Institute of Cardiovascular Disease, Key Lab for Arteriosclerology of Hunan Province, University of South China, Hengyang, 421001, Hunan, China.
Mol Cell Biochem. 2012 Jan;359(1-2):347-58. doi: 10.1007/s11010-011-1028-6. Epub 2011 Aug 17.
This paper investigated the effects of ox-LDL on PCSK9, and the molecular mechanisms of PCSK9 siRNA-inhibited apoptosis induced by ox-LDL in human umbilical vein endothelial cells (HUVECs), to clarify the role of PCSK9 in atherosclerogenesis. HUVECs were incubated with ox-LDL for 24 h. The apoptosis was observed by Hoechst 33258 staining. The expression of PCSK9, LOX-1 mRNAs and proteins was detected by RT-PCR, western blot, respectively. The PCSK9 siRNAs labeled with fluorescence were transfected into HUVECs by Lipofectamine 2000. After transfection for 24 h, cells were treated with ox-LDL for 24 h, HUVECs apoptosis transfected siRNA was detected by Hoechst 33258 staining and flow cytometer. The expression of Bcl-2, Bax, caspase3, 8, 9 was detected by western blot. The activity of caspase3, 9 was detected by kits. Our results showed that apoptosis of HUVECs and the expressions of PCSK9 and LOX-1 were upregulated secondary to induction by ox-LDL in a concentration-dependent manner. However, ox-LDL-induced HUVEC apoptosis and PCSK9 expression, but not LOX-1 expression, were significantly reduced by PCSK9 siRNA. These results demonstrate a linkage between HUVEC apoptosis and PCSK9 expression. Furthermore, we detected the possible pathway involved in apoptotic regulation by PCSK9 siRNA; our results showed that the expression of Bcl-2 decreased, whereas that of Bax increased. In addition, ox-LDL enhanced the activity of caspase9 and then caspase3. Pretreatment of HUVECs with PCSK9 siRNA blocked these effects of ox-LDL. These findings suggest that ox-LDL-induced HUVECs apoptosis could be inhibited by PCSK9 siRNA, in which Bcl/Bax-caspase9-caspase3 pathway maybe was involved through reducing the Bcl-2/Bax ratio and inhibited the activation of both caspase9 and 3.
本文研究了 ox-LDL 对 PCSK9 的影响，以及 PCSK9siRNA 抑制 ox-LDL 诱导的人脐静脉内皮细胞（HUVEC）凋亡的分子机制，以阐明 PCSK9 在动脉粥样硬化形成中的作用。将 HUVEC 与 ox-LDL 孵育 24 小时。通过 Hoechst 33258 染色观察细胞凋亡。分别通过 RT-PCR 和 Western blot 检测 PCSK9、LOX-1mRNA 和蛋白质的表达。用 Lipofectamine 2000 将荧光标记的 PCSK9siRNA 转染入 HUVEC。转染 24 小时后，用 ox-LDL 处理细胞 24 小时，通过 Hoechst 33258 染色和流式细胞术检测转染 siRNA 的 HUVEC 凋亡。Western blot 检测 Bcl-2、Bax、caspase3、8、9 的表达。通过试剂盒检测 caspase3、9 的活性。结果表明，ox-LDL 诱导 HUVEC 凋亡及 PCSK9 和 LOX-1 表达呈浓度依赖性上调。然而，PCSK9siRNA 显著降低了 ox-LDL 诱导的 HUVEC 凋亡和 PCSK9 表达，但不影响 LOX-1 表达。这些结果表明 HUVEC 凋亡与 PCSK9 表达之间存在联系。此外，我们检测了 PCSK9siRNA 调节凋亡的可能途径；结果显示 Bcl-2 表达减少，而 Bax 表达增加。此外，ox-LDL 增强了 caspase9 的活性，进而增强了 caspase3 的活性。HUVEC 用 PCSK9siRNA 预处理可阻断 ox-LDL 的这些作用。这些发现表明，PCSK9siRNA 可抑制 ox-LDL 诱导的 HUVEC 凋亡，其中 Bcl/Bax-caspase9-caspase3 途径可能通过降低 Bcl-2/Bax 比值并抑制 caspase9 和 3 的激活而发挥作用。
