Nutr Res Pract.  2021 Dec;15(6):686-702. 10.4162/nrp.2021.15.6.686.

Schisandrae Fructus ethanol extract attenuates particulate matter 2.5-induced inflammatory and oxidative responses by blocking the activation of the ROS-dependent NFκB signaling pathway

• Lee H
• Park C
• Kwon DH
• Hwangbo H
• Kim SY
• Kim MY
• Ji SY
• Kim DH
• Jeong JW
• Kim GY
• Hwang HJ
• Choi YH

• KMID: 2522650
• DOI: http://doi.org/10.4162/nrp.2021.15.6.686

Abstract

BACKGROUND/OBJECTIVES
Schisandrae Fructus, the fruit of Schisandra chinensis Baill., has traditionally been used as a medicinal herb for the treatment of various diseases, and has proven its various pharmacological effects, including anti-inflammatory and antioxidant activities. In this study, we investigated the inhibitory effect of Schisandrae Fructus ethanol extract (SF) on inflammatory and oxidative stress in particulate matter 2.5 (PM2.5)-treated RAW 264.7 macrophages.
MATERIALS/METHODS
To investigate the anti-inflammatory and antioxidant effects of SF in PM2.5-stimulated RAW 264.7 cells, the levels of pro-inflammatory mediator such as nitric oxide (NO) and prostaglandin E₂ (PGE₂ ), cytokines including interleukin (IL)-6 and IL-1β, and reactive oxygen species (ROS) were measured. To elucidate the mechanism underlying the effect of SF, the expression of genes involved in the generation of inflammatory factors was also investigated. We further evaluated the anti-inflammatory and antioxidant efficacy of SF against PM2.5 in the zebrafish model.
RESULTS
The results indicated that SF treatment significantly inhibited the PM2.5-induced release of NO and PGE₂ , which was associated with decreased inducible NO synthase and cyclooxygenase-2 expression. SF also attenuated the PM2.5-induced expression of IL-6 and IL-1β, reducing their extracellular secretion. Moreover, SF suppressed the PM2.5-mediated translocation of nuclear factor-kappa B (NF-κB) from the cytosol into nuclei and the degradation of inhibitor IκB-α, indicating that SF exhibited anti-inflammatory effects by inhibiting the NF-κB signaling pathway. In addition, SF abolished PM2.5-induced generation of ROS, similar to the pretreatment of a ROS scavenger, but not by an inhibitor of NF-κB activity. Furthermore, SF showed strong protective effects against NO and ROS production in PM2.5-treated zebrafish larvae.
CONCLUSIONS
Our findings suggest that SF exerts anti-inflammatory and antioxidant effects against PM2.5 through ROS-dependent down-regulating the NF-κB signaling pathway, and that SF can be a potential functional substance to prevent PM2.5-mediated inflammatory and oxidative damage.

Keyword

Particulate matter; oxidative stress; inflammation; reactive oxygen species; NF-kappa B

Figure

• 

  Fig. 1 Effect of SF on the production pro-inflammatory mediators and cytokines in PM2.5-stimulated RAW 264.7 macrophages. Cells were treated with the indicated concentrations of SF for 1 h and then stimulated with 50 µg/mL PM2.5 for 24 h. (A) The NO concentration in the culture medium was determined by the Griess reaction. (B-D) The PGE2 (B), IL-6 (C), and IL-1β (D) concentration was determined using commercial ELISA kits. The absorbance was measured using a microplate reader. The error bars represent the mean ± SD of 3 independent experiments.SF, Schisandrae Fructus ethanol extract; PM, particulate matter; NO, nitric oxide; PGE2, prostaglandin E2; IL, interleukin; ELISA, enzyme-linked immunosorbent assay.***P < 0.001 vs. PM2.5-unstimulated cells; #P < 0.05, ##P < 0.01 and ###P < 0.001 vs. PM2.5-stimulated cells.

• 

  Fig. 2 Effect of SF on the expression of pro-inflammatory enzymes and cytokines in PM2.5-stimulated RAW 264.7 macrophages. Cells were treated with the indicated concentrations of SF for 1 h and then stimulated with 50 µg/mL PM2.5 for 24 h. After treatment, total RNA and protein were extracted from the cells. The expression levels of iNOS, COX-2, IL-6, and IL-1β mRNA (A) and proteins (C) were measured by RT-PCR and Western blot analysis, respectively. GAPDH and actin and were used as internal controls for the RT-PCR and Western blot analyses, respectively. (B, D) Bands were quantified using ImageJ and normalized to GAPDH and actin, and the ratio was determined. Data are expressed as the mean ± SD of 3 independent experiments.SF, Schisandrae Fructus ethanol extract; PM, particulate matter; iNOS, inducible nitric oxide synthase; COX-2, cyclooxygenase-2; IL, interleukin; RT-PCR, reverse transcription-polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.***P < 0.001 vs. PM-unstimulated cells; #P < 0.05, ##P < 0.01 and ###P < 0.001 vs. PM-stimulated cells.

• 

  Fig. 3 Inactivation of NF-κB signaling pathway by SF in PM2.5-stimulated RAW 264.7 macrophages. Cells were treated with 400 µg/mL SF alone for 24 h or pre-treated with or without 400 µg/mL SF for 1 h before 50 µg/mL PM2.5 stimulation for 1 h. (A) For Western blot analysis, nuclear and cytosolic proteins were isolated, and the expression of NF-κB and IκB-a was investigated. Protein loading was confirmed by the analysis of lamin B or actin expression in each protein extract.(B) Bands were quantified using ImageJ and normalized to lamin B and actin, and the ratio was determined. Data are expressed as the mean ± SD of 3 independent experiments. (C) The cells were subjected to immunofluorescence staining with NF-κB p65 antibody and representative fluorescence images were acquired using a fluorescence microscope. Green fluorescence indicates the localization of NF-κB p65 and blue fluorescence by DAPI staining allows visualization of the nuclei (scale bar = 200 µM).SF, Schisandrae Fructus ethanol extract; PM, particulate matter; NF-κB, nuclear factor-kappa B; DAPI, 4′,6-diamidino-2-phenylindole.***P < 0.001 vs. PM-unstimulated cells; ###P < 0.001 vs. PM-stimulated cells.

• 

  Fig. 4 Inhibition of ROS generation by SF in PM2.5-stimulated RAW 264.7 macrophages. Cells were pre-treated with 400 µg/mL SF, 10 mM NAC or 20 µM JSH-23 for 1 h and then treated with 50 µg/mL PM2.5 for 1 h. (A) The DCF-DA-stained cells were collected, and then DCF fluorescence was analyzed by flow cytometry. (B) Data are given as the mean ± SD of 3 independent experiments. (C) ROS generation was also detected by a fluorescence microscope and representative fluorescence micrographs depicting ROS generation are presented. Green fluorescence indicates the intensity of ROS generation (scale bar = 200 µM).ROS, reactive oxygen species; SF, Schisandrae Fructus ethanol extract; NAC, N-acetyl cysteine; JSH-23, 4-methyl-N 1-(3-phenyl-propyl)-benzene-1,2-diamine; DCF-DA, 5,6-carboxy-2′,7′-dichlorofluorescein diacetate; N.S., not significant; PM, particulate matter.***P < 0.001 vs. PM2.5-unstimulated cells; ###P < 0.001 vs. PM2.5-stimulated cells.

• 

  Fig. 5 Role of ROS on the inhibitory effect of SF on PM2.5-induced NF-κB activation and inflammatory response. Cells were pre-treated with 400 µg/mL SF or 10 mM NAC for 1 h and then treated with 50 µg/mL PM2.5 for 1 h (A, B) or 24 h (C, D). (A, B) Nuclear and cytosolic proteins were isolated, and the expression of NF-κB and IκB-a was investigated. Protein loading was confirmed by the analysis of lamin B or actin expression in each protein extract. (B) Bands were quantified using ImageJ and normalized to lamin B and actin, and the ratio was determined. The NO (C) and IL-6 (D) concentration in the culture medium was determined by the Griess reaction and IL-6 ELISA kit, respectively. The absorbance was measured using a microplate reader. (B-D) Data are expressed as the mean ± SD of 3 independent experiments.ROS, reactive oxygen species; SF, Schisandrae Fructus ethanol extract; PM, particulate matter; NF-κB, nuclear factor-kappa B; NO, nitric oxide; IL, interleukin; ELISA, enzyme-linked immunosorbent assay.***P < 0.001 vs. PM-unstimulated cells; ##P < 0.01 and ###P < 0.001 vs. PM-stimulated cells.

• 

  Fig. 6 Inhibition of PM2.5-induced NO and ROS generation by SF in zebrafish larvae. Zebrafish at 3 dpf were treated with 50 µg/mL PM2.5 and placed in E3 media containing the indicated concentrations of SF for 24 h. The larvae were incubated with 5 µM DAF-FM-DA (A, B) or 20 µM DCF-DA (C, D) for NO and ROS detection and visualized using the CELENA® S Digital Imaging System (scale bar = 1,000 µm). (B, D) Relative fluorescence intensities were calculated and expressed compared to the untreated control. Each value indicates the mean ± SD of 3 independent experiments. Significant differences among the groups were determined.PM, particulate matter; NO, nitric oxide; ROS, reactive oxygen species; SF, Schisandrae Fructus ethanol extract; dpf, days post-fertilized; DAF-FM-DA, 4-amino-5-methylamino-2′7′-difluorofluorescein diacetate; DCF-DA, 5,6-carboxy-2′,7′-dichlorofluorescein diacetate.***P < 0.001 vs. PM2.5-unstimulated larvae; #P < 0.05 and ###P < 0.001 vs. PM2.5-stimulated larvae.

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