Study On Immune Mechanism Of Tyrosine-rich Peptide Of Immunostimulating (Trpi)

Tyrosine-rich Peptide of Immunostimulating (Trpi) was separated from tryptic casein hydrolysates (TCH). Trpi was found to be a novel casein-derived antimicrobial peptide with obvious immunostimulating and antimicrobial properties. The purification of Trpi was proved by SDS-PAGE(Urea SDS-PAGE&Tricine-SDS-PAGE)and capillary electrophoresis(CE).The results showed that the peptide was of high purity. Agar diffusion test was used to reveal antimicrobial activities of TCH and purified Trpi. The results suggested that both of them have strong antimicrobial activities to E.coli. E.coli, treated with or without Trpi, was immobilized, double stained by uranium acetate and lead citrate and finally observed by transmission electron microscope (TEM).The results showed the cell wall and membrane rupture of E.coli and the cytoplasmic content leakge lead to the final death of cells treated with Trpi.The results of in vitro experiment (MTT) indicated that Trpi can very significantly enhance proliferation of mouse lymphocytes(p<0.001,SI=2.13).Also, oral administration of Trpi in Balb/c mice was carried out for a further study on the immunostimulating activity of Trpi. Feed intake, body weight and indices of immune organs were determined periodly. The results suggested that Trpi had no obvious effect on body weight with a low downregulation on feed intake, while no matter TCH, partially purified or fully purified Trpi can significantly promote indices of mouse spleen(p=0.004<0.01)and thymus (also p<0.01). Further more, three different administration means of Trpi in Balb/c mice, including oral administration, intraperitoneal injection and caudal vein injection, were adopted to detailedly study the different serum cytokine levels stimulated by Trpi. Cytokine ELISA kits were used to determine cytokine levels. The results suggested that Trpi can significantly increase the production of pro-inflammatory cytokines such as IL-1βand IL-6 and anti-inflammatory cytokine IL-10, also have some effect on an anti-inflammatory cytokine IL-4, but have no effect on TNF-α, IFN-γand TGF-α. The results also showed: intraperitoneal injection group>caudal vein injection group>oral administration. All of the above evidences suggested that Trpi may affect cytokine network to improve immnunity and the effect was the strongest through mucosal immune. To sum up, Trpi can reveal strong antimicrobial activity, significantly enhance proliferation of mouse lymphocytes in vitro and upregulate indices of mouse spleen and significantly increase some cytokine levels in vivo.The regulation of cytokine production to improve the immune system maybe one of its immune mechanism. The study on the immune mechanism of Trpi was based on that on antimicrobial mechanism. The further elucidation of mechanism of action of Trpi will bring forth the prospect of it as antimicrobial and immunomodulator and accelerate the application of Trpi as functional food or other medical and pharmaceutical practices in theory.