Potential Allergenicity Response to Moringa oleifera Leaf Proteins in BALB/c Mice

Abstract

The reported association of Moringa oleifera seeds and allergic disease clinically resembling occupational asthma in cosmetic manufacturing workers has resultedin the need to identify such components in the manufacturing process. However, Moringa oleifera leaves from the same plant, an important food ingredient, have limited immunotoxicity data. This study aimed to determine if Moringa oleifera leafproteins (MLP) can elicit allergic responses in BALB/c mice. The BALB/c mice were sensitized twice and challenged 10 times to evaluate the potential allergenicityof MLP in vivo. The results showed increased levels of mast cells, total and specific IgE and IgG, severe signs of systemic anaphylaxis, and reduced body temperature compared with controls. The sensitized mice serum observed enhanced levels of histamine and Th-related cytokine release. Compared with the control group, increased levels of interleukins IL-4, IL-9, and IL-17A and enhanced expression and secretion of normal T cells were found in the culture supernatant of splenocytes treated with MLP.This study suggeststhat MLPcanelicit allergic responses; this providesmore comprehensive guidance for identifying new allergen candidates and developing hypoallergenic MLP products.

Keywords: Moringa oleifera; cytokines; food allergy; potential allergenicity.

Figure 1

Mouse immunization protocol. Mice were sensitized with two intragastric administrations of MLP (MLP, 30 μg/60 μg per mouse) at a 14-day interval (days 0, 14) and challenged with MLP at a 2-day intervals (days 28, 30, 32, 34, 36, 38, 40, 42, 44, and 48) to establish a mouse model of food allergy in the MLP group or phosphate-buffered saline (PBS) in the controlgroup.

Figure 2

SDS-PAGE (A), MALDI-TOF MS (B), and Fourier transform infrared spectroscopy spectra (C) for MLP.

Figure 3

The score of hypersensitivity symptoms (A), diarrhea (B), and rectal temperature (C) in the MLP low-dose group and MLP high-dose group. Levels of specific antibodies (IgG or IgE) ((D,E), fix the background) and histamine (F) in sera from mice exposed to low-dose MLP, high-dose MLP, or PBS (control group) were detected by ELISA. The number of mast cells (G) in jejunum following MLP and saline challenges were assessed by morphometric analysis of toluidine blue-stained cells (n = 4 mice per group). Histopathological analysis of jejunum ((H), arrows point at mast cells). Each point representsdata from an individual mouse, and values are expressed as means ± SD (n ≥ 6). * p < 0.05, ** p < 0.01, *** p < 0.001,**** p < 0.0001 by two-tailed unpaired Student’s t-test where indicated or Mann–Whitney U test.

Figure 4

(A) Level of Th1 (IFN-γ), (B) Th2 (IL-4 and IL-13), and (C) Th17 (IL-17A and IL-23) in MLP-sensitized mice sera by ELISA. Significant difference (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; n ≥ 5) compared with the control group.

Figure 5

(A) Level of Th2 (IL-4), (B) Th9 (IL-9), and (C) Th17 (IL-17A)in MLP-sensitized mice splenocyte supernatant by ELISA. Significant difference (** p < 0.01; n ≥ 5) compared with the control group.

Figure 6

Histological sections (HE) of intestine ((A): jejunum; (B): ileum; (C): colon) tissues from the MLP high-dose group and MLP low-dose group.