Effects Of Phycocyanin On LPL Expression And Lipid Accumulation In Macrophages And Its Mechanism

[Background and Objective]Atherosclerosis?As?is a vascular degenerative disease caused by various factors such as lipid metabolism disorder,which seriously endangers human health.In the pathogenesis of As,the formation of macrophage-derived foam cells constitute the core link.Therefore,reducing macrophage-derived foam cell formation is important for the prevention and treatment of As.Previous studies have shown that lipoprotein lipase?LPL?is a key rate-limiting enzyme for the hydrolysis of plasma triglycerides and is essential for lowering plasma triglyceride levels.However,LPL promotes the formation of foam cells by mediating intracellular lipid accumulation in macrophages,thereby promoting the progress of As.Exploring the mechanism of regulation of LPL expression,reducing macrophage lipid accumulation is one of great significance for the prevention and treatment of As.Phycocyanin?PC?is a kind of pigment protein widely found in spirulina,which content in spirulina is about 10%to 20%.The structure is stable at room temperature.Studies have shown that phycocyanin plays a variety of biological functions in anti-oxidation,anti-tumor,immune response,but whether it affects the formation of foam cells by reducing macrophage lipid accumulation is still unclear.Mitogen-activated protein kinase kinase kinase 7?TAK1?regulates cell proliferation and apoptosis,mediates the activation of various pro-inflammatory and pro-survival signaling pathways,and participates in the pathological process of As.Stress-activated protein kinase?JNK?is a factor downstream of TAK1,and elevated expression of TAK1 increases phosphorylated JNK levels,thereby causing activation or inhibition of JNK downstream factors.Activating transcription factor 3?ATF3?,a member of the ATF/cyclic adenosine response element binding protein transcription factor family,is located downstream of JNK and is a key response gene for lipoprotein lipolysis products rich in triglycerides.The accumulation of inflammatory factors promotes the occurrence of As.However,whether the TAK1/JNK/ATF3 signaling pathway is involved in PC regulation of macrophage LPL expression and lipid accumulation is unclear.MiR-10a-5p is a newly discovered RNA molecule,which is closely related to tumorigenesis and inflammatory response.The study found that miR-10a-5p can target TAK1,thereby increasing the area of As plaque and accelerating the occurrence of As.However,whether miR-10a-5p participates in the regulation of LPL expression and lipid accumulation in macrophages by PC is still unclear.Therefore,the purpose of this study was to investigate the effects of PC on the expression of LPL and lipid accumulation in macrophages from the perspective of lipid metabolism.[Methods]THP-1 cells were cultured in 10%fetal bovine serum?PBS?in RPMI 1640 medium?containing 10 U/mL penicillin and 10U/mL streptomycin?,and the cell incubator temperature was maintained at 37°C while ensuring the inside of carbon dioxide water tank incubator5%CO₂ concentration.THP-1 cells were incubated with 160 nmol/L phorbol?PMA?for 48 hours to promoted differentiation and transformation into macrophages.Macrophages were incubated with 50?g/ml oxidized low density lipoprotein?ox-LDL?serum-free medium.At first,PC?5,10,20?g/ml?and PBS treated the cells for 6,12,24 hours,and then,LPL mRNA and expression were detected by RT-PCR and Western Blot,LPL activity was detected by LPL activity colorimetric assay kit.High Performance Liquid Chromatography?HPLC?was used to measure intracellular lipid content.Chromatin immunoprecipitation?ChIP?and luciferase reporter gene?Luciferase?were used to detect the effects of PC on the binding of transcription factor ATF3 to LPL promoter region and LPL promoter activity;using TAK1 overexpression plasmid,JNK agonist anisomycin and ATF3 overexpression after plasmid treatment,RT-PCR and Western Blot were used to detect the effects of PC on TAK1 mRNA and expression,JNK and its phosphorylation level,ATF3 mRNA and expression,LPL mRNA and expression.Verify that PC affected LPL expression via the TAK1/JNK/ATF3 signaling pathway.Using bioinformatics analysis to predict and compare the sequence,conservation and target genes of miR-10a-5p between different species,based on this,to determined the binding site of miR-10a-5p and TAK1,using the microRNA target prediction website found that the miR-10a-5p binds to TAK1 with a seed sequence and the binding free energy was low.The luciferase reporter gene detected the binding of miR-10a-5p to the TAK1 3'UTR,and the miR-10a-5p inhibitor transfected THP-1 cells for24 hours.PC was added for processing,RT-PCR and Western Blot were used to detect the expression of related genes and proteins.[Results]RT-PCR,Western Blot and high performance liquid chromatography showed that the levels of LPL mRNA and expression,total cholesterol,free cholesterol and cholesterol ester in PC-treated macrophage cells were significantly decreased in a concentration-and time-dependent manner.A bioinformatic predictive analysis revealed that there might be a binding site between the transcription factor ATF3 and the promoter of LPL.The results of luciferase reporter gene and chromatin immunoprecipitation showed that the transcription factor ATF3 could enhance the activity of the LPL promoter,and PC treatment significantly inhibited the binding of ATF3 to the LPL promoter.RT-PCR and Western Blot assays showed that PC decreased ATF3mRNA and expression,down-regulated LPL mRNA and expression,but overexpression of ATF3 reversed the inhibitory effects of PC on LPL,indicating that ATF3 is a key factor involved in the regulation of LPL.Next,the effects of PC on JNK was examined,it was found that PC inhibited the activation of JNK and restored ATF3,LPL mRNA and expression after treatment with JNK agonist anisomycin.JNK is another subclass of MAPK signaling pathway,TAK1 is the upstream of JNK,and we further examined the effects of PC on TAK1.The results showed that PC inhibited the TAK1 mRNA and expression,then after overexpression of TAK1,the phosphorylation JNK level,ATF3,LPL mRNA and expression returned to normal,proving PC through the TAK1/JNK/ATF3 signaling pathway inhibits the expression of LPL.The data provided by bioinformatics analysis indicated that miR-10a-5p is highly conserved among different species,and miR-10a-5p binds to TAK1 with a seed sequence and the binding free energy of both is low.These results suggested that miR-10a-5p has the potential to bind to the 3'untranslated region of TAK1?3'UTR?.Analysis by TargetScan found that the LPL 3'untranslated region?3'UTR?had no binding site to miR-10a-5p.PC treatment significantly increased the level of intracellular miR-10a-5p;luciferase reporter gene assay confirmed that miR-10a-5p targets the TAK1 3'untranslated region?3'UTR?.When treated with PC,it could significantly decreased the phosphorylation JNK level,and down-regulated TAK1,ATF3,LPL mRNA and expression;however,when treating with miR-10a-5p inhibitor,the effects on phosphorylation JNK level,TAK1,ATF3,LPL mRNA and expression was reversed,this indicated that miR-10a-5p was involved in the regulation of LPL expression and macrophage lipid accumulation by PC.[Conclusion]PCup-regulatesmiR-10a-5p,inhibits TAK1/JNK/ATF3 signaling pathway,and decreases LPL expression and lipid accumulation in THP-1 macrophages.