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  • br Materials and methods br Results br Discussion The

    2020-07-27


    Materials and methods
    Results
    Discussion The pathophysiology of sepsis is complex, which is a host\'s exaggerated immune response to severe infection increases mortality [13]. During the early phase, sepsis patients present with an exaggerated immune response, also described as SCH 727965 a “cytokine storm”. And this acute hyperinflammation against infection leads to multiple organ dysfunction. It has been reported that a nuclear export factor CRM1 appears to be critical in assisting HMGB1 active export from the nucleus into the cytoplasm, which is a key “late-phase” inflammatory mediator of sepsis [14]. In this study, we evaluated the effects of KPT330, a potent and specific small molecule inhibitor of CRM1, in LPS-induced sepsis model in vivo and explored the underlying mechanism by performing a series of in vitro experients, and we found that KPT330 exhibited anti-inflammation effects and protection against sepsis. 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 [15]. 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 SCH 727965 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 [16]. 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 [17]. 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.