The Effects Research Of Melamine In The Absence And Presence Of Cyanuric Acid On Allergenic Effect Of Mice And The Effect Of LPS-TLR4 Signaling Pathway

Melamine (MA) is widely used as organic chemical raw materials, and Cyanuric a cid is its homologue. At present, melamine and cyanuric acid mixtures researches focu sed mainly on the content detection, the effects of circulation, urinary and reproductiv e systems, but less research of the immune system at home and abroad.This study ai med at clarifying melamine and cyanuric acid mixtures on the effects of mice allergic reaction and mouse lipopolysaccharide (LPS) signaling pathway, in order to provide rel iable basis for improving the food safety toxicology evaluation system of melamine an d its homologues.TEST ONE The effects of melamine and cyanuric acid mixtures on the allergy reacti on of ovalbuminMethods:Male KM mice of SPF weighing 22-24g were randomly divided into ne gative control group, positive control group, melamine low, medium and high (2mg·kg-1,8mg·kg-1,32mg·kg-1) dose group and mixed with cyanuric acid in low, medium and hig(1+1mg·kg-1,4+4mg·kg-1,16+16mg·kg-1) dose of 8 experimental groups. In 0d,14d int raperitoneal injection of ovalbumin 100ug/only (except the negative control group),28 oral ovalbumin 50mg/only sensitized excitation, followed by separation of serum,the de tection of ovalbumin-specific IgE, histamine, class trypsin, IL-4, IFN-y levels.The results showed that:① melamine and cyanuric acid mixtures affected the aller gy reaction of ovalbumin in mice. The concentration of melamine was 2mg·kg-1·d-1 co uld promote ovalbumin-induced allergic reactions, and that reached 32mg·kg-1·d-1 could inhibited ovalbumin-induced allergic reactions; With cyanuric acid there were the inhi bition in the two agents over 4mg·kg-1·d-1. ② in mouse lung H.E stained sections, ea ch group showed reticular alveolar walls, the experimental groups had a lot of inflam matory exudation, alveolar wall thickening, high dose group had some alveolar damage. ③the mixtures had almost no effect on the mouse serum IFN-γ concentration, but si gnificantly (p<0.05) or highly significantly (p<0.01) decreased the concentration of seru m inflammatory inhibitor IL-4 to aggravate allergic symptoms in mice. The values of IFN-γ/IL-4 can predict the joint concentrations of melamine and cyanuric acid over 16 mg·kg-1·d-1 could induce immune unbalanced mice.④melamine and cyanuric acid in lo w doses could lead to the increased HIS, TPS significantly(p<0.05)or highly significant (p<0.01), indicating that low doses mixtures can promote the body’s mast cells to rele ase HIS and tryptase, and then exacerbate allergic reactions; At 32mg·kg-1·d-1 melamin e alone, and 16mg·kg-1·d-1 mixtures, HIS, TPS decreased significantly(p<0.01), thereby inhibiting ovalbumin sensitization effects. This indicates that low-dose mixtures can pro mote ovalbumin effect, but,high dose can inhibit.TEST TWO The mechanism study of the effects of melamine and cyanuric acid mixt ures on the allergy reaction of ovalbuminMethods:Mice for groups and treatments were the same as test one. By toluidine blue staining the number of mast cell degranulation grade level, flow detection is Thl, Th2 cells and CD40, CD40L expression, Western blot and real-time quantitative PCR detect theexpression of FcεR Ⅰ receptor, ELISA detection the content of IL-10,1L-16, MCP-1The results showed that:①melamine and cyanuric acid mixtures could increase the number of Th1 and Th2 cells, lead to immune unbalanced mice(p<0.05), that were m ainly relevant to the relatively low number of Th2 cells which suppress the inflammat ory response,and then promote ovalbumin effects.② low doses mixtures could increaset he expression of FcεR Ⅰ receptor in the mast cells and basophils surface, and the conc entration of IgE, thus exacerbating ovalbumin sensitization effects.③ mixtures could re duce expression of CD40 and CD40L in spleen, affect B cell activation and the transf orm IgM to IgE, and,thereby reducing the synthesis of ovalbumin-specific IgE.④ the mixtures could promote degranulation of mast cells in lungs for affecting ovalbumin sensitization effect, and 16mg·kg-1·d-1 mixtures even could reduce the number of mast cells in lung, decrease the concentrations of mast cells activated factor IL-16 and baso phils activated factor MCP-1, and then reduce the release of the HIS, TPS, thus suppr essing ovalbumin sensitized effects.TEST THREE The study of the effects of melamine and cyanuric acid mixtures on LPS-TLR4 signaling pathway in male miceMethods:conteracting toxic treatment with a test,28 days after the mice by intrap eritoneal injection LPS5mg·kg-1. Detect the content of mCD14 and sCD14 in periphera 1 blood, TLR4, MyD88, NF-κb relative expression in spleen and IL-1,IL-6 in serum.Results showed that:①with melamine and cyanuric acid could significantly reduce peripheral blood sCD14 content(p<0.01), increase mCD14 content (p<0.01) to affect t he signal preliminary presentation in LPS-TLR4 signaling pathway. ② the mixtures co uld increase significantly TLR4 expression in mouse spleen(p<0.01)and MyD88 express ion levels(p<0.05), thereby affecting the extracellular to intracellular signal transduction in the LPS-TLR4 signaling pathway. ③ such substances into the body could decrease the expression of NF-Kb mRNA in spleen, affect the biological effects of LPS-TLR4 signaling pathway(p<0.05), thereby reducing serum IL-1, IL-6 content(p<0.01) and affe cting the maturation and differentiation of B cells.