The anti-inflammatory effects of idazoxan on inflammatory mediator release in endotoxin-challenged mice in vivo and activated macrophages in vitro / 中华危重病急救医学

ABSTRACT

Objective To study the anti-inflammatory effects of idazoxan (IDA) on endotoxin lipopolysaccharide (LPS) challenged mice in vivo and activated macrophages in vitro,and explore its potential molecular mechanisms.Methods To do the experiments in vivo,30 adult male C57BL/6 mice were randomly divided into control group,model group,and low,medium and high doses IDA groups (IDA-L,IDA-M,and IDA-H groups),n =6 in each group.The inflammatory model was reproduced by intraperitoneal injection of LPS 10 mg/kg,and the control group was injected with the same amount of normal saline.The IDA groups received LPS (10 mg/kg) and IDA 0.3,1.0 and 3.0 mg/kg,respectively.The blood samples of mice in each group were collected at 6 hours after the reproduction of the model.For the in vitro experiments,primary peritoneal macrophages were collected from 20 adult male C57BL/6 mouse cells and they were divided into control group,LPS group (10 mg/L) and LPS+IDA-L,IDA-M,IDA-H groups (10 mg/L LPS + 5,25,100 μmol/L IDA,respectively).Cell culture supernatants were collected at 24 hours after the reproduction of the model.Detection methods:enzyme linked immunosorbent assay (ELISA) was used to determine the levels of serum tumor necrosis factor-α (TNF-α),interleukin-6 (IL-6),monocyte chemotactic protein-1 (MCP-1) and nitric oxide (NO).Western Blot was used to determine the effect of IDA on the expression levels of nuclear factor-κB (NF-κB) in macrophages.Results ① For the in vivo experiment,the serum levels of TNF-α and IL-6 were significantly elevated in the model group as compared with those in the control group [TNF-o (ng/L)403.96 ± 40.98 vs.17.50 ± 8.68;IL-6 (ng/L)61 400.31 ± 7 826.61 vs.2 436.30 ± 448.89;both P ＜ 0.01].IDA treatment could inhibit the elevation of inflammatory cytokines in a dose-dependent manner,with the most significant decrease in LPS+IDA-H group [TNF-α (ng/L)170.09 ± 28.53 vs.403.96 ± 40.98,IL-6 (ng/L)16 570.81 ± 1 083.65 vs.61 400.31± 7 826.61;both P ＜ 0.01].② For the in vitro experiment,the levels of TNF-α,IL-6,MCP-1,and NO secreted by LPS-stimulated macrophages were distinctly higher in the LPS group than those in the control group [TNF-α (ng/L)7 259.14 ± 320.70 vs.28.50±27.08,IL-6 (ng/L)14809.60±5852.73 vs.1 113.47±465.53,MCP-1 (ng/L)20847.37± 1 788.33 vs.447.37± 395.69,NO (μmol/L)1 900.00 ± 144.31 vs.603.03 ± 102.18;all P ＜ 0.01].However,IDA intervention could lower the secretion of TNF-α,IL-6,MCP-1 and NO in a dose-dependent manner,with the most notable decrease in the LPS+IDA-H group [TNF-α (ng/L)784.40±281.90 vs.7259.14±320.70,IL-6 (ng/L)1 802.96± 1 534.18 vs.14 809.60± 5 852.73,MCP-1 (ng/L)2005.26± 1 534.28 vs.20847.37 ± 1 788.33,NO (μ mol/L)654.54± 150.21 vs.1 900.00 ± 144.31;all P ＜ 0.05].In addition,IDA at the concentration of 100 μmol/L could promote the translocation of NF-κBp65 in macrophages into the nucleus 15 minutes early and lead to increased NF-κBp65 expression (gray value18.70 ± 2.29 vs.1.09 ± 0.36,P ＜ 0.05),hut significantly reduce the expression levels of NF-κBp50 in the nucleus at 45 minutes after treatment (gray value1.99 ± 0.14 vs.2.94 ± 0.54,P ＜ 0.05).Conclusions IDA could significantly reduce inflammation of mice challenged with LPS and inhibit inflammatory cytokines and mediators secreted by macrophage in a dose-dependent manner.High concentration of IDA (100 μmol/L) exhibited the greatest anti-inflammatory effects.The anti-inflammatory effect of IDA may be worked through NF-κB signaling pathway.