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OBJECTIVE To observe the effect of mannitol on oxidative stress of human kidney tubular epithelial cells(HK-2). METHODS MTT assay was applied to detect HK-2 cell viability after ex?posure to different concentrations(50,100,150,200,250,300,350 and 400 mmol · L-1)of mannitol for 4,10,24,48 and 72 h. DCFH-DA method was used to determine the level of reactive oxygen species (ROS)after HK-2 cells were exposed to mannitol 100 and 250 mmol · L-1 for 4 h. Furthermore,cell morphology and indexes of oxidative stress such as malondialdehyde(MDA) content,superoxide dismutase(SOD )activity and glutathione(GSH) content were observed at different time points. RESULTS HK-2 cell viability decreased by about 50%after being treated with mannitol 250 mmol · L-1 for 72 h (P<0.05). ROS production in mannitol 250 mmol · L-1 treated group (68.7 ± 3.6) was higher than in solvent control group(50.3 ± 4.6)(P<0.05). HK-2 cells exhibited morphological changes after treatment with mannitol 250 mmol · L-1 for 4-72 h,including cell swelling,vacuoles and fall off. After treatment with mannitol 250 mmol · L-1 for 4,10,24,48 ahd 72 h,the MDA content increased signifi?cantly(P<0.05),while the activity of SOD and the content of GSH decreased significantly compared with solvent control group(P<0.05). CONCLUSION Over-production of ROS in HK-2 cells induced by high concentration(250 mmol·L-1)of mannitol may cause lipid peroxidation and injure the ability of an?tioxidation,which may contribute to mannitol induced cytotoxicity.
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[ ABSTRACT] AIM:To observe the effect of the metabolites generated from oxidative deamination of methylamine ( MA) or benzylamine ( BZA ) catalyzed by semicarbazide-sensitive amine oxidase ( SSAO ) on 3T3-L1 adipocytes. METHODS:3T3-L1 preadipocytes were induced to differentiation.SSAO activity was determined by high performance liquid chromatography ( HPLC) at different differentiation time points.MTT assay was applied to detect cell vitality after exposure to different concentrations of MA or BZA.Fluorescence probe DCFH-DA was used to determine the production of reactive oxygen species after incubation of 3T3-L1 adipocytes with MA or BZA.After exposure to 0.5 mmol/L MA or BZA for 4 h, malondialdehyde ( MDA) , total superoxide dismutase ( T-SOD) and glutathione ( GSH) in the adipocytes or prea-dipocytes were measured.RESULTS:SSAO activity increased with the increase in the differentiation days, and reached a maximum at the 8th day.Incubation of the cells with different concentrations of MA or BZA for 4 h did not significantly de-creased the cell vitality (P>0.05).After exposure to 0.5 mmol/L MA or BZA, the reactive oxygen species in adipocytes significantly increased, and were about 3 to 4 times as compared with control group (P0.05 ) .CONCLUSION: MA or BZA induces oxidative stress in the mature adipocytes, which might result from the deamination products catalyzed by SSAO.
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AIM To study whether inhibition of semicarbazide (Sem)-sensitive amine oxidases (SSAO) attenuates myocardial ischemia-reperfusion (I-R) injury. METHODS Male Sprague-Dawley rats were randomly divided into 4 groups. Each group consisted of 10 rats. Sham group: the ligature was placed under the left coronary artery (LCA), but not ligated. Sham+Sem group: Sem (30 mg·kg-1, ip) was given 10 min prior to the experiment, the LCA ligature was not ligated. I-R group: the LCA was occluded for 30 min and reperfused for 180 min. I-R+Sem group: Sem (30 mg·kg-1, ip) was given 10 min prior to the experiment, and then 30 min ischemia followed by 180 min reperfusion. Myocardial infarct size was determined by using nitroblue tetrazolium staining. Plasma creatine kinase (CK) and myeloperoxidase (MPO) activities, and malondialdehyde (MDA) and hydroxyl radicals levels were determined by spectrophotometer. Plasma SSAO activity was determined by high performance liquid chromatography. HE staining was used for histopathological evaluation. RESULTS There were no significant differences on each index between sham and sham+Sem groups. Plasma MPO and SSAO activities, and MDA and hydroxyl radials levels significantly increased in I-R group, compared with sham group. Myocardial infarct size was remarkably smaller in I-R+Sem group (27.7±3.7)%, compared with I-R group (43.2±3.1)%. Plasma MDA level, MPO activity and hydroxyl radical level were lower in I-R+Sem group than those in I-R group, from (27.5±9.3) μmol·min-1·L-1,(2.6±0.4)mmol·L-1 and (628±50)mmol·min-1·L-1 down to (14.2±5.6)μmol·min-1·L-1,(1.7±0.5)mmol·L-1 and (503±88)mmol·min-1·L-1, respectively. Histological results showed that inhibition of SSAO activity significantly attenuated leukocyte infiltration. CONCLUSIONPlasma SSAO activity is increased in myocardial I-R injury and inhibition of SSAO can attenuate myocardial I-R injury.
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AIM To study whether chronic administration of methylamine may induce elevation of semicarbazide-sensitive amine oxidase (SSAO) activity and initiate the injury of cardiovascular endothelium. METHODS New Zealand rabbits were treated with methylamine hydrochloride (100 mg·kg-1) by ig, once a day for 6 months. The rabbits were weighed every other week and the dosage was adjusted according to the body weight. The number of circulating endothelial cells (CEC) in the arterial blood, nitric oxide (NO) concentration in the serum and ultrastructure of endothelial cells of aorta were assessed. The plasma SSAO activity and formaldehyde concentration were assessed by liquid chromatography. RESULTS The number of CEC, NO concentration, levels of SSAO activity and formaldehyde concentration in the methylamine group were increased significantly, compared with the control group. Ultrastructure of endothelial cells in the methylamine group showed inordinate morphological changes (multiple intranuclear inclusions, karyopyknosis and karyorrhexis). CONCLUSIONChronic administration of methylamine can induce the elevation of SSAO activity and initiate the injury of cardiovascular endothelium.