Effects Of Lactoferrin On The Expression Of TLR4Signaling Pathway-associated Membrane Molecule On LPS-activated U937Macrophages

【Objective】To investigate the effects of recombinant humanlactoferrin (rhLF) on the mRNA and protein expression of TLR4signalingpathway-associated membrane molecule(TLR4, CD14and MD2)onLPS-activated U937macrophages.【Methods】1. U937cells was induced to maturation by10ng/mlPMA and then divided into three groups: control group, LPS group andrhLF treatment group(given50μg/ml,100μg/ml,200μg/ml,500μg/mlrhLF respectively).2. The mRNA expression of TLR4, CD14and MD2were determined by RT-PCR.3. The protein expression of TLR4, CD14and MD2were determined by western blot.【Results】1. U937cells differentiated into monocyte/macrophage-like cells following incubation with10ng/ml PMA for24h, stoppedproliferating and altered their morphology from non-adherent to adherentcells after differentiation.2. The basal mRNA expressions of TLR4, CD14and MD2were very low in control group, whereas the mRNA expressionincreased significantly in the LPS group (P<0.05). When compared with LPS group, the mRNA expression of TLR4, CD14and MD2showed nosignificant change in50μg/ml rhLF treatment group (P>0.05), but reducedsignificantly in100μg/ml,200μg/ml and500μg/ml rhLF treatment groupwith a dose-dependent relationship (P<0.05).3.The basal proteinexpressions of TLR4, CD14and MD2were also very low in control group,whereas the protein expression increased significantly in the LPS group(P<0.05). When compared with LPS group, the expression of TLR4, CD14and MD2protein showed no significant change in50μg/ml rhLF treatmentgroup (P>0.05), but reduced significantly in100μg/ml,200μg/ml and500μg/ml rhLF treatment group with a dose-dependent relationship(P<0.05). Consistent change trend were found between the proteinexpression and the mRNA expression of TLR4, CD14and MD2.【Conclusion】1.U937cells differentiated into monocyte/macrophage-like cells in the presence of PMA.2. A relatively small amount of TLR4,CD14and MD2mRNA and protein could be measured in control group.3.The mRNA and protein expression of TLR4, CD14and MD2increasedsignificantly in the LPS group, it may indicate that during sepsis, TLR4,CD14and MD2expression levels may control the extent of LPSresponsiveness with over-expression triggering uncontrolled pro-inflammatory responses.4. The mRNA and protein expression of TLR4,CD14and MD2reduced significantly in rhLF treatment group with adose-dependent relationship, that may suggest that rhLF might have a potential protective effect in LPS induced sepsis through down-regulatingthe mRNA and protein expression of TLR4,CD14and MD2in activatedU937cells, resulting in suppressing the transmembrane signal transductionpathway of TLR4, which may lead to potentially life-threatening overactiveinflammatory responses during sepsis. These results may be a solidtheoretical basis for the treatment of neonatal sepsis with LF treatment.