Niacinamide (NIA) is a water-soluble vitamin that is widely used in the treatment of skin diseases. Moreover, NIA displays antioxidant effects and helps repair damaged DNA. Recent studies showed that particulate matter 2.5 (PM2.5) induced reactive oxygen species (ROS), causing disruption of DNA, lipids, and protein, mitochondrial depolarization, and apoptosis of skin keratinocytes. Here, we investigated the protective effects of NIA on PM2.5-induced oxidative stress in human HaCaT keratinocytes. We found that NIA could inhibit the ROS generation induced by PM2.5, as well block the PM2.5-induced oxidation of molecules, such as lipids, proteins, and DNA. Furthermore, NIA alleviated PM2.5-induced accumulation of cellular Ca2+, which caused cell membrane depolarization and apoptosis, and reduced the number of apoptotic cells. Collectively, the findings show that NIA can protect keratinocytes from PM2.5-induced oxidative stress and cell damage.
Niacinamide (NIA), also known as nicotinamide, is a hydrophilic amide of vitamin B3 that is an important component in various cosmetics and medicines. NIA is found in a wide array of foods such as fish, mushroom, and nuts (Damian, 2010). As a typical medicine for treating pigmentary disorders, NIA blocks the melanosome migration between melanocytes and keratinocytes and suppresses skin pigmentation (Bissett
Due to excessive consumption of fossil fuels, air pollution has become a major health hazard for humans (Park
In this study, we focused on particulate matter <2.5 μm (PM2.5), which can attach to epidermal skin due to its small size. PM2.5 is known to stimulate the generation of reactive oxygen species (ROS) in keratinocytes (Hyun
NIA (Fig. 1A) was purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA) and dissolved in dimethylsulfoxide (DMSO). Diesel particulate matter NIST SRM 1650b (PM2.5, Sigma-Aldrich) was dissolved in DMSO and stored at a concentration of 25 mg/mL (Piao
HaCaT human keratinocytes from Cell Lines Service (Heidelberg, Germany) were cultured in Dulbecco’s Modified Eagle’s Medium (Life Technologies Co., Grand Island, NY, USA) with 10% heat-inactivated fetal calf serum at 37°C with 5% CO2.
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to test the cytotoxicity of NIA. Cells were seeded into a 24-well plate with 12.5, 25, 50, 100, or 200 μM of NIA. Then, MTT solution was added to each well and samples were incubated for 4 h. Finally, the solutions from DMSO and yield formazan crystals were detected at 540 nm using the scanning multi-well spectrophotometer (Piao
To investigate anti-oxidative stress effect of NIA, we used 2′,7′-dichlorofluorescein diacetate (DCF-DA, Sigma-Aldrich) staining. ROS generation was induced using PM2.5 (50 μg/mL). After 16 h of incubation, the cells were treated with 12.5, 25, 50, 100, or 200 μM of NIA and PM2.5, followed by addition of 25 μM DCF-DA. The fluorescence of DCF-DA was measured using a flow cytometer (Becton Dickinson, Mountain View, CA, USA) (Piao
To determine the ratio of intracellular NADP and NADPH, we used NADP/NADPH assay kit (Abcam, Cambridge, UK) according to the manufacturer’s instructions.
To detect superoxide levels in cells, we measured the DHE oxidation. Cells were treated with NIA and PM2.5 and incubated with DHE (10 μM) for 30 min. After incubation, the fluorescence intensity was analyzed by a confocal microscope.
Lipid oxidative stress was investigated using two methods. The cells were dyed with 5 μM diphenyl-1-pyrenylphosphine (DPPP, Sigma-Aldrich) and fluorescence was analyzed using the confocal microscope. Harvested cells were subjected to analysis using the 8-Isoprostane ELISA kit (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer’s instructions (Zhen
The harvested cells were used for detecting protein oxidation with an OxiselectTM Protein Carbonyl ELISA kit (Cell Biolabs, San Diego, CA, USA) according to the manufacturer’s instructions.
To quantify oxidative DNA damage, we determined the level of 8-oxoG through the Bioxytech 8-OHdG ELISA kit (OXIS Health Products, Portland, OR, USA) according to the manufacturer’s instructions. For image analysis, the cells were fixed on a chamber slide and stained with avidin-tetramethylrhodamine isothiocyanate (TRITC) (1:200) conjugate (Sigma-Aldrich), and fluorescence images were obtained using a confocal microscope (Piao
The comet assay was used to detect DNA damage induced by PM2.5. Harvested cells were dispersed in low-melting agarose (1%). Then, the mixtures were solidified on microscopic slides, and the slides were immersed in lysis buffer (2.5 M NaCl, 100 mM Na-EDTA, 10 mM Tris, 1% Trion X-100, and 10% DMSO, pH 10) for 1 h at 4°C. After electrophoresis, the slides were stained with ethidium bromide, and the percentage of the comet tail fluorescence and the tail length (50 cells per slide) was determined using a fluorescence microscope equipped with an image analysis software (Kinetic Imaging, Komet 5.5, Liverpool, UK) (Park
To determine the intracellular Ca2+ in viable cells, the cells were co-cultured with 10 μM fluo-4-acetoxymethyl ester (Fluo-4-AM, Sigma-Aldrich) dye for 30 min, and green fluorescence in the confocal micrographs was quantified.
To visualize the changes in membrane potential, the cells were stained with 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylben zimidazolylcarbocyanine iodide (JC-1, Invitrogen, Carlsbad, CA, USA) and were analyzed by confocal microscopy.
To observe apoptotic bodies, we used the nuclear-specific dye, Hoechst 33342 (Sigma-Aldrich). Cells were pre-treated with 100 μM NIA, treated with PM2.5 for 24 h, and then stained with Hoechst 33342 (10 μM). Nuclei were visualized under a fluorescence microscope equipped with a Cool SNAP-Pro color digital camera (Media Cybernetics, Silver Spring, MD, USA) (Han
Data are shown as mean ± standard error, and were analyzed using the Tukey’s test and analysis of variance (ANOVA) by the Sigma Stat (v12) software (SPSS, Chicago, IL, USA).
Here, we used PM2.5 at a concentration of 50 μg/mL for ROS induction. This concentration was selected based on the findings of our recent study investigating whether PM2.5 induced oxidative stress. In that study, we measured ROS generation at various concentrations (25–100 μg/mL) of PM2.5, and 50 μg/mL PM2.5 was found to be optimal concentration to clearly observe oxidative stress-induced cell damage and inflammatory response (Piao
Evaluation of the NADPH oxidase (NOX) activity (NADP/NADPH ratio) showed that PM2.5 increased the oxidation of NADPH, which was reversed by NIA pre-treatment (Fig. 2A). In the DCF-DA staining assay, NIA inhibited PM2.5-induced fluorescence in the cells, demonstrating that NIA protected cells from PM2.5-induced ROS generation (Fig. 2B). Similarly, DHE staining revealed that superoxide generation induced by PM2.5 was blocked by NIA (Fig. 2C). These results further indicated that NIA exerted antioxidant effects in keratinocytes.
The detection of phosphine oxide (DPPP oxide) using the DPPP probe indicated that PM2.5 significantly induced lipid peroxidation, and pretreatment with NIA decreased the fluorescence emitted by the probe (Fig. 3A). Moreover, the levels of 8-isoprostane, a specific indicator of lipid peroxidation, revealed that NIA suppressed PM2.5-induced peroxidation (Fig. 3B). These results showed that NIA rescued cells from PM2.5-induced lipid oxidation.
The levels of protein carbonylation, a specific indicator of protein oxidation, revealed that NIA significantly inhibited PM2.5-induced oxidative protein modification (Fig. 4). These results showed that NIA rescued cells from PM2.5-induced protein carbonylation.
The 8-OHdG assay was used to assess oxidative damage on DNA caused by PM2.5 (Fig. 5A). Notably, NIA reduced 8-OHdG generation induced by PM2.5. Furthermore, confocal microscopy showed that the level of 8-oxoG in PM2.5-exposed cells was the highest, suggesting severe DNA lesions formed via avidin-TRITC binding. Consistent with other results, NIA ameliorated the DNA lesions (Fig. 5B). The protective effect of NIA on DNA damage was also observed in the comet assy. Length of comet tails and the percentage of tail fluorescence were increased by PM2.5, but decreased following NIA pretreatment (Fig. 5C). Collectively, these results illustrated that NIA protected against DNA damage induced by PM2.5.
Intracellular Ca2+, tracked by Fluo-4-AM, was detected by confocal microscopy. The image analysis revealed that PM2.5 stimulated excessive Ca2+, which could be reduced by NIA treatment (Fig. 6A). JC-1 staining was used to determine Δψm, with red and green fluorescence representing polarization and depolarization, respectively. The images obtained from confocal microscopy showed that Δψm polarization and depolarization were decreased and increased by PM2.5 treatment, respectively; however, they were reversed by NIA pretreatment (Fig. 6B). PM2.5 also promoted apoptotic bodies, when observed using Hoechst 33342 staining, and NIA pretreatment reduced their numbers (Fig. 6C). These results proved that PM2.5 disrupted the homeostasis of intracellular Ca2+ levels and accelerated cell apoptosis, but NIA exerted cytoprotective effects against these PM2.5-induced damages.
The skin is the outermost organ and acts as the first protective layer from air pollution. Current studies indicate that air pollutants damage skin via two main routes. The first route is from the outside to inside, whereby PM penetrates the skin (including keratinocytes), and the second route is from the inside to the outside, whereby toxic effects in the lung subsequently influence the skin (Krutmann
A previous study showed that ROS is involved in various biological processes, including oxygen sensing, cell growth, cell differentiation, and cell death (Touyz
Next, we examined the effect of PM2.5 on the three main molecules in cells, namely lipids, proteins, and DNA. ROS is known to damage lipids and proteins via lipid peroxidation and protein carbonylation, respectively (Hyun
Calcium plays a key role in cell survival, as well as it can improve apoptosis. Ca2+ mediated pro-apoptotic action is a response to many endogenous organelles, including mitochondria (Hajnoczky
Taken together, our findings show that PM2.5 notably aggravated skin cell damage by inducing ROS generation, disrupting cellular components, and activating apoptotic pathways. However, cells pre-treated with NIA were protected from the ROS-induced lipid peroxidation, protein carbonylation, and DNA damage (Fig. 7). NIA also inhibited PM2.5-induced apoptosis by maintaining both Ca2+ levels and mitochondrial membrane potential in a steady state. These results suggest that NIA can protect against PM2.5-induced skin damage.
The authors declare that there are no conflicts of interest.
This work was supported by grant from the Basic Research Laboratory Program (NRF-2017R1A4A1014512) by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP). This research was supported by the 2019 scientific promotion program funded by Jeju National University.