Study Of Idazoxan On Organs Protection In Septic Mice And Its Anti-oxidative Stress Molecular Mechanism

Objective:To investigate the protective effects of idazoxan(IDA)on organ dysfunction in septic mice and its anti-oxidative stress effects and molecular mechanism.Methods:(1)Sixty mice were randomly divided into control group(n=12),LPS group(n=12,with intra-peritoneal injection of LPS 10mg/kg),treatment group with low/ medium/ high dose IDA(n=12,with intra-peritoneal injection of LPS 10mg/kg and IDA 1,2,4 mg/kg,respectively).All mice were sacrificed at 24 h after injection,serum,livers,lungs and kidneys were harvested.Serum samples were collected for determination of levels of ALT and AST by automatic biochemistry analyzer.The levels of MDA and enzyme activities of SOD,CAT and GPx in liver were measured by using chemical colorimetry.The pathological changes of liver,lung and kidney were detected by H&E staining.The protein levels of HO-1,NQO-1 and Nrf2 in liver were determined by western blot.The expressions of Nrf2 in liver and kidney were detected by immunohistochemistry.(2)RAW264.7 cells were cultivated in vitro,and randomly divided into four groups: control group,LPS group(10?g/ml LPS),LPS+IDA group(10?g/ml LPS+100?M IDA)and IDA group(100?M IDA).The reactive oxygen species(ROS)levels were determined with 2,7-d ichlorofluoresce in diacetate(DCFH-DA)as fluorescence probe by flow cytometry and fluorescence microscope.The protein expressions of NQO-1 and Nrf2 were determined by western blot.The protein levels of HO-1 were detected by western blot and immunofluorescence.Results:(1)The serum levels of ALT and AST in LPS group were significantly higher than those in control group [ALT(U/L): 88.10±12.05 vs.35.93 ± 3.02;AST(U/L): 311.97 ± 75.04 vs.136.77 ± 10.80;P<0.01],while IDA treatment remarkably decreased serum ALT and AST [ALT(U/L): low dose group 68.40 ± 5.17 vs.88.10 ± 12.05(P=0.06),medium dose group 51.10 ± 5.66 vs.88.10 ± 12.05(P<0.01),high dose group 44.27 ± 9.10 vs.88.10 ± 12.05(P?0.01);AST(U/L)low dose group 257.73 ± 51.97 vs.311.97 ± 75.04(P=0.36),medium dose group 194.37 ± 20.84 vs.311.97 ± 75.04(P=0.06),high dose group 161.13 ± 27.91 vs.311.97 ± 75.04(P?0.05)].After 24 h of LPS injection,the hepatic levels of MDA in LPS group were significantly increased as compared with control group [MDA(nmol/mg): 6.37 ± 1.45 vs.1.13 ± 0.25(P<0.01)],and the enzyme activities of SOD,CAT and GPx were decreased at the same time [SOD(U/mg): 0.97 ± 0.35 vs.3.83 ± 0.71(P<0.01),CAT(U/mg): 30.67 ± 7.02 vs.70.33 ± 8.50(P<0.01),GPx(U/mg): 8.33 ± 2.52 vs.30.33 ± 5.51(P<0.01)].While comparing to LPS group,IDA treatment decreased the high levels of MDA [low dose group(4.83 ± 0.80)nmol/mg vs.(6.37 ± 1.45)nmol/mg(P=0.18),medium dose group(3.20 ± 0.62)nmol/mg vs.(6.37 ± 1.45)nmol/mg(P<0.05),high dose group(2.30 ± 0.46)nmol/mg vs.(6.37 ± 1.45)nmol/mg(P?0.01)] and improved the enzyme activities of SOD,CAT and GPx in liver homogenates in a dose-dependent manner [SOD(U/mg): low dose group 1.47 ± 0.31 vs.0.97 ± 0.35(P=0.14),medium dose group 2.36 ± 0.47 vs.0.97 ± 0.35(P<0.05),high dose group 3.27 ± 0.70 vs.0.97 ± 0.35(P?0.01);CAT(U/mg): low dose group 50.33 ± 13.50 vs.30.67 ± 7.02(P=0.09),high dose group 67.67 ± 13.50 vs.30.67 ± 7.02(P?0.05),high dose group 80.67 ± 13.01 vs.30.67 ± 7.02(P?0.01);GPx(U/mg): low dose group 15.67 ± 4.04 vs.8.33 ± 2.52(P=0.06),medium dose group 21.33 ± 4.51 vs.8.33 ± 2.52(P?0.05),high dose group 35.33 ± 9.07 vs.8.33 ± 2.52(P?0.01)].Histological examination demonstrated that IDA remarkably improved the histological changes in liver,lung and kidney caused by LPS.Besides,the protein levels of HO-1and NQO-1 in liver were s ignificantly increased,and the expressions of Nrf2 in liver and kidney were also up-regulated.(2)In vitro,LPS significantly increased the production of ROS in RAW264.7 cells(P?0.01),while IDA remarkably decreased the high levels of ROS caused by LPS(P?0.05).Besides,IDA also increased the protein levels of HO-1,NQO-1 and Nrf2.Conclusion:IDA activated Nrf2 signaling pathway in macrophage,exerted anti oxidative stress effects,thus attenuated multiple organ dysfunction in septic mice.