| Chapter 1 Effect of Adenovirus-mediated GPX gene transfection of oxidative injury and apoptosis of endothelial cells induced by cigarette smoking extractObjective To elucidate the effect of CSE on oxidative injury and apoptosis of endothelial cells,and whether the transfection of Adenovirus-mediated GPX to endothelial cells could partly inhibited the effect.Methods Study the transfection efficiency of ECV304 transfected by 0,30,50,70MOI Ad-LacZ.Study the AI of ECV304 interfered by different concentration of CSE for different time.ECV304 were cultured in vitro and divided into control group,10%CSE group,LacZ group and GPX group.The last three groups were interfered by 10%CSE, 10%CSE+LacZ,10%CSE+GPX respectively.The cells and supematants of each group were collected after 24 hours.MDA and SOD were tested in the supematants,AI of ECV304 were measured by TUNEL.Results1.The transfection efficiency of Ad-LacZ was above 90%.2.The AI of 10%CSE interfered ECV304 group was higher than the 0%CSE and 5%CSE interfered groups(p＜0.05).24 hours after 10%CSE interfered ECV304,the AI was higher than the 0h,6h and 12h groups (p＜0.05).3.The AI and the concentrations of MDA in the 10%CSE group were higher than the control group,while the activity of SOD was lower than the control group(p＜0.05).4.Adenovirus-mediated GPX gene transfection could decrease the concentration of MDA in supematant and AI of ECV304,and increase the activity of SOD(p＜0.05).5.There was a positive correlation between the concents of MDA in supematant and the AI of ECV304(r=0.913,p＜0.05).There was a negative correlation between the activity of SOD and the AI of ECV304 (r=-0.660,p＜0.05).Conclusion1.CSE can induce oxidative injury and apoptosis of endothelial cells.2.CSE can dose-dependently and time-dependently induce the apoptosis of ECV304.3.Transfection of Adenovirus-mediated GPX gene to endothelial cells could partly inhibit the oxidative injury and apoptosis.4.The oxidative injury of ECV304 was positive correlated with apoptosis.Chapter 2 Oral N-acetylcysteine attenuates pulmonary emphysema and alveolar septa apoptosis in smoking-induced COPD ratsObjective To study the effects of the antioxidant N-acetylcysteine (NAC)in the development of emphysema and the alveolar septal cell apoptosis in smoking-induced COPD rats.Methods Wistar rats(n=48)were randomly divided into 4 groups:normal group,COPD group,sham-interfered group and NAC-interfered group.All rats in the last three groups were exposed to cigarette smoking daily for 80 days to establish COPD models.Rats in the NAC-interfered group and the sham-interfered group were instillated by NAC or saline solution respectively daily after 30 days of smoking exposure.The effects of treatment were assessed by measuring contents of vascular endothelial growth factor(VEGF)in bronchoalveolar lavage fluid(BALF)by ELISA,VEGF and VEGFR2 protein expression in lungs by Western blotting,MDA by TBA,SOD by xanthine oxidase method, apoptotic index(AI)of alveolar septal cells by TUNEL,a histopathology evaluation[ie,mean linear intercept(MLI)and mean alveolar numbers(MAN)]and lung function.Results 1.FEV0.3/FVC and PEF in the COPD group[(65.1±8.4)%and (18.8±1.6)ml/s]were lower than the normal group[(85.6±5.9)% and(47.2±7.3)ml/s]and the NAC-interfered group[(75.7±6.7)% and(37.7±4.9)ml/s](p＜0.05).FEV0.3/FVC and PEF in sham-interfered group[(60.8±12.9)%and(16.9±1.8)ml/s]were lower than those in the NAC-interfered group(p＜0.05).2.The MLI of the COPD group[(77.3±28.7)×10-6m]was significantly higher than those of the normal group[(44.0±7.2)×10-6m] and the NAC-interfered group[(53.9±12.4)×10-6m],while the MAN of the COPD group[(232±97)×10-6/m2]was significantly lower than those of the normal group[(393±24)×10-6/m2]and the NAC-interfered group [(315±74)×10-6/m2](p＜0.05).Significant differences of MLI and MAN had been found between the sham-interfered group[(73.5±13.8)×10-6m and(227±71)×10-6/m2]and the NAC-interfered group(p＜0.05).3.The AI of alveolar septal cells in the normal group[(2.50±0.28)%] was significantly lower than those in the COPD group[(9.60±0.79)%],the sham-interfered group[(9.65±0.39)%]and the NAC-interfered group [(5.59±0.62)%],and the AI in the NAC-interfered group was significantly lower than those in the COPD group and the sham-interfered group(p＜0.05).4.The contents of MDA in serum and BALF in COPD group [(4.69±0.52)μmol//L,(2.38±0.28)μmol/L]were significantly higher than those of the normal group[(2.79±0.31)μmol/L,(1.28±0.17)μmol/L](p＜0.05).The contents of MDA in serum and BALF in the NAC-interfered group[(2.93±0.40)μmol//L,(1.52±0.21)μmol/L]were significantly lower than those of the COPD group and the sham-interfered group [(4.59±0.66)μmol/L,(2.36±0.27)μmol/L](p＜0.05).5.The activity of T-SOD and MnSOD in serum of COPD group [(102±17)U//L,(41.4±8.4)U//L]were significantly lower than those of the normal group[(172±20)U/L,(68.5±10.8)U/L](p＜0.05).The activity of T-SOD and MnSOD in serum of NAC-interfered group[(163±13)U//L, (63.6±7.9)U//L]were significantly higher than those of the COPD group and the sham-interfered group[(98±21)U/L,(38.8±9.5)U/L](p＜0.05). 6.Total white cell counts,neutrophil counts and proportion, macrophage counts and proportion in BALF were higher in COPD group than those of the normal group and the NAC-interfered groups(p＜0.05). Total white cell counts,neutrophil and macrophage counts were also higher in the NAC-interfered group than those of the sham-interfered group(p＜0.05).7.The contents of VEGF in BALF in the normal group [(1500±374)pg/ml]were significantly higher than those in the COPD group[(851±136)pg/ml],the sham-interfered group[(840±131)pg/ml] and the NAC-interfered group[(967±68)pg/ml],and the contents of VEGF in the NAC-interfered group were also higher than those of the COPD group and the sham-interfered group(p＜0.05).8.The VEGF and VEGFR2 protein expression in the COPD group were significantly decreased compared with the normal group and the NAC-interfered group by Western blotting.9.There was a negative correlation between the contents of VEGF in BALF and the AI of alveolar septal cells(r=-0.731,p＜0.05).ConclusionsNAC attenuates oxidative stress,lung function damage,pulmonary emphysema and alveolar septa apoptosis by partly reversing the decrease of VEGF and VEGFR2 expression and alveolar septal cell apoptosis in smoking-induced COPD rats.
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