[Effect and mechanism of PCSK9 on lectin-like oxidized low-density lipoprotein receptor-1 mediated oxidized low-density lipoprotein uptake by THP-1…
Authors of this article are:
Bao HL, Liao FJ, Fang L, Zhong F, Liu W, Li JQ.
A summary of the article is shown below:
in English, Chinese目的： 探讨前蛋白转化酶枯草溶菌素9（PCSK9）对单核巨噬细胞THP-1源性巨噬细胞血凝素样氧化低密度脂蛋白受体-1（LOX-1）介导的氧化低密度脂蛋 白（ox-LDL）摄取的影响及机制。 方法： 用丙二醇甲醚醋酸酯孵育THP-1单核细胞48 h诱导分化成巨噬细胞，人重组PCSK9蛋白预处理巨噬细胞1 h后，与ox-LDL孵育24 h诱导为泡沫细胞，油红O染色观察对照组（泡沫细胞）与不同浓度人重组PCSK9蛋白孵育组细胞内脂质蓄积情况，酶法测定细胞内胆固醇含量，实时荧光定量 聚合酶链反应（real-time PCR）与Western blot法检测LOX-1 mRNA和蛋白表达。荧光显微镜观察对照组（巨噬细胞）、PCSK9蛋白单独孵育组和PCSK9蛋白与抗LOX-1抗体、IgG抗体共孵育组对Dil标记 氧化低密度脂蛋白（Dil-ox-LDL）的摄取情况。检测对照组（泡沫细胞）与PCSK9蛋白组对Toll样受体4（TLR4）、核因子κB（NF- κB）、环氧合酶-2（COX-2）mRNA和蛋白表达影响，以及PCSK9蛋白与TLR4抑制剂（TAK-242）共同孵育组NF-κB mRNA和蛋白表达影响，与NF-κB抑制剂（PDTC）共同孵育组COX-2 mRNA和蛋白表达影响，与COX-2抑制剂（NS-398）或还原型烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶抑制剂（DPI）共同孵育组活性氧簇 （ROS）生成的影响。检测对照组（PCSK9蛋白预处理泡沫细胞）和PCSK9蛋白分别与TAK-242、PDTC、NS-398、DPI共同孵育组 LOX-1 mRNA和蛋白表达。 结果： （1）PCSK9组细胞内脂滴综合光密度、总胆固醇、胆固醇酯和胆固醇酯/总胆固醇比值均高于对照组（P均<0.05），PCSK9组LOX-1 mRNA和蛋白表达均高于对照组（P均<0.05）。（2）PCSK9组与PCSK9+IgG抗体组巨噬细胞Dil-ox-LDL荧光强度高于对照 组（P均<0.05），PCSK9+抗LOX-1抗体组荧光强度低于PCSK9组与PCSK9+IgG抗体组（P均<0.05）。 （3）PCSK9组TLR4、NF-κB、COX-2 mRNA和蛋白表达均高于相应对照组（P均<0.05），PCSK9+TAK-242组TLR4、NF-κB与PCSK9+PDTC组NF-κB、 COX-2 mRNA和蛋白表达均低于相应PCSK9组（P均<0.05）。PCSK9组ROS水平高于对照组（P<0.05），PCSK9+NS- 398和PCSK9+DPI组ROS水平均低于PCSK9组（P均<0.05）。（4）TAK-242、PDTC、NS-398、DPI分别与 PCSK9蛋白共孵育组的LOX-1 mRNA和蛋白表达均低于PCSK9蛋白单独孵育组（P均<0.05）。 结论： PCSK9可能通过THP-1源性巨噬细胞TLR4/NF-κB/COX-2/ROS途径调节LOX-1介导的ox-LDL摄取。.Objective: To investigate the effect and mechanism of proprotein convertase subtilisin type 9 (PCSK9) on lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) mediated oxidized low-density lipoprotein (ox-LDL) uptake by mononuclear macrophage (THP-1) derived macrophages. Methods: THP-1 monocyte was incubated with PMA for 48 hours to induce the differentiation into macrophages. Macrophages were pretreated with human recombinant PCSK9 protein for 1 hour and incubated with ox-LDL for 24 hours to induce foam cells. Oil red O staining was used to observe the accumulation of lipid in the control group (foam cells) and groups treated with different concentrations of recombinant PCSK9 protein, and the intracellular cholesterol content was measured by enzyme method, and mRNA and protein expressions of LOX-1 were detected by real-time PCR and Western blot. The uptake of Dil-labeled oxidized low density lipoprotein (Dil-ox-LDL) was observed by fluorescence microscopy in control group (macrophage), PCSK9 protein treated group and PCSK9 protein plus anti-LOX-1 antibody and IgG antibody treated group. mRNA and protein expression of toll-like receptor 4 (TLR4), nuclear factor-kappa B (NF-κB), cyclooxygenase-2 (COX-2) were detected in control and PCSK9 protein treated group in the absence and presence of TLR4 inhibitor (TAK-242), NF-κB inhibitor (PDTC). In addition, reactive oxygen species (ROS) production was evaluated in the absence or presence of COX-2 inhibitor (NS-398) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor (DPI). The mRNA and protein expression of LOX-1 in the control group (PCSK9 protein pretreated foam cells) and PCSK9 protein group in the absence or presence of TAK-242, PDTC, NS-398 and DPI respectively. Results: (1) The total optical density of intracellular lipid droplets, total cholesterol level, cholesterol ester level and cholesterol ester/total cholesterol ratio as well as expression of LOX-1 were significantly higher in PCSK9 group than those in control group (all P<0.05). (2) The fluorescence intensity of Dil-ox-LDL was significantly higher in PCSK9 group and PCSK9+IgG antibody group than in the control group (all P<0.05). The fluorescence intensity was significantly lower in PCSK9+anti-LOX-1 antibody group than in PCSK9 group and PCSK9+IgG antibody group (all P<0.05). (3) The expressions of TLR4, NF-κB and COX-2 were significantly higher in PCSK9 group than in control group (all P<0.05). The expressions of TLR4, NF-κB and COX-2 were significantly lower in PCSK9+TAK-242 group and PCSK9+PDTC group than in PCSK9 group (all P<0.05). The ROS level was significantly higher in PCSK9 group than in the control group (P<0.05). The ROS levels were significantly lower in PCSK9+NS-398 and PCSK9+DPI groups than in PCSK9 group (all P<0.05). (4) The expressions of LOX-1 mRNA and protein were lower in respective PCSK9 protein plus TAK-242, PDTC, NS-398 or DPI group than in PCSK9 protein alone (all P<0.05). Conclusion: PCSK9 may regulate LOX-1 mediated ox-LDL uptake by the THP-1 derived macrophage via TLR4/NF-κB/COX-2/ROS pathway.
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This article is a good source of information and a good way to become familiar with topics such as: Atherosclerosis; Lipoproteins, LDL; Proprotein convertase subtilisin type 9; Toll-like receptor 4.