LPS is the major toxic component of the outer membrane
LPS is the major toxic component of the outer membrane of gram-negative bacteria and high dose of LPS challenge in animal can induce a rapid systemic inflammatory response which resembles septic shock in clinical . We demonstrated that administration of KPT330 significantly attenuated lung injury including interstitial oedema, haemorrhage, alveolar collapse, inflammatory cell infiltration and increased survival rate in LPS-induced septic mice. By analyzing serum samples from septic mice, we showed that the levels of inflammatory cytokines TNF-α, IL-6 and HMGB1 were significantly increased at 12 h after sepsis induction, while treatment with KPT330 reduced the production of these proinflammatory cytokines. Moreover, innate phagocytes including PMNs and macrophages form the first line in the host defense against microbial infection. The recruitment inflammatory Safingol mg from the circulation into the infectious site plays a key role in eradicating the invaded microbial pathogens and successful clearance of bacterial infection has been shown to rely on a rapid and efficient macrophage and polymorphonuclear neutrophils (PMN) migration into the infectious site such as peritoneal cavity in several experimentally established murine polymicrobial sepsis models . We observed decreased macrophage and PMN subpopulations in peritoneal exudate from LPS-challenged mice, which indicated that KPT330 treatment after LPS injection substantially attenuated LPS-induced inflammatory cell infiltration. Proinflammatory cells, mainly activated macrophages, are responsible for most of the cellular and molecular pathophysiology of sepsis by producing cytokines and other proinflammatory molecules such as TNF-α, IL-6 and HMGB1, which further augment systemic inflammation . Based on KPT330\'s effective in LPS-induced sepsis model, we then sought to the potential anti-inflammatory effects of KPT330 in vitro using macrophage cell culture system. Our results demonstrated that KPT330 treatment inhibited LPS-induced TNF-α, IL-6 and HMGB1 with a dose-dependent manner in murine macrophages. To further elucidate the molecular mechanism, the mRNA level of these proinflammatory cytokines were examined. We found exposure of LPS for 8 h significantly increased mRNA expression of TNF-α and IL-6, but not HMGB1 mRNA level. And the necleocytoplasmic translocation of HMGB1 was obviously induced under LPS stimulation. KPT330 treatment significantly suppressed TNF-α and IL-6 mRNA expression and inhibited HMGB1 trafficking from the nucleus to the cytoplasm. As CRM1 is an export receptor for intracellular transport mediated by the nuclear export signal, we examined the influence of KPT330 on CRM1 distribution in LPS-stimulated macrophages. Our results provided us evidence indicating that KPT330 prevented LPS-induced HMGB1 translocation through inhibiting CRM1 re-localization. During sepsis, massive cytokine production can be triggered by exogenous pathogen-associated molecular pattern molecules (PAMPs) from invading microorganisms, or by endogenous damage-associated molecular pattern molecules (DAMPs) released from injured host cell. Both PAMPs and DAMPs bind to various types of receptors in immune cells and activate several signaling pathways, and in particular the NF-κB and MAPKs pathway, which are the two important signal pathway to regulate the production of broad range of cytokines . Previous studies showed that specific inhibiting of CRM1-mediated nuclear protein export, further increases the nuclear accumulation of IκBα and reduces TNF-α release [9,19]. Our further Western Blot analyses showed that KPT330 antagonized LPS induced the phosphorylation of p65, degradation of IκBα and p65 nuclear translocation. In addition, we examined activation of MAPKs pathway. Our data showed KPT330 treatment reduced the LPS-evoked phosphorylation level of p38, but not of JNK and ERK1/2, with a dose-dependent manner. Taken together, in the current study we demonstrate the potent anti-inflammation effects of KPT330, a potent selecitve inhibitor of CRM1 in vivo and in vitro. KPT330 protects mice against LPS associated septic death and ameliorates LPS-induced lung injury, with suppressed levels of TNF-α, IL-6 and HMGB1 in the circulation and decreased macrophage and PMN subpopulations in peritoneal cavity. We have further revealed that KPT330 treatment significantly suppressed TNF-α and IL-6 expression and inhibited HMGB1 necleocytoplasmic translocation by inhibiting CRM1 distribution. In addition, in vitro investigation data provide evidence that KPT330 exerted anti-inflammation effects by inhibiting the production of pro-inflammatory cytokines through suppressing activation of NF-κB and p38 signaling.