Cigarette Smoke Extract Promotes Proliferation Of Airway Smooth Muscle Cells In Asthmatic Rats Via Regulating CyclinD1 Expression

Objective:Bronchial asthma is one of the most familiar chronic diseases in the world.At present,domestic and international studies suggest that asthma is characterized by chronic airwayinflammation,which is the pathophysiological basis of airway hyperresponsiveness andairway remodeling.The proliferation of ASMCs play a key role in airway remodeling,aswell as the significant pathology basis of the irreversible airflow obstruction andpersistent airway hyperresponsiveness.At present,the regulatory mechanism ofproliferation of airway smooth muscle in asthma remains unclear.Smoking is an important risk factor involved in the development and progression ofasthma,it can increase the incidence and severity of symptom of asthma.Current studieshave found smoking can reduce the therapeutic effect of corticosteroids on asthma patients.Cigarette smoke contains more than 6000 kinds of chemical substances,including a varietyof toxic substances.The cigarette smoke can increase the acute allergic inflammationcaused by ovalbumin on guinea pig.In past investigations,we found CSE can promotethe ASMCs proliferation in asthmatic rat.However,the mechanism has not beenelaborated.Domestic and international studies have shown cell division and increase in thenumber of cells must be through the cell cycle,in the cell cycle must be involved in cellcycle protein,This kind of protein regulate the cell cycle.CyclinD 1 is the key factor in theregulation process from G₁ phase to S phase in cell cycle,and it also participate in cellcycle regulation of numerous cells.At present,little has been reported about whether CSE take part in the regulation of the ASMCs proliferation through the expression of cyclinD 1.In this experiment we study the effect of CSE on airway smooth muscles cellsproliferation and the influence of CSE on cyclinD 1 expression in cell level through thefoundation of model in bronchial asthma rats and culture of ASMCs in vitro,and whether aPKC-cyclinD 1 signal pathway takes part in the proliferation of ASMCs in response to CSEin order to discuss the probable mechanisms of CSE promoting ASMCs proliferation.Thiswould provide a theoretical basis in order to understand the pathogenesis of asthma and inorder to find effective treatmentsMethod:1.The rats model of bronchial asthma was established,and the ASMCs in rats werecultured from primary generation.CSE and GW8510 were used to intervene ASMCs inasthmatic rats.The proliferation of ASMCs was examined with MTT colorimetric assay,cell cycle analysis and proliferating cell nuclear antigen (PCNA) immunocytochemicalstaining.The expression of cyclinD 1 was detected by reverse transcriptase polymerasechain reaction (RT-PCR) and western blotting,respectively.This study investigated theinfluence of CSE on ASMCs proliferation and the expression of cyclinD 1 based on theobject of ASMCs in asthmatic rat.2.The rats model of bronchial asthma was established,and the ASMCs in rats werecultured from primary generation.CSE and antisense cyclinD1 expression plasmid wereused to intervene ASMCs in asthmatic rats.The proliferation of ASMCs was examined withMTT colorimetric assay,cell cycle analysis and proliferating cell nuclear antigen (PCNA)immunocytochemical staining.The expression of cyclinD1 was detected by reversetranscriptase polymerase chain reaction (RT-PCR) and western blotting,respectively.Thisstudy investigated the influence of CSE and antisense cyclinD1 expression plasmid onproliferation of ASMCs in asthmatic rats.3.The rats model of bronchial asthma was established,and the ASMCs in rats werecultured from primary generation.CSE,PKC agonist PMA and inhibitor Ro-31-8220 were used to intervene ASMCs in asthmatic rats.The proliferation of ASMCs was examined withMTT colorimetric assay,cell cycle analysis and proliferating cell nuclear antigen (PCNA)immunocytochemical staining.The expression of cyclinD1 and PKC-αwas detected byreverse transcriptase polymerase chain reaction (RT-PCR) and western blotting,respectively.This study investigated whether a PKC-cyclinD1 signal pathway takes part inthe proliferation of ASMCs in response to CSE.Results:1.Compared with control group,the proportion of cells at G₀/G₁ phase was decreasedevidently in CSE group,while the proportion of cells at S+G₂M phase was increasedsignificantly.The absorbance A value and expression rate of PCNA also were increasedsignificantly.The expression of CyclinD1 mRNA and protein were increased significantly.Compared with control group,the proportion of cells at G₀/G₁ phase was decreasedevidently in asthma group,while the proportion of cells at S+G₂M phase was increasedsignificantly.The absorbance A value and expression rate of PCNA also were increasedsignificantly.The expression of CyclinD 1 mRNA and protein were increased significantly.After intervened by GW8510,the proportion of cells at G₀/G₁ phase was increasedsignificantly in asthma group,while the proportion of cells at S+G₂M phase was decreasedevidently.The absorbance A value and expression rate of PCNA also were decreasedevidently.The expression of CyclinD 1 mRNA and protein were decreased significantly.2.Compared with control group,the proportion of cells at G₀/G₁ phase was increasedsignificantly in antisense plasmid group,while the proportion of cells at S+G₂M phase wasdecreased evidently.The absorbance A value and expression rate of PCNA also weredecreased evidently.The expression of CyclinD1 mRNA and protein were decreasedsignificantly.Compared with CSE+pcDNA3.1-ascyclinD1 group,the proportion of cells atG₀/G₁ phase was decreased evidently in CSE group,while the proportion of cells at S+G₂Mphase was increased significantly.The absorbance A value and expression rate of PCNAalso were increased significantly.The expression of CyclinD1 mRNA and protein were increased significantly.3.Compared with CSE group,the proportion of cells at G₀/G₁ phase was increasedsignificantly in CSE+Ro-31-8220 group,while the proportion of cells at S+G₂M phase wasdecreased evidently.The absorbance A value and expression rate of PCNA also weredecreased evidently.The expression of CyclinD1 and PKC-αmRNA and protein weredecreased significantly.Compared with CSE+PMA+pcDNA3.1-ascyclinD1 group,theproportion of cells at G₀/G₁ phase was decreased evidently in CSE+PMA group,while theproportion of cells at S+G₂M phase was increased significantly.The absorbance A valueand expression rate of PCNA also were increased significantly.The expression of CyclinD 1and PKC-αmRNA and protein were increased significantly.Conclusions:1.Proliferation activity of ASMCs in asthmatic rats was enhanced significantly.CSEcould promoted the proliferation of ASMCs in asthmatic rats.CyclinD and its subtypecyclinD 1 played a very important in proliferation of ASMCs in asthmatic rats.2.Antisense cyclinD 1 expression plasmid can inhibit the proliferation of ASMCs inasthmatic rats and this impairment can not be reversed or recovered even stimulated byCSE.CSE may increase proliferation of ASMCs in asthmatic rats via regulating cyclinD1expression.3.A PKC signal pathway might take part in the proliferation of ASMCs in response toCSE.PKC promotes proliferation of airway smooth muscle cells in asthmatic rats viaregulating cyclinD1 expression.CSE promotes proliferation of airway smooth muscle cellsthrough PKC-cyclinD 1 cascade in asthmatic rats.