Germinated Rhynchosia nulubilis Fermented with Lactobacillus pentosus SC65 Reduces Particulate Matter Induced Type II Alveolar Epithelial Apoptotic Cell Death

Abstract

Particulate matter (PM) is a significant environmental pollutant that promotes respiratory diseases, including lung injury and inflammation, by inducing oxidative stress. Rhynchosia nulubilis (black soybean) is traditionally used to prevent chronic respiratory disease via inducing antioxidant and anti-inflammatory effects. To investigate the effects of Lactobacillus pentosus SC65 fermented GR (GR-SC65) and Pediococcus pentosaceus ON81A (GR-ON81A) against PM-induced oxidative stress and cell death in A549 cells, we performed the 2-7-dichlorodihydrofluorescein diacetate and cell counting kit-8 assays, as well as Hoechst 33342 and propidium iodide staining and western blotting. GR-SC65 showed the highest total polyphenolic contents and 1,1-diphenyl-2-picrylidrazil radical scavenging activity among lactic acid bacteria-fermented GRs (p < 0.001 vs. GR). Four soy peptides, β-conglycinin breakdowns (INAENNQRNF, ISSEDKPFN, LAFPGSAQAVEK, and LAFPGSAKDIEN), were detected in GR-SC65, but not in GR. In GR-SC65, PM-induced A549 cell death was less than that observed in GR-ON81A and GR (p < 0.001 vs. PM-treated group). GR-SC65 significantly decreased intracellular reactive oxidative species (ROS) when compared with PM (*** p < 0.001 vs. PM). GR-SC65 decreased the levels of BAX, active caspase-9, -3, and poly ADP-ribose polymerase (PARP) proteins (^#p < 0.01, ^###p < 0.001 vs. PM), while increasing the level of BCL-2 protein, a mitochondrial anti-apoptotic protein (^###p < 0.001 vs. PM). Our findings indicate that GR-SC65 inhibited PM-induced cell death by suppressing the levels of ROS, active caspase-9 and -3, and PARP proteins, while enhancing the level of BCL-2 protein in type II alveolar epithelial A549 cells. Therefore, GR-SC65 might be a potential therapeutic and preventive agent against PM-induced lung injury.

Keywords: A549 cells; PM < 11; ROS; apoptosis; cell death; germinated Rhynchosia nulubilis fermented with Lactobacillus pentosus SC65.

Figure 1

Comparison of diphenyl picrylhydrazyl (DPPH) radical scavenging activity in GR fermented with different lactic acid bacteria strains (L. pentosus SC65 and P. pentosaceus ON81A). Results from three independent experiments are expressed as means ± standard deviation (SD). Statistically significant differences between groups were analyzed using one-way ANOVA/Dunnett’s t-test (*** p < 0.001 vs. GR).

Figure 2

Effects of particulate matter (PM) on the viability and morphology of A549 cells. (A) A549 cells were exposed to control or 25, 50, and 100 μg/mL of PM in dimethyl sulfoxide (DMSO). The viability of A549 cells exposed to 25, 50, and 100 μg/mL PM (<11 μm) was determined by the cell counting kit-8 (CCK-8) assay. Values are expressed as mean value ± standard deviation (SD), based on triplicate samples from three independent experiments. Statistically significant differences between groups were analyzed using one-way ANOVA/Dunnett’s t-test (* p < 0.05, ** p < 0.01, and *** p < 0.001 vs. Control). (B) PM-exposed A549 cells were observed under the microscope and charge-coupled device (CCD) camera after exposure to PM (<11 μm). Arrows indicate morphological changes. Morphology of A549 cells exposed to PM was examined using Nikon Eclipse Ti microscopy (Nikon Instruments Incorporated, Melville, NY, USA) and a CCD camera (Point Grey Research Inc., Richmond, BC, Canada) (Magnification 100×, Scale bar 100 μm).

Figure 3

Effect of GR-SC65 on A549 cell viability exposed to particulate matter (PM). (A) Effect of GR, GR-SC65, and GR-ON81A on the viability of cultured A549 cells exposed to PM (100 μg/mL). Based on the CCK-8 assay results, 100 μg/mL PM decreases the viability of A549 cells when compared with the untreated cells, but pretreatment with 100 μg/mL GR-SC65 for 1 h increases the viability of the A549 cells exposed to PM (100 μg/mL). Data are expressed as the mean value ± standard deviation (SD). All values are means obtained three independent experiments. Statistically significant differences between groups were analyzed using one-way ANOVA/Dunnett’s t-test (^### p < 0.001 vs. untreated-A549 cells), (*** p < 0.001 vs. PM-treated A549 cells). Statistically significant differences between two groups were analyzed using the two-sample t-test. (B) Viability of the cultured A549 cells exposed to 30–300 μg/mL GR, GR-SC65, and GR-ON18A was determined by the CCK-8 assay. The CCK-8 assay results revealed that incubation for 48 h with 300 μg/mL GR and 300 μg/mL GR-ON18A decreases the viability of A549 cells. Data are expressed as the mean ± standard deviation (SD). All values are means obtained from three independent experiments. Statistically significant differences between groups were analyzed using one-way ANOVA/Dunnett’s t-test (^### p < 0.001 vs. untreated-A549 cells).

Figure 4

Scavenging effect of GR-SC65 on PM-induced intracellular ROS in A549 cells. (A) Intracellular ROS were detected by fluorescence microscopy (Nikon Eclipse Ti microscope, Point Grey Research, Richmond, BC, Canada) after 2-7-dichlorodihydrofluorescein diacetate (DCF-DA) staining. Tert-Butyl hydroperoxide (TBHP), a ROS inducer, was used as a positive control. (B) ROS scavenging effect of 30−300 μg/mL of GR and GR-SC65 on PM-induced ROS in A549 cells. The ROS degeneration ability was measured using DCF-DA, as described in the Methods section, at a wavelength of 485/535 (Ex/Em). Statistically significant differences between groups were analyzed using one-way ANOVA/Dunnett’s t-test (^### p < 0.001 vs. untreated-A549 cells and *** p < 0.001 vs. PM). PM, particulate matter; ROS, reactive oxygen species; DCF-DA, 2,7-dichloro-dihydro-fluorescein diacetate.

Figure 5

GR-SC65 attenuates the particulate matter (PM)-induced apoptotic cell death in A549 cells. (A) A549 cells were treated with each sample for 24 h. Apoptotic cells were detected by Hoechst 33342 and propidium iodide (PI) nuclear staining. Apoptotic cells stained by Hoechst 33342 and PI were detected by confocal microscopy with Metamorph software (Universal Imaging, West Chester, PA, USA; Magnification = 200×, Scale bar = 100 μm). White arrows indicate the apoptotic, shrunken, and hyperchromic cells. Statistically significant differences between groups were analyzed using one-way ANOVA/Dunnett’s t-test (^### p < 0.001 vs. untreated-A549 cells), (*** p < 0.001 vs. PM-treated A549 cells). (B) The purple merged images with both Hoechst 33342 and PI dye indicate late apoptotic cells or necrotic cells. (C) Relative apoptotic or necrotic cells were calculated as a percentage of the total number of cells. Relative apoptotic cells (%) = (the number of Hoechst 33342 positive with chromatin condensation and PI-positive cells/total cells counted with Hoechst 33342 staining) × 100. Relative necrotic cells (%) = (the number of Hoechst 33342 positive with nucleus swelling and PI-positive cells/total cells counted with Hoechst 33342 staining) × 100. Data are expressed as the mean value ± standard deviation (SD). All values are means obtained from three independent experiments. Statistically significant differences between groups were analyzed using one-way ANOVA/Dunnett’s t-test (^### p < 0.001 vs. control), (*** p < 0.001 vs. PM).

Figure 6

GR-SC65 inhibits particulate matter (PM)-induced A549 cell apoptosis through apoptotic pathways. A549 cells were pre-treated with 100 μg/mL GR or GR-SC65 for 1 h, followed by treatment with 100 μg/mL PM for 24 h. Whole-cell lysates were processed for western blot analysis and probed with indicated antibodies. Active caspase-3, active caspase-9, active PARP, BAX, and BCL-2 protein expression levels in A549 cells were detected using western blotting. Relative intensity is shown as the means ± standard deviation (SD) of three independent experiments. ^# p < 0.05 and ^### p < 0.001 using ANOVA test for the comparison between control and PM-treated A549 cells. *** p < 0.001, ** p < 0.01, and * p < 0.05 using ANOVA test for the comparison between PM-treated A549 cells and other GR or GR-SC65 treated A549 cells. PARP, poly (ADP-ribose) polymerase.

Figure 7

Soy peptide qualification in GR and GR-SC65 by LC/MS analysis (A) LC/MS chromatogram of GR and GR-SC65. Peak numbers refer to (B). (B) Peptides quantified by in GR and GR-SC65 by LC/MS. LC/MS, liquid chromatography-mass spectrometry.

Figure 8

GR-SC65 suppresses the PM-induced ROS-dependent apoptosis pathway. GR-SC65 suppresses PM-induced ROS (hydrogen peroxide, superoxide anions, and hydroxyl radicals). The levels of BCL-2 proteins are upregulated by GR-SC65. GR-SC65 also inhibits the activation of BAX, caspase-9, caspase-3, and PARP, all downstream signaling molecules. The brown arrow indicates the inhibitory activity of GR-SC65 through the PM-induced ROS-mediated apoptosis pathway. PM, particulate matter; ROS, reactive oxygen species; PAHs, polycyclic aromatic hydrocarbons; PARP, poly (ADP-ribose) polymerase; NOX2, NADPH oxidase 2.

Figure 9

Schematic diagram of PM extraction. PM sampling was performed at the Functional Food Laboratory at Gachon University for 12 months (June 2016–June 2017; Seoungnam si, Gyeonggi-do, Republic of Korea). PM samples were filtered using an 11 μm pore-size filter paper (1001−110, Whatman, Maidstone, Kent, England).