[Effect and mechanism of PCSK9 on lectin-like oxidized low-density lipoprotein receptor-1 mediated oxidized low-density lipoprotein uptake by THP-1 derived macrophages].

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.