Experimental Study Of Zinc Finger Protein A20 Inhibits Oxidized Low Density Lipoprotein Induced Damages In Smooth Muscle Cell

Objective: To investigate the effects of ox-LDL on the expression of LOX-1 and TLR-4 mRNA, the nuclear translocation of NF-κB and the migration of rat aortic vascular smooth muscle cells(VSMC) with and without exogenous zinc finger protein A20 gene and to explore the possible effects and mechanisms that zinc finger protein A20 inhibits oxidized low density lipoprotein induced damages in smooth muscle cell.Methods: Sprague-Dawley(SD) rats'thoracic aorta VSMC were primarily cultured in vitro and were randomly divided into four groups: A group (control group, VSMC were cultured with normal DMEM medium with 10% fetal bovine serum for 48 hours); B group (ox-LDL intervention group, VSMC were cultured in normal DMEM medium with 10% fetal bovine serum for 24 hours, and then they were incubated with ox-LDL (50μg/ml)for 24 hours); C group(A20 group, VSMC were transfected with plamid containing A20 gene and were cultured for 48 hours) and D group(ox-LDL and A20 intervention group, VSMC were transfected with plamid containing A20 gene and were cultured for 24 hours, then they were incubated with ox-LDL (50μg/ml)for 24 hours). All of the cells were collected. TLR4 and LOX-1 mRNA expression were measured by RT-PCR. The nuclear translocation of NF-κB was measured by nuclear protein Western blot analysis. The migration of VSMC were measured by Boyden chamber.Results: The expression of LOX-1 and TLR-4 mRNA, the activation of NF-κB and the migration of rat aortic VSMC after were incubated with ox-LDL(50μg/ml)for 24 hours. However, when VSMC were transfectedwith plamid containing A20 gene, the expression of TLR-4 mRNA and the migration of rat aortic VSMC reached normal levels. Even the expression of LOX-1 mRNA and the NF-κB nuclear translocation of VSMC reduced to lower than normal level after VSMC were transfected with plamid containing A20 gene.Conclusion:1. ox-LDL can significantly activate NF-κB signaling system and enhance migration in VSMC. The activation may induce the inflammatory response in arterial wall, the migration of VSMC to the intima, and triggering the process of atherosclerosis.2. ox-LDL can stimulate LOX-1 and TLR-4 mRNA expression and migration of VSMC. As receptors of transmembrane signal transduction, LOX-1 and TLR-4 induce the development of atherosclerosis. Therefore, the effect of ox-LDL may play the role of positive feedback and further expand the damaging effects of ox-LDL, and then promote the development of atherosclerosis.3. Transfecting plasmid containing A20 gene completely inhibits the ox-LDL triggering effect on inflammation, meanwhile completely blocks its stimulating effects on LOX-1 and TLR-4 mRNA expression, and migration of VSMC. What is more it may thus blocking the continuous expansion of the cascading effect of positive feedback, which may thoroughly prevent the development of atherosclerosis. Meanwhile because A20 effects through inhibiting NF-κB signaling, from the other hand, ox-LDL effects at least in part by activating NF-κB signaling.