Molecular Mechanism Of TLR4Signaling Pathway In Activated Macrophage Induced By Recombinant RTB

Ricin, a protein which extracted from the seed of the castor bean plant, is considered asone of the most toxic nature poisions. It is a glycoprotein composed of two different subunits,containing RTA and RTB and has a molecular weight about64kDa. RTA is active chain, playsa role by inhibiting synthesis of protein. RTB has lectin activity, is generally considered toplay its biological function by helping RTA transfer into the cell. Recent studies have foundthat RTB can combine with galactose or mannose receptor on the cell surface, and has the theability to enter cells that any known type. Macrophage is specific immune response cell with arole in antigen presentation, and is wildly studied immune cells. When it is stimulated by thepathogenic microorganisms, macrophage transforms from unactivated state to activated state,and releases NO and cytokines. Activated macrophage can perform different immunefunctions. Our group show that the recombinant RTB has the ability to activate macrophageswhich demonstrated related with NF-κB signaling pathways, promotes IκB-α proteinphosphorylated and nuclear translocation of NF-κB, and activates NF-κB signal transductionway. But for NF-κB signaling pathway upstream of Toll-like receptor signaling pathway hasnot been explored, and there is no coverage at home and abroad, and is currently itsmechanism is not yet clear.In this study, using different concentrations of recombinant RTB in vitro stimulatesRAW264.7cells, explore the effects of TLR4signaling pathway and its function ofdownstream signaling molecules and related molecular mechanisms by recombinant RTB, andprovide foundation for the reaserch on ubsequent application experiment of RecombinantRTB.The aspects of this research as follow:1. Toll-like receptor family were screened to determine the role of recombinant protein RTBon Toll signaling pathway.Using different concentrations of recombinant RTB proteins in vitro stimulatedRAW264.7cells, extracted total RNA, screened using TLR1-TLR9PCR primers to determinethe expression of Toll-like receptor signaling pathway, and prepared for the next experiment.2. Further to determine the effects of recombinant RTB protein on expression of TLR4mRNA. Using different concentrations of recombinant RTB proteins in vitro stimulatedRAW264.7cells, extracted total RNA, LPS as a positive control, using RT-PCR and qRT-PCRmethod to determined the role of TLR4signaling pathway by recombinant RTB protein3. Ensure the effects of recombinant RTB proteins on the expression of TLR4mRNAsignaling pathway downstream signaling moleculesExplore the effects of recombinant RTB proteins on the expression of TLR4mRNAsignaling pathway downstream signaling molecules MyD88, TRIF, TRAM and TRAF6byRT-PCR and qRT-PCR methods4. Determine the effects of recombinant RTB protein on TLR4protein and signaling moleculeprotein downstream TLR4signaling pathwaysUsing different concentrations of recombinant RTB proteins in vitro stimulatedRAW264.7cells, extracted total protein, LPS was considered as a positive control, ensure theeffects of recombinant RTB protein on TLR4signaling pathway and its downstream signalingmolecule protein. And to expore whether the effects of recombinant RTB protein on TLR4signaling pathway through MyD88-dependent pathway or through MyD88-independentpathway.The above results demonstrate that the recombinant RTB protein can promote mousemacrophage RAW264.7activating through TLR4signaling pathway activation; recombinantRTB proteins induced TLR4signaling pathway activated through MyD88-dependent andMyD88-independent pathway. Provide foundation for the immune regulation and themechanism of the recombinant RTB proteins and its clinical applications, and provided newdata for the recombinant RTB protein.