Effects Of Melamine In The Absence And Presence Of Cyanuric Acid On Humoral Immunity In Male Mice

Melamine (MA) is widely used as organic chemical raw materials in the production and life of human. In the face of strong acid and strong alkali, melamine amidogen is replaced by hydrolyzed and it generates cyanuric acid. The combined toxicity of melamine and cyanuric acid increases in animal body mainly causing kidney toxicity in animals. At present, little information is available on the toxicity of compounds of melamine and cyanuric acid to the immune system in animals. In 2004, Lazarova reported that some people were allergic to cause textile dermatitis by exposuring to ethylene urea melamine formaldehyde and melamine formaldehyde, which makes the author think deeply about damagement of this compounds on animal immune system. The aim of this paper is to provide a theoretical basis for the evaluation data about the immune toxicity of melamine and cyanuric acid by researching melamine and co-administration of melamine and cyanuric acid effect on the humoral immunity in male mice.TEST ONE Effects of melamine and its combination with cyanuric acid on the resistance of male mice suffered E.coli poisoning attack treatmentMethod:Male KM mice were divided into eight groups:the control group, E.coli positive control group, melamine high (50 mg/kg), medium (10 mg/kg) and low (2 mg/kg) dose groups, as well as, co-administration of melamine and cyanuric acid high(25+25 mg/kg), medium (5+5 mg/kg) and low(1+1 mg/kg) dose groups. The test groups were fed every other day for 15 days. After 48h of intraperitoneal injection with E.coli, we detected the spleen B lymphocyte transformation rate, and hemolysin index optical density values, etc.Result:①There was no significant differences of the mice spleen index (P>0.05); ② Compared with the control group and Escherichia coli positive control group, the melamine group and combined toxic groups made the B lymphocyte transformationrate decrease, and the mixed high dose conversion was the lowest (P<0.05).③ The combination of the two chemical compounds made the mice serum hemolysin OD value decrease significantly (P<0.01).④ The melamine and co-administration groups both showed a downward trend in the T lymphocyte transformation rate as the dose increasing, but did not show significant diffrence (P>0.05). ⑤ Thickness growth of mice toe decreased significantly in melamine medium, high dose and co-administration high group (P<0.01).TEST TWO Effects of melamine and its combination with cyanuric acid on mice spleen plasma cells and its associated immune globulin and complementMethod:The male mice (18-22g) were randomly divided into 7 groups, the control group, melamine low, middle and high group, co-administration of melamine and cyanuric acid low, middle and high group. the dose is as same as test one. Flow cytometry was used to detect the mouse spleen cell surface Marker CD 138+ content, and western blot is used to detect the expresson of Blimp-1 protein in mouse spleen. Also, ELISA is used to detect the content of serum antibody IgG, sIgA and complement C3.Result:①Compared with the control group, melamine and co-administration of melamine and cyanuric acid both made the expression of Blimp-1 protein decrease, and Blimp-1 content of co-administration of melamine and cyanuric acid high group decreased significantly (P<0.01).These data shows that the compounds decreased cells surface Marker CD 138+protein by reducing the mouse splenic Blimp-1 regulatory protein to a certain extent, and it may interference the activity of plasma cell.② Low dose of melamine reduced the content of IgG (P<0.05), and co-administration of melamine and cyanuric acid dose also made the sIgA content decrease(P<0.05). Co-administration of melamine and cyanuric acid reduced the content of complement C3. Among them, the differences in medium dose group was significant (P<0.05), and high dose group was very significant(P<0.01). It suggests these compounds can affect the level of humoral immunity.TEST THREE Effects ofmelamine and its combination with cyanuric acid on helper T cells, Tfh cells and Th2 cell subsetsand related cytokines protein in miceMice for groups and treatments were the same as test two. The flow cytometry was used to detect the content of Marker CD4+CXCR5+ on the surface of mice spleen Tfh cells, and ELISA was used to detect the serum IL-4, IL-6, IL-10, IL-21 in mice. Also, western blot was used to detect the the expression of GATA-3 protein in mice spleen. qRT PCR was used to detect the GATA-3 gene expression level of mRNA.Result:① Melamine and co-administration of melamine and cyanuric acid had no effect of mice spleen cell surface marker CD4+CXCR5+ content (P>0.05), but combined with high dose group made IL-21 protein content in serum decrease significantly(P<0.01).② Serum IL-4, IL-6, IL-10 displays:the combinational groups made the content of IL-4 decrease significantly (P<0.05 or P<0.01), and the combination of the two conpounds made the IL-4 content decrease. With the increase of the dose of melamine and cyanuric acid, there was a decreasing trend (P<0.01). At the same time, melamine alone and combined with cyanuric acid reduced the IL-6 content in mice serum, and melamine alone declined significantly (P< 0.01); compared with the control group, the poison attacked group made the content of IL-10 significantly decreased (P<0.01).③ GATA-3 protein and GATA-3 expression level of mRNA displays:when the combination of the two conpounds dose increased, and the amount of GATA-3 protein decreased gradually, while there-was no significant difference (P>0.05). The expression level of the GATA-3 mRNA in the attacked groups were significantly decreased. It shows that the combinational dose of melamine and cyanuric acid in high dose group could reduce Gata-3 protein by suppressing GATA-3 expression of mRNA gene. This suggests that th2 cells are affected, further interferences Th2 cells related cytokines IL-4, IL-6, IL-10, resulting in inhibiting the humoral immunity.