Effects Of Olaparib On The Atherosclerosis Process Of THP-1 Cells And Its Relationship With NLRP3 Inflammasome Activation

Background&ObjectiveActivation of NLRP3 inflammasomes is a major contributor to atherosclerosis.Macrophage uptake of oxidized low-density lipoprotein(Ox-LDL)activates NLRP3 inflammasomes and subsequent secretion of proinflammatory mediators and foam cell formation.Poly(ADP-ribose)polymerase(PARP)is a molecular sensor of DNA damage,abound nuclear enzyme present in many eukaryotic cells including monocytes/macrophages.Evidence shows that PARP overactivation is detrimental to various inflammatory diseases including atherosclerosis.This project is to investigate whether or not 1)Ox-LDL activates PARP activation in THP-1 cells;2)inhibition of PARP by olaparib,a recently FDA approved anti-cancer drug,attenuates NLRP3 inflammasome activity and subsequent secretion of proinflammatory cytokines,foam cell formation,and cell adhesion;3)olaparib-mediated inhibition of NLRP3 inflammasomes involves the two signal steps of priming and activating on NLRP3 inflammasome activation.Methods1.THP-1 cells,widely used a human monocytic cell line,are performed for most of the experiments throughout this project.This cell line is cultured in suspension with RPMI 1640 medium containing 10%FBS at 37 ℃.5%CO2.Cells,treated with olaparib or MCC950 without or with following Ox-LDL,were divided into seven(7)groups in most experiments:Control group,Olaparib group,MCC group,Ox-LDL group,Olaparib+Ox-LDL group,MCC+Ox-LDL group,and Olaparib+MCC+Ox-LDL group.2.The western blot was extensively used in this project for the detection of protein expression and semi-quantitative assessment of protein expression based on the intensity of a protein band on the blot membrane.3.The enzyme-linked immunosorbent assay(ELISA)was used to test the amount of interleukin(IL)-1β and IL-18 secreted into culture medium,followed activation of NLRP3 inflammasomes.4.Oil Red-O staining was used to asses the foam cell formation.After fixation with formalin solution,THP-1 cells stained with filtered freshly prepared Oil Red-O working solution.Positive foam cells were quantified from images photographed using Canon DS126431 camera under an inverted microscope.5.Cell adhesion assay was used to evaluate the adhesion of THP-1 cells and human umbilical vein endothelial cells(HUVEC),mimicking the adhesion of monocytes adhesion to vascular endothelium,a very early step for monocytes migration to atherosclerotic lesion sites.6.Co-immunoprecipitation(Co-IP),a standard method commonly used to identify protein-protein interaction,was used to assess the NF-κB dynamic activity involved in the priming of NLRP3 inflammasomes using specific antibodies against NF-κB complexes in conjunction with Protein A/G affinity beads.Results1.Ox-LDL increased protein expression of NLRP3 inflammasome components(NLRP3,ASC,caspase-1)and cleaved PARP-1 in a dose-and time-dependent manner.2.PARP inhibition by olaparib inhibits Ox-LDL-induced cleaved PARP-1 protein expression but not the total PARP-1 protein expression.Olaparib also decreased the Ox-LDL-enhanced protein expression of NLRP3 inflammasome components(NLRP3,ASC,caspase-1).As expected,MCC950 decreased the protein expression of Ox-LDL-mediated NLRP3 inflammasome components as well,but olaparib or MCC950 alone did not change the NLRP3 activity compared to that of vehicle treatment.3.Ox-LDL increased the protein expression of interleukin(IL)-1β and IL-18;olaparib decreased the Ox-LDL-enhanced protein expression of IL-1β and IL-18,but olaparib alone did not change their protein expression compared to that of vehicle treatment.Consistently,Ox-LDL increased the secretion of IL-1β and IL-18 into cell culture medium detected by ELISA assay;olaparib significantly decreased the Ox-LDL-enhanced IL-1β and IL-18 concentration in the supernatant,but olaparib alone did not change their IL-1β and IL-18 concentration compared to that of vehicle treatment.4.Protein expression of LOX-1 and CD36 scavenger receptor was remarkably decreased with the treatment of olaparib plus Ox-LDL compared to that treated with Ox-LDL alone.Olaparib and MCC950 alone did not change the protein expression of scavenger receptors compared to that of vehicle treatment.Consistently,foam cell formation had remarkably increased with Ox-LDL treatment,and olaparib significantly decreased the foam cell formation stained by Oil Red-O,while olaparib and MCC950 alone did not change the foam cell formation compared to that of vehicle treatment.5.Protein expression of VC AM-1 was remarkably decreased with the treatment of olaparib plus Ox-LDL compared to that treated with Ox-LDL alone;Olaparib and MCC950 alone did not change the VCAM-1 protein expression compared to that of vehicle treatment.Consistently,cell adhesion had remarkably increased with Ox-LDL treatment,and olaparib significantly decreased the cell adhesion of monocytes to the HUVEC cells,while olaparib and MCC950 alone did not change the cell adhesion compared to that of vehicle treatment.6.Ox-LDL increased mitochondrial ROS production in THP-1 cells,detected by MitoSox Red using FASC flow cytometry.When cells treated with either olaparib or MCC950 or combined with both olaparib and MCC950,mitochondrial ROS production was significantly reduced compared to Ox-LDL alone treated cells.7.Ox-LDL increased protein expression of IKK-α,accordingly the increase in the protein expression of phospho-IκBα,and olaparib decreased the Ox-LDL-induced protein expression of IKK-a and phospho-IκBα,but olaparib or MCC950 alone had no effect on this phosphorylation change.Antibodies against anti-IκBP were precipitated p65 and p50,and p65 was precipitated by both anti-p50 and anti-IκBα as well.Fractions of co-immunoprecipitation of IκBα with p65 and p50 were significantly decreased by Ox-LDL treatment,which was abrogated by olaparib treatment,while fractions of coimmunoprecipitation of p65 with p50 were not affected by treatment with olaparib and Ox-LDL alone or in combination.8.Antibodies against anti-IκBα were precipitated RelB and p52,and Rel B was precipitated by both anti-p52 and anti-IκBα as well.Fractions of co-immunoprecipitation of IKBa with RelB and p52 were significantly decreased by Ox-LDL treatment,which was abrogated by olaparib treatment(olaparib+Ox-LDL group),while fractions of co-immunoprecipitation of RelB with p52 were not affected by treatment with olaparib and Ox-LDL alone or in combination.Conclusion1.Ox-LDL increased NLRP3 inflammasome and PARP activity,and inhibition of PARP by olaparib inhibited Ox-LDL-induced PARP activity,and Ox-LDL-mediated NLRP3 inflammasome activity as well.2.Inhibition of PARP by olaparib inhibited an Ox-LDL-mediated increase in IL-1β and IL-18 protein expression and secretion.3.Inhibition of PARP by olaparib inhibited Ox-LDL-induced foam cell formation and suppressed Ox-LDL-induced adhesion of monocytes to endothelial cells.4.Mitochondrial reactive oxygen species(ROS)was involved in olaparib-mediated inhibition of NLRP3 inflammasome activation.5.NF-Kb activation participated in olaparib-mediated NLRP3 inflammasome inhibition,and both the canonical and non-canonical pathways were involved in this olaparib-mediated inhibition of Ox-LDL-enhanced NLRP3 inflammasome activity.