Published online May 20, 2022.
https://doi.org/10.5021/ad.2022.34.3.163
Effects of <10-µm Particulate Matter on Cultured Human Sebocytes and Outer Root Sheath Cells and Usefulness of Siegesbeckia Herba Extract
Abstract
Background
Particulate matter (PM) is one of the air pollutants that can damage human skin; the recent increase in the amount of PM may be detrimental to skin health.
Objective
We aimed to investigate the effects of PM on cultured human sebocytes and outer root sheath (ORS) cells and the effects of Siegesbeckia Herba extract (SHE) on PM-treated cultured cells.
Methods
Sebocytes and ORS cells were cultured. The cultured cells were treated with various concentrations of PM of <10 µm in size (PM10) (10 µg/ml, 25 µg/ml, 50 µg/ml, and 100 µg/ml) for 24 h. Real-time polymerase chain reaction, measurement of reactive oxygen species (ROS), small interfering (si) RNA transfection, Oil Red O and Nile red staining, and immunofluorescence staining were performed to analyze the presence of inflammatory cytokines, matrix metalloproteinases (MMPs), aryl hydrocarbon receptor (AhR), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), ROS, and lipid production. In addition, PM10 (100 µg/ml)-treated cultured cells were treated with 10 mg/ml of SHE.
Results
PM10 upregulates the expression of inflammatory cytokines, MMPs, AhR, NF-κB, and ROS in cultured human sebocytes and ORS cells. The production of ROS was dramatically reduced in AhR siRNA-transfected cells. In addition, PM10 upregulates sebum production in cultured sebocytes. SHE inhibited the upregulation of inflammatory cytokines, MMPs, AhR, NF-κB, ROS, and sebum production in cultured human sebocytes and/or ORS cells by PM10.
Conclusion
Effects of PM10 on cultured human sebocytes and ORS cells can be regulated by SH.
INTRODUCTION
Particulate matter (PM) in the urban area consists of complex mixtures of components of various sizes originating from factories, power plants, automobiles, construction activities, and natural windblown dust1, 2. PM of <10 µm in size (PM10) is one of the most harmful components of air pollutant. PM10 serves as a mediator for toxic compounds, such as transitional metals, endotoxins, organic chemicals, and ultrafine components. PM10 can reach the lungs and damage the respiratory and cardiovascular system. In addition, PM10 has a negative effect on the skin3.
PM10 attaches or penetrates into the skin epidermis and hair follicles, and then, it can damage the skin via the synthesis of reactive oxygen species (ROS). The generation of oxidative stress promotes skin aging, such as wrinkles, pigmentation, and telangiectasia4, 5, 6. Furthermore, inflammatory skin conditions including atopic dermatitis, acne, and psoriasis are associated with PM107. Antioxidants have been used to inhibit oxidative stress and improve inflammatory skin conditions. Several polyphenolic compounds have anti-oxidative and anti-inflammatory effects8, 9, 10. Also, plants are reported as a potential antioxidant and certain phytochemicals are expected to reduce cellular oxidative stress directly or indirectly. Siegesbeckiae Herba (SH), used as traditional medicine in Korea, Japan, China, and Vietnam, has been recently studied for their phytochemicals and biological efficacy11.
Although it is known that PM10 induces skin inflammation, insufficient research on the impact of PM10 on human sebocytes and outer root sheath (ORS) cells is available. Here, we aimed to investigate the effects of PM10 on the inflammatory cytokines, matrix metalloproteinases (MMPs), aryl hydrocarbon receptor (AhR), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), ROS and sebum of sebocytes and ORS cells.
MATERIALS AND METHODS
Sebocyte and ORS cell culture
Patients provide the occipital non-balding scalp specimens through hair transplantation surgery. The study was conducted according to the Declaration of Helsinki Principles. Patients signed informed written consent of their own accord. The Medical Ethical Committee of the Kyungpook National University Hospital (Daegu, Korea) approved all of the described studies (IRB no. KNU 2018-0155).
The sebaceous glands of human occipital hair were transferred to a tissue culture dish. The isolated cells were cultivated in Sebocyte Basal Medium (Cell Application, San Diego, CA, USA) containing Sebocyte Growth Supplement in a 5% CO2 incubator at 37℃. After 2 weeks of isolation, the cells were harvested with 0.25% trypsin/10 mM ethylenediaminetetraacetic acid in Hank’s balanced salt solution and subcultured at EpiLife (Gibco BRL, Rockville, MD, USA).
For the cultivation of ORS cells, contamination with other cells was prevented by cutting off the hair shaft and hair bulb regions of the hair follicle. Trimmed hair follicles were immersed in Dulbecco’s Modified Eagle’s medium (Hyclone, Logan, UT, USA) supplemented with 20% fetal bovine serum (Hyclone). On the third day of culture, the medium was changed to EpiLife keratinocyte growth medium-containing supplement. These experiments used sebocytes and ORS cells after the second passage.
Siegesbeckiae Herba extract (SHE) and its solvent fractions
Dried Siegesbeckiae Herba was purchased from Sinsun Herb (Seoul, Korea) and the leaves (140 g) were ground and extracted with 0.9 L water at 90℃ for 1 h. The extracted solution was evaporated under reduced pressure to obtain the crude extract (9 g). The SHE was dispersed in 150 ml water and partitioned sequentially with an equal volume of methylene chloride (MC), ethyl acetate (EA), and n-butyl alcohol (BA). Evaporation of the organic solvents yielded MC fraction (0.27 g), EA fraction (0.16 g), and BA fraction (0.46 g). The aqueous layer was filtered to remove insoluble material (1.15 g) and then evaporated to obtain a water fraction (6.64 g).
MTT assay
We used the MTT assay to check cell viability. We seeded the sebocytes and ORS cells in 96-well collagen-coated plates at a density of 5,000 cells/well (Becton Dickinson, Franklin, NJ, USA) for 24 h. We added various concentrations (5 µg/ml, 10 µg/ml, 25 µg/ml, 50 µg/ml, and 100 µg/ml) of PM10 (Sigma-Aldrich, St. Louis, MO, USA) and various concentrations (0.5 mg/ml, 1 mg/ml, 10 mg/ml, 30 mg/ml, and 50 mg/ml) of SHE to the well plates of the sebocytes and ORS cells for 1 day or 3 days each and the MTT solution (3-[4,5]dimethylthiazol-2,5-diphenyltetrazolium bromide) at 70 µg/well for 3 h. We solubilized the formazan produced with dimethyl sulfoxide (DMSO) and measured the optical density at 570 nm.
Real-time polymerase chain reaction
Cells were treated with a variety of concentrations of PM10 (10 µg/ml, 25 µg/ml, 50 µg/ml, and 100 µg/ml) for 24 h. PM10 (PM10-like, European reference material ERM-CZ120) was obtained from Sigma-Aldrich. PM10 (100 µg/ml)-treated cells were also treated with 10 mg/ml of SHE for 24 h. Total RNA was isolated using an RNeasy Mini Kit (Qiagen, Hilden, Germany), and cDNA was synthesized from 3 µg of total RNA with a cDNA synthesis kit containing ImProm-II™ reverse transcriptase and oligo (dT) primers based on the protocol suggested by the manufacturer (Promega, Madison, WI, USA).
Real-time polymerase chain reaction (PCR) was conducted with Step One Plus Real-Time PCR Assay (Applied Biosystems, Foster City, CA, USA). All reactions were conducted with Power SYBR Green premix (Applied Biosystems) using 50 ng cDNA and 10 pM primers. The cycling conditions for amplification were as follows: 95℃ for 10 min, 40 cycles at 95℃ for 15 s, and 60℃ for 60 s. The products of the PCR were evaluated with the StepOnePlus Real-time PCR analysis software (Applied Biosystems). The primers used in the study are shown in Supplementary Table 1.
Measurement of ROS
The cellular production of ROS was assessed by measuring 2′,7′-dichlorodihydrofluorescein diacetate. The cells were plated onto 6-well collagen coated plates (Becton Dickinson) at 5×105 cells/well for 24 h. Cells were pre labeled with 10 µM DCF-DA (Invitrogen, Carlsbad, CA, USA) for 30 min. Plates were washed with phosphate-buffered saline (PBS) and then treated with various concentrations of PM10 (25 µg/ml, 50 µg/ml, and 100 µg/ml) or with PM10 (100 µg/ml) and SHE (10 mg/ml) and incubated at 37℃ for 1 h protected from light. Cells were extracted with 20 mM Tris-Cl buffer containing 1% sodium dodecyl sulfate and 2.5 mM ethylenediamine-N,N,N’,N’-tetraacetic acid. The extracts were centrifuged at 13,000 rpm for 15 min, and the supernatants were detected using a fluorescence microplate reader (Molecular Devices, Sunnyvale, CA, USA) with excitation at 485 nm and emission at 538 nm.
Small interfering RNA transfection
The small interfering (si) RNAs used in this study were purchased from Bioneer (Daejeon, Korea). Cultured sebocytes and ORS cells were transfected with either control siRNA or a mixture of AhR siRNAs at a final concentration of 10 nmol/L in the presence of Lipofectamine™ RNAiMAX reagent (Invitrogen) for 48 h. After 2 days, the cells were pre labeled with 10 µM DCF-DA (Invitrogen) for 30 min and treated with 100 µg/ml PM10 for 1 h. The level of ROS production was visualized by fluorescent microscopy.
Quantification of lipid production: Oil Red O and Nile red
The Oil Red O staining kit (Abcam, Cambridge, UK) was used according to the manufacturer’s instructions. The slides were incubated in propylene glycol for 2 min and in Oil Red O solution for 30 min. The slides were immersed in 85% propylene glycol for 1 min and washed twice in water and stained hematoxylin for 2 min. Then, the slides were washed with water and mounted.
AdipoRed assay reagent (Lonza, Walkersville, MD, USA) was used for Nile red. The slides were incubated in Adipo red solution (1:100 dilution) for 10 min at dark, washed with water, and mounted.
Immunofluorescence staining
The hair follicle specimens were maintained in 100 µg/ml Williams E media without phenol red (Sigma, St. Louis, MI, USA) at 37℃ in a humidified atmosphere of 5% CO2 for 2 days.
Sebocytes and ORS cells were seeded into eight chambers at a density of 30,000 cells per well (Nuck, Roskilde, Denmark) for 24 h. The cells were treated with or without 100 µg/ml PM10 for 24 h. The fixation and blocking processes were the same as above. The cells were incubated with a rabbit anti-interleukin (IL)-1α antibody (1:100 dilution; Abcam), anti-IL-1β antibody (1:100 dilution; Abcam), anti-IL-6 antibody (1:100 dilution; Abcam), anti-IL-8 antibody (1:100 dilution; MybioSource), anti-MMP-1 antibody (1:100 dilution; Abcam), anti-MMP-3 (1:100 dilution; Abcam), anti-MMP-12 antibody (1:100 dilution; Abcam), anti-p-NF-κB (1:100 dilution; Cell signaling, Beverly, MA, USA), goat anti-tumor necrosis factor (TNF)-α antibody (1:100 dilution; R and D) and mouse anti-AhR (1:100 dilution; Santa Cruz, Delaware, CA, USA) at 4℃ overnight. Then, these were washed three times with PBS and incubated with Alexa Flour 488-labeled donkey anti-rabbit secondary antibody (Molecular Probes), 555-labeled donkey anti-goat secondary antibody (Molecular Probes) or 555-labeled donkey anti-mouse secondary antibody (Molecular Probes) for 1 h. The slides were counterstained with 4′,6-diamidino-2-phenylindole.
Statistical analysis
Data are expressed as follows: means±standard deviation. Experiment was done three times independently. Statistical analysis for data was done with analysis of variance (SPSS Statistics ver. 18.0; SPSS Inc., Chicago, IL, USA). p-values of <0.05 was considered statistically significant.
RESULTS
MTT assay to determine PM10 concentration and SHE concentration
Effects of PM10 and SHE on the cell viability of the human sebocytes and ORS cells were shown in MTT assay according to varying concentrations (5 µg/ml, 10 µg/ml, 25 µg/ml, 50 µg/ml, and 100 µg/ml) of PM10 (Fig. 1A) and according to varying concentrations (0.5 mg/ml, 1 mg/ml, 10 mg/ml, 30 mg/ml, and 50 mg/ml) of SHE (Fig. 1B).
Fig. 1
(A) Effects of particulate matter (PM) 10 in human-cultured sebocytes and outer root sheath (ORS) cells. We treated the cells with PM10 (5 µg/ml, 10 µg/ml, 25 µg/ml, 50 µg/ml, and 100 µg/ml) for 1 day, using the 3-[4,5]dimethylthiazol-2,5-diphenyltetrazolium bromide assay to measure cell viability. (B) We treated sebocytes and outer root sheath cells with varying concentrations (0.5 mg/ml, 1 mg/ml, 10 mg/ml, 30 mg/ml, and 50 mg/ml) of Siegesbeckia Herba extract (SHE). Data in the bar graphs represent the means±standard deviation from the three independent experiments (*p<0.05).
PM10 upregulates the expression of inflammatory cytokine in cultured human sebocytes and ORS cells and SHE inhibits the effects of PM10
The cells were treated with various concentrations of PM10 for 24 h to investigate the effects of PM10 on inflammatory responses in the human primary sebocytes and ORS cells. The expressions of IL-1α, IL-1β, IL-6, IL-8, and TNF-α were increased in PM10-treated sebocytes and ORS cells for 24 h in real-time PCR (Fig. 2A, B). A concentration-dependent increase was observed in the expression of inflammatory cytokines. SHE (10 mg/ml) decreased the upregulation of IL-1α, IL-1β, IL-6, IL-8, and TNF-α in PM10 (100 µg/ml)-treated sebocytes and ORS cells for 24 h in real-time PCR (Fig. 2C, D).
Fig. 2
(A) Particulate matter of <10 µm in size (PM10) upregulates the expression of inflammatory cytokines in cultured sebocytes in real-time polymerase chain reaction (PCR). (B) PM10 upregulates the expression of inflammatory cytokines in outer root sheath (ORS) cells in real-time PCR. (C) Siegesbeckia Herba extract (SHE) (10 mg/ml) decreased the upregulation of inflammatory cytokines in PM10 (100 µg/ml)-treated sebocytes. (D) SHE (10 mg/ml) decreased the upregulation of inflammatory cytokines in PM10 (100 µg/ml)-treated ORS cells. (E, F) Immunostaining showed the expression of inflammatory cytokines were increased in sebocytes and ORS cells after treatment with 100 µg/ml PM10 for 24 h. The data in the bar graphs represent mean±standard deviation from three independent experiments (*p<0.05). IL: interleukin, TNF: tumor necrosis factor.
Next, the protein levels of inflammatory cytokines in the human primary sebocytes and ORS cells were analyzed using immunostaining. The expression of inflammatory cytokines showed an increase in sebocytes and ORS cells after treatment with 100 µg/ml PM10 for 24 h (Fig. 2E, F).
PM10 upregulates the expression of MMPs in cultured human sebocytes and ORS cells and SHE inhibits the effects of PM10
The MMP family is known to play a critical role in skin aging. The cells were treated with various concentrations of PM10 for 24 h to investigate the effects of PM10 on the expression of MMP-1, MMP-3, and MMP-12. The expressions of MMP-1, MMP-3, and MMP-12 were increased in PM10-treated sebocytes and ORS cells for 24 h in real-time PCR (Fig. 3A, B). A concentration-dependent increase was observed in the expression of MMP genes. SHE (10 mg/ml) decreased the upregulation of MMP-1, MMP-3, and MMP-12 in PM10 (100 µg/ml)-treated sebocytes and ORS cells for 24 h in real-time PCR (Fig. 3C, D).
Fig. 3
(A) Particulate matter of <10 µm in size (PM10) upregulates the expression of matrix metalloproteinases (MMPs) in cultured sebocytes. (B) PM10 upregulates the expression of MMPs in outer root sheath (ORS) cells. (C) Siegesbeckia Herba extract (SHE) (10 mg/ml) decreased the upregulation of MMPs in PM10 (100 µg/ml)-treated sebocytes. (D) SHE (10 mg/ml) decreased the upregulation of MMPs in PM10 (100 µg/ml)-treated ORS cells. (E, F) Immunostaining showed the expression of MMP-1, MMP-3, and MMP-12 were increased in sebocytes and ORS cells after treatment with 100 µg/ml PM10 for 24 h. The data in the bar graphs represent mean±standard deviation from three independent experiments (*p<0.05).
Immunostaining showed that the expression of MMP-1, MMP-3, and MMP-12 were increased in sebocytes and ORS cells after treatment with 100 µg/ml PM10 for 24 h (Fig. 3E, F).
PM10 upregulates the expression of AhR and NF-κB in cultured sebocytes and ORS cells and SHE inhibits the effects of PM10
PM contains a significant amount of polycyclic aromatic hydrocarbons, which can induce ROS production via AhR. Oxidative stress can also activate NF-κB. We next assessed whether PM10 treatment increased AhR and NF-κB expression levels in cultured human sebocytes and ORS cells. The expressions of AhR and NF-κB were increased in PM10-treated sebocytes and ORS cells for 24 h in real-time PCR (Fig. 4A). A concentration-dependent increase was observed in the expression of AhR and NF-κB. In addition, nuclear AhR and NF-κB were increased by treatment with 100 µg/ml PM10 for 24 h by immunostaining (Fig. 4B). These results demonstrated that PM10 treatment causes nuclear activation of AhR and NF-κB pathway. SHE (10 mg/ml) decreased the upregulation of AhR and NF-κB in PM10 (100 µg/ml)-treated sebocytes and ORS cells for 24 h in real-time PCR (Fig. 4C, D).
Fig. 4
(A) Particulate matter of <10 µm in size (PM10) upregulates the gene expression of aryl hydrocarbon receptor (AhR) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in cultured sebocytes and outer root sheath (ORS) cells. (B) PM10 upregulates protein expression of AhR and NF-κB in cultured sebocytes and ORS cells. (C) Siegesbeckia Herba extract (SHE) (10 mg/ml) decreased the upregulation of AhR in PM10 (100 µg/ml)-treated sebocytes. (D) SHE (10 mg/ml) decreased the upregulation of NF-κB in PM10 (100 µg/ml)-treated ORS cells. The data in the bar graphs represent mean±standard deviation from three independent experiments (*p<0.05).
PM10 upregulates the production of ROS in cultured sebocytes and ORS cells and SHE inhibits the effects of PM10
The production of ROS was increased in a concentration-dependent manner (Fig. 5A, B). Next, we investigated whether ROS induction is mediated by AhR. The production of ROS was dramatically reduced in AhR siRNA-transfected cells (Fig. 5C, D). These results demonstrated that ROS production is mediated by AhR in cultured sebocytes and ORS cells. SHE (10 mg/ml) decreased the upregulation of ROS in PM10 (100 µg/ml)-treated sebocytes and ORS cells (Fig. 5E).
Fig. 5
(A) Particulate matter of <10 µm in size (PM10) upregulates the production of reactive oxygen species (ROS) in cultured sebocytes. (B) PM10 upregulates the production of ROS in outer root sheath (ORS) cells. (C) The upregulation of ROS was inhibited in cultured sebocytes by aryl hydrocarbon receptor (AhR) siRNA. (D) The upregulation of ROS was inhibited in ORS cells by AhR siRNA. (E) Siegesbeckia Herba extract (SHE) (10 mg/ml) decreased the upregulation of ROS in PM10 (100 µg/ml)-treated sebocytes and PM10 (100 µg/ml)-treated ORS cells. The data in the bar graphs represent mean±standard deviation from three independent experiments (*p<0.05).
PM10 upregulates sebum production in cultured sebocytes and SHE inhibits the effects of PM10
The increased expressions of peroxisome proliferator-activated receptor (PPAR)-γ, sterol regulatory element-binding protein 1a (SREBP1a), SREBP1c, and stearoyl-CoA desaturase (SCD) were observed in PM10-treated sebocytes in real-time PCR for 24 h (Fig. 6A). A concentration-dependent increase was observed in the expression of sebum production-related genes. We examined the expression of lipids through Oil Red O staining and Nile red staining after PM10 treatment of human scalp skin for 48 h to verify the production of sebum-related genes in sebocytes. Increased sebum production was observed in the sebaceous glands of the human scalp skin organ culture after treatment with 100 µg/ml of PM10 in Oil Red O and Nile red staining (Fig. 6B). SHE (10 mg/ml) downregulated the expressions of PPAR-γ, SREBP1a, SREBP1c, and SCD in PM10 (100 µg/ml)-treated sebocytes (p<0.05) (Fig. 6C).
Fig. 6
Particulate matter of <10 µm in size (PM10) upregulates sebum production in cultured sebocytes. (A) PM10 upregulates the expression of sebum production-related genes in cultured sebocytes. (B) Sebum production was increased in sebaceous glands of the human scalp skin organ culture after treatment with 100 µg/ml of PM10 in Oil Red O staining and Nile red staining (×20). (C) Siegesbeckia Herba extract downregulated these expressions in the cultured sebocytes (*p<0.05). PPAR: peroxisome proliferator-activated receptor, SREBP: sterol regulatory element-binding protein, SCD: stearoyl-CoA desaturase.
DISCUSSION
Nitrogen dioxide (NO2) stimulates AhR and activates human keratinocytes resulting in the release of increased amounts of proinflammatory cytokines through the creation of free radicals. In this study, the synthesis of ROS was increased in the sebocytes and ORS cells by PM10. AhR was upregulated in the sebocytes and ORS cells in this study. We found that the production of ROS was decreased after the inhibition of AhR with siRNA. AhR is a receptor for PM10 resulting in the production of ROS. SHE also reduced the upregulation of AhR and ROS in the cultured sebocytes and ORS cells. ROS then subsequently stimulates the release of proinflammatory mediators12. Oxidative stress induces complex biological processes that lead to the activation of transcription factors, such as activator protein 1 and NF-κB, cell growth and differentiation, and the degradation of the connective tissue of the dermis. The expression of NF-κB was increased in the sebocytes and ORS cells by PM10 in this study. SHE also reduced the upregulation of NF-κB in the cultured sebocytes and ORS cells. PM10 increases the expression of proinflammatory cytokines and MMP-1 in human epidermal keratinocytes13. In this study, the expression of proinflammatory mediators, such as IL-1α, IL-1β, IL-6, IL-8, and TNF-α, was upregulated in the sebocytes and ORS cells. Furthermore, the expression of MMPs in the cultured sebocytes and ORS cells was increased. Some studies have indicated that PM2.5 activated NF-κB and the induction of inflammatory genes, such as IL-1α, IL-1β, IL-6, and IL-814, 15. SHE also reduced the upregulation of IL-1α, IL-1β, IL-6, IL-8, and TNF-α and MMPs in the cultured sebocytes and ORS cells.
Interestingly, the present study showed that PM10 increased the production of sebum and the expression of sebum-related transcription factors, such as PPAR-γ, SREBP1a, SREBP1c, and SCD. Furthermore, SHE reduced the expression of the sebum production-related biomarkers in the cultured sebocytes. Increased exposure to PM2.5 was associated with an increased sebum secretion in human skin16. Some studies revealed that the prevalence of oily skin was considerably higher in areas with a high level of air pollution in China and Mexico17. In addition, some air pollutants decreased sebum secretion18. PM10 showed fluctuations in the sebum levels. Some studies highlighted that PM2.5 reduced sebum synthesis in human SZ95 sebocytes19.
Based on this study, PM10 can be considered in clinical area including hair loss or hair disease. Jun et al.20 reported particulate matters induce apoptosis through increased production of ROS and inflammatory cytokines in human hair follicular keratinocytes, leading to impairment of hair growth. Antioxidant and anti-inflammatory agents such as SHE may be useful for PM-related clinical areas. Lee and Lee21 described that susceptibility to environmental factors may provide a clinical evidence to support the pathogenesis of alopecia areata. New research, which was presented at the 28th European Academy of Dermatology and Venereology (EADV) Congress in Madrid, revealed air pollution can be linked to hair loss.
In conclusion, PM10 can affect the biologic process of sebocytes and ORS cells, including inflammation and sebum production and SHE can inhibit the effects of PM10 on sebocytes and ORS cells.
SUPPLEMENTARY MATERIALS
Supplementary data can be found via http://anndermatol.org/src/sm/ad-
PCR primers used in this studySupplementary Table 1
CONFLICTS OF INTEREST:The authors have nothing to disclose.
FUNDING SOURCE:This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute and funded by the Ministry of Health and Welfare, Republic of Korea (grant number: HP20C0004).
DATA SHARING STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.
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