J Bacteriol Virol.  2019 Dec;49(4):153-161. 10.4167/jbv.2019.49.4.153.

Aralia cordata Extract Activates NF-κB and MAPK Signaling Pathways and Induces Pro-inflammatory Changes in RAW264.7 Macrophages

Affiliations
  • 1Gyeongbuk Institute for Bio industry, Andong-si, Gyeongbuk 36618, South Korea.
  • 2Department of Animal Science and Technology, Suncheon National University, 255 Jungang-ro, Suncheon-si, Jeollanam-do 57922, South Korea.
  • 3Division of Biotechnology, College of Environmental & Bioresources, Jeonbuk National University, Iksan-si, Jeollabukdo 54596, South Korea. leesangm@jbnu.ac.kr

Abstract

Macrophages play essential roles in innate immune responses by producing various immune mediators. Therefore, modulating macrophage function is an attractive strategy to treat immune disorders. Aralia cordata var. continentalis (AC), known as "Dokwhal" in Korea, possesses various biological and medicinal functions, including immunomodulation. The present study investigated the effect of the hot water extract of AC (HAC) on RAW264.7 murine macrophages. When these cells were treated with HAC, nitric oxide production and inducible nitric oxide synthase expression was induced dose-dependently. In addition, HAC treatment triggered the secretion of innate immune cytokines, such as TNF-α and IL-6. Phagocytosis, measured by FITC-dextran internalization showed that HAC stimulated the phagocytic activity of macrophages. Furthermore, HAC promoted the production of reactive oxygen species in RAW264.7 cells, determined by CM-H2DCFDA. In addition, the immunoblot analysis of intracellular signaling proteins revealed that NF-kB and MAPK signaling pathways, which are important signaling mediators of inflammation, are upregulated by HAC. In conclusion, these findings suggested that HAC can stimulate macrophage activity, and NF-kB and MAPK signaling pathways might be involved in the immunostimulatory effects of HAC.

Keyword

Aralia cordata; RAW264.7; Immunostimulatory effect; NF-κB; MAPK

MeSH Terms

Aralia*
Cytokines
Immune System Diseases
Immunity, Innate
Immunomodulation
Inflammation Mediators
Interleukin-6
Intracellular Signaling Peptides and Proteins
Korea
Macrophages*
NF-kappa B
Nitric Oxide
Nitric Oxide Synthase Type II
Phagocytosis
Reactive Oxygen Species
Water
Cytokines
Inflammation Mediators
Interleukin-6
Intracellular Signaling Peptides and Proteins
NF-kappa B
Nitric Oxide
Nitric Oxide Synthase Type II
Reactive Oxygen Species
Water

Figure

  • Figure 1 Effect of HAC on cell viability, nitrite accumulation, and iNOS expression in RAW264.7 cells. (A) Cells were treated with different concentrations of HAC for 24 h, and their viability was assessed by the MTT assay. The results of independent experiments were averaged and are expressed as the percentage of cell viability relative to the untreated control group. (B) Nitric oxide production was measured in the culture medium by the Griess reaction, and NaNO2 was used as a standard. (C) iNOS expression was determined in RAW264.7 cells treated with LPS or HAC by immunoblotting. Data are presented as the mean ± SEM of three independent experiments. **p<0.01, ***p<0.001 indicate significant difference from the untreated control group by the Student's t-test.

  • Figure 2 Effects of HAC on cell morphology, cytokine and ROS production, and phagocytosis. RAW264.7 cells were plated and then treated with the indicated concentration of HAC for 24 h. (A) Cell morphology was observed by light microscopy. Images were obtained with a 100×objective. (B) IL-6 and TNF-α levels were determined using an ELISA kit, as described in the text. (C) After incubation, the cells were washed twice with PBS and labeled with 20 µM CM-H2DCFDA for 30 min. The intracellular ROS level was analyzed by flow cytometry. (D) The cells were incubated with DMEM containing 1 mg/ml FITC-dextran, 2% FBS, and the indicated concentration of HAC for 6 h. After incubation, phagocytosis activity was measured by flow cytometry. The results are expressed as mean ± SEM of three independent experiments. *p<0.05, *p<0.01, ***p<0.001 indicate significant difference from the untreated control group by the Student's t-test.

  • Figure 3 Effects of HAC on NF-κB and MAPK pathways. RAW264.7 cells were incubated at a density of 1×106 cells/well in a 12-well plate and stimulated with 200 µg/ml of HAC or 1 µg/ml of LPS for different times. Total cell lysates were collected and hybridized with a specific antibody detecting the phosphorylated form or total amount of NF-κB and MAPK. The results are representative of three independent experiments.


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