Resveratrol prevents nanoparticles-induced inflammation and oxidative stress via downregulation of PKC-α and NADPH oxidase in lung epithelial A549 cells

Resveratrol, bovine serum albumin (BSA), apocynin, diphenylene iodonium (DPI), N-acetyl cysteine (NAC), L-NG-Nitroarginine Methyl Ester (L-NAME), dimethyl sulfoxide (DMSO), 2′,7′-dichloro-dihydrofluorescein diacetate (H₂DCF-DA), 12-(2-Cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole (Gö6976), (+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate (MK-801), verapamil, 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT), Hoechst 33,342, Triton X-100, and mouse antibody against β-actin and iNOS were obtained from Sigma–Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, amphotericin B, streptomycin, trypsin-EDTA, and 5,5′,6,6′-tetrachloro- 1,1′,3,3′-tetraethyl benzimidazolylcarbocyanine iodide (JC-1) were obtained from Invitrogen (Carlsbad, CA, USA). All materials for SDS–PAGE were obtained from Bio-Rad (Hercules, CA, USA). NS-398, mouse antibodies against COX-2, rabbit antibody against PKC-α, p67^phox, and all horseradish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse antibody against Nox2 was obtained from BD Bioscience (San Jose, CA, USA). Enhanced chemiluminescence reagent and polyvinylidene difluoride (PVDF) membranes were purchased from PerkinElmer Life and Analytical Sciences (Boston, MA, USA). The LDH cytotoxicity assay kit was purchased from G-Biosciences (St. Louis, MO, USA).

The CBNPs (Printex90®, 14 nm) were obtained from Evonik Industries/Degussa (Frankfurt, Germany). Stock suspensions of the CBNPs were made at a concentration of 1 mg/mL in DMEM, sonicated for 30 min before used.

Human epithelial carcinoma cell line (A549) was purchase from Food Industry Research and Development Institute (Hsin-Chu, Taiwan). Cells were cultured in DMEM containing 10% (v/v) heat-inactivated FBS, 2 mM glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, and 0.25 mg/mL amphotericin B at 37 °C in a humidified incubator under 5% CO₂ and 95% air.

To investigate the effects of CBNPs on A549 cells, cells were treated with CBNPs (1–100 μg/mL) for 24 h. To investigate the mechanisms of CBNPs-induced inflammation and oxidative stress, cells were pre-treated with verapamil (100 μM), MK801 (100 μM), Gö6976 (10 μM), DPI (10 μM), apocynin (500 μM), NAC (5 mM), L-NAME (50 μM), or NS398 (1 μM) for 1 h before addition of CBNPs (25 μg/mL) for 24 h. To investigate the protective effects of resveratrol in CBNPs-treated A549 cells, cells were pre-treated with resveratrol (1, 5, and 10 μM) for 1 h before addition of CBNPs (25 μg/mL) for 24 h. The control group in this study were cells without any treatment.

The cell viability was determined by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay and lactate dehydrogenase (LDH) assay as previous described [26]. The principle of MTT assay is based on the cleavage of tetrazolium ring of MTT by dehydrogenases in active mitochondria of living cells as an estimate on cell viability. In MTT assay, cells were treated with CBNPs (1–100 μg/mL) for 24 h or pre-treated with resveratrol (1, 5, and 10 μM) for 1 h before addition of CBNPs (25 μg/mL) for 24 h, the medium was replaced with MTT at a final concentration of 0.5 mg/mL for 3 h at 37 °C and 5% CO₂. The formazan crystals in the cells were solubilized with 100 μL DMSO. Absorbance was read at 560 nm on a microplate reader. Besides, LDH is a soluble cytosolic enzyme in cells that can be released into culture medium when cell death occurs; therefore, the presence of this enzyme in the culture medium can be used as a cell death marker. In LDH assay, culture medium was collected and assayed for LDH activity using a cytotoxicity detection kit. Briefly, the release of LDH was measured with a coupled enzymatic reaction that results in the conversion of a tetrazolium salt into red-colored formazan. The amount of formazan formed correlated with LDH activity. The formazan product was measured with a microplate reader at 490 nm.

The change in ΔΨm was evaluated by JC-1 fluorescence staining assay [27] which was determined by the ratio between red and green fluorescence. The red JC-1 fluorescence were detected by excitation/emission at 540/570 nm, and green JC-1 fluorescence were detected by excitation/emission at 495/520 nm by Coulter CyFlow Cytometer (Partec, Germany). The production of ROS was detected by fluorescence assay that involved H₂DCF-DA staining [28]. After 24 h of drug treatments, the A549 cells were stained with 10 μM H₂DCF-DA at 37 °C. After 30 min of incubation, the A549 cells were detached by trypsin-EDTA and washed with PBS. One hundred thousand (1 × 10⁵) cells were analyzed and the DCF fluorescence was detected by excitation at 495 nm and emission of 520 nm by Coulter CyFlow Cytometer (Partec, Germany).

Nitrite generation was an indicator of nitric oxide (NO) release measured using the Griess reagent (1% sulfanilamide and 0.1% N-1-naphthylethylenediamide in 5% phosphoric acid) [29]. Briefly, the medium from treated cells was collected. and 50 μL was incubated for 10 min at room temperature under dark conditions with 50 μL of Griess reagent. The absorbance was measured at 540 nm (OD540) and the concentration was calculated using a sodium nitrite standard reference curve [29]. The PGE₂ production in the supernatant was detected using EIA kit (Cayman Chemical, USA), following the manufacturer’s guide [30]. Supernatant was collected after 24 h of CBNPs (1, 10, or 25 μg/mL) treatment with or without Gö6976 (10 μM), DPI (10 μM) or resveratrol (1, 5, or 10 μM) pretreatment, and the absorbance measured at 405 nm (OD405).

After 24 h of CBNPs exposure, cells were washed with PBS and fixed with 4% paraformaldehyde for 30 min, and further incubated in a permeabilizing solution (0.1% Triton X-100) for 5 min. Cells were then stained with 5 μg/mL Hoechst 33,342 for 10 min to detect DNA condensation, and nuclear fragmentation, which were features of apoptotic cells [31]. The cells were observed under fluorescence microscopy (Nikon, Japan).

Western blotting analysis was used to determine protein expressions. After CBNPs (1, 10, or 25 μg/mL) treatment with or without verapamil (100 μM), MK801 (100 μM), Gö6976 (10 μM), DPI (10 μM), apocynin (500 μM), NAC (5 mM), L-NAME (50 μM), NS398 (1 μM), or resveratrol (1, 5, or 10 μM) pretreatment, cytosolic or membrane protein extracts of A549 cells were collected by using lysis buffer (Thermo Scientific, Waltham, MA, USA) or commercial kit (BioVision, Mountain View, CA, USA) respectively as described previously [30]. The lysates were centrifuged at 15000×g for 30 min at 4 °C and the supernatant containing proteins was collected. The proteins were reversed by SDS-polyacrylamide gel electrophoresis after using the Bio-Rad protein assay kit to determine the total protein concentration, then the protein bands were transferred onto polyvinylidene difluoride (PVDF) membranes and incubated with TBST (50 mM Tris–HCl, pH 7.6, 150 mM NaCl, 0.1% Tween 20) containing 5% non-fat milk for 1 h at room temperature to block the non-specific binding sites. Following that, the PVDF membranes were incubated overnight at 4 °C with one of the following specific primary antibodies: rabbit anti-PKC-α (1: 1000), rabbit anti-Nox2 (1: 1000), rabbit anti-p67^phox (1: 1000), mouse anti-iNOS (1:500), goat anti-COX-2 (1: 1000), and mouse anti-β-actin (1: 10,000). After six wash cycles with TBST at 5 min intervals, membranes were incubated with one of the following secondary antibodies: goat anti-rabbit IgG-HRP (1: 1000), goat anti-mouse IgG HRP (1: 1000), or donkey anti-goat IgG HRP (1: 5000) for 1 h at room temperature. After six wash cycles with TBST, the protein bands were stained with the enhanced chemiluminescence reagent.

Data are shown as the mean ± standard error of the mean (S.E.M) from six independent experiments (n = 6). A one-way analysis of variance (ANOVA) followed by Tukey’s test was used for all pair comparisons. A value of P < 0.05 was considered as statistically significant. Data were analyzed with the Statistical Package for Social Sciences (SPSS, Chicago, IL, USA).

To examine the cytotoxic effects of CBNPs on lung epithelial cells, cultured A549 were treated with various concentrations of CBNPs for 24 h. Results from MTT (Fig. 1a) and LDH (Fig. 1b) assay indicated that CBNPs (10–100 μg/mL) significantly decreased cell viability of A549 cells. CBNPs also decreased ΔΨm of A549 cells (Fig. 1c). Moreover, CBNPs-induced nuclear condensation of A549 cells was observed by using Hoechst 33,342 staining (Fig. 1d).

Next, we examined the effects of CBNPs on PKC-α and Nox activation. As shown in Fig. 2a, CBNPs produced its peak effect on PKC-α activation at 1 h (421.33 ± 27.75% of control). Accordingly, cells were incubated with different concentrations (1, 10 and 25 μg/mL) of CBNPs for 1 h, and the results suggested that CBNPs induced PKC-α activation occurred in a concentration-dependent manner (Fig. 2b). Also, CBNPs (10 and 25 μg/mL) enhanced epithelial Nox2 expression (Fig. 2c) and induced p67^phox membrane translocation (Fig. 2d).

As shown in Fig. 3, CBNPs up-regulated inflammatory protein expression of iNOS (Fig. 3a) and COX-2 (Fig. 3c), and increased their downstream products NO (Fig. 3b) and PGE₂ (Fig. 3d) respectively. The CBNPs-induced ROS production was also measured by using H₂DCF-DA staining and detecting the DCF fluorescence. Results indicated that CBNPs significantly increased epithelial ROS production (Fig. 3e). We further examined the role of PKC-α and Nox on CBNPs-induced inflammation and oxidative stress via measuring NO, PGE₂, and ROS production. Results demonstrated that both PKC-α inhibitor Gö6976 (10 μM) and Nox inhibitor DPI (10 μM) attenuated CBNPs-induced production of NO (Fig. 3b) and PGE₂ (Fig. 3d). CBNPs-induced production of ROS was also inhibited by Gö6976 and DPI pretreatment (Fig. 3e).

To clarify the role of PKC-α on CBNPs-induced Nox activation, L-type Ca²⁺ channel blocker verapamil, and NMDA receptor antagonist MK-801 were added to A549 cells 1 h before CBNPs (25 μg/mL) treatment. Results indicated that both verapamil and MK-801 treatments attenuated PKC-α expression induced by CBNPs (Fig. 4a). Moreover, PKC-α inhibition (by verapamil, MK-801, and Gö6976) attenuated CBNPs-induced Nox2 expression (Fig. 4b). However, Nox inhibitors (DPI and apocynin) could not attenuate PKC-α activation caused by CBNPs (Fig. 4c).

To examine the roles of Nox and ROS on CBNPs-induced inflammation, Nox inhibitor DPI and ROS scavenger NAC were used. The present results indicated that DPI (10 μM) could attenuate CBNPs-induced expression of iNOS (Fig. 5a) and COX-2 (Fig. 5b). Similarly, CBNPs-induced expression of iNOS and COX-2 could be attenuated by NAC (5 mM) pretreatment (Figs. 5a and b). Moreover, CBNPs-induced ROS production could be attenuated by DPI, L-NAME (NOS inhibitor) and NS-398 (COX-2 inhibitor) (Fig. 5c). However, L-NAME and NS-398 could not significantly attenuate CBNPs-induced Nox2 activation (Fig. 5d).

Further, we investigated the potential protective effects of the known anti-inflammatory and anti-oxidative agent, resveratrol, on CBNPs-induced epithelial cytotoxicity. A549 cells were treated with resveratrol for 1 h before the addition of CBNPs (25 μg/mL) for 24 h. Results from MTT and LDH assays indicated that resveratrol decreased cytotoxicity induced by CBNPs (Figs.6a and b).

We next evaluated the effects of resveratrol on CBNPs-induced epithelial inflammation. Results indicated resveratrol not only decreased iNOS expression (Fig. 7a) and NO production (Fig. 7b), but also decreased COX-2 expression (Fig. 7c) and PGE₂ production (Fig. 7d) in CBNPs-treated A549 cells.

We further investigated the protective effects of resveratrol on CBNPs-induced PKC-α expression, Nox2 activation and ROS production on A549 cells. Resveratrol attenuated PKC-α expression (Fig. 8a), Nox2 expression (Fig. 8b), p67^phox membrane translocation (Fig. 8c), and ROS production (Fig. 8d) induced by CBNPs.