Protection And Mechanisms Of Nicaraven On LPS-induced Septic Shock In Mice

Objective:Sepsis is a systemic inflammatory response syndrome(SIRS)which is caused by bacterial infection,including severe sepsis and septic shock.Since septic shock is still the main reason for mortality in ICU patients without effective drug for treatment of this syndrome,it is necessary to develop a novel anti-shock drug with apparent therapeutic efficacy and low side effects.Nicaraven(AVS)is a new type of synthetic hydroxyl free radical scavenger,with obvious protective effects for multiple organs.The aim of this study is to investigate the protective effects of AVS on septic shock in vivo and in vitro,as well as the possible mechanisms.Methods: 1.Examine the protection of AVS on septic shock in mice: The cecal ligation and puncture(CLP)model and endotoxin(LPS)-induced sepsis model were employed for evaluating the anti-septic shock of AVS in mice.AVS(100 mg/kg)was intraperitoneally administrated and the mortality was monitored within 3 days and 7 days,respectively.Pathological analyses of the mouse liver,lung,spleen,and kidney were performed on the day 3 after treated with LPS.2.Evaluate the therapeutic efficacy of the combination of AVS and dexamethasone on PLS-induced septic shock in mice: A combination of AVS and dexamethasone with an ineffective dose was used to treat the LPS-induced septic shock in mice and the survival rates were examined after LPS challenge.Moreover,the proteins from different tissues were extracted and levels of cytokines including TNF-?,IL-1?,and IL-6 were detected by ELISA.3.Access the mechanisms of anti-shock of AVS in vivo: The thymus and the spleen were dissected from healthy mice to determine the effects of AVS on immune system.RAW264.7 cells,a macrophages cell line,were used for elucidating the mechanisms of the anti-shock of AVS.The inflammatory responses of the cells were induced with LPS stimulation.The nitric oxide(NO)and the reactive oxygen species(ROS)were detected after AVS treatment.The mRNA expressions and the protein levels of inflammatory factors were evaluated by real-time PCR and ELISA.Western Blot was used to determine the effects of AVS on Sirt1/NF-?B,AMPK signal pathways and the nitrogen synthase levels.Results: 1.AVS alone improved the survival rate in LPS-induced septic shock of mice: the results showed that protections of AVS on LPS-induced septic shock in mice were observed,seen as the mouse mortality was reduced.Histopathological results showed that AVS effectively protected the vital organs of mice from LPS-induced injuries.In addition,AVS significantly inhibited the migration of inflammatory factors including TNF-?,IL-1? and IL-6 in the organs.2.A combination of AVS and dexamethasone markedly increased the survive rate of LPS-induced septic shock in mice: our results showed that a combination of AVS and dexamethasone with an inefficient dose significantly inhibited the mortality of LPS-induced septic shock in mice.Although the combination also significantly inhibited the migration of the inflammatory factors into the organs of mice there was no significant improvement for the combination compared with AVS alone group.3.The mechanisms of anti-septic shock of AVS may be mainly involved in its anti-inflammatory actions: In comparison with dexamethasone,AVS did not display an obvious immunosuppression.In the LPS-induced inflammatory response,AVS can significantly inhibit NO production and ROS generation in RAW264.7 cells.It effectively inhibited the expression of multiple pro-inflammatory genes and reduced the migrations of TNF-?,IL-1?,and IL-6 to the organs.AVS increased Sirt1 levels,inhibited the activation of NF-?B signal pathway,and enhanced AMPK phosphorylation after LPS stimulation.Moreover,AVS inhibited the expression of nitric oxide synthase.Conclusions: Our study demonstrated that AVS was effectively resistance to LPS-induced septic shock in mice.The protective effects may be involved in inhibiting releases of inflammatory cytokines from macrophages,attenuating migration of the cytokines into the organs and activating the antioxidant signaling pathways.