PCSK9 SiRNA Inhibits HUVECs Apoptosis Induced By OxLDL Via Mitochondrial Pathway

BackgroundEndothelial injury is an initiating factor of atherosclerogenesis. Endothelial cells apoptosis is easier than necrosis leading to the occurrence of atherosclerosis. Studies have shown that smooth muscle cell apoptosis induced by oxLDL may be related to upregulate the expression of P53 and downregulate the expression of Bcl-2. Other studies have confirmed that oxLDL can bind LOX-1 receptor, reduce the expression of Bcl-2 and can inhibit apoptosis induced by a mitochondrial-dependent apoptotic signaling pathway.PCSK9 as a neural apoptosis-regulated convertase 1, plays an important role in liver regeneration, the differentiation of cortical neurons and lipid metabolism, and other multiple roles by impacts the level of low density lipoprotein receptor (LDLR) in liver.However, excessive accumulation of intracellular LDL affects the clearance of plasma LDL,then resulting in hypercholesterolemia.It has been confirmed that hypercholesterolemia is an independent atherosclerotic risk factor. Scientists have discovered that PCSK9 facilitated the neuronal differentiation in the process of embryonic development, and found to participate in the apoptosis of nerve cells in vitro, apoptotic regulatory factors Caspase3 and co-regulated death receptor 6 may be regulate this process. Our research have showed that PCSK9 siRNA inhibited oxLDL induced THP-1 derived-macrophage apoptosis, but the mechanism is unclear. Whether PCSK9 siRNA also impact apoptosis of endothelial cells have not been reported.ObjectiveIn order to examine the effect of oxLDL on PCSK9/NARC-1 expression in HUVECs, and examine the role of PCSK9 siRNA on oxLDL induced apoptosis of HUVECs and the expression of apoptotic-related proteins. Materials and MethodsHUVECs were incubated with diffenrent concentration of oxLDL for 24h.The apoptosis of HUVECs was observed by staining with Hoechst33258; RT-PCR,western blot were conducted to detect the expression of PCSK9,LOX-1 mRNAs and proteins respectively. The PCSK9 siRNAs gene were transfected into HUVECs by positive ion liposome Lipofectamine 2000. Transfection efficiency was assessed by fluorescence microscope assay. The most efficient siRNA was selected to transfected into HUVECs,after transfection for 24 h, cells were treated with oxLDL for 24 h, the rate of HUVECs apoptosis transfected siRNA was detected by staining with Hoechst 33258 and flow cytometer. Western blot was conducted to detect the expression of apototic proteins Bcl-2,Bax,Caspase3,8,9 expressions. The activity of Caspase3,9 was detected by Enzyme linked immunosorbent assay.ResultsThe results showed that a number of cells with nuclear condensation induced by 80μg/ml oxLDL for 24h increased significantly,Hoechst33258 staining showed a number of cells apoptosis. PCSK9 was upregulated with increasing concentration of oxLDL in HUVECs, while 80μg/ml oxLDL increased significantly. Moreover,RT-PCR,western blot showed that LOX-1,PCSK9 mRNAs and proteins both increased.The different concentration of PCSK9 siRNAs were transfected into HUVECs,the most effective dose of siRNA was selected by RT-PCR and western blot.HUVECs pretreated with PCSK9 siRNA for 24h , then cells treated with oxLDL for 24h, oxLDL-induced apoptosis was significantly reduced in HUVECs transfected with siRNA detected by staining with Hoechst33258 and flow cytometer. PCSK9 siRNA upregulated Bcl-2 protein expression while Bax expression was decreased. Compared with control,PCSK9 siRNA supressed Caspase3,9 proteins expression,but had on effect on Caspase8. Moreover,activity of Caspase 3,9 both decreased by PCSK9 siRNA.Apopotosis of HUVECs decreased significantly by PCSK9 siRNA,but the expression of LOX-1 between siRNA and oxLDL treatment had no difference. Conclusions1.These results revealed that the expression of PCSK9 mRNA and protein were increased by oxLDL. PCSK9 gene expression could be effectively suppressed by siRNA. So oxLDL induced apoptosis of HUVECs could be effectively suppressed by PCSK9 siRNA.2.Our study demostrated that PCSK9 siRNA inhibits HUVECs apoptosis via Bcl/Bax-Cyt C-Caspase 9-Caspase3 mitochondrial pathway.