Zinc Finger Protein A20 Gene Transfection Inhibits Inflammatory Reaction Mediated By Toll Like Receptor-4 Activated In Human Monocytes

Objective:To explore the protection and potential mechanism of zinc finger protein A20 on human monocytes inflammatory reaction , the TLR4/NF-κB signaling system was observed.The human monocytes were intervened with purified lipopolysaccharide(LPS) and gene transfection with exogenous A20 gene.in vitro.The expression of TLR4 and A20 were detected.The pro-inflammatory factors including tumor necrosis factor alpha(TNF-α) and interleukin12(IL-12) and the anti-inflammatory interl- eukin10 (IL-10) secreted by monocytes were also analysed. on this basis, we further studed the ratio between pro-inflammation factor and anti- inflammation factor that were TNF-α/ IL-10 and IL-12/ IL-10 effected by exogenous zinc fing protein A20.Methods: The Ficoll separating medium was used to separate the single nuclear cell from perpheral blood anticoagulanted by EDTA.The monocytes were planted in culture plank to culture with serum-free medium at density of 1pice/ml approximately and divided randomly into four groups: A group (control group, monocytes were cultured without any intervention); B group (LPS intervention group, monocytes were cultured with serum-free medium for 48 hours and then stimulated for 24 hours by LPS(1mg/l) );C group(A20 group, monocytes were cultured with serum-free medium for 24 hours fllowed by being transfected with plamid containing A20 gene for 48 hours.) and D group(LPS and A20 intervention group, monocytes were stimulated for 24 hours by LPS(1mg/l) before transfected with plamid containing A20 gene and cultured for 24 hours). All of the cells were collected by trypsin digestion method. immunofluorescence and immuno- histochemistry were altogether employed to analyze the expression zinc finger protein A20.RT-PCR was used to detected TLR4 and A20 mRNA expression.Flow cytometry was also used to determine the level expression TLR4 in monocytes.The level of TNF-α,IL-12 and IL-10 was measured by ELISA.Results:1.The monocyte receive LPS(1mg/L) activation, its own acceptor TLR4, the endogenous A20 expression and the promoting inflammation factor TNF-α,IL-12 and the anti-inflammation factor IL-10 expression obviously increases; Simultaneously, the ratio of TNF-α/IL-10 and IL-12/IL-10 obviously elevates.2.Transfection with exogenous A20 gene to monocytes doesn't obviously change the expression of the TLR4,endogenous A20 and inflammation factor TNF-α,IL-12 and IL-10 ; the ratio of TNF-α/IL-10 and IL-12/IL-10 was changed not obviously too.3.The monocyte of exogenous A20 overexpression after receiving the LPS (1mg/L) stimulation, its TLR4, and promoting inflammation factor TNF-α,IL-12 expression obviously reduces, but the anti-inflammation factor IL-10 expression obvious increases; Simultaneously, the ratio of TNF-α/IL-10 and IL-12/IL-10 drops obviously.Conclusion:1.TLR4 mediates the inflammation reaction of monocyte, also adjusts self- receptor TLR4 and endogenous protein A20 in a positive feedback fashion; A20 participates in the inflammation reaction mediated by TLR4, its expression increasment is related with the TLR4 expression increasment.2.Simple enhancement of the A20 expression has little einfluences to TLR4 and the signal passage of monocyte not being activated , this prompts A20 doesn't cause the inflammation reflection directly, its action possesses the dependence of TLR4 activation.3.The overexpression of A20 has been possible to suppress the inflammation reflection of monocyte mediated by TLR4 activation,its mechanism is the negative feedback fashion by which could suppresses expression of TLR4, then down-regrualted the expression of the promoting inflammation factor, increases the anti-inflammation factor expression, improves the balance relations of the promoting inflammation/anti- inflammation factor, thus achieves the function suppressing the inflame- mation response.4.This research suggests: through the gene transfection increases the A20 expression, this may suppress the inflammation reaction of the monocyte participating in the inflammation processes. This research provides important academic and practice accordings for the inflammation disease such as the gene treatment of atherosclerosis.