Panax ginseng-derived fraction BIOGF1K reduces atopic dermatitis responses via suppression of mitogen-activated protein kinase signaling pathway

Abstract

Background: BIOGF1K, a fraction of Panax ginseng, has desirable antimelanogenic, anti-inflammatory, and antiphotoaging properties that could be useful for treating skin conditions. Because its potential positive effects on allergic reactions in skin have not yet been described in detail, this study's main objective was to determine its efficacy in the treatment of atopic dermatitis (AD).

Methods: High-performance liquid chromatography was used to verify the compounds in BIOGF1K, and we used the (3-4-5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide method to determine its cytotoxicity in RBL-2H3 and HMC-1 cell lines. RBL-2H3 cells were induced using both anti-DNP-IgE/DNP-BSA and calcium ionophore (A2187) treatments, whereas HMC-1 cells were induced using A2187 alone. To measure mast cell degranulation, we performed histamine (enzyme-linked immunosorbent assay) and β-hexosaminidase assays. To quantify interleukin (IL)-4, IL-5, and IL-13 levels in RBL-2H3 cells, we performed quantitative polymerase chain reaction (PCR); to quantify expression levels of IL-4 and IL-13 in HMC-1 cells, we used semiquantitative reverse transcription polymerase chain reaction (RT-PCR). Finally, we detected the total and phosphorylated forms of extracellular signal-regulated kinase, p-38, and c-Jun N-terminal kinase proteins by immunoblotting.

Results: BIOGF1K decreased the AD response by reducing both histamine and β-hexosaminidase release as well as reducing the secretion levels of IL-4, IL-5, and IL-13 in RBL-2H3 cells and IL-4 and IL-13 in HMC-1 cells. In addition, BIOGF1K decreased MAPK pathway activation in RBL-2H3 and HMC-1 cells.

Conclusions: BIOGF1K attenuated the AD response, hence supporting its use as a promising and natural approach for treating AD.

Keywords: Atopic dermatitis; BIOGF1K; Compound K; MAPK; P. ginseng.

Fig. 1

Cell viability of RBL-2H3 and HMC-1 cells after BIOGF1K treatment. (A and B) RBL-2H3 and HMC-1 cells were incubated with BIOGF1K in concentrations from 0 to 50 μg/mL for 24 h. MTT assay was used to determine their cell viability. (C) HPLC analysis and profile of the phytochemical compounds present in BIOGF1K. MTT, (3-4-5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide; HPLC, high-performance liquid chromatography.

Fig. 2

BIOGF1K decreased the effect of mast cell degranulation. A density of 5 × 10⁵ cells per mL was used to seed RBL-2H3 cells in a 12-well plate. (A, C) After 24 h of incubation time, RBL-2H3 cells were pretreated for 30 min with BIOGF1K (0-50 μg/mL), then induced with 1 μg/mL of A2187 for 24 h. (B, D) RBL-2H3 cell sensitization was performed overnight with IgE (0.1 μg/mL), washed with PBS, pretreated for 30 min with BIOGF1K (0-50 μg/mL), and treated with 1 μg/mL of DNP-BSA for 2 h. (A, B) For the β-hexosaminidase secretion assay, 60 μL of the supernatant was mixed with 60 μL of p-NAG and kept for 2 h in a 96-well plate at 37°C. Glycine buffer (120 μL) was added, and the absorbance was measured at 405 nm. (C, D) 50 μL of supernatant was collected and a histamine release assay (ELISA) was performed using the manufacturer's specifications. ^##p < 0.01 versus a normal (untreated) group, **p < 0.01 versus a control (induced) group. DNP-BSA, 2,4-dinitrophenyl albumin from bovine serum; p-NAG, 4-nitrophenyl n-acetyl-β-d-glucosaminide; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay.

Fig. 3

Downregulating effect of BIOGF1K on atopic dermatitis–related cytokines expression. A density of 5 × 10⁵ cells per mL was used for seeding RBL-2H3 cells in a 12-well plate. (A, C, and E) After 24 h, RBL-2H3 cells were pretreated for 30 min with BIOGF1K (0-50 μg/mL), then treated with 1 μg/mL of A2187 for 24 h. (B, D, and F) RBL-2H3 cell sensitization was performed overnight with IgE (0.1 μg/mL), then the cells were washed with PBS, pretreated for 30 min with BIOGF1K (0-50 μg/mL), and finally induced using 1 μg/mL of DNP-BSA for 2 h. The secretion level of IL-4, IL-5, IL-13, and GADPH were determined by quantitative PCR. (G) A density of 1 × 10⁶ cells per mL was used for seeding HMC-1 cells in a 6-well plate. After 24 h, cells were pretreated for 30 min with BIOGF1K (0-50 μg/mL), then treated with 1 μg/mL of A2187 for 24 h. RT-PCR was used to quantify the expression of IL-4 and IL-13 and GADPH. ^##p < 0.01 versus a normal (untreated) group, **p < 0.01 versus a control (induced) group. DNP-BSA, 2,4-dinitrophenyl albumin from bovine serum; IL, interleukin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PBS, phosphate-buffered saline.

Fig. 4

Downregulation effect of BIOGF1K on the atopic dermatitis–activated MAPK pathway. A density of 5 × 10⁵ cells per mL was used to seed RBL-2H3 cells in a 12-well plate. (A) After 24 h of harvesting time, RBL-2H3 cells were pretreated for 30 min with BIOGF1K (0–50 μg/mL) and induced with A2187 (1 μg/mL) for 24 h. (B) RBL-2H3 cell sensitization was performed overnight with IgE (0.1 μg/mL), and then the cells were washed with PBS, pretreated for 30 min with BIOGF1K (0-50 μg/mL), and treated with DNP-BSA (1 μg/mL) for 2 h. (C) A density of 1 × 10⁶ cells per mL was used for seeding HMC-1 cells in a 6-well plate. Cells were incubated for 24 h, pretreated for 30 min with BIOGF1K (0-50 μg/mL) and then treated with A2187 (1 μg/mL) for 24 h. Phosphorylated and total forms of ERK, p-38, and JNK proteins were determined using immunoblotting. ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; DNP-BSA, 2,4-dinitrophenyl albumin from bovine serum; PBS, phosphate-buffered saline; MAPK, mitogen-activated protein kinase.

Fig. 5

Schematic pathway summarizing the effect of BIOGF1K on atopic dermatitis induction. Antigen/IgE cross-linking with the mast cell–specific receptor FCεRI and calcium ionophore (A23187) induction activates the MAPK pathway. BIOGF1K decreases MAPK pathway protein activation, decreasing intracellular calcium signaling and therefore diminishing mast cell degranulation, histamine release, and interleukin production. DNP-BSA, 2,4-dinitrophenyl albumin from bovine serum; IL, interleukin; MAPK, mitogen-activated protein kinase.