| Aim: To investigate the protection of mycophenolate mofetil(MMF)for septic mice and the underlying mechanisms.Methods: 8~10-week C57BL-6J mice received SHAM operation or cecal ligation and puncture(CLP),and some of the CLP mice were treated either with or without MMF in different dose.Survival rate was monitored for 7 consecutive days.Depending on the result of the above experiment,60mg/kg MMF was given via gavage at the beginning of CLP.6h and 24 h following CLP,the mice were sacrificed and lungs,heart,liver,peripheral blood and peritoneal fluids were obtained.H&E staining,lung wet/dry ratio,serum levels of creatine kinase(CK)and alanine aminotransferase(ALT)were used to assess organ injuries.Sera and peritoneal interleukin(IL)-1β,IL-6,tumor necrosis factor(TNF)-α,monocyte chemoattractant protein(MCP)-1,transforming growth factor(TGF)-β,and IL-10 were calculated via enzyme linked immunosorbent assay(ELISA).The expression of toll like receptor(TLR)-4 and the phosphorylation of NF-κB,JNK,ERK,p38,SHP-2 were analyzed using western blot(WB).In addition,blood and peritoneal fluids were dilutd and cultured for bacterial count.Immunohistochemistry was used to detect Casepase3 and programmed death 1(PD-1)expression in the spleen.And flow cytometry was conducted to analyze cell apoptosis,PD-1,and major histocompatibility complex(MHC)II expression on peritoneal macrophages.GFP-labled fluorescent beads and lipopolysaccharides(LPS)were used in vitro to judge the immune response of peripheral blood mononuclear cells(PBMCs)and peritoneal macrophages to pathogeny stimuli.And finally,natural PD-1 ligand,PD-L1,was administered to MMF mice to test its abrogation of MMF’s protection.Result: At the beginning of CLP,60 mg/kg MMF administered by gavage significantly protected septic mice with elevated 7d survival.6h following CLP,lung wet/dry ratio,sera ALT and some of the cytokines in sera and peritoneal fluids were significantly decreased by MMF,while cell apoptosis of the spleen and peritoneal macrophages were not obviously affected,neither were the in vivo bacterial loads.24h following CLP,pathological changes of the lungs,heart and liver were significantly reversed by MMF and the entire 6 kinds of cytokines listed above were decreased in either sera or peritoneal fluids.MMF reduced the in vivo bacterial loads,attenuated apoptosis of the spleen and peritoneal macrophages,and downregulated their PD-1 expression,meanwhile MHCII expression on peritoneal macrophages were upregulated.Phosphorylation of NF-κB,ERK,p38 and SHP-2 were dramatically prevented in the MMF spleen and peritoneal macrophages,while TLR4 expression and JNK phosphorylation were not observed changed.Peritoneal macrophages from the MMF mice were more phagocytotic than the CLP cells.And PBMCs,as well as peritoneal macrophages,produce more cytokines when challenged with LPS in vitro.In addition,PD-L1 abolished the protective effects of MMF and resulted in similar bacterial loads in the MMF mice and CLP mice.Conclusin: MMF is protective to septic mice,probably in association with the dual inhibition of hyper-inflammation,characterized by cytokines over production,and immunosuppression,mediated by PD-1 expression. |
