assay buffer, 50 µL of PG E 2 tracer solution, and 50 µL of PG E 2 monoclonal antibody solution were added, and the mixtures were incubated at 25 • C for 2 h.The well was rinsed 3 times with wash buffer and 200 µL of p-nitrophenyl phosphate substrate solution was added to initiate the phosphatase reaction.After 45 min of incubation at 25 • C, absorbances were measured at 405 nm with a SPECTROstar Nano microplate reader (BMG LABTECH GmbH).The amount of PG E 2 in the samples was estimated from a standard curve.

Quantitative Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR) Analysis
The mRNA level was analyzed by qRT-PCR, as described in a previous study [18].Cells were treated with various test materials for 24 h and the total cellular RNA from the treated cells was used in the preparation of complementary DNA.The qRT-PCR was run using the gene-specific primers, whose nucleotide sequences are shown in Table 1.The mRNA level of each target gene was compared to that of the internal reference, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), using the comparative Ct method [42].Data are presented as a percentage of the control group.

Gene Name GenBank Accession Number Primer Sequences
Ref.

Western Blotting
The primary antibodies for cPLA2 (#2832) and phospho-cPLA2 (Ser505) (#2831) were purchased from Cell Signaling Technology (Danvers, MA, USA), and β-actin (#47778) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).The anti-rabbit IgG (#7074) secondary antibody was purchased from Cell Signaling Technology.Protein samples were denatured by mixing them with a Laemmli sample buffer and heating them at 95 • C for 5 min.Equal amounts of proteins (20 µg) were resolved using 10% SDSpolyacrylamide gel electrophoresis at 80 V and were then electrically transferred to a polyvinylidene difluoride membrane (Amersham Pharmacia, Little Chalfont, UK) at 4 • C overnight.After blocking the incubation of the membrane with TBST (137 mM of sodium chloride, 20 mM of Tris, 0.1% Tween 20, pH of 7.6.)containing 5% skim milk, it was incubated with the primary antibody in TBST containing 5% skim milk at 4 • C overnight; this was followed by incubation with the secondary antibody in TBST containing 5% skim milk at room temperature (25 • C) for 1 h.The target protein bands were visualized with a chemiluminescence method using the picoEPD Western Reagent kit (ELPIS-Biotech, Daejeon, Republic of Korea).The captured blot images were analyzed using the Image J program provided by the U.S. National Institute of Health (Bethesda, MD, USA).

Statistical Analysis
Experimental data were analyzed using SigmaStat v.3.11software (Systat Software Inc., San Jose, CA, USA) and presented as mean ± standard deviation (SD).The presence of group means that were significantly different from other groups was determined using a one-way analysis of variance (ANOVA) at the p < 0.05 level.Then, Duncan's multiple range test was subsequently run to compare all groups to each other.

Results
The dried leaves and stems of OJ were used in the preparation of the total hot water extract of OJ and its MC, EA, BA, and WT fractions.HPLC-DAD analysis was performed to compare the overall composition of the total OJ extract and its solvent fractions.Chlorogenic acid and caffeic acid were used as the standards, as these compounds are known to be the main components of OJ [29][30][31].As shown in Figure 1, two of the main peaks observed in the total OJ extract and its solvent fractions were identified as chlorogenic acid and caffeic acid by comparing their retention times and absorption spectra with those of the standards.The total OJ extract contained a substantial level of chlorogenic acid, while its caffeic acid content was relatively lower.Chlorogenic acid was highly enriched in the BA fraction and caffeic acid was enriched in the EA fraction, and their contents were relatively lower in the MC fraction and the WT fraction.
As shown in Figure 2, in quantities up to 300 µg mL −1 , PM 10 decreased the viability of HaCaT cells in a concentration-dependent manner.The effects of the total OJ extract and each fraction on cell viability were evaluated at 10-1000 µg mL −1 in the absence and presence of 200 µg mL −1 PM 10 .The total extract of OJ significantly reduced cell viability at concentrations above 300 µg mL −1 in the absence of PM 10 , but had no additional effect on cell viability in the presence of PM 10 .None of the solvent fractions showed significant cytotoxicity up to 100 µg mL −1 , but they did show reduced cell viability at 300 µg mL −1 in the order of the EA fraction, MC fraction, BA fraction, and WT fraction.In the presence of 200 µg mL −1 of PM 10 , the water fraction had no additional effect on cell viability, but the other fractions enhanced the cytotoxic effects of PM 10 at their high concentrations (300 or 1000 µg mL −1 ).In the following experiments, cells were treated with the total OJ extract or each fraction at 30 and/or 100 µg mL −1 within non-cytotoxic ranges.As shown in Figure 2, in quantities up to 300 μg mL −1 , PM10 decreased the viability of HaCaT cells in a concentration-dependent manner.The effects of the total OJ extract and each fraction on cell viability were evaluated at 10−1000 μg mL −1 in the absence and presence of 200 μg mL −1 PM10.The total extract of OJ significantly reduced cell viability at concentrations above 300 μg mL −1 in the absence of PM10, but had no additional effect on cell viability in the presence of PM10.None of the solvent fractions showed significant cytotoxicity up to 100 μg mL −1 , but they did show reduced cell viability at 300 μg mL −1 in the order of the EA fraction, MC fraction, BA fraction, and WT fraction.In the presence of 200 μg mL −1 of PM10, the water fraction had no additional effect on cell viability, but the other fractions enhanced the cytotoxic effects of PM10 at their high concentrations (300 or 1000 μg mL −1 ).In the following experiments, cells were treated with the total OJ extract or each fraction at 30 and/or 100 μg mL −1 within non-cytotoxic ranges.The effects of chlorogenic acid and caffeic acid, identified as major components of OJ, on cell viability were examined at 10-1000 µM in the absence and presence of 200 µg mL −1 of PM 10 , and were compared with that of NAC, a positive control.As shown in Figure 3, caffeic acid decreased cell viability at concentrations above 100 µM, and chlorogenic acid and NAC decreased cell viability at concentrations above 300 µM.Chlorogenic acid and caffeic acid at 1000 µM enhanced the cytotoxic effects of PM 10 , but NAC did not.In the following experiments, cells were treated with each of these compounds at 30 and/or 100 µM.
The effects of the total OJ extract, solvent fractions, and compounds on PM 10 -induced ROS production were examined using a fluorescent probe.Cells were treated with the total extract and each fraction at 30 and 100 µg mL −1 , or with chlorogenic acid, caffeic acid, and NAC at 30 and 100 µM in the absence and presence of 200 µg mL −1 PM 10 .As shown in Figure 4, PM 10 at 100-300 µg mL −1 increased ROS production in a concentrationdependent manner.The total extract, MC fraction, EA fraction, BA fraction, and WT fraction at 30-100 µg mL −1 did not affect ROS production in the absence of PM 10 .However, the BA fraction (30-100 µg mL −1 ), EA fraction (30-100 µg mL −1 ), MC fraction (30-100 µg mL −1 ), and WT fraction (100 µg mL −1 ) attenuated the PM 10 -induced ROS production, in that order.Chlorogenic acid, caffeic acid, and NAC at 30-100 µM did not affect the basal level of ROS production, but reduced the PM 10 -induced ROS production, in that order.The images of cells fluorescing due to ROS confirmed that the basal ROS production was not affected by the total OJ extract (100 µg mL −1 ), BA fraction (100 µg mL −1 ), chlorogenic acid (100 µM), and NAC (100 µM), but that the PM 10 -induced ROS production was reduced by chlorogenic acid (100 µM), BA fraction (100 µg mL −1 ), and NAC (100 µM), in that order.The effects of chlorogenic acid and caffeic acid, identified as major components of OJ, on cell viability were examined at 10−1000 μM in the absence and presence of 200 μg mL −1 of PM10, and were compared with that of NAC, a positive control.As shown in Figure 3, caffeic acid decreased cell viability at concentrations above 100 μM, and chlorogenic acid and NAC decreased cell viability at concentrations above 300 μM.Chlorogenic acid and following experiments, cells were treated with each of these compounds at 30 and/or 100 μM.The effects of the total OJ extract, solvent fractions, and compounds on PM10-induced ROS production were examined using a fluorescent probe.Cells were treated with the total extract and each fraction at 30 and 100 μg mL −1 , or with chlorogenic acid, caffeic acid, and NAC at 30 and 100 μM in the absence and presence of 200 μg mL −1 PM10.As shown in Figure 4, PM10 at 100−300 μg mL −1 increased ROS production in a concentration-dependent manner.The total extract, MC fraction, EA fraction, BA fraction, and WT fraction at 30−100 μg mL −1 did not affect ROS production in the absence of PM10.However, the BA fraction (30−100 μg mL −1 ), EA fraction (30−100 μg mL −1 ), MC fraction (30−100 μg mL −1 ), and WT fraction (100 μg mL −1 ) attenuated the PM10-induced ROS production, in that order.Chlorogenic acid, caffeic acid, and NAC at 30−100 μM did not affect the basal level of ROS production, but reduced the PM10-induced ROS production, in that order.The images of cells fluorescing due to ROS confirmed that the basal ROS production was not affected by the total OJ extract (100 μg mL −1 ), BA fraction (100 μg mL −1 ), chlorogenic acid (100 μM), and NAC (100 μM), but that the PM10-induced ROS production was reduced by chlorogenic acid (100 μM), BA fraction (100 μg mL −1 ), and NAC (100 μM), in that order.The effects of the total OJ extract, BA fraction, chlorogenic acid, and NAC on PM 10induced lipid peroxidation were examined by measuring MDA production.As shown in Figure 5, PM 10 at 100-300 µg mL −1 increased lipid peroxidation in a concentrationdependent manner.The total OJ extract (100 µg mL −1 ), BA fraction (100 µg mL −1 ), chlorogenic acid (100 µM), and NAC (100 µM) did not affect the basal level of lipid peroxidation in the absence of PM 10 .The BA fraction (100 µg mL −1 ) and chlorogenic acid (100 µM) suppressed the lipid peroxidation induced by PM 10 (200 µg mL −1 ), whereas the total OJ extract (100 µg mL −1 ) and NAC (100 µM) had no significant effects.The increases in the MDA levels due to PM 10 exposure were smaller in groups treated with the BA fraction (100 µg mL −1 ) or chlorogenic acid (100 µM) compared to the control group.
The effects of the total OJ extract, BA fraction, chlorogenic acid, and NAC on protein carbonylation were examined in HaCaT cells exposed to PM 10 .As shown in Figure 6, PM 10 at 100-300 µg mL −1 increased the protein carbonyl content in a concentration-dependent manner.The total OJ extract (100 µg mL −1 ), BA fraction (100 µg mL −1 ), chlorogenic acid (100 µM), and NAC (100 µM) slightly increased the protein carbonyl content of cells in the absence of PM 10 , but attenuated an increase in the protein carbonyl contents due to the exposure of cells to PM 10 (200 µg mL −1 ).(100 μM), and NAC (100 μM) did not affect the basal level of lipid peroxidation in the absence of PM10.The BA fraction (100 μg mL −1 ) and chlorogenic acid (100 μM) suppressed the lipid peroxidation induced by PM10 (200 μg mL −1 ), whereas the total OJ extract (100 μg mL −1 ) and NAC (100 μM) had no significant effects.The increases in the MDA levels due to PM10 exposure were smaller in groups treated with the BA fraction (100 μg mL −1 ) or chlorogenic acid (100 μM) compared to the control group.
Figure 5. Effects of the total OJ extract, its BA fraction, chlorogenic acid, and NAC on lipid peroxidation in HaCaT keratinocytes exposed to PM 10 .In (A), cells were treated with PM10 at different concentrations for 48 h.In (B,C), cells were treated with the total OJ extract (100 μg mL −1 ), BA fraction (100 μg mL −1 ), chlorogenic acid (100 μM), or NAC (100 μM), alone or in combination with PM10 (200 μg mL −1 ), for 48 h.Lipid peroxidation was determined by measuring malondialdehyde (MDA) levels in whole-cell lysates and the values were normalized to the protein contents (nmole mg protein −1 ) (B).The % change in the MDA values due to PM10 exposure in each group was calculated using the following equation: change (%) = ( − M)/M  100, where  is a value in the presence of PM10 and M is the mean value of each group in the absence of PM10 (C).Data are presented as mean ± SD (n = 4).Groups that do not share the same lowercase alphabet letters (a-c) are considered to have significantly different means at the p < 0.05 level.
Figure 5. Effects of the total OJ extract, its BA fraction, chlorogenic acid, and NAC on lipid peroxidation in HaCaT keratinocytes exposed to PM 10 .In (A), cells were treated with PM 10 at different concentrations for 48 h.In (B,C), cells were treated with the total OJ extract (100 µg mL −1 ), BA fraction (100 µg mL −1 ), chlorogenic acid (100 µM), or NAC (100 µM), alone or in combination with PM 10 (200 µg mL −1 ), for 48 h.Lipid peroxidation was determined by measuring malondialdehyde (MDA) levels in whole-cell lysates and the values were normalized to the protein contents (nmole mg protein −1 ) (B).The % change in the MDA values due to PM 10 exposure in each group was calculated using the following equation: change (%) = (α − M)/M × 100, where α is a value in the presence of PM 10 and M is the mean value of each group in the absence of PM 10 (C).Data are presented as mean ± SD (n = 4).Groups that do not share the same lowercase alphabet letters (a-c) are considered to have significantly different means at the p < 0.05 level.
The effects of the total OJ extract, BA fraction, chlorogenic acid, and NAC on the secreted level of PG E 2 in HaCaT cells exposed to PM 10 were examined using ELISA.As shown in Figure 7, PM 10 at 100-300 µg mL −1 increased the secreted level of PG E 2 up to 14-fold in a concentration-dependent manner.The total OJ extract (100 µg mL −1 ), BA fraction (100 µg mL −1 ), chlorogenic acid (100 µM), and NAC (100 µM) did not affect the secreted level of PG E 2 in the absence of PM 10 .The BA fraction (100 µg mL −1 ) and chlorogenic acid (100 µM) inhibited the increase in the secreted level of PG E 2 in cells stimulated by PM 10 (200 µg mL −1 ), whereas the total OJ extract (100 µg mL −1 ) and NAC (100 µM) had no significant effects.carbonylation were examined in HaCaT cells exposed to PM10.As shown in Figure 6, PM10 at 100−300 μg mL −1 increased the protein carbonyl content in a concentration-dependent manner.The total OJ extract (100 μg mL −1 ), BA fraction (100 μg mL −1 ), chlorogenic acid (100 μM), and NAC (100 μM) slightly increased the protein carbonyl content of cells in the absence of PM10, but attenuated an increase in the protein carbonyl contents due to the exposure of cells to PM10 (200 μg mL −1 ). Figure 6.Effects of the total OJ extract, its BA fraction, chlorogenic acid, and NAC on the protein carbonyl content in HaCaT keratinocytes exposed to PM 10 .In (A), cells were treated with different concentrations of PM10.In (B,C), cells were treated with the total OJ extract (100 μg mL −1 ), BA fraction (100 μg mL −1 ), chlorogenic acid (100 μM), and NAC (100 μM), alone or in combination with PM10 (200 μg mL −1 ), for 48 h.Protein carbonyl contents were normalized to the protein contents and presented in nmole mg protein −1 (B).The % change in the protein carbonyl contents due to PM10 exposure in each group was calculated using the following equation: change (%) = ( − M)/M  100, where  is the value in the presence of PM10 and M is the mean value of each group in the absence of PM10 (C).Data are presented as mean ± SD (n = 3).Groups that do not share the same lowercase alphabet letters (a-f) are considered to have significantly different means at the p < 0.05 level.
The effects of the total OJ extract, BA fraction, chlorogenic acid, and NAC on the secreted level of PG E2 in HaCaT cells exposed to PM10 were examined using ELISA.As shown in Figure 7, PM10 at 100−300 μg mL −1 increased the secreted level of PG E2 up to 14fold in a concentration-dependent manner.The total OJ extract (100 μg mL −1 ), BA fraction (100 μg mL −1 ), chlorogenic acid (100 μM), and NAC (100 μM) did not affect the secreted level of PG E2 in the absence of PM10.The BA fraction (100 μg mL −1 ) and chlorogenic acid (100 μM) inhibited the increase in the secreted level of PG E2 in cells stimulated by PM10 Figure 6.Effects of the total OJ extract, its BA fraction, chlorogenic acid, and NAC on the protein carbonyl content in HaCaT keratinocytes exposed to PM 10 .In (A), cells were treated with different concentrations of PM 10 .In (B,C), cells were treated with the total OJ extract (100 µg mL −1 ), BA fraction (100 µg mL −1 ), chlorogenic acid (100 µM), and NAC (100 µM), alone or in combination with PM 10 (200 µg mL −1 ), for 48 h.Protein carbonyl contents were normalized to the protein contents and presented in nmole mg protein −1 (B).The % change in the protein carbonyl contents due to PM 10 exposure in each group was calculated using the following equation: change (%) = (α − M)/M × 100, where α is the value in the presence of PM 10 and M is the mean value of each group in the absence of PM 10 (C).Data are presented as mean ± SD (n = 3).Groups that do not share the same lowercase alphabet letters (a-f) are considered to have significantly different means at the p < 0.05 level.
The effects of the total OJ extract, BA fraction, chlorogenic acid, and NAC on the mRNA expression levels of the genes involved in PG E 2 production were examined.The qRT-PCR was run to analyze the mRNA expression levels of phospholipase A2 group IIA (PLA2G2A), phospholipase A2 group IVA (PLA2G4A), prostaglandin-endoperoxide synthase 1 (PTGS1), prostaglandin-endoperoxide synthase 2 (PTGS2), prostaglandin E synthetase 1 (PTGES1), and prostaglandin E synthetase 2 (PTGES2).As shown in Figure 8, PM 10 at 200 µg mL −1 decreased the mRNA expression level of PLA2G2A, but increased that of PLA2G4A.The PM 10 -induced increase in PLA2G4A mRNA was reduced by chlorogenic acid (100 µM) and NAC (100 µM), whereas the total OJ extract (100 µg mL −1 ) and BA fraction (100 µg mL −1 ) had no effects.PM 10 increased the mRNA expression levels of PTGS1 and PTGS2; these changes were not affected by being treated with the total OJ extract, BA fraction, chlorogenic acid, or NAC.PM 10 and other test materials had little effect on the mRNA expression levels of PTGES1 and PTGES2.
Figure 7. Effects of the total OJ extract, its BA fraction, chlorogenic acid, and NAC on the secreted level of prostaglandin (PG) E2 in HaCaT keratinocytes exposed to PM 10 .In (A), cells were treated with different concentrations of PM10.In (B), cells were treated with the total OJ extract (100 μg mL −1 ), BA fraction (100 μg mL −1 ), chlorogenic acid (100 μM), and NAC (100 μM), alone or in combination with PM10 (200 μg mL −1 ), for 48 h.The secreted level of PG E2 was determined using a PG E2 enzyme-linked immunosorbent assay (ELISA).Data are presented as mean ± SD (n = 3).Groups that do not share the same lowercase alphabet letters (a-d) are considered to have significantly different means at the p < 0.05 level.
The effects of the total OJ extract, BA fraction, chlorogenic acid, and NAC on the mRNA expression levels of the genes involved in PG E2 production were examined.The qRT-PCR was run to analyze the mRNA expression levels of phospholipase A2 group IIA (PLA2G2A), phospholipase A2 group IVA (PLA2G4A), prostaglandin-endoperoxide synthase 1 (PTGS1), prostaglandin-endoperoxide synthase 2 (PTGS2), prostaglandin E synthetase 1 (PTGES1), and prostaglandin E synthetase 2 (PTGES2).As shown in Figure 8, PM10 at 200 μg mL −1 decreased the mRNA expression level of PLA2G2A, but increased that of PLA2G4A.The PM10-induced increase in PLA2G4A mRNA was reduced by chlorogenic acid (100 μM) and NAC (100 μM), whereas the total OJ extract (100 μg mL −1 ) and BA fraction (100 μg mL −1 ) had no effects.PM10 increased the mRNA expression levels of PTGS1 and PTGS2; these changes were not affected by being treated with the total OJ extract, BA fraction, chlorogenic acid, or NAC.PM10 and other test materials had little effect on the mRNA expression levels of PTGES1 and PTGES2.Effects of the total OJ extract, its BA fraction, chlorogenic acid, and NAC on the secreted level of prostaglandin (PG) E 2 in HaCaT keratinocytes exposed to PM 10 .In (A), cells were treated with different concentrations of PM 10 .In (B), cells were treated with the total OJ extract (100 µg mL −1 ), BA fraction (100 µg mL −1 ), chlorogenic acid (100 µM), and NAC (100 µM), alone or in combination with PM 10 (200 µg mL −1 ), for 48 h.The secreted level of PG E 2 was determined using a PG E 2 enzyme-linked immunosorbent assay (ELISA).Data are presented as mean ± SD (n = 3).Groups that do not share the same lowercase alphabet letters (a-d) are considered to have significantly different means at the p < 0.05 level.The effects of the total OJ extract, BA fraction, chlorogenic acid, and NAC on the protein expression levels of cPLA2 and its phosphorylated active form (phospho-cPLA2) were examined by Western blotting.As shown in Figure 9, PM10 increased the protein levels of cPLA2 and phospho-cPLA2.The total OJ extract (100 μg mL −1 ), BA fraction (100 μg mL −1 ), chlorogenic acid (100 μM), and NAC (100 μM) did not affect the basal levels of the cPLA2 protein.However, the BA fraction, chlorogenic acid, and NAC lowered the levels of cPLA2 protein under PM10-exposed conditions.In addition, chlorogenic acid and Figure 8. Effects of the total OJ extract, its BA fraction, chlorogenic acid, and NAC on the mRNA expression levels of phospholipase A2 group IIA (PLA2G2A) (A), phospholipase A2 group IVA (PLA2G4A) (B), prostaglandin-endoperoxide synthase 1 (PTGS1) (C), prostaglandin-endoperoxide synthase 2 (PTGS2) (D), prostaglandin E synthetase 1 (PTGES1) (E), and prostaglandin E synthetase 2 (PTGES2) (F) in HaCaT keratinocytes exposed to PM 10 .Cells were treated with the total extract (100 µg mL −1 ), BA fraction (100 µg mL −1 ), chlorogenic acid (100 µM), and NAC (100 µM), alone or in combination with PM 10 (200 µg mL −1 ), for 24 h.The mRNA expression level of each gene was analyzed by the quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH).Data are presented as % of control (means ± SD, n = 3).Groups that do not share the same lowercase alphabet letters (a-c) have statistically different means at the p < 0.05 level.

Discussion
This study, for the first time, has examined the antioxidant and anti-inflammatory effects of the total OJ extract and its solvent fractions on skin cells exposed to PM10.Neither the total extract nor the solvent fraction of OJ significantly reversed HaCaT cell death due to a high level of PM10 (200 μg mL −1 ), but some solvent fractions within a non-cytotoxic range significantly reduced the PM10-induced cellular ROS production.When the effect of reducing ROS production was compared between the solvent fractions, the BA fraction was more effective than the EA fraction and other fractions.The effects of the total OJ extract, BA fraction, chlorogenic acid, and NAC on the protein expression levels of cPLA2 and its phosphorylated active form (phospho-cPLA2) were examined by Western blotting.As shown in Figure 9, PM 10 increased the protein levels of cPLA2 and phospho-cPLA2.The total OJ extract (100 µg mL −1 ), BA fraction (100 µg mL −1 ), chlorogenic acid (100 µM), and NAC (100 µM) did not affect the basal levels of the cPLA2 protein.However, the BA fraction, chlorogenic acid, and NAC lowered the levels of cPLA2 protein under PM 10 -exposed conditions.In addition, chlorogenic acid and NAC lowered the levels of phospho-cPLA2 protein under basal and PM 10 -exposed conditions.

Discussion
This study, for the first time, has examined the antioxidant and anti-inflammatory effects of the total OJ extract and its solvent fractions on skin cells exposed to PM 10 .Neither the total extract nor the solvent fraction of OJ significantly reversed HaCaT cell death due to a high level of PM 10 (200 µg mL −1 ), but some solvent fractions within a non-cytotoxic range significantly reduced the PM 10 -induced cellular ROS production.When the effect of reducing ROS production was compared between the solvent fractions, the BA fraction was more effective than the EA fraction and other fractions.
The BA fraction contained chlorogenic acid (49.0 mg g −1 ), whereas the EA fraction contained caffeic acid (18.7 mg g −1 ) as a major phenolic component.These two phenolic compounds did not significantly alleviate the cell death caused by PM 10 , but effectively reduced the cellular ROS production, and their effects were comparable to that of NAC, a positive control antioxidant.Thus, we speculated that chlorogenic acid and caffeic acid might have been the active components of OJ that reduced ROS levels in cells exposed to PM 10 .Chlorogenic acid showed a stronger effect than the same molar concentration of caffeic acid in the inhibition of cellular ROS production.These results explain why the BA fraction that was rich in chlorogenic acid was more effective than the EA fraction, which was rich in caffeic acid, in reducing cellular ROS levels under PM 10 exposure.
Because plant-derived substances may have various effects on cell physiology, it is important to optimize preparations for targeting biological activity.In a previous study comparing the phytochemical composition and antioxidant activity of a 70% ethanol extract of OJ and its solvent fractions, the EA fraction showed a higher content of total phenolic compounds and flavonoids, and a higher free radical scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, 2,2 -azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) radical, and hydroxyl radical in vitro, compared to the BA fraction [29].Another study showed that the BA fraction of the hot water extract of OJ was more effective than other fractions in reducing the blood ethanol concentration after the oral administration of ethanol in ICR mice [34].In the current study, the BA fraction would be more effective and potentially useful in attenuating oxidative stress in human keratinocytes under PM 10 -exposed conditions.
Therefore, additional experiments were performed focusing on the antioxidant and anti-inflammatory effects of the BA fraction and its major component, chlorogenic acid.The results showed that the BA fraction (100 µg mL −1 ) and chlorogenic acid (100 µM) were effective in inhibiting lipid peroxidation and PG E 2 secretion in cells exposed to PM 10 .The results suggest the potential utility of the BA fraction of OJ in attenuating the PM 10induced oxidative damage to and inflammatory responses of cells.It is also suggested that chlorogenic acid may be an active component that is responsible for the antioxidant and anti-inflammatory effects of the BA fraction of the OJ extract.
The arachidonic acid that is released from membrane phospholipids via the enzymatic action of phospholipases A2 (PLA2) is converted to PG G 2 and then to PG H 2 in a reaction that is catalyzed by prostaglandin-endoperoxide synthases (PTGS), also known as cyclooxygenase (COX); further, the conversion of PG H 2 to PG E 2 is catalyzed by prostaglandin E synthetases (PEGS) [48,49].The PLA2G4A gene encodes a cytosolic phospholipase A2 (cPLA2), while the PLA2G2A gene encodes a secretory phospholipase A2 (sPLA2) [43,44].Interestingly, PM 10 decreased the mRNA expression of PLA2G2A and increased that of PLA2G4A.It also increased the mRNA expression of PTGS1 and PTGS2, which encode COX1 and COX2, respectively, but had no significant effects on the mRNA expression of PT-GES1 and PTGES2, which encode microsomal prostaglandin E synthetase 1 (mPEGS1) and mPEGS2.This suggests that the increase in PG E 2 production by PM 10 could be mediated by cPLA2, COX1, and COX2 proteins.Since chlorogenic acid attenuated the expressions of PLA2G4A mRNA and cPLA2 protein induced by PM 10 , this is considered to be one of chlorogenic acid's main mechanisms of action regarding its ability to reduce PM 10 -induced PG E 2 production, as depicted in Figure 10.The cPLA2 is activated by airborne agricultural particulate matter and mediates inflammatory responses in human lung epithelial A549 cells [51].The differential regulation of the gene expression of PLA2G4A versus PLA2G2A by PM10 observed in the present study suggests that each isozyme may play a distinct role under different cellular contexts.The cPLA2 is a calcium-dependent enzyme that is additionally activated by phosphorylation at Ser505 and other sites [52].In the present study, PM10 increased the total cPLA2 protein and phospho-cPLA2 (Ser505), and these changes were reduced by chlorogenic acid and NAC.A more expanded study is needed to elucidate the differential role of phospholipase A2 isoenzymes in association with the PM10-induced inflammatory reaction.
Previous studies demonstrated the anti-inflammatory effects of OJ extracts on the attenuation of the NF-kB-mediated expression of COX2, and on the secretion of TNF- and PG E2 in lipopolysaccharide-stimulated RAW 264.7 macrophages [35,36].The topical application of OJ extracts alleviated the decrease in skin collagen caused by UV irradiation in male ICR mice, and this effect was associated with the restoration of the expression of collagen types I and III, MMP1, MMP3, TNF-, and COX2 [37].The present study additionally showed that the BA fraction purified from the OJ extract has anti-inflammatory effects, by reducing the secretion of PG E2 in keratinocytes stimulated by PM10.Taken together, data from these previous studies and the current study suggest that OJ extract is a useful natural product to alleviate skin inflammation caused by UV rays, atmospheric fine Although the ability of chlorogenic acid to reduce the PM 10 -induced mRNA expression of PTGS1 and PTGS2, which encode COX1 and COX2, respectively, was not observed in the present study, it is worth recalling that chlorogenic acid is a potent inhibitor of COX2 enzyme activity (IC 50 , 8.1 µM) [50].Chlorogenic acid could reduce PG E 2 production through the direct inhibition of COX-2 enzymatic activity as an additional mechanism of action.
The cPLA2 is activated by airborne agricultural particulate matter and mediates inflammatory responses in human lung epithelial A549 cells [51].The differential regulation of the gene expression of PLA2G4A versus PLA2G2A by PM 10 observed in the present study suggests that each isozyme may play a distinct role under different cellular contexts.The cPLA2 is a calcium-dependent enzyme that is additionally activated by phosphorylation at Ser505 and other sites [52].In the present study, PM 10 increased the total cPLA2 protein and phospho-cPLA2 (Ser505), and these changes were reduced by chlorogenic acid and NAC.A more expanded study is needed to elucidate the differential role of phospholipase A2 isoenzymes in association with the PM 10 -induced inflammatory reaction.
Previous studies demonstrated the anti-inflammatory effects of OJ extracts on the attenuation of the NF-kB-mediated expression of COX2, and on the secretion of TNF-α and PG E 2 in lipopolysaccharide-stimulated RAW 264.7 macrophages [35,36].The topical application of OJ extracts alleviated the decrease in skin collagen caused by UV irradiation in male ICR mice, and this effect was associated with the restoration of the expression of collagen types I and III, MMP1, MMP3, TNF-α, and COX2 [37].The present study additionally showed that the BA fraction purified from the OJ extract has anti-inflammatory effects, by reducing the secretion of PG E 2 in keratinocytes stimulated by PM 10 .Taken together, data from these previous studies and the current study suggest that OJ extract is a useful natural product to alleviate skin inflammation caused by UV rays, atmospheric fine dust, or a combination thereof.In addition, it is suggested that the efficacy can be further improved by purifying the OJ extract and increasing the content of chlorogenic acid, one of its active ingredients.
Chlorogenic acid has been previously reported to attenuate oxidative stress and inflammatory responses in various in vitro and in vivo models [53,54].In our recent study, chlorogenic acid was identified as a major component the Siegesbeckiae herba extract, which has now been shown to mitigate PM 10 -induced cytotoxicity by activating the NRF 2 pathway [25].It has also been shown to suppress the production of nitric oxide (NO) and PG E 2 by inhibiting the expression of inducible nitric oxide synthase (iNOS) and COX2 in lipopolysaccharide-stimulated RAW 264.7 macrophages [55], and in human chondrocytes stimulated by IL-1β [56].In the present study, chlorogenic acid was shown to reduce the production of PG E 2 in human keratinocytes induced by PM 10 .Thus, there is consolidating evidence that chlorogenic acid exhibits antioxidant and anti-inflammatory activities in cells.
OJ contains a variety of compounds that exhibit anti-inflammatory activity [27].In previous studies, ten biphenyl derivatives were isolated from the cyclohexane fraction of an 85% ethanol extract of the aerial part of OJ, and some significantly inhibited COX2 activity in vitro [57].Thirteen kinds of phenylpropanoids were isolated from the EA fraction of the extract, and some of these compounds exhibited anti-inflammatory activities that inhibited NO production in RAW 264.7 macrophages [58].Therefore, it should not be overlooked that OJ has various components that exhibit anti-inflammatory capabilities via various mechanisms, in addition to chlorogenic acid and caffeic acid.
In this study, OJ extract and its fractions showed different degrees of cytotoxicity at high concentrations in the absence of fine dust.Currently, the identity and mechanism of action of these toxic components are unknown.Nonetheless, it is desirable to remove toxic components as much as possible, while leaving active components, when using OJ extract to protect cells.Since even active components can be toxic at high concentrations, it is important to find the optimal concentration range, considering both toxicity and efficacy.

Conclusions
This study demonstrated that the BA fraction of OJ extract reduces PM 10 -induced ROS production in HaCaT cells most effectively among the solvent fractions.Of its major phenolic components, chlorogenic acid was more effective than caffeic acid in reducing ROS levels.The BA fraction and chlorogenic acid attenuated lipid peroxidation, protein carbonylation, and PG E 2 production due to the exposure of cells to PM 10 .The PM 10induced expressions of PLA2G4A mRNA and cPLA2 protein were alleviated by chlorogenic acid, and this action of chlorogenic acid is suggested to be the main mechanism that enables a reduction in PM 10 -induced PG E 2 production.This study suggests that the chlorogenic acid contained in OJ extract may help to alleviate the oxidative damage to and inflammatory responses produced by the skin cells when exposed to air pollutants.

Figure 1 .
Figure 1.High-performance liquid chromatography with photodiode array detection (HPLC-DAD) analysis of the total extract Oenanthe javanica (Blume) DC, abbreviated OJ, and its MC, EA, BA, and WT fractions.Authentic chlorogenic acid and caffeic acid were used as the standards for comparing the retention times and absorption spectra.Chromatograms at 330 nm and UV absorption spectra of the indicated peaks are shown.

Figure 1 .
Figure 1.High-performance liquid chromatography with photodiode array detection (HPLC-DAD) analysis of the total extract Oenanthe javanica (Blume) DC, abbreviated OJ, and its MC, EA, BA, and WT fractions.Authentic chlorogenic acid and caffeic acid were used as the standards for comparing the retention times and absorption spectra.Chromatograms at 330 nm and UV absorption spectra of the indicated peaks are shown.

Figure 2 .
Figure 2. Effects of the total OJ extract and its solvent fractions on the viability of HaCaT keratinocytes exposed to particulate matter 10 (PM 10 ).In (A), cells were treated with PM 10 at different concentrations for 48 h.In (B−F), cells were treated with the total OJ extract (B), MC fraction (C), EA fraction (D), BA fraction €, or WT fraction (F) at the specified concentrations, alone or in combination with PM 10 (200 μg mL −1 ), for 48 h.Cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT).Data are presented as mean ± SD (n = 4 for A, n = 3 for B−F).Duncan's multiple range test was performed to compare all group means to each other.Groups that do not share the same lowercase alphabet letters (a-e) are considered to have significantly different means at the p < 0.05 level.

Figure 2 .
Figure 2. Effects of the total OJ extract and its solvent fractions on the viability of HaCaT keratinocytes exposed to particulate matter 10 (PM 10 ).In (A), cells were treated with PM 10 at different concentrations for 48 h.In (B-F), cells were treated with the total OJ extract (B), MC fraction (C), EA fraction (D), BA fraction €, or WT fraction (F) at the specified concentrations, alone or in combination with PM 10 (200 µg mL −1 ), for 48 h.Cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT).Data are presented as mean ± SD (n = 4 for A, n = 3 for B-F).Duncan's multiple range test was performed to compare all group means to each other.Groups that do not share the same lowercase alphabet letters (a-e) are considered to have significantly different means at the p < 0.05 level.

Figure 3 .
Figure 3. Effects of chlorogenic acid, caffeic acid, and N-acetyl cysteine (NAC) on the viability of HaCaT keratinocytes exposed to PM10.Cells were treated with chlorogenic acid (A), caffeic acid (B), or NAC (C) at the specified concentrations, alone or in combination with PM10 (200 μg mL −1 ), for 48 h.Data are presented as mean ± SD (n = 3).Groups that do not share the same lowercase alphabet letters (a-e) are considered to have significantly different means at the p < 0.05 level.

Figure 3 .
Figure 3. Effects of chlorogenic acid, caffeic acid, and N-acetyl cysteine (NAC) on the viability of HaCaT keratinocytes exposed to PM 10 .Cells were treated with chlorogenic acid (A), caffeic acid (B), or NAC (C) at the specified concentrations, alone or in combination with PM 10 (200 µg mL −1 ), for 48 h.Data are presented as mean ± SD (n = 3).Groups that do not share the same lowercase alphabet letters (a-e) are considered to have significantly different means at the p < 0.05 level.

Figure 4 .
Figure 4. Effects of the total OJ extract, its solvent fractions, chlorogenic acid, caffeic acid, and NAC on the production of reactive oxygen species (ROS) in HaCaT keratinocytes exposed to PM10.In (A), after pre-labeling with 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) for 30 min, cells were treated with PM10 at different concentrations for 60 min.In (B,C), the pre-labeled cells were treated with the total OJ extract, its solvent fractions, or compounds at the specified concentrations, alone or in combination with PM10 (200 μg mL −1 ), for 60 min.Typical images of cells fluorescing due to ROS production are shown in (D).The change in the fluorescence intensity due to cellular ROS

Figure 4 .
Figure 4. Effects of the total OJ extract, its solvent fractions, chlorogenic acid, caffeic acid, and NAC on the production of reactive oxygen species (ROS) in HaCaT keratinocytes exposed to PM 10 .In (A), after pre-labeling with 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) for 30 min, cells were treated with PM at different concentrations for 60 min.In (B,C), the pre-labeled cells were treated with the total OJ extract, its solvent fractions, or compounds at the specified concentrations, alone or in combination with PM 10 (200 µg mL −1 ), for 60 min.Typical images of cells fluorescing due to ROS production are shown in (D).The change in the fluorescence intensity due to cellular ROS production was determined.Data are presented as mean ± SD (n = 4).Groups that do not have the same lowercase alphabet letters (a-f) are considered to have significantly different means at the p < 0.05 level.

Figure 7 .
Figure 7. Effects of the total OJ extract, its BA fraction, chlorogenic acid, and NAC on the secreted level of prostaglandin (PG) E 2 in HaCaT keratinocytes exposed to PM 10 .In (A), cells were treated with different concentrations of PM 10 .In (B), cells were treated with the total OJ extract (100 µg mL −1 ), BA fraction (100 µg mL −1 ), chlorogenic acid (100 µM), and NAC (100 µM), alone or in combination with PM 10 (200 µg mL −1 ), for 48 h.The secreted level of PG E 2 was determined using a PG E 2 enzyme-linked immunosorbent assay (ELISA).Data are presented as mean ± SD (n = 3).Groups that do not share the same lowercase alphabet letters (a-d) are considered to have significantly different means at the p < 0.05 level.
Cosmetics 2023, 10, x FOR PEER REVIEW 15 of 21NAC lowered the levels of phospho-cPLA2 protein under basal and PM10-exposed conditions.

Figure 9 .
Figure 9. Effects of the total OJ extract, its BA fraction, chlorogenic acid, and NAC on the protein levels of cytosolic phospholipase A2 (cPLA2) and phospho-cPLA2 in HaCaT keratinocytes under basal and PM10-exposed conditions.Cells were treated with the total OJ extract (100 μg mL −1 ), BA fraction (100 μg mL −1 ), chlorogenic acid (100 μM), and NAC (100 μM), alone or in combination with PM10 (200 μg mL −1 ), for 48 h.The protein levels of total cPLA2 (A) and phospho-cPLA2 (B) were determined by Western blotting and compared to that of β-actin.Representative blots are shown.Data are presented as percentages of the control (mean ± SD, n = 3).Duncan's multiple range test was performed to compare all group means to each other.Groups that do not share the same lowercase alphabet letters (a-e) have significantly different means at the p < 0.05 level.

Figure 9 .
Figure 9. Effects of the total OJ extract, its BA fraction, chlorogenic acid, and NAC on the protein levels of cytosolic phospholipase A2 (cPLA2) and phospho-cPLA2 in HaCaT keratinocytes under basal and PM 10 -exposed conditions.Cells were treated with the total OJ extract (100 µg mL −1 ), BA fraction (100 µg mL −1 ), chlorogenic acid (100 µM), and NAC (100 µM), alone or in combination with PM 10 (200 µg mL −1 ), for 48 h.The protein levels of total cPLA2 (A) and phospho-cPLA2 (B) were determined by Western blotting and compared to that of β-actin.Representative blots are shown.Data are presented as percentages of the control (mean ± SD, n = 3).Duncan's multiple range test was performed to compare all group means to each other.Groups that do not share the same lowercase alphabet letters (a-e) have significantly different means at the p < 0.05 level.

Cosmetics 2023 , 21 Figure 10 .
Figure 10.Antioxidant and anti-inflammatory effects of chlorogenic acid in HaCaT keratinocytes exposed to PM10.PM10 increased ROS production and lipid peroxidation.It also increased PG E2 production through the enhanced expressions of PLA2G4A mRNA and cPLA2 protein.These changes were attenuated () by chlorogenic acid, a main component of OJ.

Figure 10 .
Figure 10.Antioxidant and anti-inflammatory effects of chlorogenic acid in HaCaT keratinocytes exposed to PM 10 .PM 10 increased ROS production and lipid peroxidation.It also increased PG E 2 production through the enhanced expressions of PLA2G4A mRNA and cPLA2 protein.These changes were attenuated (⊗) by chlorogenic acid, a main component of OJ.