The Effects And Mechanism Of5-aza-2’-deoxycytidine On Mouse Emphysema Model Induced By Intraperitoneal Injection Of Cigarette Smoke Extract

Part one The effects of5-aza-2’-deoxycytidine on mouse emphysema model induced by intraperitoneal injection of cigarette smoke extractObjective:with mouse model of emphysema induced by intraperitoneal injection of cigarette smoke extract (CSE), to investigate the effects of5-aza-2’-deoxycytidine (AZA) treatment on lung function, cellular components/classification of bronchoalveolar lavage fluid (BALF), the degree of emphysema and apoptosis in alveolar septa among those mice.Methods:using a random number table,40BALB/C inbred male mice were divided into four groups (i.e. the control group, AZA group, CSE group, CSE+AZA intervention group) with10mice in each group. CSE was made by Furong brand of cigarettes through continuous sucking to burn with the suction device. The inhaled cigarettes were entered into phosphate-buffered saline (PBS) solution (2ml PBS contain1cigarette) through three-limb tubes to become CSE-PBS solution. Only freshly prepared solutions were used in this study. Control group mice were injected intraperitoneally with PBS (0.3ml/20g) at day1,11,15,17,19and22respectively. AZA group mice were injected intraperitoneally with PBS (0.3ml/20g) at day1,11,22respectively, and with AZA (2.5mg/kg/day) at day15,17, and19respectively. CSE group mice were injected intraperitoneally with CSE (0.3ml/20g) at day1,11,22respectively, and with PBS (0.3ml/20g) at day15,17and19respectively. The CSE+AZA intervention group mice were injected intraperitoneally with CSE (0.3ml/20g) at day1,11,22respectively, and with AZA (2.5mg/kg/day) at day15,17and19respectively. The animals were sacrificed at the28th day (i.e, the last day) of the experiment. Airway resistance (Raw), dynamic lung compliance (Cdyn) and peak expiratory flow (PEF), and other related indicators were recorded by the Small-Animal Pulmonary Function Instrument. Cellular components have been counted and classified in bronchoalveolar lavage fluid (BALF) by bronchoalveolar lavage (BAL) method. After the preparation of lung histological sections stained with hematoxylin-eosin (HE), the mean liner intercepts (MLI, μm) and destructive index (DI,%) of the lung were measured to assess the severity of emphysema. Alveolar septal cell apoptosis and apoptotic index (AI) were evaluated using the terminal transferase uridyl nick end labelling (TUNEL) assay. Statistical analysis was performed using SPSS Statistical Software Package, version13.0(SPSS Inc, Chicago, IL). Data were presented as Mean±SD and analyzed using independent samples t-test. Statistical significance was defined as P<0.05.Results:(1) the study found that airway resistance (RAW) was higher in the CSE group than in the control group [(0.212±0.012) cmH2O. mL-1. min-1vs.(0.069±0.005) cmH2O. mL-1. min-1, P<0.05], dynamic lung compliance (Cdyn) was lower in the CSE group than in the control group [(0.109±0.006) mL/cmH2O vs (0.217±0.010) mL/cmH2O, P<0.05], airway resistance (RAW) was lower in the CSE+AZA intervention group than in the CSE group [(0.072±0.005) cmH2O. mL-1. min-1vs.(0.212±0.012) cmH2O. mL-1. min-1P<0.05], dynamic lung compliance (Cdyn) was higher in the CSE+AZA intervention group than in the CSE group [(0.162±0.006) mL/cmH2O vs.(0.109±0.006) mL/cmH2O, P<0.05], airway resistance (RAW) was higher in the AZA group than in the control group [(0.115±0.009) cmH2O. mL-1. min-1vs.(0.069±0.005) cmH2O. mL-1. min-1, P<0.05], and dynamic lung compliance (Cdyn) was lower in the AZA group than in the control group [(0.131±0.005) mL/cmH2O vs (0.217±0.010) mL/cmH2O, P<0.05].(2) this study revealed that the total number of cells [(4.86±0.52)×108/L vs.(1.58±0.24)×108/L], the number of macrophages (AM)[(3.26±0.32)×108/L vs.(1.21±0.16)×108/L], the number of neutrophil (N)[(7.56±0.89)×107L vs.(0.76±0.08)×107L], and the proportion of neutrophils [(13.01±2.12)%vs.(8.12±0.86)%] in BALF were significantly higher (P<0.05) in the CSE group than in the control group. The total number of cells [(3.87±0.46)×108/L vs.(4.86±0.52)×108/L], the number of macrophages (AM)[(2.96±0.20)×108/L vs.(3.26±0.32)×108/L], the number of neutrophil (N)[(4.31±0.62)×107L vs.(7.56±0.89)×107L], and the proportion of neutrophils [(11.01±1.56)%vs. (13.01±2.12)%] in BALF were significantly lower (P<0.05) in the CSE+AZA intervention than in the CSE group.There were no significant differences (P>0.05) between AZA group and control group of the total number of cells [(1.62±0.26)×108/L vs.(1.58±0.24)×108/L], the number of macrophages (AM)[(1.26±0.20)×108/L vs.(1.21±0.16)×108/L], the number of neutrophil (N)[(0.78±0.11)×107L vs.(0.76±0.08)×107L], and the proportion of neutrophils [(8.32±0.91)%vs.(8.12±0.86)%] in BALF.(3) Pathological examination found that airway wall structure can be clearly seen, the airway epithelium arranged in order with no obviously epithelial shedding in the control group mice. The AZA group mice also showed clearly airway wall structure and orderly airway epithelium but with a small amount of inflammatory cell infiltration. However, the CSE group mice showed disordered airway epithelial with some shed; hyperplasia, thickened walls, narrowed lumen occurred in part of the airway epithelial, inflammatory cell infiltration occurred around the airway. Also, the CSE group mice showed deficiency of support structure such as the missing of partly alveolus surrounded airway. CSE+AZA intervention group showed slightly disordered airway epithelial with only some shed, slightly narrowed lumen, and minor inflammatory cell infiltration around the airway.(4) Quantitative pathologic analysis showed that the mean liner intercepts (MLI) and destructive index (DI) of the lung were higher in the CSE group mice than in the control group [(52.47±1.85)μm vs.(34.26±3.22)μm,(36.69±1.81)%vs.(11.85±3.12)%, P<0.01], lower in the CSE+AZA intervention-group than in the CSE group [(40.04±1.76) μm vs.(52.47±1.58) μm,(22.55±1.43)%vs.(36.64±2.30)%, P<0.01], and were no statistical differences between the AZA group and the control group [(34.36±3.46) μm vs.(34.26±3.22)μm,(13.96±3.31)%vs.(11.85±3.12)%, P>0.01].(5) Results of alveolar septal cell apoptosis showed that the apoptotic index (AI) was higher in the CSE group than in the control group [(14.2±0.6)%vs.(4.5±0.6)%, P<0.05], lower in the CSE+AZA intervention group than in the CSE group [(8.1±0.5)%vs.(14.2±0.6)%, P <0.05], and no differences between the AZA group mice and the control group [(5.2±0.5)%vs.4.5±0.6)%, P>0.05].Conclusions:(1) mouse emphysema model was successfully established by intraperitoneal injection of CSE.(2) intraperitoneal injection of CSE induced emphysema model of mice presented airway inflammation, alveolar septal cell apoptosis, increased airway resistance (Raw) and decreased dynamic lung compliance (Cdyn).(3) AZA intervention can partially inhibit airway inflammation, improve the severity of emphysema, improve dynamic lung compliance (Cdyn), decrease airway resistance (Raw), and reduce apoptosis of alveolar septal cell for intraperitoneal injection of CSE induced emphysema model mice. Part two The effects of5-aza-2’-deoxycytidine on mtTFA and COX Ⅱ expression in mouse emphysema model induced by intraperitoneal injection of cigarette smoke extractObjective:with mouse model of emphysema induced by intraperitoneal injection of cigarette smoke extract (CSE), to measure the changes of mtTFA (Mitochondrial transcription factor A) expression, COX Ⅱ (cytochrome c oxidase subunit Ⅱ) expression and COX activity in mice lung tissue by5-aza-2’-deoxycytidine (AZA) intervention, to analyze its effects on apoptosis of alveolar septal cell, and to explore the possible contributions of mtTFA to the pathogenesis of mouse emphysema model.Methods:using a random number table,40BALB/C inbred male mice were divided into four groups (i.e. the control group, AZA group, CSE group, CSE+AZA intervention group) with10mice in each group. All lung tissue samples were collected to extract genomic DNA of lung tissue to detect the COX activity via spectrophotometry, to detect mtTFA expression and COX Ⅱ mRNA expression by ordinary RT-PCR and real-time RT-PCR, and to measure mtTFA expression and COX II protein expression by Western-blot assay. Statistical analysis was performed using SPSS Statistical Software Package, version13.0(SPSS Inc, Chicago, IL). Data were presented as Mean±SD and analyzed using independent samples t-test and Pearson correlation analysis. Statistical significance was defined as P<0.05. Results:(1) in the control group, the relative expression level of mtTFA mRNA was1.001±0.067and mtTFA protein was1.31±0.34in mice lung tissue. In the AZA group, the relative expression level of mtTFA mRNA was0.991±0.34and mtTFA protein was1.30±0.06in mice lung tissue. In the CSE group, the relative expression level of mtTFA mRNA was0.534±0.019and mtTFA protein was0.85±0.04in mice lung tissue. In the CSE+AZA intervention group, the relative expression level of mtTFA mRNA was0.723±0.055and mtTFA protein was1.14±0.06in mice lung tissue. There was lower level of mtTFA mRNA expression and mtTFA protein expression in the CSE group than in the control group [(0.534±0.019) vs.(1.001±0.067),(0.85±0.04) vs.(1.31±0.34), P<0.01]. There was higher level of mtTFA mRNA expression and mtTFA protein expression in the CSE+AZA intervention group than in the CSE group [(0.723±0.055) vs.(0.534±0.019),(1.14±0.06)vs.(0.85±0.04), P<0.01]. Level of mtTFA mRNA expression and mtTFA protein expression showed no statistical difference between the AZA group and the control group [(0.991±0.34) vs.(1.001±0.067),(1.30±0.06) vs.(1.31±0.34), P>0.01].(2) the relative expression level of COX activity, COX Ⅱ mRNA and COX Ⅱ proteins in the control group mice lung tissue were [(6.5±0.3)×10-1]U/mg,1.000±0.024, and2.99±0.13respectively. The relative expression level of COX activity, COX Ⅱ mRNA and COX Ⅱ proteins in the AZA group mice lung tissue were [(6.2±0.5)×10-1]U/mg,1.009±0.024, and2.96±0.15respectively. The relative expression level of COX activity, COX Ⅱ mRNA and COX Ⅱ proteins in the CSE group mice lung tissue were [(4.2±0.4)×10-1]U/mg,0.678±0.045and0.89±0.13respectively. The relative expression level of COX activity, COX Ⅱ mRNA and COX Ⅱ proteins in the CSE+AZA intervention group mice lung tissue were [(5.1±0.5)×10-1]U/mg,0.837±0.010and2.71±0.18respectively. The relative expression level of COX activity, COX Ⅱ mRNA and COX Ⅱ proteins were lower in the CSE group than in the control group [(4.2±0.4)×10-1] U/mg vs.(6.5±0.3)×10-1] U/mg, P<0.01;(0.678±0.045) vs.(1.000±0.024), P<0.01;(0.89±0.13) vs.(2.99±0.13), P<0.01]. The relative expression level of COX activity, COX Ⅱ mRNA and COX Ⅱ proteins were higher in the CSE+AZA intervention group than in the CSE group [(5.1±0.5)×10-1] U/mg vs.(4.2±0.4)×10-1] U/mg, P<0.01;(0.837±0.010) vs.(0.678±0.045), P<0.01;(2.71±0.18) vs.(0.89±0.13), P<0.01]. However, there were no statistical differences for the relative expression level of COX activity, COX Ⅱ mRNA and COX Ⅱ proteins between the AZA group and the control group [(6.2±0.5)×10-1] U/mg vs.(6.5±0.3)×10-1] U/mg, P>0.01;(1.009±0.024) vs.(1.000±0.024), P>0.01;(2.96±0.15) vs.(2.99±0.13) P>0.01].(3) by Pearson correlation analysis, apoptotic index (AI) of alveolar septal cell apoptosis has been negatively correlated with dynamic lung compliance (Cdyn)(r=-0.787, P<0.01), and with COX activity (r=-0.796, P<0.01). The mtTFA protein expression in mice lung tissue was positively correlated with COX Ⅱ protein expression (r=0.889, P<0.01) and with COX activity (r=0.826, P<0.01), while it was negatively correlated with apoptotic index (AI)(r=-0.758, P<0.01).Conclusions:(1) the mtTFA expression, COX Ⅱ expression and COX activity were decreased in lung tissue of emphysema mice;(2) in this mouse emphysema model, CSE may have effects on COX activity via inhibition of mtTFA expression, and then contribute to the apoptosis of alveolar septal cell;(3) AZA intervention up-regulated mtTFA expression and COX Ⅱ expression that may promote COX activity to reduce alveolar septa apoptosis in emphysema mice. Part three The effects of5-aza-2’-deoxycytidine on mtTFA gene methylation and status of genome overall methylation of lung tissue in mouse emphysema model induced by intraperitoneal injection of cigarette smoke extractObjective:with mouse model of emphysema induced by intraperitoneal injection of cigarette smoke extract (CSE), to measure the changes of the methylation in the promoter region of the mtTFA gene and changes of overall genome methylation status in lung tissue, and to explore the possible role of methylation in mtTFA gene to the mouse emphysema model.Methods:using a random number table,40BALB/C inbred male mice were divided into four groups (i.e. the control group, AZA group, CSE group, CSE+AZA intervention group) with10mice in each group. All lung tissue samples were collected to extract genomic DNA of lung tissue to detect methylation status in mtTFA gene promoter region using Sodium bisulfite genomic sequencing (BSP) method, and to detect overall genome methylation status in lung tissue. Statistical analysis was performed using SPSS Statistical Software Package, version13.0(SPSS Inc, Chicago, IL). Data were presented as Mean±SD and analyzed using One-Way ANOVA. Statistical significance was defined as P<0.05.Results:(1) using BSP method, multiple methylated CpG sites were found in mtTFA gene promoter region in the CSE group with the average methylation rate of9.40±0.66%, which was higher than3.50±2.41%in the CSE+AZA group (P<0.05). However, we failed to find methylated CpG sites in this region in the control group and AZA group.(2) the overall methylation rate in lung tissue were5.219±0.115%in the control group,4.916±0.156in the AZA group,6.317±0.102%in the CSE group and5.412±0.135%in the CSE+AZA group. It was higher in the CSE group than the control group, AZA group and CSE+AZA group[(6.317±0.102%)vs.(5.219±0.115%), P<0.05;(6.317±0.102%) vs.(4.916±0.156%), P<0.05;(6.317±0.102%))vs.(5.412±0.135%), P<0.05].Conclusion:(1) CSE may down-regulate mtTFA gene expression via inducing mtTFA methylation to inhibit COX Ⅱ expression and COX activity to increase alveolar septa apoptosis.(2) AZA may up-regulate mtTFA gene expression via depressing mtTFA methylation to active COX Ⅱ expression and COX activity to decrease alveolar septa apoptosis.