Food allergy herbal formula 2 protection against peanut anaphylactic reaction is via inhibition of mast cells and basophils

Abstract

Background: Mast cells and basophils are key effector cells of IgE-mediated anaphylactic reactions. The Chinese herbal formula, food allergy herbal formula 2 (FAHF-2), protects against peanut anaphylaxis in mice. However, the mechanisms underlying this effect are not fully elucidated.

Objective: To investigate whether FAHF-2 inhibits mast cell/basophil numbers and IgE-mediated activation.

Methods: Mice with peanut allergy (PNA mice) were treated with FAHF-2 intragastrically for 7 weeks and challenged intragastrically with peanut 1 day and 4 weeks posttreatment. Peripheral blood basophil numbers and peritoneal mast cell numbers and FcεRI expression were determined. Direct effects of FAHF-2 on the murine mast cell line MC/9, and effects of 4 fractions and 3 compounds isolated from FAHF-2 on rat basophilic leukemia cells (RBL-2H3) and human skin mast cells degranulation and on the IgE-mediated spleen tyrosine kinase signaling pathway, were determined.

Results: Although all sham-treated PNA mice developed anaphylaxis, FAHF-2-treated PNA mice were protected against anaphylaxis after peanut challenge at 1 day and 4 weeks posttherapy. Reduction of peripheral blood basophils began after 1 week of treatment and continued for at least 4 weeks posttherapy. The number and FcεRI expression of peritoneal mast cells were also significantly decreased 4 weeks posttherapy. FAHF-2-treated MC/9 cells showed significantly reduced IgE-induced FcεRI expression, FcεRI γ mRNA subunit expression, proliferation, and histamine release on challenge. Fraction 2 from FAHF-2 inhibited RBL-2H3 cell and human mast cell degranulation. Three compounds from fraction 2-berberine, palmatine, and jatrorrhizine-inhibited RBL-2H3 cell degranulation via suppressing spleen tyrosine kinase phosphorylation.

Conclusion: Food allergy herbal formula 2 reduction of basophils and mast cell numbers as well as suppression of IgE-mediated mast cell activation may contribute to FAHF-2's persistent protection against peanut anaphylaxis.

Figure 1

A. Experimental protocol. Female C3H/HeJ mice were sensitized intragastrically with freshly ground roasted peanut together with cholera toxin as indicated and treated with FAHF-2 (n = 10), or water (Sham, n = 10). All mice were orally challenged with peanut at wk14 and 18. Naïve mice were served as controls (n = 10). B. Anaphylactic symptom scores. C. Post-challenge body temperatures. D. Post-challenge plasma histamine levels. Bars in B indicates median of scores of mice from each group with combined data at wk 14 (open triangles) and wk 18 challenges (solid triangles). (Sham, n=20; FAHF-2, n=20; and naïve, n=20). Bars in C and D are means of each group of mice from combined data at wk 14 (open circles) and wk 18 (open triangles) challenges. *, p< 0.05; ***, p<0.001 vs sham.

Figure 2. FAHF-2 reduced the number of peripheral blood basophils

A. Dot plots show an increased percent of basophils in PNA mice, at wk 8 following the last boost, as compared to naïve mice. B. Histogram shows labeled FcεRI expression on cells from allergic (bold line) and naïve mice (thin line). Shaded = unstained cells; gray = isotype controls. C. Percent of basophils in peripheral blood from each group at wk 14 immediately after treatment. D. Reduction of basophil numbers as determined weekly at different time points as indicated. Numbers were normalized to and expressed as % of sham. E. FcεRI MFI of blood basophils from each group over time as in D.

Figure 3. FAHF-2 reduced the number of and FcεRI expression of peritoneal cavity mast cells

A. Data show one representative result with mast cells within the gated area. Numbers indicate percent. B. Mast cells (c-kit and FcεRI double-positive) were gated to show FcεRI levels in each group as indicated. C. FcεRI MFI. Each dot indicates one set of pooled cells from each group. Bars indicate the mean values of each group. D. Representative images of cutaneous mast cells in skin tissue of each group of mice. E. Percent of degranulated cutaneous mast cells in skin samples post challenge. Data is shown as Mean ± SEM (n=5). ***p<0.001 vs naïve.

Figure 4. FAHF-2 inhibited mast cell proliferation and degranulation

A. Mast cell proliferation. MC/9 cells were cultured and viable cells were counted at indicated time points. Cell numbers are expressed as fold increase vs day 0. Data are shown as Means ± SEM of 3 individual experiments. * p<0.05, ** p<0.01 vs. IgE alone. ##, p<0.01 vs Medium alone (Med). B. Mast cell degranulation. MC/9 cells were cultured and then challenged at different conditions as indicated. Supernatant and total cell histamine levels were determined. Data expressed as percent of histamine release (Means ± SEM of 3 individual experiments). ***p<0.001vs IgE + DNP without FAHF-2 (1^st bar).

Figure 5. FAHF-2 treatment in vitro reduced the expression of surface FcεRI and mRNA levels of FcεRI γ subunit by the mast cell line MC/9

A. Flow-cytometry detection of mast cell surface FcεRI expression. Dashed line, isotype control; Grey line, medium alone (Med); bold line, 2μg/ml of IgE (IgE only); thin lines, IgE plus 10μg/ml and 20μg/ml of FAHF-2. B. Messenger levels of FcεRI subunits of MC/9 cells in the presence or absence of anti-DNP IgE with or without FAHF-2. Data are expressed as means ± SD of 3 individual experiments (*p<0.05 vs IgE alone, # # # vs Med alone).

Figure 6. Effect of active fractions of FAHF-2 on RBL-2H3 cells and human skin mast cells

A. Effect of fractions at different doses fraction 1(0, 18, 36,72μg/ml), fraction 2 and 3(0, 9,18, 36μg/ml, ) and fraction 4(0,18, 36,72μg/ml) of FAHF-2 on β-hexosaminidase released by RBL-2H3. B. Effect of fraction 2 on human skin mast cells. β-hexosaminidase release was triggered by mAb 22E7 or C5a. Data are mean±SD of 3 experiments. *p<0.05, **p<0.01,***p<0.001vs untreated control.

Figure 7. Major alkaloid compounds in fraction 2 inhibited mast cell degranulation in RBL-2H3 Cells

A. Identification and chemical structures of berberine, palmatine and jatrorrhizine in F2 of butanol FAHF-2 (B-FAHF-2). B. Dose dependent responses to berberine, palmatine and jatrorrhizine by RBL-2H3 cells. C. Cell viability. Berberine, palmatine and jatrorrhizine toxic effects on RBL-2H3 cells using MTT assay. *p<0.05, ***p<0.001vs untreated control.

Figure 8. Berberine, palmatine and jatrorrhizine effects on Syk signaling pathway and IgE binding in RBL-2H3 cells

A. Down-regulation of Syk-phosphorylation (p-syk) in IgE-triggered RBL-2H3 cells by berberine, palmatine and jatrorrhizine combination (B+P+J). Bands are representative of three individual western blot experiments. B. Quantitative assay by densitometry of p-syk/β-actin ratio. All data are Mean ± SD of 3 individual experiments.*p<0.05, DNP+IgE+B+P+J vs DNP+IgE. C. Effects of B, P and J on IgE binding to RBL-2H3 cells. Red line=cells only without IgE, green=cells with IgE, purple, brown, blue= B, P, J +IgE.