Studies On The Effect Of The Proliferation And Apoptosis And The Protective Effect Of HSP70 In Airway Smooth Muscle Cells By Smoking

PartⅠ: Studies on the effect of possible signal pathways in the proliferation of airway smooth muscle cells by smokingAirway smooth muscle cells (ASMCs) have an important role not only on airway remodeling but also on chronic airway inflammation. Smoking can cause the inflammation, fibrosi, the hyperplasis of airway smooth muscle, the thicknesses of airway wall and the stenosis of airway in the small airway There are lots of signal pathways involved in the course of proliferation of airway smooth muscle cells. In the mitogen-activated signal pathways of airway smooth muscle cells, the extracellular signal regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI-3K) pathways appear to be key positive regulators of airway smooth muscle mitogenesis.But it remains unknown if ERK and PI-3K pathways participate in the course of proliferation of airway smooth muscle cells by smoking. So we study the effect of possible signal pathways in the proliferation of airway smooth muscle cells by smoking and their relations in vitro and vivo in this part.Article one: Effect of cigarette smoke extract on the ERKs and NF-κB in the proliferation of airway smooth muscle cellsThe airway smooth muscles were hyperplastic in the airway of COPD patients and COPD model of rats, and cigarette smoke extract can induced the proliferation of airway smooth muscle cells. In the effect factors of the cell proliferation, extracellular regulated protein(ERK) is the important member of mitogen activated protein kinase(MAPK). ERK pathway is the important approach in regulating the cell proliferation by grow factors and cytokines. The study indicated ERK pathway was the key signal in regulating the cell proliferation by mitogen. The transcription factor nuclear factor- kappaB(NF-κB) regulates various genes involved in immunoresponses, cell proliferation and apoptosis. The expression levels of NF-κB were increased in the alveolar macrophages and airway primary epithelial cells of COPD model of rats induced by smoking. NF-κB play the important role in the proliferation of airway smooth muscle cells. We study the effect of cigarette smoke extract on the ERKs and NF-κB in the proliferation of airway smooth muscle cells.Methods:The airway smooth muscle cells in Wistar rats were cultured in vitro and stimulated with CSE for 24h. Cell proliferation was determined with mitochondrial dehydrogenase activty (MTT assay). Western bloting was used to detect the expression level of ERKs, p-ERK and NF-κB.Results:1. After airway smooth muscle cells stimulating by different concentration CSE, the absorbency results in different groups: 0.247±0.027(control group), 0.298±0.038(1:32CSE), 0.353±0.017(1:16CSE) and 0.376±0.024(1:8CSE).Cell proliferation was gradually increased with 1:32, 1:16 and 1:8 of CSE compared by control group, and it was associated with the rising CSE concentration(P<0.05, n=4).2. After airway smooth muscle cells stimulating by different concentration CSE, the expression level of ERK1 in different groups: 0.586±0.032(control group), 0.801±0.067(1:32CSE), 0.842±0.067(1:16CSE) and 0.913±0.101(1:8CSE); the expression level of ERK2 in different groups: 0.589±0.061(control group), 0.765±0.046(1:32CSE), 0.836±0.054(1:16CSE) and 0.919±0.086(1:8CSE); the expression level of p-ERK in different groups: 0.579±0.058(control group), 0.773±0.051(1:32CSE), 0.848±0.075(1:16CSE) and 0.941±0.067(1:8CSE); the expression level of NF-κB in different groups: 0.629±0.069(control group), 0.879±0.058(1:32CSE), 0.901±0.066(1:16CSE) and 0.936±0.087(1:8CSE). The results indicated the expression levels of ERKs,p-ERK and NF-КB gradually were increased compared by control group, and it was associated with the rising CSE concentration (P<0.05,n=4).3. The expression level of NF-κB had strongly positive correlation with the expression level of p-ERK r=0.858 (P<0.05 ,n=4).Conclusion:It is suggested that appropriate concentration CSE can induced the proliferation of airway smooth muscle cells, and it was associated with the rising CSE concentration. The expression levels of ERKs, p-ERK, NF-κB were increased. The activated ERKs(p-ERK) may be associated with the activation of NF-κB and regulated the proliferation of airway smooth muscle cells.Article two:The Role of PI-3K in airway smooth muscle hyperplasia in COPD model rats induced by smokingSmoking is The most important risk factor for COPD. The airway smooth muscles were hyperplastic in the airway of COPD patients and COPD model of rats, and cigarette smoke extract can induced the proliferation of airway smooth muscle cells. In the effect factors of the cell proliferation, phosphoinositide3-kinases (PI-3K) pathway regulates various cellular processes, such as proliferation, growth, apoptosis and cytoskeletal rearrangement.Specific inositol lipids such as PtdIns(3, 4)P2 and PtdIns(3, 4, 5)P3 which are generated by phosphoinositide 3-kinases are specific activators to the serine/threonine protein kinase Akt that have been implicated in a plethora of cell functions and then Akt stimulates cell proliferation and suppresses apoptosis, and is implicated in cancer progression. The study indicated PI-3K can induce the synthesis of DNA and promoted the cell proliferation in human airway smooth muscle cells.So the rat models of COPD induced by smoking were made to investigated the effect of the PI-3Kexpression in the airway smooth muscle hyperplasia of rats with chronic obstructive pulmonary disease(COPD) induced by smoking.Methods:The rat models of COPD induced by smoking were made to investigate the resistance of airway and the total compliance of respiratory system.The expression of PCNA in ASMCs was determined by immuno–histochemistry, the expression of the PI-3K in ASMCs were determined by reverse transcription polymerase chain reaction(RT-PCR). Results:1. The detection of respiratory function The resistance of airway (cmH2O/L/s):2.898±0.856 (COPD group); 1.686±0.142 (control group). The result indicated the risistance of airway was increased in the COPD groups compared by control groups (P<0.05, n=8). The total compliance of respiratory system:0.386±0.046(COPD group); 0.426±0.042(control group), the result had no difference between the COPD groups and control groups(P>0.05,n=8).2. The HE stain results of lung issue in different group rats indicated that the airway smooth muscle layer was thicker in COPD groups than in control groups.3. The expressions of PCNA in ASMCs of different group rats: 0.773±0.076 (COPD group); 0.573±0.062(control group). The result indicated the expressions of PCNA in COPD groups were significantly increased compared by control groups (P<0.05, n=8).4. The expressions of PI-3K in ASMCs of different group rats: 0.872±0.068 (COPD group); 0.648±0.066 (control group). The result indicated the expressions of PI-3K in COPD groups were significantly increased compared by control groups (P<0.05, n=8).5. The expression level of PI-3K had strongly positive correlation with the expression level of PCNA(r=0.816, P<0.05, n=8).Conclusion:It is suggested that the smoking can induce the airway smooth muscle hyperplasia of rats, and increase the expression of PI-3K in the airway smooth muscle of rats.The PI-3K signal pathway may be involved in airway smooth muscle hyperplasia in COPD rats induced by smoking.PartⅡ: Studies on the possible signal pathways and the protective effect of HSP70 in apoptosis of airway smooth muscle cells by smokingThe most important risk factor for COPD is smoking. Higher concentration cigarette smoke extract (CSE) can induce the apoptosis of airway smooth muscle cells(ASMCs). C-J unN-terminal kinase (JNK) is the important member in the apoptosis course. JNK is the important member of mitogen activated protein kinase(MAPK), and it can express in airway smooth muscle cells. The activation of JNK can activate the downstream apoptosis signal pathway.The expressions of heat shock protein 70 (HSP70) were increased in airway smooth muscle cells (ASMCs) induced by the lower concentration, and it can protect the cells. Geranylgeranylacetone(GGA), an acyclic polyisoprenoid, is developed as an antiulcer drug. It has been shown that this compound is effective in protecting the gastric mucosa against several types of insults without affecting gastric acid secretion. As a non-toxic inducer, geranylgeranylacetone can induce the expression of heat shock protein 70 in cells and gastrointestinal issuses in vitro and vivo. So we increased the expressions of HSP70 induced by geranylgeranylacetone (GGA), then investigated the effect of HSP70 on the p-JNK in the apoptosis of airway smooth muscle cells induced by cigarette smoke extract .Article one: Effect of geranylgeranylacetone on the the expression of HSP70 in airway smooth muscle cellsGeranylgeranylacetone(GGA), an acyclic polyisoprenoid, is developed as an antiulcer drug. It has been shown that this compound is effective in protecting the gastric mucosa against several types of insults without affecting gastric acid secretion. As a non-toxic inducer, geranylgeranylacetone can induce the expression of heat shock protein 70 in cells and gastrointestinal issuses in vitro and vivo. So we investigated the effect of heat shock protein 70 expression in airway smooth muscle cells, (ASMCs) by GGA.Methods:The airway smooth muscle cells in Wistar rats were cultured in vitro and divided in four groups: (1) group A, control group; (2) group B, the ethanol with 2μg/mlα-tocopherol group; (3) group C, 10-6MGGA group; (4) group D, 10-5MGGA group. Western bloting was used to detect the expression level of HSP70.Results:After airway smooth muscle cells stimulating by different concentration GGA, the expression level of HSP70 in different groups: 0.252±0.018 (group A); 0.264±0.082 (group B); 0.601±0.092 (group C); 0.842±0.062 (group D). The results indicated the expression levels of HSP70 gradually were increased compared by control group, and it was associated with the rising GGA concentration (P<0.05,n=4),and it had no difference between group A and group B(P>0.05,n=4).Conclusion:It is suggested that appropriate concentration GGA can increased the expression levels of HSP70 of airway smooth muscle cells and it was associated with the rising GGA concentration.Article two: Effect of cigarette smoke extract on the p-JNK in the apoptosis of airway smooth muscle cellsThe most important risk factor for COPD is smoking. Higher concentration cigarette smoke extract (CSE) can induce the apoptosis of airway smooth muscle cells(ASMCs). C-J unN-terminal kinase (JNK) is the important member in the apoptosis course. JNK is the important member of mitogen activated protein kinase(MAPK), and it can express in airway smooth muscle cells. It remains unknowed if JNK pathways participate in the course of apoptosis of airway smooth muscle cells by cigarette smoke extract. So we study the effect of cigarette smoke extract on the p-JNK in the apoptosis of airway smooth muscle cells in this part.Methods:The airway smooth muscle cells in Wistar rats were cultured in vitro and divided in four groups: (1) control group; (2) 15%CSE group; (3) 30% CSE group; (4) 45% CSE group. Flowcytometry was used to detect the apoptosis of airway smooth muscle cells and western bloting was used to detect the expression level of p-JNK.Results:1. After airway smooth muscle cells stimulating by different concentration CSE, flowcytometry showed the apoptosis rate of airway smooth muscle cells in different groups: 5.33±0.27 (control group), 17.67±1.24 (15%CSE), 26.38±3.08 (30%CSE) and 30.42±4.28 (45%CSE).Cell apoptosis rates were gradually increased with 15%, 30% and 45% of CSE compared by control group, and it was associated with the rising CSE concentration(P<0.05, n=4). 2. After airway smooth muscle cells stimulating by different concentration CSE, the expression level of p-JNK in different groups: 0.451±0.035 (control group), 0.624±0.059 (15%CSE), 0.736±0.075 (30%CSE) and 0.886±0.079 (45%CSE). The results indicated the expression levels of p-JNK gradually were increased compared by control group, and it was associated with the rising CSE concentration (P<0.05, n=4).Conclusion:Higher concentration CSE can induced the apoptosis of airway smooth muscle cells, and it was associated with the rising CSE concentration. And higher concentration CSE can increased the expression levels of p-JNK of airway smooth muscle cells and it was associated with the rising CSE concentration. It is suggested that CSE may induced the apoptosis of airway smooth muscle cells by increasing the expression levels of p-JNK.Article three: Effect of HSP70 on the p-JNK in the apoptosis of airway smooth muscle cells induced by cigarette smoke extractThe expressions of heat shock protein 70 (HSP70) were increased in airway smooth muscle cells (ASMCs) induced by the lower concentration, and it can protect the cells. The apoptosis of ASMCs were increased in ASMCs induced by the higher concentration CSE. C-J unN-terminal kinase (JNK) is the important member of mitogen activated protein kinase(MAPK), and it can express in airway smooth muscle cells. The activation of JNK can activate the downstream apoptosis signal pathway. So we increased the expressions of HSP70 induced by geranylgeranylacetone (GGA), then investigated the effect of HSP70 on the p-JNK in the apoptosis of airway smooth muscle cells induced by cigarette smoke extract .Methods:The airway smooth muscle cells in Wistar rats were cultured in vitro and divided in four groups: (1) group A, control group; (2) group B, 10-5MGGA group; (3) group C, 30%CSE group; (4) group D, 10-5MGGA and 30%CSE group. Flowcytometry was used to detect the apoptosis of airway smooth muscle cells and western bloting was used to detect the expression level of HSP70 and p-JNK. Results:1. After airway smooth muscle cells stimulating by different concentration CSE, flowcytometry showed the apoptosis rate of airway smooth muscle cells in different groups: 5.33±0.27 (A group), 4.15±0.23 (B group), 26.38±3.08 (C group), 14.49±1.48 (D group). Cell apoptosis rates were increased in C group compared by A group (P<0.05, n=4); cell apoptosis rates in B group and D group were decreased compared by A group (P<0.05, n=4). Cell apoptosis rates in B group were lower than that in D group (P<0.05, n=4).2. The expression level of HSP70 in different groups: 0.601±0.092 (A group), 0.873±0.078 (B group), 0.434±0.082 (C group), 0.784±0.072 (D group). The results indicated the expression levels of HSP70 in C group were decreased compared by A group (P<0.05,n=4); the expression levels of HSP70 in B group and D group were increased compared by A group (P<0.05, n=4). The expression levels of HSP70 in B group were higher than that in D group (P<0.05, n=4).3. The expression level of p-JNK in different groups: 0.216±0.026 (A group), 0.157±0.018 (B group), 0.736±0.075 (C group), 0.468±0.058 (D group). The results indicated the expression levels of p-JNK in C group were increased compared by A group (P<0.05,n=4); the expression levels of p-JNK in B group and D group were decreased compared by A group (P<0.05,n=4). The expression levels of p-JNK in B group were lower than that in D group (P<0.05,n=4).Conclusion:Our study indicated HSP70 can decrease the apoptosis of cells induced by higher concentration CSE, and down-regulate the expression levels of p-JNK in cells when the expression levels of HSP70 were increased in airway smooth muscle cells. It is suggested that the increased expression levels of HSP70 may decreased the apoptosis of cells by down-regulated the expression levels of p-JNK in airway smooth muscle cells.Auxiliay: Effect of smoking on pulmonary function and the expression of IL-2mRNA in human peripheral blood T lymphocytesThe most important risk factor for COPD is smoking. Studies indicated there were chronic inflammation in the airway, lung parenchyma and pulmonary vascular vessel of COPD patients.T lymphocytes and cytokines play a important role in the course of inflammation induced by smoking.Interleukine-2 (IL-2) is a important number of T lymphocyte cytokines. It play a important role in development, activation of T lymphocyte and maintains immune response. It is unknowed the effects on pulmonary function and IL- 2mRNA by smoking. We study the effect of smoking on pulmonary function and IL- 2mRNA in human peripheral blood T lymphocytes.Methods:We randomly selected 24 healthy smokers as smoking group and 24 healthy nonsmokers as control group. To detected their pulmonary function by portable spirometers and the expression of the IL-2mRNA in T lymphocytes were determined by reverse transcription polymerase chain reaction(RT-PCR).Results:The FEV1,FEV1/ FVC % and MMEF75/25 in smoking group had significantly decreased compared with control group(P<0.05,n=24); the expression of the IL-2mRNA in T lymphocytes from smoking group had significantly increased compared with control group(P<0.05, n=24).The pulmonary function had strongly negative correlation with the expression level of IL-2mRNA(r=-0.785, P<0.05,n=24).Conclusion:Although smokers did not show clinical symptoms, their pulmonary function had been reduced; It is suggested that smoking may increased lung inflammatory response by inducing the expression level of IL-2mRNA.