Therapeutical Effect And Antioxidant Mechanism Of Agmatine On Lipopolysaccharide-induced ARDS In Rat

BackgroundAcute respiratory distress syndrome(ARDS)is an acute and diffuse lung injury caused by excessive inflammation.The main clinical manifestations of it are hypoxemia,acute or progressive dyspnea and non-cardiogenic pulmonary edema.In 2012,the new standard formulated in Berlin canceled the concept of acute lung injury(ALI)/ARDS,and the disease was collectively referred to as ARDS.As a critical clinical disease with high mortality,its prevention and treatment have attracted great attention of the world.In recent years,although there have been some progress in drug therapy,protective mechanical ventilation and airway management,but the mortality rate among ARDS patients remains high and its pathogenesis is still unclear.Oxidative stress is considered as a potential mechanism for the development of excessive inflammation in pulmonary.Tissue damages mediated by excessive oxidation are critical events of ARDS,and neutrophils and macrophages in lung are mainly responsible for the generation of reactive oxygen species(ROS).Agmatine is a cationic polyamine formed by decarboxylation of L-arginine.In our previous research,we found that agmatine treatment relieved lipopolysaccharide-mediated acute lung injury and significantly improved survival rate.On this basis,we hypothesis agmatine may attenuate the LPSmediated lung injury via regulating antioxidant effects in macrophages.Our experiments aim to definite whether agmatine shows a therapeutical effect in LPS-mediated lung injury in vivo and in vitro,explore the role of agmatine in mediating antioxidant effects in macrophages and clarify the antioxidant mechanism in it.We expect a deep understanding of the properties of endogenous biogenic amines and to provide a new target for treatment of ARDS.Materials and Methods(1)The therapeutical effect of agmatine on LPS-mediated ARDSIntratracheal injection was administrated.100 male SD rats were randomly divided into sham-operated group,LPS group,AGM treatment group and AGM group(n=25).The first dose: sham-operated group with an injection of normal saline 0.1ml,LPS group with an injection of 3mg/kg LPS 0.1ml,AGM treatment group with an injection of 3mg/kg LPS 0.1ml,AGM group with an injection of 5mg/kg AGM 0.1ml;the second dose: 30 minutes late to first;sham-operated group,LPS group and AGM group were injected with normal saline 0.1ml,AGM treatment group received 5mg/kg AGM 0.1ml.Ten rats were randomly selected in each group to accept the 4 day survival analysis to assess the protective effects on LPS-induced ARDS.Five rats in each group were randomly selected and injected with fluorescent marker in tail vein 20 minutes in advance.All rats except used for survival analysis were sacrificed after administrated for 24 hours.Bronchoalveolar lavage fluid(BALF)and lung tissues were collected.The expression levels of inflammation cytokines and myeloperoxidase(MPO)were measured to assess the condition of pulmonary inflammation.Besides,vascular permeability was determined by wet to dry ratio and FITC-BSA fluorescence labeling method in lung tissues and tissue damage was detected by HE staining of lung tissues.Additionally,the extent of oxidative stress was determined by measurement of the activity of SOD,content of MDA and expression of HO-1 in lung tissues with methods of Western blot,SOD and MDA detection kits respectively.(2)Agmatine reduces LPS-mediated excessive oxidation in RAW264.7 cellCell viability was determined by MTT assay and the appropriate concentrations for administration were selected.LPS-activated RAW264.7 cells were treated by AGM(1 mM)therapeutically.After co-incubated with AGM for 24 h,cell culture supernatants and cell proteins were collected.Chemical colorimetric detection was used to detect SOD activity and MDA content.NO expression in cell culture supernatants were measured by Griess assay.ROS levels were determined by DCFH-DA probe and iNOS protein levels were detected by Western Blot.(3)The antioxidant mechanism for AGM regulation of LPS-induced excessive oxidation in macrophagesReal-time quantitative PCR was used to detect ?2 adrenergic receptor and imidazoline receptors(I1R,I2R)to explore their roles on antioxidant effects of AGM.Furthermore,to investigate the relationship between antioxidant effects of AGM and PI3K/Akt and HO-1 signaling pathways,Akt and p-Akt protein were detected by Western blot and the expressionsof Nrf2 and HO-1mRNA were measured by quantitative PCR.LY294002 and ZnPP were administrated to block PI3K/Akt and HO-1 signaling pathways respectively.DNA binding of Nrf2 was detected by EMSA.Bilirubin,one of the final by-products of HO-1 enzyme catalysis,was measured to clarify whether they contribute to the antioxidant effects of AGM.Results(1)AGM administrated therapeutically significantly increased 4 day survival rates in LPS-mediated ARDS rats(P <0.05).Compared with LPS group,the levels of TNF-? and IL6 in BALF and lung tissues were significantly reduced,wet to dry ratio and the levels of FITC-BSA fluorescence were decreased and the extent of tissue damage was alleviated in AGM treatment group.Additionally,the levels of MPO and MDA content were decreased and SOD activity and HO-1 expression were significantly increased(P <0.05).(2)LPS significantly enhanced iNOS protein levels and increased NO and ROS generation in RAW 264.7 cells.Agmatine treatment significantly decreased the LPS-induced iNOS overexpression and excessive production of NO and ROS.(3)AGM markedly enhances Nrf2 nuclear translocation,increases nuclear Nrf2 protein level,up-regulates the expression of HO-1 and attenuates ROS generation induced by LPS.LY294002,a specific PI3K/Akt inhibitor,abolished agmatine-induced HO-1 up-regulation and ROS suppression significantly.Inhibiting HO-1 pathway significantly attenuated the antioxidant effects of agmatine which the products of HO-1 enzymatic activity contributed to.Furthermore,the common membrane receptors of agmatine,?2-adrenoceptor,I1-imidazoline receptor or I2-imidazoline receptor,are not required by the antioxidant property of agmatine.Conclusions(1)Agmatine can attenuate LPS-induced lung edema,vascular permeability increasing and neutrophil infiltration in rats and shows a therapeutical effect on acute respiratory distress syndrome.(2)Agmatine can reduce inflammatory cytokines release,enhance antioxidant enzyme SOD activity and decrease ROS and NO production in macrophages,suggesting that agmatine can exert therapeutical effects via regulating antioxidant effects in RAW264.7 cells.(3)Agmatine reduces LPS-mediated excessive oxidation via activating PI3K/Akt pathway and up-regulating Nrf-2 and HO-1 expression in macrophages and the products of HO-1 enzymatic activity also contributed to this property.