Annexin A5 Improved Inflammation By Regulating Macrophage Polarization

Annexins is a kind of calcium-based phospholipid binding proteins,which is involved in anticoagulation,apoptosis,and signal transduction.Annexin A5(Anx A5)is the most widely distributed member of Annexins,and is known as anticoagulant in the earliest.In recent years,with the in-depth study,Anx A5 was found inprocess of inflammatory response cancer,orthopedic diseases,immune diseases and neurological diseases,in addition to anticoagulant function.Macrophages are important part of the innate immune system,and have the characteristics of functional diversity and phenotypic transformation.Macrophages have functions of pathogens removal,anti-infection,tissue repair,and so on.Different micro-environment stimulation differentiate macrophages into distinct phenotypes to play diverse roles.Macrophages can be polarized into proinflammatory M1 macrophages or anti-inflammatory M2 macrophages by FNs/TLR or IL-4/IL-13 signaling pathways,respectively.This study was to investigate the effect of Anx A5 on macrophage polarization and its anti-inflammatory mechanism.We have established several in vivo inflammation model to explore the role of Anx A5.Anx A5 can effectively increased the survival rate of LPS-induced sepsis by 50%and 70%at 30 ?g/kg and 100 ?g/kg,and decreased the secretion of proinflammatory cytokines IL-6,IL-1? and TNFa in serum,inhibitd the expression of IL-6,IL-1? and TNF? mRNA in liver and lung tissue,and effectively protected LPS-induced lung injury.Moreover,Anx A5 also showed good efficacy in peritonitis caused by cecum ligation perforation(CLP),with significantly improved survival rate of CLP model.Also,we established TNBS and DSS-induced inflammatory bowel disease model,and the results showed that Anx A5 can effectively improve the mouse's inflammatory bowel disease,as maintaining the weight of mice,inhibiting the DAI index increased and improving the colon shortening and tissue lesions.With Anx A5 treatment,the expression of IL-6,IL-1?,TNF?,IFN-?,IL-12 and IL-17a mRNA in colonic tissues were reduced,and the expression of CD206,Fizz1 and IL-10 mRNA were increased in contrast.According to results of immunohistochemistry,the expression of Claudin-1 and ZO-1 were increased;CD11c,the surface marker of M1 macrophage,was decreased;and CD206,the marker of M2 macrophage,was increased by Anx A,treatment Western blots showed that Anx A5 down-regulated LPS/TLR and IFN-y/STAT1 signaling pathway,and up-regulated IL-4/STAT6 signaling pathway.The above experiments showed that Anx A5 in the mouse inflammation model had a good anti-inflammatory effect.Next,we focused to identify target cells of Anx A5.In our results,Anx A5 did not affect the proliferation of B cells and the activation of T cells.However,Anx A5 inhibited M1 macrophage polarization and enhanced M2 macrophage polarization in dose-dependent manner.What's more,Anx A5 could shift macrophages from mixed phenotype to M2-like.Immunofluorescence assay showed that Anx A5 inhibited the nuclear import of p-p65 and STAT1,and promoted the nuclear import of STAT6 in M1 macrophages.Western Blot also showed that Anx A5 reduced the expression of p-p65 and p-STAT1 and up-regulated p-STAT6 dose-dependently.These results indicated that Anx A5 exhibited the anti-inflammatory effects by the selective action in macrophages through p65,STAT1 and STAT6 signals.In conclusion,Anx A5 improved sepsis and inflammatory bowel disease model mainly through inhibition of M1 macrophage polarization and promotion of M2 macrophage polarization.Anx A5 showed significantly down-regulation in LPS/TLR and IFN-?/STAT1 signals and up-regulation in IL-4/STAT6 signals,presenting good prospects in anti-inflammatory disorders.However,there are still some shortcomings in this thesis,as the molecular target in macrophages of Anx A5 is unclear,and the mechanisms of Anx A5 in selective regulation of macrophages also need further studies.