Kinetics Of The Activation Of NF-κB And AP-1 And Their Regulations In LPS Induced Expression Of IL-6

Kupffer cells (KC) are the principal defense cells which protect host from endogenous and exogenous infections. As important effector cells, they also play pivotal roles in the pathogenesis of hepatic injury in sepsis in local and systemic inflammatory responses. It has been well documented that nuclear factors may play an essential role in LPS induced inflammation. They regulate inflammation through adjusting the expression of proinflammatory cytokine. Nuclear factor-kappa B (NF-KB) and activator protein-1 (AP-1) are two important nuclear factors in KC. The purposes of this study is to investigate by electrophoretic mobility shift assay (EMSA) and enzyme-linked immunosorbent assay (ELISA): (1) the kinetics of the activation of NF-KB and AP-1 in KC during endotoxin, i.e.lipopolysaccharides (LPS), induced hepatic injury, and the effects of antioxidant PDTC on their activation; (2) the kinetics of proinflammatory cytokine IL-6 level in liver tissues during LPS induced hepatic injury, the effect of PDTC on the level of IL-6 ,and the intrinsic relationship betweenthe level of IL-6 and the activation of NF-KB and AP-1. Through this study, we hope to confirm the principal regulating elements in the expressional regulation of inflammatory mediators and to provide bases for effective control of inflammation. The main results and conclusions are shown as follows:1. Kinetic study of the activation of NF-кB in KC found that NF-кB activation after 3 hours of LPS infusion could be detected in Img/kg LPS group, reached the highest level in 5mg/kg LPS group, and persisted in 10mg/kg LPS group; study on the time-course after 5mg/kg LPS infusion shows that the DNA-binding activity was observable at 0.5 hour after LPS infusion, increased significantly at 3 hours, and persisted for at least 8 hours; In addition, antioxidant PDTC could inhibit the activation of NF-кB significantly.2. Kinetic study of the activation of AP-1 in KC found that AP-1 activation after 3 hours of LPS infusion could be detected in Img/kg LPS group, slightly reduced in5mg/kg LPS group compared with that of the Img/kg LPS group, and significantly increased in 10mg/kg LPS group; study on the time-course of AP-1 activation after 5mg/kg LPS infusion shows that its DNA-binding activity was detectable at 0.5 hour after LPS infusion, peaked at 1 hour, reduced after 3 hours, increased after 5 hours, then reduced after 8 hours again, all exhibiting higher activity than the control but with some fluctuations; PDTC could significantly promote the activation of AP-1.3. Further investigation by ELISA on the kinetics of IL-6 level in liver tissues during LPS-induced hepatic injury shows that IL-6 level after 3 hours of infusion increased first and then reduced; the same trend was observed in the time-course on IL-6 level after LPS infusion; PDTC could significantly inhibit the release of IL-6.Correlation analyses revealed that IL-6 level was significantly and positively correlated with the activation of NF-KB, while not significantly correlated with theactivation of AP-1. These results suggest that NF-KB may have some regulation on the expression of IL-6, while AP-1 may not.4. This study shows that, as an antioxidant, PDTC could inhibit the LPS-induced activation of NF-кB and down-regulate the expression of the proinflamrnatory cytokine IL-6. Therefore, antioxidants like PDTC probably will be potential drugs for clinical inflammation therapy.