Protective mechanisms of peptides from Spirulina maxima against allergy and atherosclerosis via IgE and histamine receptor signaling pathways

Abstract

Allergic and atherosclerotic diseases are one of the major public health problems in the developed world. The discovery of novel, efficient, and safe drugs for the treatment of these diseases is an important subject in human healthy. Recently, a great deal of interest has been developed by consumers towards novel bioactive agents as ingredients in nutraceuticals and pharmaceuticals from natural resources. Among them, Spirulina has been known as a rich source of bioactive agents and it has long been used as a nutritional supplement with various health benefit effects. One of the main interesting feathers in this microalga is their richness in protein content which can be hydrolized into bioactive peptides. In the present study, Spirulina maxima have been used to purify active peptides with anti-allergic and anti-atherosclerotic activities. S. maxima was hydrolysed with gastrointestinal endopeptidases (pepsin, trypsin, and α-chymotrypsin). For the selection of active fraction during purification steps, MTT assays, histamine release from mast cells, and cytokine production from endothelial cells were performed as maker of the screening. Following several purification steps, the peptides responsible for anti-allergic and anti-atherosclerotic activities were purified and identified as P1 (LDAVNR; 686 Da) and P2 (MMLDF; 655 Da). Allergy is considered as a disorder of the immune system in which an exaggerated response occurs when a person is exposed to normally harmless environmental substances. Mast cells are well known for their important role in pathogenesis of allergic diseases. The activation of mast cells by immunoglobulin E (IgE)-mediated stimuli is considered as a central event in allergic reactions. In this regard, two peptides P1 and P2 purified from enzymatic hydrolysate of S. maxima were investigated for their capabilities against the activation of RBL-2H3 mast cell sensitized with dinitrophenyl-specific immunoglobulin E (DNP-specific IgE) antibody and stimulated by antigen dinitrophenyl-bovine serum albumin (DNP-BSA). It was found that P1 and P2 significantly inhibited RBL-2H3 mast cell degranulation via attenuating the releases of histamine and β-hexosaminnidase releases. The inhibitory activity of these peptides was accompanied by a reduction in intracellular Ca2+ elevation. Moreover, the suppressive effects of P1 and P2 on production as well as protein and mRNA expression of histamine, IL-4, IL-13 were determined. The inhibitory mechanisms of P1 and P2 on mast cell degranulation were found in different pathways. The peptides P1 inhibited mast cell degranulation due to blockade of IgE receptor (FcεRI), thus blocking a cascade of intracellular events including inhibition of the activation of Lyn, Syk, PLC, PKC, IP3R, Fyn, and Gab2, and the polymerization of microtubule. Meanwhile, the peptide P2 suppressed mast cell degranulation via inhibiting ROS production and depressing activation of PLC, PKC and IP3R. In addition, both peptides P1 and P2 exhibited the inhibitory mechanisms on newly protein and mRNA synthesis of histamine and cytokines via suppression of PI3K/Akt, MAPKs, and NF-κB signaling pathways. Accordingly, it indicates that peptide P1 and P2 are efficient inhibitors of allergic reactions. On the other hand, IL-4 released from mast cell degranulation plays an important role in development of IgE-induced allergic responses due to enhancement of the expression of IgE receptor-FcεRI on the surface of mast cells. Herein, the peptides P1 and P2 were investigated for their abilities against the FcεRI expression on RBL-2H3 mast cells induced by IL-4 and IgE. It was revealed that the up-regulation of FcεRI expression at protein and mRNA levels was suppressed by P1 and P2 treatment. Moreover, flow cytometry and microscope assays showed that the increase in the cell surface FcεRI expression was reduced in the presence of P1 or P2. As a result, P1 and P2 decreased histamine and cytokine release from mast cell degranulation which is increased by IL-4 stimulation and IgE sensitization. Especially, the inhibition of P1 and P2 on FcεRI expression was found to be due to down-regulation of the phosphorylation of ERK and expression of transcription factor including GATA-1. These results indicate that peptide P1 and P2 are able to suppress the increase of FcεRI expression induced by IL-4 and IgE on mast cells. Besides IL-4, histamine is considered as the major mediator of the acute inflammatory responses. The interaction of histamine with its cell-surface receptor on endothelial cells can cause early atherosclerotic symptoms. In this sense, the peptides P1 and P2 have been used to examine their protective effects against atherosclerotic responses induced by histamine in EA.hy926 endothelial cells. Interestingly, both P1 and P2 exhibited inhibitory activities on the production of ROS, the production and expression of IL-6, IL-8, and MCP-1 at protein and mRNA levels. Furthermore, P1 and P2 reduced the levels of PGE2 and LTB4 production by suppressing the expression of COX-2 and 5-LO. Also, P1 and P2 inhibited the production and expression of adhesion molecules including P-selectin and E-selectin. In addition, the increase in expression of TLR4 receptor induced by histamine in endothelial cells was suppressed by P1 and P2 treatment. Notably, the inhibitory mechanism of both peptides P1 and P2 was found to block histamine receptor 1, thus inhibiting the activation as well as expression of many molecules in signaling pathway including PKC, MAPKs (ERK, p38, and JNK), and transcript factors such as c-fos and Egr-1. These results suggest that peptides P1 and P2 are potential agents for control of atherosclerotic responses in endothelial cells. Taken together, both peptides P1 and P2 derived from gastric enzymatic hydrolysate of microalga Spirulina maxima have a great potential to be used in pharmaceuticals and nutraceuticals as protective agents against allergic and atherosclerotic diseases.