The Role And Mechanisms Of LXRs In The Generation Of THP-1 Macrophage Microparticles Induced By LPS

Background: ALI / ARDS is a common and serious disease, excessive inflammatory response is one of the characteristic Clinical features of ALI / ARDS. Microparticles as biomarkers of inflammation play important roles in the onset and progression of ALI / ARDS. Macrophages increase rapidly in the ALI/ARDS and have important influence on it. liver X receptors can alleviate the ALI / ARDS and its, inflammatory response, but the specific mechanisms are still not clear, and further researches of the mechanisms may have great significance to the prevention and treatment of ALI / ARDS.Objective: in vitro experiments, observing the roles of LXRs in the generation of THP-1 macrophage microparticls induced by LPS as well as the relationship between THP-1 macrophage microparticls and inflammatory reaction of THP-1 macrophages induced by LPS.Methods: 1, Firstly, THP-1 macrophages were stimulated differentiation by PMA100 nM, and then replacing medium with fresh medium. Secondly, pretreating THP-1 macrophages with LXRs agonist T0901317(0μM, 5μM, 10μM, 20μM). After 6 hours, replacing medium with fresh medium. Thirdly, treating THP-1 macrophages with 0.1 μg / ml of LPS for 24 hours. The concentration of TLR4 protein was detected by western blot. The concentrations of TNF-α, IL-6, IL-1β and microparticls(instead by the concentration of phosphatidylserine) were measured by enzyme linked immunosorbent assay. 2, Firstly, THP-1 macrophages were stimulated differentiation by PMA100 nM, and then replacing medium with fresh medium. Secondly, pretreating THP-1 macrophages with LXRs agonist T0901317 10μM for(0h, 6h, 12 h, 24h). again replacing medium with fresh medium. Thirdly,treating THP-1 macrophages with 0.1 μg / ml of LPS for 24 hours. The concentration of TLR4 protein was detected by western blot. The concentrations of TNF-α, IL-6, IL-1β and microparticls(instead by the concentration of phosphatidylserine) were measured by enzyme linked immunosorbent assay. 3, Firstly, THP-1 macrophages were stimulated differentiation by PMA100 nM, and then replacing medium with fresh medium. Secondly, THP-1 macrophages were divided into three groups, they were treated by nothing, LXRs agonist T0901317(10μM), TLR4 inhibitor TAK-242(3μM). After 6 hours, replacing the medium with fresh medium Thirdly,treating THP-1 macrophages with 0.1 μg / ml of LPS for 24 hours. The concentration of TLR4 protein was detected by western blot. The concentrations of TNF-α, IL-6, IL-1β and microparticls(instead by the concentration of phosphatidylserine) were measured by enzyme linked immunosorbent assay.Result: 1, LXRs agonist T0901317 could significantly inhibit the expression of TLR4 protein and reduce the concentrations of THP-1 macrophage microparticles, TNF-α, IL-6 and IL-1β. The degree of inhibition increased with the concentration of LXRs agonist. compared with LXRs agonist T0901317 in 0μM, LXRs agonist T0901317 played a significant role in 5μM, The concentrations of TLR4 protein and TNF-α, IL-6, IL-1β, microparticles were the most significant reduction(P <0.001) in 20μM of LXRs agonist. 2, LXRs agonist T0901317 could significantly inhibit the expression of TLR4 protein and alleviate the concentrations of THP-1 macrophage microparticles, TNF-α, IL-6 and IL-1β. The degree of inhibition increased with the time of THP-1 macrophages treated by LXRs agonist. Compared with LXRs agonist T0901317 in 0h, LXRs agonist T0901317 played a significant role in 6h, and the concentrations of TLR4 protein and TNF-α, IL-6, IL-1β, microparticles were the most significant reduction(P <0.001) in 24 h. 3, The concentration of THP-1 macrophage microparticles were positively correlated with the concentrations of TLR4 protein, TNF-α, IL-6, IL-1β. 4, TLR4 inhibitor(TAK-242) and LXRs agonist T0901317 could both effectively reduce TLR4 protein expression as well as the concentrations of THP-1 macrophage microparticles, TNF-α, IL-6 and IL-1β,and the degree of TLR4 inhibitor(TAK-242) is more obvious than LXRs agonist T0901317.Conclusion: 1, LXRs agonists could inhibit the generation of THP-1 macrophage microparticles induced by LPS. 2, LXRs may regulate macrophage inflammatory responses through regulating the generation of THP-1 macrophage microparticles by TLR4 / MYD88 pathway...