Gu-4 Targets Integrin CD11b In The Treatment Of Sepsis And The Regulation Of HMGB1 In Late Media

Sepsis,a systemic inflammatory responses mediated by infection or injury,is still one of the important causes of death in clinical patients. In recent years, growing evidence demonstrated that High mobility group box 1 (HMGB1) plays a critical role in the generation and development of sepsis by acting as a key "late-phase" mediator.Here, we investigated the therapeutic effects of Gu-4, a lactosyl derivative, on sepsis and the underlying molecular mechanisms.Firstly, we show that Gu-4 administration prominently attenuated lung injury and improved the survival of the CLP-induced septic animals, which was positively correlated with the decrease of the serum HMGB1 level. By detecting the dynamic changes of levels of HMGB1 and TNF-a in the sera of CLP rats, we found that Gu-4 possesses a therapeutic potential in the treatment of sepsis probably via inhibiting the CLP-induced release of HMGB1 and via suppressing the pro-inflammatory activity of HMGB1.Secondly, the detailed molecular mechanisms of the inhibition of Gu-4 in CLP-induced HMGB1 release were further investigated in vitro. We found that antagonist of CDllb like Gu-4 or CDllb blocking antibody could inhibit HMGB1 release from LPS-stimulated macrophages. Meanwhile, CDllb knockdown by RNAi suppressed LPS-induced HMGB1 cytoplasmic translocation and active release. And the same result was found in peritoneal macrophages from CD11b-/- mice. Moreover,our data form Co-immunoprecipitation and Western Blot revealed that the downregulation of CDllb blocked HMGB1 nucleo-cytoplasmic translocation by inhibiting interaction with CRM1 and phosphorylation of HMGB1 by cPKC. These results indicated integrin CD11b is required for LPS-induced HMGB1 release and translocation.Thirdly, we found Gu-4 not only dose-dependently attenuated recombinant human HMGB1 (rhHMGB1)-induced production of TNF-α, IL-6, and IL-1β in THP-1 cells, but also greatly inhibited the adhesion of rhHMGB1-challenged THP-1 cells to HUVECs. Analyses of flow cytometry demonstrated that Gu-4 could effectively reduce the activation of CDllb elicited by rhHMGB1. Western blot analyses revealed that Gu-4 targeted CD1 lb to partially block the rhHMGB1-induced activation of ERK and NF-κB signalings.In conclusion, our study indicates Gu-4 owns a dual-role in suppressing HMGB1 secretion and blocking HMGB1 pro-inflammatory activity during sepsis. Moreover,our data reports integrin CDllb is required for LPS-induced HMGB1 release from macrophages for the first time. Therefore, targeting CD11b could be an alternative choice for the intervention of HMGB1-mediated inflammation.