Extract of Boehmeria nivea Suppresses Mast Cell-Mediated Allergic Inflammation by Inhibiting Mitogen-Activated Protein Kinase and Nuclear Factor-κB

Abstract

Mast cells are effector cells that initiate allergic inflammatory immune responses by inducing inflammatory mediators. Boehmeria nivea (Linn.) Gaudich is a natural herb in the nettle family Urticaceae that possesses numerous pharmacological properties. Despite the various pharmacological benefits of Boehmeria nivea, its effects on allergic inflammation have not yet been determined. Here, we investigated the effect of the ethanol extract of Boehmeria nivea (BNE) on degranulation rat basophilic leukemia (RBL)-2H3 mast cells stimulated with anti-dinitrophenyl (anti-DNP) and bovine serum albumin (BSA) during immunoglobulin E (IgE)-mediated allergic immune response. The results showed inhibition of the release of β-hexosaminidase and histamine from the cells. BNE suppressed pro-inflammatory cytokines (Tumor necrosis factor (TNF)-α, Interleukin (IL)-1β, and IL-6) and reduced T helper (Th)2 cytokine IL-4 expression and/or secretion correlated with the downregulation of p38, extracellular signal-regulated kinases (ERK) mitogen-activated protein kinase (MAPK), and nuclear factor-κB (NF-κB) signaling pathways in treated RBL-2H3 mast cells. In passive cutaneous anaphylaxis, treatment with BNE during IgE-mediated local allergic reaction triggered a reduction in mouse ear pigmentation and thickness. Taken together, these results indicated that BNE suppressed mast cell-mediated inflammation, suggesting that BNE might be a candidate for the treatment of various allergic disorders.

Keywords: Boehmeria nivea; allergic inflammation; cytokines; immunoglobulin E; mast cells.

Figure 1

Effects of BNE on the degranulation of mast cells. (A) HPLC analysis of phenolic compounds. The peaks shown in the HPLC chromatogram results: rutin, luteorin-7-glucoside, naringin, and hesperidin. (B) Rat basophilic leukemia (RBL)-2H3 cells were treated with indicated concentrations of BNE for 12 h. The cell viability was measured using the MTT assay. (C,D) RBL-2H3 (6 × 10⁵ cells/well) cells were pre-sensitized with anti-DNP IgE (100 ng/mL) overnight. The cells were treated with or without indicated doses of BNE for 1 h and then challenged with anti-DNP-BSA for 4 h. The histamine (C) and β-hexosaminidase (D) levels in cultured media were measured as described in the Materials and Methods section. Data represent the mean ± standard error of the mean (SEM) from three independent experiments. * p < 0.05; *** p < 0.001, Tukey’s test, significantly different from Anti-DNP IgE plus DNP-BSA-treated group. Anti-DNP IgE, anti-dinitrophenyl immunoglobulin E; DNP-BSA, dinitrophenyl-Bovine serum albumin; BNE, Boehmeria nivea extract.

Figure 2

Effects of BNE on the expression and/or secretion of inflammatory cytokines. (A,B) RBL-2H3 cells were pre-sensitized with anti-DNP IgE (5 × 10⁵ cells/well). The cells were activated with anti-DNP-BSA by pretreating with or without DNE, and qPCR was used to quantify the expression of inflammatory cytokine (TNF-α, IL-1β, IL-6, and IL-4) genes (A). (B) Analysis of cytokines in the cultured supernatant for 6 h by ELISA. Data represent the mean ± SEM from three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001, Tukey’s test, significantly different from Anti-DNP IgE plus DNP-BSA-treated group. Anti-DNP IgE, anti-dinitrophenyl immunoglobulin E; DNP-BSA, dinitrophenyl-Bovine serum albumin; BNE, Boehmeria nivea extract; TNF-α, tumor necrosis factor-alpha; IL, interleukin.

Figure 3

BNE inhibited MAPKs and the nuclear factor-κB (NF-κB) signaling pathway. (A,B) RBL-2H3 cells were pre-sensitized with anti-DNP IgE (1 × 10⁶ cells/well). Cells were pre-sensitized with anti-DNP IgE (100 ng/mL) overnight. The cells were treated with or without indicated doses of DNE for 1 h and then challenged with anti-DNP-BSA for 15 min in RBL-2H3. Whole cell lysates or nuclear extracts were analyzed by western blotting: MAPKs (p38, ERK, and JNK) (A) and NF-κB (B) pathways. Protein levels were normalized to total form or β-actin and quantified using Image-J software (right panel). Data represent the means ± SEM from three independent experiments. ** p < 0.01; *** p < 0.001, Tukey’s test, different from the Anti-DNP IgE plus DNP-BSA-treated group. Anti-DNP IgE, anti-dinitrophenyl immunoglobulin E; DNP-BSA, dinitrophenyl-Bovine serum albumin; BNE, Boehmeria nivea extract; MAPKs, mitogen-activated protein kinase; NF-κB, nuclear factor-κB; IκB, I kappa-B alpha.

Figure 4

Effects of DNE on IgE-mediated passive cutaneous anaphylaxis. (A) The ear skin of mice (n = 6/group) was sensitized by intradermal injection of anti-DNP IgE (0.5 mg/site) for 48 h. DNE (100 and 200 mg/kg) or dexamethasone (10 mg/kg) was orally administered before the intravenous (i.v.) injection of a DNP-BSA and 4% Evans blue (1:1) mixture. After 30 min, the thickness of both ears was measured. (B) Ear thickness was measured with a dial thickness gauge. (C) The dye was extracted and quantified using a spectrophotometer. Data represent the mean ± SEM from three independent experiments. *** p < 0.001, Tukey’s test, significantly different from Anti-DNP IgE plus DNP-BSA with PBS-treated group. Anti-DNP IgE, anti-dinitrophenyl immunoglobulin E; DNP-BSA, dinitrophenyl-Bovine serum albumin; BNE, Boehmeria nivea extract; Dexa, dexamethasone.