Nutr Res Pract.  2022 Dec;16(6):685-699. 10.4162/nrp.2022.16.6.685.

Immunostimulatory activity of hydrolyzed and fermented Platycodon grandiflorum extract occurs via the MAPK and NF-κB signaling pathway in RAW 264.7 cells

• Jung JI
• Lee HS
• Kim SM
• Kim S
• Lim J
• Woo M
• Kim EJ

• KMID: 2536504
• DOI: http://doi.org/10.4162/nrp.2022.16.6.685

Abstract

BACKGROUND/OBJECTIVES
Platycodon grandiflorum (PG) has long been known as a medicinal herb effective in various diseases, including bronchitis and asthma, but is still more widely used for food. Fermentation methods are being applied to increase the pharmacological composition of PG extracts and commercialize them with high added value. This study examines the hydrolyzed and fermented PG extract (HFPGE) fermented with Lactobacillus casei in RAW 264.7 cells, and investigates the effect of amplifying the immune and the probable molecular mechanism.
MATERIALS/METHODS
HFPGE’s total phenolic, flavonoid, saponin, and platycodin D contents were analyzed by colorimetric analysis or high-performance liquid chromatography. Cell viability was measured by the MTT assay. Phagocytic activity was analyzed by a phagocytosis assay kit, nitric oxide (NO) production by a Griess reagent system, and cytokines by enzyme-linked immunosorbent assay kits. The mRNA expressions of inducible nitric oxide synthase (iNOS) and cytokines were analyzed by reverse transcription-polymerase chain reaction, whereas MAPK and nuclear factor (NF)-κB activation were analyzed by Western blots.
RESULTS
Compared to PGE, HFPGE was determined to contain 13.76 times and 6.69 times higher contents of crude saponin and platycodin D, respectively. HFPGE promoted cell proliferation and phagocytosis in RAW 264.7 cells and regulated the NO production and iNOS expression. Treatment with HFPGE also resulted in increased production of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, C-X-C motif chemokine ligand10, granulocytecolony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and monocyte chemoattractant protein-1, and the mRNA expressions of these cytokines. HFPGE also resulted in significantly increasing the phosphorylation of NF-κB p65, extracellular signalregulated kinase, and c-Jun N-terminal kinase.
CONCLUSIONS
Taken together, our results imply that fermentation and hydrolysis result in the extraction of more active ingredients of PG. Furthermore, we determined that HFPGE exerts immunostimulatory activity via the MAPK and NF-κB signaling pathways.

Keyword

Platycodon grandiflorum; immunostimulation; cytokines; chemokines; NF-κB

Figure

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  Fig. 1 Effect of PGE and HFPGE on the viability of RAW 264.7 cells. Cells were seeded at density of 1 × 105 cells/well in 24-well plates and incubated in DMEM supplemented with 100 mL/L FBS. After 24 h, cells were treated with varying concentrations of PGE (A) and HFPGE (B), and incubated for 48 h. Cell viability was determined by the MTT assay. Each bar expresses the mean ± SEM (n = 6). Means without the same letter differ, P < 0.05.DMEM, Dulbecco’s Modified Eagle’s Medium; FBS, fetal bovine serum; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; PGE, Platycodon grandiflorum extract; HFPGE, hydrolyzed and fermented Platycodon grandiflorum extract.

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  Fig. 2 Effect of PGE and HFPGE on the phagocytic activity in RAW 264.7 cells. Cells were seeded at density of 1 × 105 cells/well in a 96-well plate and incubated. After 24 h, cells were treated with varying concentrations of PGE (A) and HFPGE (B), and incubated for 24 h. The phagocytic activity was measured using the Phagocytosis assay (zymosan substrate) kit. Each bar expresses the mean ± SEM (n = 6). Means without the same letter differ, P < 0.05.PGE, Platycodon grandiflorum extract; HFPGE, hydrolyzed and fermented Platycodon grandiflorum extract; LPS, 0.1 μg/mL lipopolysaccharide.

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  Fig. 3 Effect of HFPGE on NO production and iNOS mRNA expression in RAW 264.7 cells. Cells were seeded at a density of 1 × 105 cells/well in a 24-well plate and incubated. After 48 h, cells were serum-starved with serum-free DMEM for 2 h, and treated with the indicated concentration of HFPGE in serum-free DMEM for 24 h. The 24-h conditioned media were collected. (A) NO production was determined using the Griess reagent system. (B) Total RNA was extracted, reverse-transcribed, and real-time PCR was performed. iNOS mRNA expression was normalized to GAPDH and presented relative to the 0 μg/mL group. Each bar expresses the mean ± SEM (n = 6). Means without the same letter differ, P < 0.05.DMEM, Dulbecco’s Modified Eagle’s Medium; HFPGE, hydrolyzed and fermented Platycodon grandiflorum extract; LPS, 0.1 μg/mL lipopolysaccharide; NO, nitric oxide; iNOS, inducible nitric oxide synthase; PCR, polymerase chain reaction; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

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  Fig. 4 Effect of HFPGE on various cytokines production in RAW 264.7 cells. Cells were seeded and treated with HFPGE as described in Fig. 3. Levels of (A) TNF-α, (B) IL-1β, (C) IL-6, (D) CXCL10, (E) G-CSF, (F) GM-CSF, and (G) MCP-1 in 24 h-conditioned media were measured using the relevant ELISA kit. Each bar expresses the mean ± SEM (n = 6). Means without the same letter differ, P < 0.05.HFPGE, hydrolyzed and fermented Platycodon grandiflorum extract; LPS, 0.1 μg/mL lipopolysaccharide; TNF, tumor necrosis factor; IL, interleukin; CXCL, C-X-C motif chemokine ligand; G-CSF, granulocyte-colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; MCP, monocyte chemoattractant protein; ELISA, enzyme-linked immunosorbent assay.

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  Fig. 5 Effect of HFPGE on mRNA expressions of various cytokines in RAW 264.7 cells. Cells were seeded and treated with HFPGE as described in Fig. 3. Total RNA was extracted, reverse-transcribed, and real-time PCR was performed. The mRNA expressions of (A) TNF-α, (B) IL-1β, (C) IL-6, (D) CXCL10, (E) G-CSF, (F) GM-CSF, and (G) MCP-1 were normalized to GAPDH and presented relative to the 0 μg/mL group. Each bar expresses the mean ± SEM (n = 6). Means without the same letter differ, P < 0.05.HFPGE, hydrolyzed and fermented Platycodon grandiflorum extract; LPS, 0.1 μg/mL lipopolysaccharide; PCR, polymerase chain reaction; TNF, tumor necrosis factor; IL, interleukin; CXCL, C-X-C motif chemokine ligand; G-CSF, granulocyte-colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; MCP, monocyte chemoattractant protein; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

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  Fig. 6 Effect of HFPGE on MAPK and NF-κB signaling pathways in RAW 264.7 cells. Cells were seeded and treated with HFPGE as described in Fig. 3. Cell lysates were analyzed by Western blotting using the corresponding antibodies. (A, C, E) Images of chemiluminescent detection of the blots, which are representative of 3 independent experiments. The relative expression ratio of p-p65 to p65 (B), p-ERK to ERK (D), and p-JNK to JNK (F). Protein band were quantified, with the control levels set at 100. Each bar expresses the mean ± SEM (n = 3). Means without the same letter differ, P < 0.05.HFPGE, hydrolyzed and fermented Platycodon grandiflorum extract; LPS, 0.1 μg/mL lipopolysaccharide; NF, nuclear factor; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase.

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  Fig. 7 Summary of the effects of HFPGE on the immune response in RAW 264.7 cells.HFPGE, hydrolyzed and fermented Platycodon grandiflorum extract; TNF, tumor necrosis factor; IL, interleukin; CXCL, C-X-C motif chemokine ligand; G-CSF, granulocyte-colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; MCP, monocyte chemoattractant protein; TLR, toll-like receptor; TRAF, tumor necrosis factor receptor-associated factor.

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