Effect Of Pim-3 On Proliferation Of Airway Smooth Muscle Cells In COPD And Its Role As A Potential Therapeutic Target For COPD Rats

Background:Chronic Obstructive Pulmonary Disease(COPD)is a common,preventable and treatable disease that is characterized by persistent respiratory symptoms and airflow limitation that is due to airway and(or)alveolar abnormalities usually caused by significant exposure to noxious particles or gases.COPD is a leading cause of morbidity and mortality worldwide that induces an economic and social burden that is both substantial and increasing.However,the present pharmacological treatments including bronchodilators,corticosteroids,phosphodiesterase-4 inhibitors,and so on can not actually stop nor reverse the progression of COPD.It is urgent to learn more about the pathogenesis of COPD to find new therapeutic targets.The pathogenesis of COPD is extremely complicated and is not completely understood.Previous studies suggested that,under the condition of chronic irritants(including noxious particles or gases),repeated injury and repair in airways result in structural changes,which persists after smoking cessation.Structural changes in airway include fibrous tissue proliferation,airway smooth muscle proliferation,basement membrane thickening,extracellular matrix deposition and mucosal goblet cell hyperplasia in small airway wall,which also called airway remodeling.Airway remodeling increase airflow resistance,aggravate airflow limitation in small airways,and airway collapse.In COPD,airflow limitation in small airways is the characteristic physiological abnormality,and airway remodeling is a major pathological change.Airway remodeling is a complicated pathological process,of which airway smooth muscle proliferation is a crucial component.By now,several negative regulator was found to inhibit airway smooth muscle cells(ASMCs)proliferation,such as aminophylline,leukotriene receptor antagonist,glucocorticoids and salbutamol.To date,these routine medications,as pharmacologic therapy for COPD,fail to modify the decline in lung function.Proviral integration of moloney murine leukemia virus(Pim)belong to the Ca2+/calmodulin-dependent protein kinase group.Three members of the Pim family have been identified:Pim-1,Pim-2,and Pim-3.Pim-3 can enhance cell proliferation and promote cell survival,by phosphorylating serine residues of critical proteins in cell growth pathways.Similar to other members of the Pim family(including Pim-1 and Pim-2),Pim-3 can prevent apoptosis and promote cell survival and protein translation,thereby enhancing cell proliferation of normal and malignant cells.Based on these previous studies,we proposed the hypothesis that Pim-3 might be involved in the process of ASMCs proliferation in COPD.In the present study,we selected lung tissue from patients who underwent partial resection of the lungs.The patients were divided into control group and COPD group according to pulmonary function test.We investigated thickness of airway wall and airway smooth muscle,and detected the expression of Pim-3.We assessed the role of Pim-3 in proliferation of ASMCs which were cultured in vitro by RNA interference technique.Through smoke exposure,We established rat COPD model,some of which received intragastric administration of AZD1208,to explore the therapeutic potential of Pim-3 in rat model with COPD.Chapter 1 The expression of Pim-3 in lung tissue of COPD patientsObjective:To investigate the expression of Pim-3 in lung tissue of COPD patients.Subjects and Methods12 patients who underwent partial resection of the lungs were recruited.The patients were assigned to control group(n?6)and COPD group(n?6),according to diagnostic criteria of GOLD 2014,defined as an FEV1:FVC ratio of less than 0.70 after bronchodilator use.Total RNA and protein were extracted from lung tissue that were obtained away from lesions,and the expression of Pim-3 was detected by Real time PCR or Western blot,and expression location of Pim-3 was detected by immunofluorescence.Morphological observation for lung,bronchus/bronchioles was performed under a microscope.The basement membrane perimeter(Pbm),total wall area(WAt),and wall area of smooth muscle(WAm)were measured by image analysis software.The results of WAt and WAm were adjusted by Pbm,WAt/Pbm and WAm/Pbm stand for thickness of airway wall and airway smooth muscle,respectively.Results:1.PIM3 expressed in lung tissue in control group as well as in COPD group.The PIM3 expression level increased in COPD group compared to control group(T=21,P=0.002).2.Pim-3 expressed in lung tissue of control group and COPD group.Compared to control group,Pim-3 expression increased in COPD group(T=21,P?0.002).3.Immunofluorescence detection showed Pim-3 was located mainly among submucosal airway smooth muscle layer.4.The structure of bronchus/bronchioles is clear and eumorphism in control patients.In contrast,bronchial airway wall and airway smooth muscle thickening were observed in patients with COPD,and mucosal folds which caused by epithelial hyperplasia,filled bronchial lumen.5.Two independent sample t test was used to analysis of WAt/Pbm and WAm/Pbm between COPD patients and control patients.The results indicated a statistically significant difference of WAt/Pbm between two groups[(41.24±2.28)?m2/?m vs(21.30± 1.98)?m2/?m,t=22.87,P<0.001].For WAm/Pbm,there was a statistically significant difference between COPD group and control group[(10.46 ± 0.67)?m2/?m vs(5.58 ± 0.63)?m2/?m,t=18.40,P<0.001].These results revealed that the thickness of airway wall and airway smooth muscle in COPD group increase,compared to control group.Conclusion:Pim-3 expression increased in lung tissue in COPD group,and Pim-3 was located mainly among airway smooth muscle layer.Chapter 2 Effect of Pim-3 on cell proliferation of ASMCs in vitroObjective:To investigate the effect of Pim-3 on airway smooth muscle cells(ASMCs)proliferation in vitro.Subjects and MethodsSurgically resected lung tissue was obtained from patients who were assigned to control group and COPD group meeting diagnostic standard of COPD.The ASMCs were cultured from primary generation using a tissue culture method.The cultured cells were observed under phase contrast microscope and immunostaining for a-actin,which were used to identified the cell types.According to four types of treatment,ASMCs were divided into four groups:control group(cells from control patients,without siRNA transfection)± COPD group(cells from COPD patients ± without siRNA transfection),COPD + NC-siRNA group(cells from COPD patients,with NC-siRNA transfection),COPD + PIM3-siRNA group(cells from COPD patients,with PIM3-siRNA transfection).We took the PIM3 gene as a target,transfected cells by Lipofectamine TM 2000.Real time PCR detected the expression of PIM3 in each group,and Western blot detected the expression of Pim-3 and Cyclin D1.The cell survival was assessed by MTT,and the cell cycle distribution of ASMCs was detected by flow cytometry.Results:1.The cultured cells demonstrated the typical 'hill and valley" appearance under phase contrast microscopy.After inmmunostaining for a-actin,brown positive reaction was observed in the cytoplasm.There were over 94%cultured cells identified to be ASMCs.2.Analysis of expression of PIM3 mRNA,Pim-3 protein and Cyclin D1 in primary ASMCs in each group.2(1)Kruskal-Wallis test of PIM3 mRNA expression in all group resulted in ?2 =17.65,P = 0.001;Further multiple comparison revealed that expression of PIM3 mRNA of ASMCs in COPD group were significant greater than that of control group(P = 0.005),and expression of PIM3 mRNA in COPD + PIM3-siRNA group decreased significantly(P = 0.002)compared to COPD group(silence efficiency over 60%),and the differences in COPD+NC-siRNA group and COPD group was not of statistically significance(P>0.05).Cyclin D1:(2)Kruskal-Wallis test of expression of Pim-3 and Cyclin D1 in all group resulted in ?2=18.06(?<0.001)and ?2=20.72(P<0.001),respectively.Multiple comparison showed that the differences of expression of two proteins in COPD + NC-siRNA group and COPD group was not of statistically significance(P>0.05),expression of Pim-3 and Cyclin D1 of ASMCs in COPD group increased significantly than that of control group(P<0.05),and expression of Pim-3 and Cyclin D1 in COPD+PIM3-siRNA group decreased significantly(P<0.05)compared to COPD group.These results revealed that expression of Pim-3 of ASMCs increased in COPD group compared to control group.PIM3 gene was effectively silenced by PIM3-siRNA transfection.3.Analysis of optical density(OD)of ASMCs in each group,F= 104.69,P<0.001.The OD for COPD control was greater than control group(COPD vs.Control:0.95 ± 0.04 vs.0.64 ± 0.03,P<0.001),Which meant that the cell proliferation capacity in COPD group was higher than that in control group(inhibition ratio for control group 35.90%±6.68%).Compared to COPD group,OD for COPD +PIM3-siRNA group decreased(COPD + PIM3-siRNA vs.COPD:0.71 ± 0.02 vs.0.95 ±0.04,P<0.001),which suggested that the cell proliferation capacity of ASMCs in COPD +PIM3-siRNA group was inhibited(inhibition ratio 28.58%± 2.72%),when transfected with PIM3-siRNA.The differences of OD for ASMCs in COPD?NC-siRNA group and COPD group was not of statistically significance(COPD vs.COPD+NC-siRNA:0.95±0.04 vs.0.92±0.04,P=0.26).4.Flow cytometry detected the cell cycle distribution of ASMCs.Analysis of proliferation rate of ASMCs in four groups,F = 104.69,P<0.001.The proliferation rate(%)of ASMCs in COPD group increased(COPD vs Control:24.86 ? 3.08 vs 8.19±1.25,P<0.001),compared to control group,which indicated that the cell proliferation capacity in COPD group is higher than that in control group.When transfected with P/M3-siRNA,the rate(%)of proliferation of ASMCs in COPD +PIM3-siRNA group was lower than that in COPD group(COPD?PIM3-siRNA vs COPD:15.21 ±2.62 vs 24.86 ± 3.08,P<0.001).The differences of proliferation rate(%)in COPD+NC-siRNA group and COPD group was not of statistically significance(COPD vs COPD+NC-siRNA:24.86 ±3.08 vs 24.82 ? 3.27,P=0.97).Conclusion:Down-reguration of Pim-3 inhibit proliferation of ASMCs in COPD patient small airways in vitro.Chapter 3 Pim-3 as a potential therapeutic target for COPD ratsObjective:To investigate the role of Pim-3 as a potential therapeutic target for COPD rats.Subjects and Methods24 SPF male Sprague-Dawley rats were randomly assigned to four groups:COPD group(smoke exposure),COPD+ AZD1208 group(smoke exposure and intragastric administration of AZD1208),COPD + DMSO group(smoke exposure and intragastric administration of DMSO)and normal control group(normal air exposure and without intragastric administration).Real time PCR detected the expression of PIM gene of lung tissue in COPD group.Morphological observation for pathological changes of alveoli and small airways was performed under a microscope.The mean linear intercept(MLI),basement membrane perimeter(Pbm),total wall area(WAt),and wall area of smooth muscle(WAm)were measured by image analysis software.MLI,WAt/Pbm and WAm/Pbm stand for alveolar destruction,thickness of airway wall and airway smooth muscle,respectively.Results:1.Detection of PIM families in rat COPD lung tissue by Real time PCR showed that PIMl1,PIM2 and PIM3 were detected and PIM3 was abundantly expressed.2.The alveolar structure was mostly intact in control group in morphology.By contrast,pulmonary emphysema including alveolar wall thinning,alveolar structure disruption and alveolar space enlargement or fusion was observed in COPD group and COPD + DMSO group.Pulmonary emphysema in COPD+ AZD1208 group alleviated after intragastric administration of AZD1208.3.MIL in normal control group,COPD group,COPD + DMSO group and COPD+AZD1208 group was 73.15±7.99(?m),112.15±12.23(?m),113.9±12.53(?m)and 87.46 ± 10.00(?m),respectively.Kruskal-Wallis test of MIL in all group resulted in ?2=177.25,P<0.001;Multiple comparison revealed that MIL in COPD group was significant greater than that of control group(P<0.001),and MIL in COPD+AZD1208 group decreased after treatment of AZD1208,compared to COPD group(P<0.001),and the differences of MIL in COPD + DMSO group and COPD group was not of statistically significance(P=0.748).4.The pathological morphology showed that airway wall were complete and eumorphism in normal control group,while characteristic pathological changes of bronchitis were observed in COPD group and COPD + DMSO group,and pathological changes in COPD + AZD1208 group alleviated,compared to COPD group.5.Immunofluorescence detection showed that,thickness of airway smooth muscle layer in small airways in COPD group or COPD + DMSO group increased,compared to normal control group,and airway smooth muscle thickening alleviated in COPD + AZD1208,compared to COPD group,while thickness of airway smooth muscle layer in COPD group and COPD+DMSO group did not change significantly.6.Analysis of WAt/Pbm and WAm/Pbm in each group.(1)WAt/Pbm in normal control group,COPD group,COPD+DMSO group and COPD + AZD1208 group were 8.66±1.17(?m2/?m),23.22± 1.23(?m2/?m),22.91 ± 1.19(?m2/?m)and 13.61 ± 1.05(?m2/?m),respectively.One-way ANOVA of WAt/Pbm in all group resulted in F = 456.73,P<0.001;Multiple comparison revealed that WAt/Pbm in COPD group was significant greater than that of control group(P<0.001),and WAt/Pbm in COPD+AZD1208 group decreased,compared to COPD group(P<0.001),and the differences of WAt/Pbm in COPD + DMSO group and COPD group was not of statistically significance(P=0.52).(2)WAm/Pbm in normal control group,COPD group,COPD + DMSO group and COPD+AZD1208 group were 3.03±0.48(?m2/?m),8.96±0.62(?m2/?m),8.73 ±0.76(?m2/?m)and 4.95±0.55(?m2/?m),respectively.One-way ANOVA of WAm/Pbm in all group resulted in F = 270.2,P<0.001.Further multiple comparison suggested that,WAm/Pbm in COPD group were greater than that of control group(P<0.001),and WAm/Pbm in COPD+AZD1208 group decreased,compared to COPD group(P<0.001),while the differences of WAm/Pbm in COPD+DMSO group and COPD group was not of statistically significance(P=0.37).Conclusion:Pim-3 may play a role as a potential therapeutic target for thickening of airway smooth muscle layer and airway wall in COPD rats.