| BackgroundIn recent years, a lot of research shows atherosclerosis is a chronic inflammation disease. There are not only a lot of fat, but also a large number of infiltrated inflammatory cells in atherosclerosis lesion, which is especially characterized by the accumulation of monocytes/ macrophages cell and lymphocytes. During the occurrence and development of atherosclerosis, these inflammatory cells secrete lots of inflammatory factors, which involve in the process of cell proliferation or apoptosis, extracellular matrix synthesis and degradation, vascular regeneration and so on.PCSK9, a new gene, was discovered by the application of bio-information method and DNA array technology, which is related to the regulation of blood cholesterol metabolic. Recent research indicates that PCSK9, a secretory protein, playing a critical role in the degradation of LDLR and regulation of cholesterol metabolism, may still have some other biology function. We've already found that pcsk9 is highly expression in rabbit atherosclerosis plaque and THP-1 macrophages, and is up-regulated by LDL and ox-LDL in macrophages. Targeted interference of PCSK9 RNA can effective inhibit ox-LDL-induced apoptosis of THP-1 cells, which indicates that the ox-LDL biology function can be influenced by PCSK9 in macrophages. An important biology effect of ox-LDL is ox-LDL-induced expression and secretion of inflammatory factors in macrophages. Whether PCSK9 can intervene the secretion of inflammatory factors has not been reported.ObjectiveTo investigate the effect of PCSK9-siRNA on inflammatory factors expression in ox-LDL-induced THP-1 macrophage cells. Materials and MethodsHuman THP-1 cells were differentiated into macrophages by the addition of 160 nmol/L phorbol 12-myristate 13-acetate (PMA) for 24h. Macrophages were incubated with diffenrent concentrations of ox-LDL (0,10,20,40,80μg/ml) for different times(0,6,12,24,48h). RT-PCR was conducted to detect IL-1α, IL-6 and TNF-αmRNA. ELISA was used for analysis of IL-1α, IL-6 and TNF-αprotein level. RT-PCR and Western Blot were conducted to detect the expression of PCSK9.The siRNA for PCSK9 were designed and synthesized, then transfected into THP-1 derived macrophages by positive ion liposome Lipofectamine 2000. Transfection efficiency was assessed by fluorescence microscope assay. The most efficient siRNA was selected to transfected into THP-1 derived macrophages, after transfection for 24h, cells were treated with ox-LDL for 24h. RT-PCR detect the expression of IL-1α, IL-6 and TNF-αmRNA, ELISA analyse IL-1α, IL-6 and TNF-αprotein level, the nuclear translocation of NF-κB was detect by Western Blot.ResultsIn THP-1 derived macrophages, ox-LDL increased IL-1α, IL-6 and TNF-αmRNA and protein expression. PCSK9 was upregulated with increasing concentration of ox-LDL, while 80μg/ml ox-LDL increased significantly. The RNA interference experiment showed that PCSK9 siRNA was successfully transfected into cells and 80 nmol/L as effectively suppressed dose of siRNA was selected by RT-PCR and Western Blot. Compared with 80μg/ml ox-LDL treatment group,the suppression of inflammation factors and NF-κB nuclear translocation in THP-1 derived macrophages transfected with 80 nmol/L siRNA for 24h and incubated with 80μg/ml ox-LDL for another 24h was detected by RT-PCR ,ELISA and Western Blot.ConclusionsPCSK9 siRNA can inhibit ox-LDL induced inflammatory factor expression, such as IL1-α,IL-6 and TNF-αin THP-1 macrophage. Its mechanism is related to the inhibition of NF-κB nuclear translocation, which indicate that PCSK9 may be involved in the regulation of inflammatory reaction. |
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