| The lung has a rather close relationship with the oxygen. There is growing evidence showing that reactive oxygen speicies(ROS) and oxygen free radicals(OFR) can cause the lung injure with various lung cells as targets, especially the vascular endothelial cells.When cell injure occures, introcellular calcium concentration will increase,which will reactivate the chloride channels, and it will do harm to the membrane, chondriosome and DNA or accelerate irreversible cell death, but the underlying mechanism is still not clear. The chloride channels located on the cardiac muscle cells, smooth muscle cells, renal cells are relatively studied more , but those on lung cells are studied less, and the reports are even less on protective effects to the oxidant damaged cells with chloride channel blocker administration.The operation has been conducted that renal tubular epithelial cells injure caused by the H2O2 were treated with the chloride channel blocker, and the cells were protected.but it has not been determined whether the chloride channels participate in the physiological and pathologic process of the injure to the cells and whether the chloride channel blocker can protect the cells from oxidating injure.In this experiment, we observed the variations of cell injure in two groups: one is mere treatment of H2O2 the other group is the treatment with sequence of chloride channel blocker and H2O2.to investigate the protective role of the chloride channel blocker and the mechanism.Method : Four groups: group A(control group );group B: H2O2 treatmentηial group (B1, B2, 83 with different concentrations of H2O2 );group C : H2O2 +5-nitro-2-(3-phenylprophlamino)-benzoate(NPPB) trial group(C1, C2 with different concentrations of NPPB );group D: H2O2 + niflumic acid(NFA) trial group(D1, D2 with different concentrations of NFA ). Heal thy young bovine pulmonary artery endothelial cells were procured as the primary culture,after subcultured twice and frozen, the second to fourth generation cells are applied. Cultured PAEC cells exposed to H2O2 were used to observe the effect of Cl-channel blocker stress on cells' vitality, membrane fluidity ,LDH release, ATP content, [Ca2+]j and DNA damage.MTT colorimetry were employeed to measure cell activity, different levels of activity vary with different MTT values.The injure profiles were observed .the protective effects of the NFA and NPPB can be determined. Fluorescence polarization technique were applied to measure the variations of membrane fluidity ,and to determine the injure effects of H2O2 and protective effects of NPPB and NFA.Ultraviolet spectrophotometer and fluorescence photometer were employeed to measure LDH releasing and ATP content.,to observe the injure effects of H2O2 to chondriosome of PAEC and protective effects of chloride channel blocker. Agarose gel electrophoresis were employeed to observe the DNA fragments,to analysis the injure effects of H2O2 to DNA and protective effects of chloride channel blocker. Laser scanning confocal microscopy were employeed to observe variations of intracellular calcium concentration after administration of H2O2 , then NPPB and NFA,to establish the relationship of the protective effects mechanism and Ca2+.Results: 1.Variations of cell activity : Group A :MTT value 0.623 + 0.021, group 8:0.249±0.027; group C :0.419±0.021; group D :0.459±0.023.Group B MTT value is statistically lower than group A(P<0.01). Activity of group C and group D are statistically higher than group B (P<0.01). 2. Membrane fluidity: micro-glutinosity ( η): Group A:1.606±0.010;group B: 3.417±0.013;groupC:2.306±0.012;group D:2.169±0.014, Group Bη value is statistically higher than group A(P<0.01). η value of group C and group D are statistically lower than group B (P<0.05).it is concluded that NPPB and NFA have the protective effects to membrane. 3. LDH releasing:Group A:3.1%±0.7%;group B: 36.9%± 1.4%, group C: 12.4% ±0.4%; group D: 6.4% ±0.6%; LDH releasing of Group B is statistically higher than group A(P<0.01).LDH releasing of... |
