Study Of Idazoxan On Survival Protection Of Septic Mice And Its Anti-inflammatory Effects

ObjectTo study the survival protection of idazoxan on septic mice and its anti-inflammatory effects and molecular mechanisms.Methods⑴ Adult male C57BL/6 mice were randomly divided into three groups, containing control group(normal saline),model group(intraperitoneal injection of sterile LPS,40mg/kg) and IDA treatment group(IDA 1mg/kg +LPS 40mg/kg). Respectively observe mice survival condition and timely record the time of death. Observe the survival of mice in each group at any time and record the time of death in time.⑵ Adult Male C57BL/6 mice were randomly divided into control group(normal saline), model group(LPS 10mg/kg), IDA with low/medium/high dose group(IDA 0.1, 1and 3 mg / kg). Mice in each group were sacrificed at 6 hours after modeling and blood samples were collected, and serum were separated. The contents of tumor necrosis factor-α(TNF-α), interleukin-6(IL-6) in serum were determined by enzyme linked immunosorbent assay(ELISA). At 24 hours after molding, serum and liver tissue in all the groups were collected. Serum aspartate transaminase(AST), alanine aminotransferase(ALT) and lactic dehydrogenase(LDH) were determinated by automatic biochemistry analyzer and the level of inflammatory cytokines in liver tissue were detected by ELISA.⑶ Primary peritoneal macrophages and RAW264.7 cells and LPS were used to establish inflammatory model in vitro. The control group do not any treatment, LPS group were treated with LPS 10μg/ml, IDA group were treated with LPS 10μg/ml and different doses of IDA(5, 25, 100 m). Cell were placed in culture incubator with 37 ℃and 5% CO2. At 24 h after LPS challenging, the supernatant was collected to determine the content of TNF-α and IL-6 and monocyte chemotactic factor-1(MCP-1) by ELISA.Nitrate reductase method was used to detect the NO content in the supernatant. RT-PCR was used to determine the m RNA level of inflammatory cytokines(TNF-α、IL-6、IL-1β)and western blot test was used to determine the effect of IDA on the content change of nuclear factor-κB(NF-κB) in macrophages.Result⑴ Mice in control group were in good condition with 100% survival rate. Mice challenged with LPS gradually died at 4 hours and the survival rate is only 18.75%.however, treatment with IDA 30 min before LPS challenging significantly improved mouse survival rate(56.25% vs. 18.75%, P < 0.05)⑵ At 6 hours after challenged by LPS, serum TNF-α, IL-6 levels were significantly elevated than those in the control group [TNF-α(ng/L): 403.96 ± 40.98 vs.17.50 ± 8.68; IL-6(ng/L):61400.31±7826.61 vs. 2436.30±448.89;all P<0.001] and idazoxan treatment could dose-dependent inhibit the increase of inflammatory cytokines[TNF-α(ng/L):low-dose group 379.67 ± 52.32 vs. 403.96 ± 40.98(P=0.407), middle dose group 273.17 ± 24.77 vs. 403.96 ± 40.98(P = 0.001), high dose group 170.09 ±28.53 vs. 403.96 ± 40.98(P <0.001); IL-6(ng / L):low-dose group 45516.55 ± 5422.18 vs. 2436.30 ± 448.89(P = 0.001), middle dose group 30705.32 ± 1785.33 vs. 2436.30 ±448.89(P <0.001), high dose group 16570.81 ± 1083.65 vs. 2436.30 ± 448.89(P<0.001)]. The abnormal increase of serum transaminase can be inhibited by idazoxan:[AST(U/L):255.14±11.24 比 455.89±40.87;ALT(U/L):53.62±5.02 vs. 85.51±5.10,P﹤0.01;LDH(U/L):700.33±53.47 vs. 2184.03±107.22, P﹤0.01]. At the same time,idazoxan can reduce the inflammatory cytokines of the live tissue: [TNF-α(pg/mg):2353.44±367.03 比 3670.87±258.27(P ﹤ 0.01); IL-6( ng/L): 3526.60±167.16 比4618.79±352.94( P﹤0.01)].⑶ The secretion of TNF-α、IL-6、MCP-1 and NO of macrophage at 24 hours after attacking by LPS were distinctly higher compared with the level in control group[TNF-α(ng/L):7259.14±320.70 vs.28.50±27.08(P<0.001);IL-6(ng/L):14809.60±5852.73 vs.1113.47±465.53(P=0.001);MCP-1(ng/L):20847.37±1788.33 vs.447.37±395.69(P<0.001);NO(μmol/L):1900.00±144.31 vs.603.03±102.18(P<0.001)].However, idazoxan intervention could depress the secretion of TNF-α、IL-6、MCP-1 and NO of macrophage and the degree of inhibition was related to the concentration of idazoxan [TNF-α(ng/L):low-dose group 4695.92±435.51 vs. 7259.14±320.70(P<0.001),middle dose group 3999.66±316.05 vs. 7259.14 ± 320.70(P<0.001),high dose group784.4±281.90 vs. 7259.14±320.70(P<0.001); IL-6(ng/L):low-dose group 12400.64 ±5270.22 vs. 14809.60 ± 5852.73(P=0.451), middle dose group 5522.79 ± 2492.45 vs.14809.60 ± 5852.73, P <0.013; high dose group 1802.96 ± 1534.18 vs.14809.60±5852.73(P = 0.002); MCP-1(ng/L): low-dose group 19457.90 ± 1126.71 vs. 20847.37 ±1788.33(P = 0.214), middle dose group 11373.68 ± 1110.21 vs. 20847.37 ±1788.33(P<0.001),high dose group 2005.26 ± 1534.28 vs. 20847.37 ± 1788.33(P<0.001); NO(μmol/L):low-dose group 1739.40 ± 100.14 vs. 1900 ±144.31(P=0.188),middle dose group 1390.91 ± 181.60 vs.1900 ± 144.31(P=0.001), high dose group 654.54 ± 150.21 vs. 1900 ± 144.31(P<0.001)].⑷ Idazoxan can reduce of inflammatory cytokines TNF-α,IL-6 and IL-1β m RNA expression of macrophage challenged by LPS. In addition, idazoxan could promote macrophage NF-κBp65 into nuclear in advance(18.70±2.29 vs.1.09±0.36,P<0.05),but reduce intranuclear p50 level(2.94±0.54 vs.1.99±0.14, P<0.05).ConclusionIn this report, idazoxan showed a protective effect on the survival of septic mice. In addition, idazoxan could significantly reduce inflammatory response in mice challenged with LPS and inhibit the secretion of inflammatory cytokines of macrophage. In a certain range, low concentration of IDA(5μmol/L) showed a certain inhibitory effect,high concentration(100μmol/L) showed the best anti-inflammatory effect. In short,idazoxan showed a potential anti-inflammatory effect, which may be associated with NF-κB signaling pathway.