The Evaluation Of The Impact Of A20 Deletion On GlcN Induced Vascular Protection In Vivo

Object:This study aims to analyze the impact of A20 deletion on neointima after vascular injury, furthermore, aims to discuss the impact of A20 deletion on GlcN inducing the efficacy of vascular protection via integral animal experiment.Methods:(1) The packaging, amplification and effectiveness of recombinant adenovirus:① AD-shA20-eGFP and AD-NULL-eGFP were packaged by liposome transfection, and the recombinant adenovirus was amplified and collected.② Cultivation of rat aortic smooth muscle and transfer of culture, adenovirus used to inoculate the rat’s aortic smooth muscle, fluorescence microscope used to observe the effectiveness, and extract the cellular proteins, detect of A20 protein level, verify the validity of the A20 in subtractive adenovirus.(2) Construction of rat left carotid artery balloon injury model and the experimental group: healthy male SD rats weighing 350-400 grams,72, divided for three groups, (n=24);① AD-shA20 group (balloon injury of the left carotid artery+transfected AD-shA20-eGFP);② AD-NULL group (balloon injury of the left carotid total artery+transfected AD-NULL-eGFP; ③ physiological saline group (balloon injury of the left carotid artery+normal saline.Each large group divided into two different types of processing:Vehicle group and GlcN treatment group (0.3mg·g-1·day-1 ip), Continuous injection 14d.(3) On the fifth day of the operation,3 rats of each group were extracted, remove the left common carotid artery injury segment and made frozen sections and fluorescence microscope observation of the content of green fluorescent protein, to evaluate the transfection efficiency of AD-shA20-eGFP, AD-NULL-eGFP.(4) On the fourteenth day after the operation, the common carotid artery and the right side of the left side of each group were taken as control, and HE staining was done to make the morphological analysis of the intimal hyperplasia.(5) The expression of A20, P-P65 and O-GlcN in the left balloon injured vascular tissue was detected by WB method.Results:(1) The expression of A20 was reduced in the RASMCs transfected by AD-shA20, showed that AD-shA20-eGFP has a role in reducing the role of A20, proved the effective packaging of adeno virus.(2) Under the fluorescence microscope observation of frozen section, you can see the vascular intima and the film has a large number of green fluorescent distribution, proved balloon strain AD-shA20-eGFP,AD-NULL-eGFP transfection success.(3) When AD-NULL group and saline group O-GlcNAc modification levels increase, neointimal proliferation was obviously inhibited; When improving the group AD-shA20 O-GlcNAc modification level, neointimal proliferation inhibition significantly decreased; group AD-shA20 without any increase in O-GlcNAc modification levels, neointimal hyperplasia is more significant than in the other groups.(4) When improve AD-NULL group and saline group O-GlcNAc modification level, the expression of P-P65 were significantly suppressed. In group AD-shA20, the expression of A20 significantly decreased, and when improve o-glcnacylation level expression P-P65 cannot be significantly inhibited; Compared with other groups, the expression of P-P65 in AD-shA20 group (Vehicle group) was significantly higher than that in the other groups, and the expression of was significantly higher.Conclusion:(1) Local transfection of AD-shA20 after carotid artery balloon injury in rats can reduce the expression of A20 in local tissues;(2) When improving the O-GlcNAc level of the body, the proliferation of the blood vessel after balloon injury was inhibited;(3) The lack of A20 can weaken the protective effect of GlcN on the vascular intima after balloon injury.