A Study On The Bidirectional Immunoregulatory Effects Of Ovotransferrin And Its Enzymatic Hydrolysate On RAW264.7 Macrophages

Egg white is rich in a variety of active proteins,and ovotanserrin(OVT)is one of the main active proteins,which has antioxidant,antibacterial,antiviral and immuno-regulatory activities.Studies have reported that the small peptides formed after the digestion of proteins in the gastrointestinal tract can stimulate immune cells,activate the body’s immune response,and play an immunomodulatory role.However,there are few studies on the two-way immunomodulatory effect of OVT enzymatic hydrolysates.In order to explore the immunomodulatory mechanism of OVT and its enzymatic hydrolysis,resting RAW264.7 and LPS activated RAW264.7 cells were used as models.And cell viability,inflammatory mediator NO,inflammatory factors and chemokine secretion and expression were detected by CCK method,Griess,ELISA and RT-PCR.Inverted fluorescence microscope was used to observe cell changes and NF-κB nuclear translocation.The possible signaling pathways of OVT and its hydrolysates were analyzed by RNA seq and Western blotting.The channel provides a theoretical basis for the development and utilization of OVT and fumed as functional food ingredients.The experimental research results are as follows:(1)50-200 μg/m L OVT and 1-4 mg/m L OHPT have no toxic effects on normal RAW264.7 cells.(2)A high concentration of OVT(200 μg/m L)activates the normal RAW264.7through the TLR4 receptor-mediated My D88 independent pathway,which promotes the expression of NF-κB and MAPK signaling pathway proteins,thereby increasing the release and expression of inflammatory mediators NO and cytokines TNF-α,IL-6 and IL-10.It plays a pro-inflammatory effect.(3)Low-concentration OVT(50 μg/m L)and LPS co-incubating RAW264.7 slow down the production and expression of inflammatory factors by inhibiting the phosphorylation and expression of NF-κB and MAPK mediated by My D88,and it plays an anti-inflammatory effect.(4)High concentration of OHPT(4 mg/m L)makes normal RAW264.7 cells differentiate and enhances the Fcy R-mediated phagocytic ability of macrophages in RAW264.7 cells.The secretion of NO,TNF-α,IL-6,MCP-1 and MIP-2 and the expression of iNOs are promoted by activating PI3 K,MAPK and NF-κB signal pathways,and OHPT have immuno-enhancing activity.(5)Low-concentration OHPT(1 mg/m L)improves LPS-induced cell morphology changes and decreased FcγR-mediated phagocytosis of LPS-induced RAW264.7.It inhibited NO,TNF-α,IL-6,MCP-1 and MIP-2 secretion and iNOs expression by inhibiting MD2-PI3K-NF-κB,MD2-TRIF-NF-κB,Ras-ERK and p38/JNK MAPK signal pathway protein expression,playing an anti-inflammatory effect.