The Role Of Deregulated Bcl-2Promoter Methylation Via Pulmonary Endothelial Apoptosis In Chronic Obstructive Pulmonary Disease

The First Part The relationship between deregulation of Bcl-2promoter methylation and pulmonary endothelia apoptosis in chronic obstructive pulmonary diseaseObjective:Observe and analyze the Bcl-2methylaiton loci, Bcl-2expression level, and endothelia apoptosis status in COPD patients, asympotomous smokers and non-smokers, to investigate the possible role of deregulated Bcl-2promoter methylation in COPD pathogenesis.Method:31cases of peripheral lung cancer patients were enrolled, who were defined pathologic character as NSLC, devied into3groups as follow,11cases of COPD patients,10cases of asympotomous smokers and non-smokers. TUNEL assay was used to assess apoptosis status, immunohistochemistry to detect the Bcl-2expression in pulmonary endothelia, Western-blot and realtime-RT PCR to measure the Bcl-2, expression in lung tissue. BSP was used to assess the methylation satatus and sequence the methylation. Analyze the correlationship with regression test. Data was presented with Mean±SD, Anova and LSD-t analized the normality data, and Kruskal-Wallis H and Nemenyi analized the non-normality data. P<0.05brings the statistics significant.Result:Apoptosis index of endothelia in COPD group was(22.79±5.30)%, which was higher than the other two non-COPD groups (P<0.01). The positive rate of Bcl-2expression in endothelia from COPD group was (5.18±3.76)%, obviously lesser than the two non-COPD groups (P<0.01). Bcl-2expression in lung tissure from COPD patients was0.31±0.11(Western-blot),1.40×10-4±0.16×10-4(realtime-RT PCR) P<0.01, which was lower than the two non-COPD groups (P<0.01). Moreover the methylation lever of Bcl-2promoter in COPD group was (12.23±4.39)%, which was higer than other groups. In addition the deregulated methylation of Bcl-2has relationship with endothelia apoptosis, Bcl-2expression and smoker index.Conclusions:1. Increased endothelia apoptosis and Bax expression were found in COPD patients, with decreased Bcl-2expression.2. The decreased Bcl-2expression might stimulate the mitochondria depend apoptosis pathway, and lead to endothelia apoptosis and participate the COPD pathogenesis.3. There are abnormal methylation of Bcl-2promoter in COPD, and the-127bp C was the most common methylation loci.4. Smoke might induce bcl-2promoter abnormal methylaiton, and lead to deregulate Bcl-2expression, which might play a role in COPD pathogenesis.5. Different clones of people might show different suscepticity of abnormal methylation in Bcl-3promotor. The Second Part The relationship between deregulation of Bcl-2promoter methylation and pulmonary endothelia apoptosis in emphysematous modelObjective:Observe and analyze the Bcl-2methylaiton loci, Bcl-2expression level, and endothelia apoptosis status in CSE-induced emphysematous models, control models and demethylation models, to investigate the possible role of deregulated Bcl-2promoter methylation in COPD pathogenesis.Method:Forty BALB/C mice were devied into4groups as follow, PBS group was intraperitoneally injected with PBS in the1st,11th,15th,17th,19th,22nd days. CSE group was intraperitoneally injected as PBS group, except in the1st,11th,22nd day changed to CSE from PBS. CSE+AZA group was intraperitoneally injected as the CSE group, except changed PBS to AZA in the15th,17th,19th days. AZA group also was injected as the same, just changed the CSE to PBS in the15th,17th,19th days. TUNEL assay was used to assess apoptosis status, immunohistochemistry to detect the Bcl-2expression in pulmonary endothelia, Western-blot and realtime-RT PCR to measure the Bcl-2, expression in lung tissue. BSP was used to assess the methylation satatus and sequence the methylation. Analyze the correlationship with regression test. Data was presented with Mean±SD, Anova and LSD-t analized the normality data, and Kruskal-Wallis H and Nemenyi analized the non-normality data. P<0.05brings the statistics significant.Result:Apoptosis index of endothelia in CSE group was(25.88+7.55)%, which was higher than the other three groups (P<0.01). The positive rate of Bcl-2expression in endothelia from CSE group was0.35±0.11)%, obviously lesser than the other three groups (P<0.01). Bcl-2expression in lung tissure from COPD patients was0.49±0.10(Western-blot),2.46x10-3±0.16×10-3(realtime-RT PCR), P<0.01, which was lower than the other three groups (P<0.01). The methylation lever of Bcl-2promoter in CSE group was (35.68±5.99)%, which was higer than other groups. The most common methylated loci were-1884bp and-1740bp, almost26.67%of methylation were occurred in the two loci. The lung tissue from CSE group also present higher level of Bax and plasmic cyt C expression than control group (P<0.01). Moreover, the demethylation reagent, AZA, could improve the lung function and apoptosis with increased Bcl-2and decreased Bax. In addition, the deregulated methylation of Bcl-2has relationship with endothelia apoptosis and Bcl-2expression. On the other hand, Bcl-2protein expression also has a negative relationship with plasmic cyt C, the sign of mitochondrial-depend apoptosis pathway.Conclusions:1. Increased endothelia apoptosis and Bax expression with decreased Bcl-2expression were found in CSE-induced emphysema models.2. The decreased Bcl-2expression, induced by CSE injection, might stimulate the mitochondria-depend apoptosis pathway, and lead to endothelia apoptosis and participate the COPD pathogenesis.3. There are abnormal methylation of Bcl-2promoter in CSE-induced emphysematous models, and the-1884bp and-1740bp were the most common methylation loci.4. Demethylation treatment, AZA injection, could prevent the abnormal methylation of Bcl-2promoter, decreased expression of Bcl-2, and increased apoptosis of endothelia.5. Smoke induce bcl-2promoter abnormal methylaiton, and lead to deregulate Bcl-2expression, which might stimulate the cyt C released by mitochondrial, then enter the mitochondrial-depend apoptosis pathway, and play a role in COPD pathogenesis. The Third Part The role of deregulated Bcl-2promoter methylation in CSE-induced HUVEC apoptosisObjective:To investigate the possible mechanism in CSE-induced HUVEC apoptosis, and discuss the effect of deregulated Bcl-2promoter methylation in this apoptosis programme.Method:Treat HUVEC with different concentrates (0-20%) of CSE during different time (0-12h). Detect the apoptosis rate by flow cytometry with double die (Annexin V-FITC/PI). Immunocytochemistry、 Western-blot and realtime-RT PCR were used to assess Bcl-2expression after different concentrate of CSE treatment. Furthermore, divided cells into four groups:control group, CSE(5%)group, CSE+AZA group, AZA group, give each group treatments respectively. Measure methylation of Bcl-2promoter, Bcl-2expression, Bax expression, plasmic cyt C and apoptosis rate with BSP, immunocytochemistry, Western-blot, realtime-RT PCR and flow cytometry respectively. Data was presented with Mean±SD, Anova and LSD-t analized the normality data, and Kruskal-Wallis H and Nemenyi analized the non-normality data. P<0.05brings the statistics significant.Result:After the first2h treatment, the CSE-induced HUVEC apoptosis was increased depend time and concentrate. However, if the concentrate was too high, more than10%, the necrosis cells would be the most dead cell, rather than apoptosis. The highest apoptosis rate was46.13±5.07%after10%CSE treat last for12h. After12h of5%CSE treatment, the Bcl-2protein relative expression was0.16±0.05, obviously lower than0.0%and2.5%CSE group (P<0.01). AZA could improve the Bcl-2expression and apoptosis(P<0.01). BSP found that CSE induced abnormal methylation of Bcl-2promoter in HUVEC, though AZA could prevent this deregulation. The most common methylaltion loci was the+5bp C, almost33.33%methylation occurred in this loci. The regression analysis found that Bcl-2expression had positive relationship with plasmic cyt C, which showed a positive relationship with apoptosis, and a negative relationship with methylation level.Conclusion:1. CSE-induced HUVEC apoptosis might depend on the mitochondrial apoptosis pathway.2. CSE induce abnormal methylation of Bcl-2promoter, which might decrease the expression of Bcl-2, destruct the mitochondrial membrane, lead cyt C release to cell plasma, stimulate the mitochondrial-depend apoptosis pathway. At last, increase the apoptosis.3. Cigarette exposure was a risk factor for many diseases, and endothelia apoptosis also participates in many pathogenesis of diseases. Because the HUVEC can mimic the human endothelia, it is might be possible and inspiriting that the CSE-induced abnormal methylation of Bcl-2promoter might play a role in many endothelia relative diseases, such as COPD, coronary disease, rheumatic disease.