Regulation Of Monocyte/Macrophage Inflammatory Response And Its Lipid Phagocytosis By PCSK9 Through Liver-derived MIF

Objective: Targeted expression of the proprotein convertase subtilisin/kexin type 9(PCSK9)in the liver induces hepatic macrophage migration inhibitory factor(MIF);Targeted therapy(MIF)expression and release,while lipid loading and inflammatory response of monocytes/macrophages are key components of atherosclerosis(AS)formation.This paper aims to explore the regulatory role and molecular mechanisms of PCSK9 interaction with MIF on macrophage inflammatory response and lipid phagocytosis at the cellular level.Methods:(1)Using Si RNA-mediated MIF gene transfection of THLE-3 cells,the experiment was divided into MIF low expression group(Si-MIF-THLE-3)and MIF normal expression group(Con-THLE-3),and real-time fluorescence quantitative PCR(quantitative real-time PCR,RT-q PCR)was used to detect PCSK9 and MIF m RNA in hepatocytes.PCSK9 and MIF m RNA expression were detected in hepatocytes by quantitative real-time PCR(RT-q PCR).(2)In the Si-MIF group and Con group,PCSK9 protein intervention was administered and not administered,and PCSK9 and MIF protein and m RNA expression in hepatocytes were detected by Western Blot and q PCR,and the contents of PCSK9 and MIF in the culture medium were measured by ELISA.And the culture medium of the above hepatocytes was collected.(3)THP-1cells were transfected with Si RNA-mediated MIF gene,and the experiment was divided into MIF low expression group(Si-MIF-THP-1)and MIF normal expression group(Con-THP-1).western Blot was used to detect the protein expression of MIF.(4)The collected hepatocyte cultures were added to Si-MIF-THP-1 and Con-THP-1 groups for 48 h.The PCSK9 intervention group was additionally equipped with TLR4-specific inhibitor TAK-242(1 M).Western Blot to detect the expression of TLR4-NF-κB signaling pathway in the cell lines.(5)Add 50 μg/m L of ox-LDL to the above culture system,continue to culture for 48 h,and observe the changes of intracellular lipid droplets by oil red O staining;quantify the lipid phagocytosis of THP-1 cells by reading the OD value with an enzyme marker.Result:(1)MIF m RNA expression was significantly lower in the Si-MIF-THLE-3 group compared with the Con-THLE-3 group,while the m RNA expression of PCSK9 was elevated(both P< 0.05).(2)MIF protein and m RNA expression as well as levels in the culture medium were elevated in the group given PCSK9 protein compared to the group not given,while protein and m RNA expression of PCSK9 as well as levels in the culture medium were unchanged(all P < 0.05).(3)MIF protein expression was significantly lower in the Si-MIF-THP-1group compared with the Con-THP-1 group(all P < 0.05).(4)The expression of inflammatory factors was lower in the Si-MIF-THP-1 group compared with the Con-THP-1 group,and the expression of inflammatory factors was higher in the group given PCSK9 protein compared with the group not given and the group given PCSK9 protein with TLR4 inhibitor;the expression of M1 phenotype was lower in the Si-MIF-THP-1 group compared with the Con-THP-1 group,and the expression of inflammatory factors was higher in the group given PCSK9 protein compared with the group not given and the group given M1 phenotype expression was higher in the Si-MIF-THP-1 group than in the Con-THP-1 group,and m RNA and protein expression of TLR4 was higher in the Si-MIF-THP-1 group than in the Con-THP-1 group,and m RNA and protein expression of TLR4 was higher in the Si-MIF-THP-1 group than in the Con-THP-1 group(both P < 0.05).)Conclusion: PCSK9 can induce the synthesis and secretion of MIF in hepatocytes,and MIF secreted by hepatocytes can activate pro-inflammatory macrophages in the circulatory system and bind to the macrophage surface receptor TLR4 to activate the downstream NF-κB inflammatory signaling pathway and promote the secretion of TNF-,IL-6,IL-1β,MCP-1 and other inflammatory factors to stimulate inflammatory effects,while MIF can promote lipid phagocytosis by macrophages and promote their foaminess.