PCB118 Induces Pyroptosis Through ROS-NLRP3 Inflammasome Pathway In HUVECs

Objective:Vascular endothelial cell pyroptosis,which may alter the integrity of the endothelium and lead to plaque instability,plays a critical role in the development and pathogenesis of atherosclerosis.In our present study,we explored whether PCB118 exposure could induce pyroptosis and the mechanism involved in human umbilical vein endothelial cells(HUVECs).Hope to provide a new target to prevent and treat atherosclerosis and cardiovascular disease.Methods:1)The HUVECs were exposed to different concentrations(0.1,0.5,1.0,5.0 or 10?M)of PCB118 for 48 h.(1)We evaluated the cell viability and the integrity of cell membrane in HUVECs using CCK-8 and LDH detection kits after exposed to PCB118 for 48 hours.(2)To identify whether necroptosis,a programmed cell death that depends on receptor-interacing Ser/ Thr-protein kinase 1(RIPK1),or pyroptosis,are involved in PCB118-induced cell death,RIP-1 kinase inhibitor Necrostatin-1(NEC,20?M)and pyroptosis inhibitor Gl-ycine(Gly,5mM)were used.(3)Because the cell death is Caspase-1-dependent,so we confirmed whether PCB118-induced cell death was caspase-1-dependent pyroptosis,HUVECs were cultured with 10?g/ml Caspase-1 inhibitor(AC-YVAD)and 10?M PCB118 for 48 h.Then,Western blotting and LDH detections were performed;2)We performed small interfering RNA(siRNA)to knockdown NLRP3 and the NLRP3 silencing HUVECs were treated with 10?M PCB118 for 48 h.Then we performed Western blottingting,CO-Immunoprecipitation and LDH detections to determine whether PCB118-induced pyroptosis is dependent on the activation of NLRP3 inflammasome;3)To evaluate whether the PCB118-induced pyroptosis is dependent on excessive ROS,an antioxidant,Vitamin E,was used.Then Western blottingting,LDH,ROS and CO-Immunoprecipitation detections were performed;Results:1)Exposure to10?M PCB118 sinificantly decreased the cell viability and impaired the cell membrane integrity in HUVECs: Upon exposure to10?M PCB118,cell viability decreased by 30% and LDH release increased signifi-cantly than that in the control group;2)Impaired cell membrane integrity was observed in HUVECs exposed to10?M PCB118: 20?M Necrostatin-1 could not reduce the release of LDH significantly,but 5mM Glycine reduced the release of LDH by about 30%;3)PCB118-induced pyroptosis was dependent on the activation of Caspase-1: exposure to PCB118 increased the level of activated Caspase-1 and IL-1? in HUVECs;10?g/ml Caspase-1 inhib-itor AC-YVAD decreased the levels of activated Caspase-1 and IL-1?,while the release of LDH reduced by about 25%;4)PCB118-induced pyroptosis was dependent on NLRP3 inflammasome activation: exposure to PCB118 increased the expres-sion of NLRP3,and higher levels of NLRP3 and Caspase-1 which were associated with TMS1/ASC significantly increased in HUVECs treated with 10?M PCB118.Furthermore,PCB118 failed to induce activation of Caspase-1 and IL-1? maturation in NLRP3 silencing endothelial cells.PCB118-induced LDH release significantly decreased in NLRP3 silencing cell by about 30% than that in the wild type HUVECs;5)PCB118-induced NLRP3-dependent pyroptosis was dependent on he ROS pathway: Intracellular ROS level was significantly increased in HUVECs treated with 10?M PCB118 than that in the control cells.VitaminE,which significantly decrease the intracellular ROS production,significantly decrease LDH release than that in the control group,while the expression of NLRP3,activated Caspase-1 and IL-1? were significantly decreased.Furthermore,the levels of NLRP3 and Caspase-1,which were pull downed by TMS1/ASC antibody also significantly decreased.Conclusions:1)Exposure to 10?M PCB118 significantly decreased cell viability and impaired the cell membrane integrity in HUVECs;2)Exposure to 10?M PCB118 could induce Caspase-1-dependent pyroptosis in HUVECs;3)PCB118-induced pyroptosis was dependent on NLRP3 inflammasome pathway;4)PCB118-induced NLRP3-dependent pyroptosis was mediated by ecessive ROS production.