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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 value:18.70 ± 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 value:1.99 ± 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.
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[ ABSTRACT ] AIM: To study the effect of idazoxan on the permeability of inflammatory blood-brain barrier ( BBB) model in vitro and the expression of tight junction protein ZO-1.METHODS:In vitro BBB model was established by murine brain endothelial cell line bEnd.3 incubated for 7 d.The cells were treated with TNF-α(10 nmol/L) for addi-tional 24 h to establish the inflammatory BBB model, which was pretreated with IDA at doses of 50, 100 and 200μmol/L, respectively.The permeability was measured using fluorescein isothiocyanate-conjugated dextran (FD-40, MW 40,000), the expression of ZO-1 was detected by Western blot analysis, the distribution of ZO-1 was observed by immunofluores-cence, and the mRNA expression of MMP-9/TIMP-1 was measured by RT-PCR.RESULTS:After incubated for 7 d, b. End3 cells converged to be confluent monolayer with low permeability.The inflammatory BBB model induced by TNF-αtreatment displayed much higher permeability with decreased expression of tight junction protein ZO-1, destroyed distribu-tion of ZO-1 and increased mRNA expression of MMP-9.When pretreated with IDA, the permeability was greatly de-creased, the expression of ZO-1 was greatly increased, the abnormal distribution of ZO-1 was greatly ameliorated and the mRNA expression of MMP-9 was obviously reduced.The effect was most significant in IDA ( 200 μmol/L )-pretreated group (P<0.01).CONCLUSION:IDA directly acts on brain endothelial cells to reduce the expression of MMP-9, in-crease the expression of tight junction protein ZO-1 and ameliorate the destroyed distribution of ZO-1 in the inflammatory BBB, thus reversing the abnormally elevated permeability in a inflammatory BBB model in vitro induced by TNF-α.
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Objective The aim of this study was to observe the anesthetic effect of xylazine hydrochloride Injection on Beagle dogs.Methods Anaesthetizing 30 healthy Beagles with xylazine hydrochloride injection, some physiological indexes of the dogs, such as body temperature (T), respiration (R), heart rate (P), and blood pressure (systolic pressure SBP, diastolic pressure DBP), were monitored.Results After using xylazine hydrochloride injection, the body temperature, respiration, heart rate, blood pressure, and peripheral vascular resistance were decreased in the Beagles.Conclusion Xylazine hydrochloride injection can keep a stable induction period, and has advantages including powerful and quick effect, simple method and operation, safe and insignificant toxicity and side effects,and has a better effect of anaesthesia.
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[ ABSTRACT] AIM:To study the effect of idazoxan ( IDA) on the permeability of blood-brain barrier ( BBB) and the expression of matrix metalloproteinase 9 (MMP-9) and tissue inhibitor of metalloproteinase 1 (TIMP-1) in mouse ex-perimental autoimmune encephalomyelitis (EAE).METHODS: Female C57BL/6 mice (n=36) were randomly divided into control group, EAE group and IDA group, with 12 mice in each group.EAE was induced by myelin oligodendrocyte glycoprotein 35-55 ( MOG35-55 ) .IDA (2 mg/kg, ip, bid) was administered for 15 d after immunization.The neurological defects of the mice were observed daily and scored.The pathological changes were observed under microscope with HE stai-ning and LFB myelin staining.The BBB permeability was detected by Evans blue extravasation.The expression of MMP-9 and TIMP-1 in the brain of EAE mice was determined by Western blotting.RESULTS: Compared with EAE group, the score of neurological defects in IDA group was decreased, the inflammation was relieved, the BBB permeability was re-duced, and the expression MMP-9 and the ratio of MMP-9/TIMP-1 were decreased ( P<0.05 ) .CONCLUSION: The neuroprotective effect of IDA on mouse EAE might be related to the down-regulation of MMP-9 and the ratio of MMP-9/TIMP-1, thus reducing the degradation of BBB and the permeability of BBB, and ameliorating the pathologic process of EAE.
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Yohimbine (0. 5~4mg ? kg-1, sc) and idazoxan (2~8 mg ? kg-1,sc) showed a dose related antagonistic action on the inhibitory effect of xylazine (4mg ? kg-1,sc) on the gastrointestinal propulsive motility in mice. Xylazine (2mg,4mg ? kg-1,sc),clonidine(120 ?g ? kg-1,sc) exhibited the inhibitory effect of gastrointestinal basic electrical rhythm on the stomach, duodenum, jejunum and ileum in rats.The inhibitory effects of these drugs were the strongest at 30 min after administration of Compounds. Yohimibine (4mg ? kg-1,sc) was found to antagonize the inhibitory effect of xylazine (4mg ? kg-1,sc) on the gastrointestinal basic electrical Rhythm.
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AIM To investigate the effects of idazoxan on the viability and DNA integrity of clonal BRIN-BD11 cells. METHODS MTS reduction was used to check the viability of BRIN-BD11 cells. FACS cell cycle analysis was employed to determine the effects of idazoxan on DNA integrity of BRIN-BD11 cells. RESULTS ①Idazoxan markedly reduced the viability of BRIN-BD11 cells and resulted in DNA damage in time-and dose-dependent manner.②Genistein, 5-HT 1A antagonist NAN190 and PKG inhibitor-KT5823 had no protective effects against idazoxan-induced damage of BRIN-BD11 cells. CONCLUSION Idazoxan has cytotoxic effect and induces apoptosis of clonal pancreatic ?-cells.
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Objective:To explore effects of Idazoxan(IDA) on changes of neuroglial cells in spinal cords of rats with experimental autoimmune encephalomyelitis(EAE).Methods:Rat EAE was induced by immunization with spinal cord homogenates of Geania pigs.EAE clinical manifestations were assessed in terms of the scoring standards.Histological investigation and immunohistochemistry were observed for the inflammatory dedmylinative lesion of CNS and morphology of glial cells.Results:Ida could not decrease the incidence of EAE,but alleviate its clinical manifestation and histological changes.On day 15 after immunization,astrocytes in and around the inflammatory dedmylinative lesion of CNS in EAE rats treated with Ida increased in number and size,on the contrary,microglia decreased in number and size.Conclusion:Ida has protective effects on EAE and its functional mechanism may be concerned with modulation of immunological mechanism of CNS.