The Effect And Mechanism Of TLR4 Gene Silencing On Angiogenesis And Apoptosis In Rabbit Carotid Atherosclerotic Plaque

Atherosclerosis(AS) is the pathological basis of coronary artery and cerebrovascular disease, responsible for 29% of deaths worldwide,and will become the leading cause of death by 2020. Thus the prevention and treatment of atherosclerosis has a significant economic and social benefits.Atherosclerosis is a multi-factorial inflammatory disease, and the clinical complications of atherosclerosis, including myocardial infarction and stroke, are mainly caused by thrombus formation, which results from rupture of an atherosclerotic plaque.Toll-like receptor 4(TLR4)as a pattern recognition receptor takes part in the formation and development of AS. Vulnerable plaque usually consists of thin fibrous cap, large liquid core, inflammatory cell infiltration, and influenced by hemodynamic. Recent research showed that angiogenesis and cellulaar apoptosis play a crucial role in vulnerable plaque rupture. Thereby inhibiting plaque angiogenesis and reducing apoptosis are important means of preventing plaque rupture.Objevtives:This study was to use siRNA silencing Toll-like receptor 4(TLR4) gene,and observe angiogenesis and apoptosis in rabbit carotid atherosclerotic plaque, in order to provide new mechanisms and methods for the prevention and treatment of atherosclerosis.Methods:1. We built rabbit carotid artery atherosclerosis model through high-fat diet combined with rabbit carotid artery balloon injury, and carried out si RNA-TLR4 gene with adenoviral vector during operation. We seperated the animals into control group,model group, si RNA-negative group and si RNA-TLR4 group.And observe changes of TLR4, NF-κB, hypoxia-inducible factor-1α(HIF-1α), vascular endothelial growth factor(VEGF) signaling pathway and apoptosis in gene and protein levels.2. By transfection of sh RNA-TLR4 with lentivirus vector in human vein umbilical vein endothelial cell(HUVEC), we analyze the influence of TLR4 gene on growth,proliferation, migration and apoptosis of endothelial cells, and investigate the molecular biological mechanism of TLR4 gene.Results:1.(1) 81.8% of rabbit carotid artery atherosclerosis model was successfully established.(2)The serum lipids levels increased after high-fat diet, the lipids levels of experimental groups were higher than those of control group, but there were no significant difference between the experimental groups(p>0.05);(3)HE staining showed that the intima-media thickness ratio, the percentage of plaque area of si RNA-TLR4 group was smaller compared to model group and si RNA-negative group(p<0.05);Immunohistochemistry indicated that there were CD31, HIF-1αexpression in carotid artery therosclerotic plaque,and the expression of CD31,HIF-1a in si RNA-TLR4 group was smaller compared to model group and si RNA-negative group(p<0.05);TUNEL staining suggested there was apoptosis occurrence in therosclerotic plaque,and the apoptosis rate of si RNA-TLR4 group was smaller compared to model group and si RNA-negative group(p<0.05);(4)RT-PCR, Western-blot showed that the m RNA and protein levels of TLR4, NF-κB,HIF-1α, and VEGF in si RNA-TLR4 group decreased(p<0.05).2.(1)The TLR4 activation of HUVEC after LPS stimulaion was time and concentration dependent,the results showed that TLR4 m RNA peaked at 24 h after LPS stimulation at a concentration 1μg/ml;(2)Flow cytometry analysis showed that apoptosis reached peak at 6h after LPS stimulation at a concentration 1μg/ml;(3)RT-PCR, Western-blot showed that the m RNA and protein levels of TLR4, NF-κB,and VEGF in sh RNA-TLR4 group decreased(p<0.05), there was no difference for HIF-1α between groups(p>0.05);(4)MTT showed that the cell proliferation ability of sh RNA- TLR4 group was inferior to control group(p<0.05),the wound healing experiment showed that there was no difference for migration between groups(p>0.05), the tube formation experiment showed that the tube formation ability of sh RNA-TLR4 group was weaker than control group(p<0.05);(5)The apoptosis rate in sh RNA-TLR4 group dereased(p<0.05).Conclusions:1.TLR4 gene silencing can reduce the downstream HIF-1α and VEGF expression through inhibiting the TLR4, NF-κB signal pathway activation, which resulted in plaque stable via decreasing the neovascularization occurrence and cellular apoptosis in atherosclerotic plaque.2.The TLR4 activation of HUVEC was time and concentration dependent, and cellular apoptosis could reach peak after LPS stimulaion; TLR4 gene silencing could decrease cellular apoptosis through inhibiting the TLR4, NF-κB signal pathway activation; The VEGF can promote neovascularization formation and development through TLR4,NF-κB signal pathway activation,and sh RNA-TLR4 can attenuate the effect but not through HIF-1α; TLR4 gene inhibit cell proliferation,and not influence migration.