Research On J774Cell Oxidative Damage Induced By Pseudomon Asaeruginosa And The Protective Influences Of N-acetylcysteine

ObjectiveTo demonstrate J774cell oxidative damage induced by pyocyanin (PCN) andthe protective influences of N-acetylcysteine (NAC) on it.MethodsGroup of experiments: J774cells are divided into three groups randomly,(1)normal cell group to which any medicine is not added;(2) According to reference[1],model group which is infected by PCN with different densities (10,25,50μg·ml-1) for24hours and the oxidative damage of J774cells is observed. Thebest density is decided on MTT cell toxicity test results and the J774cell oxidativedamage induced is modeled.(3) According to reference[2], NAC-interrupted groupwhich is preprocessed by NAC with different densities (2.5,5,10and20mmol·L-1)for1hour, and then infected by PCN with the best density for24hours. The effectof NAC with different densities on J774cell oxidative damage induced by PCN isobserved. Combined with determined by MTT cell toxicity experiments, selectingthe best concentration of NAC.The effect of NAC with best density on J774celloxidative damage induced by the best density PCN is observed at6h,12h and24h. The cell toxicity is measured with MTT. The Reactive oxygen species (ROS)content is measured with flow cytometry method. The leakage volume of Lactatedehydrogenase (LDH), the content of Malondialdehyde (MDA), the viability ofsuperoxide dismutase (SOD) and Catalase (CAT) are measured by reagentboxes, respectively, and then the albumen content of CAT and Manganesesuperoxide dismutase (MnSOD) are measured with Western blot method. Results(1) It shows from cytotoxicity test by MTT method that infecting J774cell for24h, PCN （5、10、25、50、75、100μg·ml-1）has no effect on cell vitality. OnlyPCN of100μg·ml-1results in vitality loss of cell of4-5%, But it is of no statisticalsignificance. So densities of50μg·ml-1and below are selected for infectionexperiment; NAC（2.5、5、10、20mmol/L）is used for J774cell for1hour, and thenPCN（10、25、50μg·ml-1）is used for J774cell for6,12and24hours. The resultsshow that each group of cells still hold their vitality.(2) The results of the ROS level measured by flow cytometry method:Compared with the normal cell group, the ROS level in the model group increases.With the increase of the density of PCN, ROS level increases gradually, which isdensity dependent. Compared with the model group, the ROS level in theNAC-interrupted group decreases. With the increase of time, ROS leveldecreases gradually, which is time dependent. The compare difference betweenexperiment groups is of statistic meaning (P<0.01).(3) The results detected by reagent boxes: Compared with the normal cellgroup, the leakage volume of LDH and MDA content in the model group increase,and SOD viability and CAT viability decrease, which is density dependent.Compared with the model group, the LDH leakage and MDA content in theNAC-interrupted group decrease, and SOD viability and CAT viability increase,which is time dependent. The compare difference between experiment groups isof statistic meaning (P<0.01).(4) The results of Western blot: Compared with the normal cell group, thealbumen contents of MnSOD and CAT in the model group decrease. With theincrease of the density of PCN, albumen contents of MnSOD and CAT decreasegradually, which is density dependent. Compared with the model group, thealbumen contents of MnSOD and CAT in the NAC interrupted group increase.With the increase of time, albumen contents of MnSOD and CAT increasegradually, which is time dependent. The compare difference between experimentgroups is of statistic meaning (P<0.01).Conclusions1.PCN can induce J774cell oxidative damage with density dependence. 2. NAC can protect J774cells from oxidation injure induced by PCN with timedependence.