ERK信号通路对慢性哮喘大鼠气道平滑肌细胞增殖与凋亡的调控作用
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摘要
支气管哮喘是以气道高反应性、气道慢性炎症及气道重建为特征的慢性疾病。气道重建可导致不可逆的气道阻塞和持续性的气道高反应性[1-3],它包括上皮下胶原沉积,黏液细胞增生/化生,平滑肌的增生和肥厚等,是目前难治性哮喘的重要病理基础之一。已有研究证实气道平滑肌细胞(airway smooth muscle cell, ASMC)的增殖是气道重建(airway remodelig)的关键环节,ASMC作为气道重建的主要成分,通过自身的异常增殖参与整个过程[4-6]。因此,探讨支气管哮喘ASMC的异常增殖的调控机制是研究支气管哮喘气道重建发生机制的重要方向,抑制ASMC的异常增殖可成为治疗难治性哮喘的重要思路。但既往的研究多集中在探讨各种生物刺激(如生长因子、细胞因子等)对ASMC功能的影响及最终产生的生物学效应,有关生物信息在ASMC内传递机制的研究则相对较少。
细胞外信号调节蛋白激酶(extracellular signal-regulated kinase, ERK)通道是一种重要的细胞内信号转导通道,联系着细胞外信号与细胞核反应间的信息转导,参与多种细胞异常增殖与分化的信号调控[7]。ERK是该通道中的关键酶,它有多种亚型,在平滑肌细胞中主要为ERK1、ERK2(ERK1/2)两种亚型分布。研究表明,在探讨血管重建机制的研究中发现,ERK1/2通道是介导血管平滑肌细胞(VSMC)异常增殖的重要信号通道[8]。它参与调控VSMC的表型变化、迁移、增殖、凋亡和分泌等活性[9,10]。那么,哮喘气道重建中是否也存在血管重建中的类似机制,即哮喘ASMC内ERK1/2的活性是否也介导了ASMC异常增殖生物信号转导?目前国内外尚少见研究报道。本实验拟通过建立大鼠慢性哮喘模型,体外培养ASMC,探讨哮喘ASMC增殖活性的变化及ERK1/2在该增殖变化中的作用,从细胞内信号转导的角度研究ERK通道在哮喘ASMC增殖调控中的意义,为进一步阐明哮喘气道重建的病理生理学机制提供理论依据,为寻求更为有针对性的治疗方法提供实验依据。
研究内容:
1.建立大鼠慢性哮喘模型,病理图像分析慢性哮喘大鼠气道重建,免疫组化法检测ERK和增殖细胞核抗原(PCNA)在肺内表达,激光共聚焦显微镜分析ERK1/2、磷酸化ERK1/2(p-ERK)和PCNA在气道平滑肌上共表达,免疫印迹检测气道平滑肌上ERK和PCNA蛋白的表达,原位杂交检测气道平滑肌中ERK和PCNA的mRNA表达,从而探讨ERK是否在慢性哮喘大鼠气道平滑肌上表达。
2.建立大鼠慢性哮喘模型,体外培养哮喘ASMC,以ERK激动剂表皮生长因子(EGF)和抑制剂PD98059作为工具药物,采用流式细胞仪、四甲基偶氮唑盐(MTT)法、3H-TdR掺入法、PCNA免疫组织化学观察ASMC的增殖,免疫细胞化学法检测细胞周期蛋白(cyclin) D1和细胞周期蛋白依赖性激酶(CDK)2表达,逆转录-聚合酶链测定(RT-PCR)和Western免疫印迹检测ERK mRNA和ERK1/2、p-ERK1/2蛋白的表达。从而探讨哮喘ASMC增殖活性的变化及ERK1/2在该增殖变化中的作用。
3.体外培养哮喘ASMC,以ERK激动剂表皮生长因子(EGF)和抑制剂PD98059作为工具药物,用原位末端标记法和Annexin-V FITC PI双染色法观察ASMC凋亡,免疫细胞化学法检测凋亡相关基因bcl-2和bax的表达,用Western免疫印迹检测半胱天冬酶(caspase)-3蛋白的表达。从而探讨哮喘ASMC凋亡变化及ERK1/2在该凋亡变化中的作用。
4.设计合成ERK反义寡核苷酸(ODN),体外培养哮喘ASMC,利用脂质体将正义、反义及错配ERK1 ODNs导入ASMC,用流式细胞仪、MTT法、3H-TdR掺入法检测不同寡核苷酸对ASMC增殖的抑制作用,原位末端标记法和Annexin-V FITC PI双染色法检测细胞凋亡变化,RT-PCR和Western免疫印迹检测ERK mRNA和ERK1/2、p-ERK1/2、PCNA蛋白的表达。观察反义核酸对哮喘ASMC增殖和凋亡的干预。
结果:
1.病理图像分析显示慢性哮喘大鼠有气道平滑肌层增厚,出现结构重建。免疫组化法检测显示ERK和PCNA在肺内表达增强,激光共聚焦显微镜分析ERK1/2、p-ERK1/2和PCNA在气道平滑肌上共表达,免疫印迹和原位杂交检测显示在气道平滑肌上有ERK和PCNA蛋白与mRNA表达增加。
2.与对照组ASMC比较,哮喘组ASMC的G0/G1期所占细胞的比例明显减少,S + G2/M期细胞所占比例增高,吸光度值( A_(490) )、细胞DNA合成量、PCNA阳性表达量、cyclin D1和CDK2蛋白表达量均明显增加。与对照组ASMC比较,哮喘组ASMC的ERK mRNA、ERK1/2蛋白、p-ERK1/2蛋白的表达量以及ERK活化率(%)均显著增高。经PD98059干预之后,哮喘组ASMC的S + G2/M期细胞所占比例、A490值、细胞DNA合成量、PCNA阳性表达量、cyclin D1和CDK2蛋白表达量明显降低,ERK mRNA、ERK1/2蛋白、p-ERK1/2蛋白的表达量以及ERK活化率(%)均显著下降。经EGF干预之后,哮喘组ASMC的S + G2/M期细胞所占比例、A490值、细胞DNA合成量、PCNA阳性表达量、cyclin D1和CDK2蛋白表达量进一步增高,而这一作用可以被PD98059所抑制。
3.与对照组ASMC比较,哮喘组ASMC凋亡指数、早期凋亡细胞百分率明显下降,ERK mRNA、ERK1/2蛋白、p-ERK1/2蛋白的表达量以及ERK活化率(%)亦显著增高。经PD98059干预之后,哮喘组ASMC的凋亡指数与早期凋亡细胞百分率、bax蛋白表达量和caspase-3蛋白含量明显增高,ERK mRNA、ERK1/2蛋白、p-ERK1/2蛋白的表达量以及ERK活化率(%)亦显著降低。经EGF干预之后,哮喘组ASMC凋亡指数与早期凋亡细胞百分率进一步下降,而这一作用可以被PD98059所抑制。
4.反义ODNs组的G0/G1期所占细胞的比例明显增高,S + G2/M期细胞所占比例明显减少,A490值、细胞DNA合成量、PCNA蛋白表达量、ERK mRNA、ERK1/2蛋白、p-ERK1/2蛋白的表达量以及ERK活化率(%)均明显减少,与哮喘组各值比较差异均有显著性(P<0.05)。反义ODNs组的凋亡指数和早期凋亡细胞百分率明显增高,与哮喘组各值比较差异均有显著性(P<0.05)。而正义和错配ERK1 ODNs没有上述作用。
结论:
1.慢性哮喘大鼠有气道平滑肌层增厚,出现结构重建。慢性哮喘大鼠气道重建模型复制成功;
2.慢性哮喘大鼠中ERK和PCNA在肺内表达增强,同时在气道平滑肌上有ERK和PCNA蛋白与mRNA表达增加。提示ERK可能是介导慢性哮喘气道重建中平滑肌增殖的重要信号通路之一。
3.慢性哮喘ASMC内源性增殖`活性增加,同时ERK1/2表达及活化率均显著增强。ERK1/2通过诱导cyclinD1和CDK2蛋白高表达,使细胞从G1期向S期发展,细胞增殖。ERK信号通道在哮喘气道重建的ASMC增殖调控中具有重要作用。
4.慢性哮喘组大鼠ASMC内源性增殖活性增加的同时,伴有凋亡活性下降。ERK1/2参与慢性哮喘ASMC凋亡调控,其机制与bcl-2家族和caspace-3有关。
5.反义ERK1 ODNs可通过抑制ERK mRNA表达和翻译来抑制慢性哮喘大鼠ASMC的增殖,促进其凋亡。
由此可见,ERK1/2信号通道是介导哮喘ASMC异常增殖的重要信号通道,对哮喘ASMC增殖与凋亡起调控作用。ERK信号通道可能通过细胞周期、bcl-2家族以及caspace-3对哮喘ASMC增殖与凋亡起调控作用。这为研究哮喘气道重建的发病机理提供新的理论,也为将来治疗难治性哮喘提供一定的新思路。
Asthma is characterized by airway hyperresponsiveness, chronic inflammation and airway remodeling. Severe asthmatics develop irreversible airway obstruction and persistent airway hyperresponsiveness[1-3] . The airways include a variety of structural changes such as subepithelial fibrosis, mucous cell hyperplasia/metaplasia, and increased smooth muscle mass, which are collectively referred to as airway wall remodeling. It has been previously reported that proliferation of airway smooth muscle cells (ASMC) was an important contributor to the airway remodeling by abnormal proliferation[4-6]. So, investigation on abnormal proliferation of ASMC is very important to understand the pathogenesis of airway remodeling in asthmatics, inhibition of abnormal proliferation of ASMC may be one of the effective methods to reduce airway wall remodeling. We had previously demonstrated that the functional influence and biological effect of asthmatic ASMC by various mitogens such as growth factor, cytokine and so on. But little is known about various of bioinformation involved in the regulation of asthmatic ASMC intracellular signaling transduction.
A critical role for extracellular signal-regulated kinase(ERK) in cell proliferation and differentiation has been suggested in many cell types, which is an important intracellular signaling pathway and plays a key role in regulating signaling transduction between extracellular signal and nuclear reaction[7]. ERK, being a key enzyme in this pathway, has a variety of subtypes. ERK kinase 1 and 2 (ERK1/2) have been demonstrated to be primarily distributed in smooth muscle cells. ERK signaling pathway has also been reported as a vital signaling pathway mediating vascular smooth muscle cel(lVSMC)abnormal proliferation[8]. ERK are the regulators in VSMC phenotype variation, migration, proliferation, apoptosis and externalization[9,10]. Based on those reports, it is speculated that ERK signaling pathway may be involved in regulating the abnormal proliferation of chronic asthmatic ASMC. So far, little is known about the regulation of ERK1/2 in asthmatic ASMC abnormal proliferation. In this study, we focused on the endogenous proliferation activity and the regulation of cell proliferation by ERK signaling pathway in ASMC from chronic asthmatic rats with the chronic asthmatic model and cultured ASMC in vitro. Thus the results further provided experimental evidence for the significance of ERK signaling pathway in chronic asthmatic ASMC proliferation. This study was therefore undertaken to explore the role of ERK signaling pathway in pathogenesis of asthmatic airway remodeling from the view point of the intracellular signal transduction, and the results should be helpful for further finding the new therapic targets to aim directly at airway remodeling in asthma.
Methods
1. Airway remodeling was detected in chronic asthmatic rats by using image analysis system. The expressions of ERK and PCNA in lung tissue from chronic asthmatic rats were observed by immuocytochemistry staining. The expressions of ERK1/2, phosphorylated of ERK1/2(p-ERK1/2)and PCNA were detected in airway smooth muscle (ASM) by immunofluorescence double staining with confocal microscopy, and the expressions of protein or mRNA of ERK and PCNA in ASM were also detected by immunoblotting and hybridization in situ,respectively. Thus this study was designed to investigate the expressions of ERK in ASM in rat model of chronic asthma.
2. The rat model of chronic asthma was established. Extracellular signal-regulated kinase (ERK) agonist epidermal growth factor (EGF) and inhibitor PD98059 were used to investigate the regulative effect of ERK signaling pathway on airway smooth muscle cells (ASMC) proliferation in chronic asthmatic rats. The ASMC proliferations were examined with flow cytometry analysis, methyl thiazolyl tetrazolium (MTT) colorimetric assay, [3H]thymidine incorporation and proliferating cell nuclear antigen (PCNA) immunocytochemical staining. The expressions of cyclin D1 and CDK2 were detected by immunocytochemical staining. The expressions of ERK mRNA, ERK protein, p-ERK1/2 protein were observed by RT-PCR and Western blotting. Thus this study was designed to investigate the endogenous proliferation activity and the regulation of cell proliferation by ERK signaling pathway in ASMC from chronic asthmatic rats.
3. Primary cultures of ASMC were established and cells between passages 4 and 7 were used for experiments. ASMC were treated with ERK activator epidermal growth factor (EGF) and inhibitor PD 98059. Apoptosis of ASMC were detected by Situ end labeling and Annexin-V FITC PI double staining. The expressions of bcl-2 and bax were detected by immunocytochemical staining. The levels of caspase-3 protein were detected by Western blotting. The expressions of ERK mRNA, ERK protein, p-ERK1/2 protein were observed by RT-PCR and Western blotting. Thus this study was designed to investigate the regulation of cell apoptosis by ERK signaling pathway in ASMC from chronic asthmatic rats.
4. ASMC were transfected with ERK sense, antisense and mismatched oligodeoxynucleotides (ODN). Proliferations of ASMC were detected by flow cytometry analysis, MTT colorimetric assay, and [3H] thymidine incorporation. Apoptosises of ASMC were detected by in situ end labeling and Annexin-V FITC PI double staining. The expressions of ERK mRNA, ERK protein, p-ERK1/2 protein and PCNA protein were observed by RT-PCR and Western blotting, respectively. Thus this study was designed to investigate the effect of ERK antisense oligodeoxynucleotides on proliferation and apoptosis of airway smooth muscle cells (ASMCs) from chronic asthmatic rats.
Results
1. The thickening of smooth muscle and structural remodeling in airway were observed in chronic asthmatic rats by image analysis. The enhanced expressions of ERK and PCNA appeared obviously increased in same lung tissue and the expressions of protein or mRNA of ERK and PCNA were significantly increased in ASM.
2. Compared with control group, the percentage of G0/G1 phase were significantly decreased and the percentage of S + G2/M phase were significantly increased, absorbance ( A490 ) value, DNA synthesis value,the expression of PCNA protein,the expression of cyclin D1 protein and the expression of CDK2 protein in ASMC from chronic asthmatic group were significantly increased. The expression of ERK mRNA, ERK1/2 protein, p-ERK1/2 protein and the activation ratio of ERK in ASMC from chronic asthmatic group were significantly increased compared to those in control group. After treatment with PD98059,the percentage of S + G2/M phase, A490 value, DNA synthesis value,the expression of PCNA protein,the expression of cyclin D1 protein and the expression of CDK2 protein in ASMC from chronic asthmatic group were significantly decreased,the expression of ERK mRNA, ERK1/2 protein, p-ERK1/2 protein and the activation ratio of ERK in ASMC from chronic asthmatic group were significantly decreased compared to those before treatment. After treatment with EGF, the percentage of S + G2/M phase, A490 value, DNA synthesis value,the expression of PCNA protein,the expression of cyclin D1 protein and the expression of CDK2 protein in ASMC from chronic asthmatic group were significantly increased compared to those before treatment. PD98059 inhibited markedly the effect of EGF.
3. Compared with control group, the apoptotic index and the percentage of the early apoptotic cells in ASMC from chronic asthmatic group were significantly decreased. The expression of ERK mRNA, ERK1/2 protein, p-ERK1/2 protein and the activation ratio of ERK in ASMC from chronic asthmatic group were significantly increased compared to those in control group. After treatment with PD98059,the apoptotic index, the percentage of the early apoptotic cells, the expression of bax and the expression of caspase-3 protein in ASMC from chronic asthmatic group were significantly increased compared to those before treatment, the expressions of ERK mRNA, ERK1/2 protein, p-ERK1/2 protein and the activation ratio of ERK were also significantly down-regulated. Compared with those before treatment, apoptotic index and the percentage of the early apoptotic cells were significantly increased in EGF-treated cells. PD98059 inhibited markedly the effect of EGF.
4. The percentage of G0/G1 phase were significantly increased, the percentage of S + G2/M phase, A490 value, DNA synthesis value, the expression of PCNA protein, the expression of ERK mRNA, ERK1/2 protein, p-ERK1/2 protein and the activation ratio of ERK in ASMC from antisense oligodeoxynucleotides group were significantly decreased compared to those of chronic asthmatic group. The apoptotic index and the percentage of the early apoptotic cells in ASMC from antisense oligodeoxynucleotides group were significantly increased compared to those of chronic asthmatic group. However, the sense and the mismatched did not have these effects.
Conclusions
1. The thickening of smooth muscle and structural remodeling in airway were observed in asthmatic rat model. It indicated the rat model of chronic asthma was established.
2. The enhanced expressions of ERK and PCNA appeared obviously in same lung tissue and the expressions of protein or mRNA of ERK and PCNA were significantly increased in ASM from asthmatic rat model. It indicated ERK signaling pathway might be an important pathway in regulating cell proliferation of ASM resulting in asthmatic airway remodeling.
3. The endogenous proliferation activity of ASMC from chronic asthmatic rats was significantly increased and the expressions of protein or mRNA of ERK and the activation ratio of ERK in chronic asthmatic rats were significantly increased compared with that from normal ones. ERK1/2 participate in this process by inducing enhanced expression of cyclinD1 and CDK2,promote asthmatic ASMC from G0/G1 into S phase, leading to cell proliferation.ERK signaling pathway might play an important role in regulating ASMC proliferation, leading to asthmatic airway remodeling.
4. Apoptotic activation is significantly decreased in ASMC from chronic asthmatic group while the endogenous proliferation activation is significantly increased. The regulation of cell apoptosis was possibly related to ERK signaling pathway. The bcl-2 family and caspase-3 may participate in the regulation mechanism of cell apoptosis by ERK signaling pathway in ASMC from chronic asthmatic group.
5. ERK antisense ODN could inhibit the proliferation and increase the apoptosis in cultured ASMC from chronic asthmatic rats.
So, our results suggest that ERK signaling pathway is crucial for the regulation of ASMC abnormal proliferation. It plays a pivotal role on regulation of the proliferation and apoptosis. ERK1/2 are involved in the regulation of asthmatic ASMC proliferation and apoptosis via cell cycle, bcl-2 family and caspace-3. The results should be helpful for further providing the novel clue for further elucidating the pathogenesis in asthma and finding the treatment in severe asthmatics.
细胞外信号调节蛋白激酶(extracellular signal-regulated kinase, ERK)通道是一种重要的细胞内信号转导通道,联系着细胞外信号与细胞核反应间的信息转导,参与多种细胞异常增殖与分化的信号调控[7]。ERK是该通道中的关键酶,它有多种亚型,在平滑肌细胞中主要为ERK1、ERK2(ERK1/2)两种亚型分布。研究表明,在探讨血管重建机制的研究中发现,ERK1/2通道是介导血管平滑肌细胞(VSMC)异常增殖的重要信号通道[8]。它参与调控VSMC的表型变化、迁移、增殖、凋亡和分泌等活性[9,10]。那么,哮喘气道重建中是否也存在血管重建中的类似机制,即哮喘ASMC内ERK1/2的活性是否也介导了ASMC异常增殖生物信号转导?目前国内外尚少见研究报道。本实验拟通过建立大鼠慢性哮喘模型,体外培养ASMC,探讨哮喘ASMC增殖活性的变化及ERK1/2在该增殖变化中的作用,从细胞内信号转导的角度研究ERK通道在哮喘ASMC增殖调控中的意义,为进一步阐明哮喘气道重建的病理生理学机制提供理论依据,为寻求更为有针对性的治疗方法提供实验依据。
研究内容:
1.建立大鼠慢性哮喘模型,病理图像分析慢性哮喘大鼠气道重建,免疫组化法检测ERK和增殖细胞核抗原(PCNA)在肺内表达,激光共聚焦显微镜分析ERK1/2、磷酸化ERK1/2(p-ERK)和PCNA在气道平滑肌上共表达,免疫印迹检测气道平滑肌上ERK和PCNA蛋白的表达,原位杂交检测气道平滑肌中ERK和PCNA的mRNA表达,从而探讨ERK是否在慢性哮喘大鼠气道平滑肌上表达。
2.建立大鼠慢性哮喘模型,体外培养哮喘ASMC,以ERK激动剂表皮生长因子(EGF)和抑制剂PD98059作为工具药物,采用流式细胞仪、四甲基偶氮唑盐(MTT)法、3H-TdR掺入法、PCNA免疫组织化学观察ASMC的增殖,免疫细胞化学法检测细胞周期蛋白(cyclin) D1和细胞周期蛋白依赖性激酶(CDK)2表达,逆转录-聚合酶链测定(RT-PCR)和Western免疫印迹检测ERK mRNA和ERK1/2、p-ERK1/2蛋白的表达。从而探讨哮喘ASMC增殖活性的变化及ERK1/2在该增殖变化中的作用。
3.体外培养哮喘ASMC,以ERK激动剂表皮生长因子(EGF)和抑制剂PD98059作为工具药物,用原位末端标记法和Annexin-V FITC PI双染色法观察ASMC凋亡,免疫细胞化学法检测凋亡相关基因bcl-2和bax的表达,用Western免疫印迹检测半胱天冬酶(caspase)-3蛋白的表达。从而探讨哮喘ASMC凋亡变化及ERK1/2在该凋亡变化中的作用。
4.设计合成ERK反义寡核苷酸(ODN),体外培养哮喘ASMC,利用脂质体将正义、反义及错配ERK1 ODNs导入ASMC,用流式细胞仪、MTT法、3H-TdR掺入法检测不同寡核苷酸对ASMC增殖的抑制作用,原位末端标记法和Annexin-V FITC PI双染色法检测细胞凋亡变化,RT-PCR和Western免疫印迹检测ERK mRNA和ERK1/2、p-ERK1/2、PCNA蛋白的表达。观察反义核酸对哮喘ASMC增殖和凋亡的干预。
结果:
1.病理图像分析显示慢性哮喘大鼠有气道平滑肌层增厚,出现结构重建。免疫组化法检测显示ERK和PCNA在肺内表达增强,激光共聚焦显微镜分析ERK1/2、p-ERK1/2和PCNA在气道平滑肌上共表达,免疫印迹和原位杂交检测显示在气道平滑肌上有ERK和PCNA蛋白与mRNA表达增加。
2.与对照组ASMC比较,哮喘组ASMC的G0/G1期所占细胞的比例明显减少,S + G2/M期细胞所占比例增高,吸光度值( A_(490) )、细胞DNA合成量、PCNA阳性表达量、cyclin D1和CDK2蛋白表达量均明显增加。与对照组ASMC比较,哮喘组ASMC的ERK mRNA、ERK1/2蛋白、p-ERK1/2蛋白的表达量以及ERK活化率(%)均显著增高。经PD98059干预之后,哮喘组ASMC的S + G2/M期细胞所占比例、A490值、细胞DNA合成量、PCNA阳性表达量、cyclin D1和CDK2蛋白表达量明显降低,ERK mRNA、ERK1/2蛋白、p-ERK1/2蛋白的表达量以及ERK活化率(%)均显著下降。经EGF干预之后,哮喘组ASMC的S + G2/M期细胞所占比例、A490值、细胞DNA合成量、PCNA阳性表达量、cyclin D1和CDK2蛋白表达量进一步增高,而这一作用可以被PD98059所抑制。
3.与对照组ASMC比较,哮喘组ASMC凋亡指数、早期凋亡细胞百分率明显下降,ERK mRNA、ERK1/2蛋白、p-ERK1/2蛋白的表达量以及ERK活化率(%)亦显著增高。经PD98059干预之后,哮喘组ASMC的凋亡指数与早期凋亡细胞百分率、bax蛋白表达量和caspase-3蛋白含量明显增高,ERK mRNA、ERK1/2蛋白、p-ERK1/2蛋白的表达量以及ERK活化率(%)亦显著降低。经EGF干预之后,哮喘组ASMC凋亡指数与早期凋亡细胞百分率进一步下降,而这一作用可以被PD98059所抑制。
4.反义ODNs组的G0/G1期所占细胞的比例明显增高,S + G2/M期细胞所占比例明显减少,A490值、细胞DNA合成量、PCNA蛋白表达量、ERK mRNA、ERK1/2蛋白、p-ERK1/2蛋白的表达量以及ERK活化率(%)均明显减少,与哮喘组各值比较差异均有显著性(P<0.05)。反义ODNs组的凋亡指数和早期凋亡细胞百分率明显增高,与哮喘组各值比较差异均有显著性(P<0.05)。而正义和错配ERK1 ODNs没有上述作用。
结论:
1.慢性哮喘大鼠有气道平滑肌层增厚,出现结构重建。慢性哮喘大鼠气道重建模型复制成功;
2.慢性哮喘大鼠中ERK和PCNA在肺内表达增强,同时在气道平滑肌上有ERK和PCNA蛋白与mRNA表达增加。提示ERK可能是介导慢性哮喘气道重建中平滑肌增殖的重要信号通路之一。
3.慢性哮喘ASMC内源性增殖`活性增加,同时ERK1/2表达及活化率均显著增强。ERK1/2通过诱导cyclinD1和CDK2蛋白高表达,使细胞从G1期向S期发展,细胞增殖。ERK信号通道在哮喘气道重建的ASMC增殖调控中具有重要作用。
4.慢性哮喘组大鼠ASMC内源性增殖活性增加的同时,伴有凋亡活性下降。ERK1/2参与慢性哮喘ASMC凋亡调控,其机制与bcl-2家族和caspace-3有关。
5.反义ERK1 ODNs可通过抑制ERK mRNA表达和翻译来抑制慢性哮喘大鼠ASMC的增殖,促进其凋亡。
由此可见,ERK1/2信号通道是介导哮喘ASMC异常增殖的重要信号通道,对哮喘ASMC增殖与凋亡起调控作用。ERK信号通道可能通过细胞周期、bcl-2家族以及caspace-3对哮喘ASMC增殖与凋亡起调控作用。这为研究哮喘气道重建的发病机理提供新的理论,也为将来治疗难治性哮喘提供一定的新思路。
Asthma is characterized by airway hyperresponsiveness, chronic inflammation and airway remodeling. Severe asthmatics develop irreversible airway obstruction and persistent airway hyperresponsiveness[1-3] . The airways include a variety of structural changes such as subepithelial fibrosis, mucous cell hyperplasia/metaplasia, and increased smooth muscle mass, which are collectively referred to as airway wall remodeling. It has been previously reported that proliferation of airway smooth muscle cells (ASMC) was an important contributor to the airway remodeling by abnormal proliferation[4-6]. So, investigation on abnormal proliferation of ASMC is very important to understand the pathogenesis of airway remodeling in asthmatics, inhibition of abnormal proliferation of ASMC may be one of the effective methods to reduce airway wall remodeling. We had previously demonstrated that the functional influence and biological effect of asthmatic ASMC by various mitogens such as growth factor, cytokine and so on. But little is known about various of bioinformation involved in the regulation of asthmatic ASMC intracellular signaling transduction.
A critical role for extracellular signal-regulated kinase(ERK) in cell proliferation and differentiation has been suggested in many cell types, which is an important intracellular signaling pathway and plays a key role in regulating signaling transduction between extracellular signal and nuclear reaction[7]. ERK, being a key enzyme in this pathway, has a variety of subtypes. ERK kinase 1 and 2 (ERK1/2) have been demonstrated to be primarily distributed in smooth muscle cells. ERK signaling pathway has also been reported as a vital signaling pathway mediating vascular smooth muscle cel(lVSMC)abnormal proliferation[8]. ERK are the regulators in VSMC phenotype variation, migration, proliferation, apoptosis and externalization[9,10]. Based on those reports, it is speculated that ERK signaling pathway may be involved in regulating the abnormal proliferation of chronic asthmatic ASMC. So far, little is known about the regulation of ERK1/2 in asthmatic ASMC abnormal proliferation. In this study, we focused on the endogenous proliferation activity and the regulation of cell proliferation by ERK signaling pathway in ASMC from chronic asthmatic rats with the chronic asthmatic model and cultured ASMC in vitro. Thus the results further provided experimental evidence for the significance of ERK signaling pathway in chronic asthmatic ASMC proliferation. This study was therefore undertaken to explore the role of ERK signaling pathway in pathogenesis of asthmatic airway remodeling from the view point of the intracellular signal transduction, and the results should be helpful for further finding the new therapic targets to aim directly at airway remodeling in asthma.
Methods
1. Airway remodeling was detected in chronic asthmatic rats by using image analysis system. The expressions of ERK and PCNA in lung tissue from chronic asthmatic rats were observed by immuocytochemistry staining. The expressions of ERK1/2, phosphorylated of ERK1/2(p-ERK1/2)and PCNA were detected in airway smooth muscle (ASM) by immunofluorescence double staining with confocal microscopy, and the expressions of protein or mRNA of ERK and PCNA in ASM were also detected by immunoblotting and hybridization in situ,respectively. Thus this study was designed to investigate the expressions of ERK in ASM in rat model of chronic asthma.
2. The rat model of chronic asthma was established. Extracellular signal-regulated kinase (ERK) agonist epidermal growth factor (EGF) and inhibitor PD98059 were used to investigate the regulative effect of ERK signaling pathway on airway smooth muscle cells (ASMC) proliferation in chronic asthmatic rats. The ASMC proliferations were examined with flow cytometry analysis, methyl thiazolyl tetrazolium (MTT) colorimetric assay, [3H]thymidine incorporation and proliferating cell nuclear antigen (PCNA) immunocytochemical staining. The expressions of cyclin D1 and CDK2 were detected by immunocytochemical staining. The expressions of ERK mRNA, ERK protein, p-ERK1/2 protein were observed by RT-PCR and Western blotting. Thus this study was designed to investigate the endogenous proliferation activity and the regulation of cell proliferation by ERK signaling pathway in ASMC from chronic asthmatic rats.
3. Primary cultures of ASMC were established and cells between passages 4 and 7 were used for experiments. ASMC were treated with ERK activator epidermal growth factor (EGF) and inhibitor PD 98059. Apoptosis of ASMC were detected by Situ end labeling and Annexin-V FITC PI double staining. The expressions of bcl-2 and bax were detected by immunocytochemical staining. The levels of caspase-3 protein were detected by Western blotting. The expressions of ERK mRNA, ERK protein, p-ERK1/2 protein were observed by RT-PCR and Western blotting. Thus this study was designed to investigate the regulation of cell apoptosis by ERK signaling pathway in ASMC from chronic asthmatic rats.
4. ASMC were transfected with ERK sense, antisense and mismatched oligodeoxynucleotides (ODN). Proliferations of ASMC were detected by flow cytometry analysis, MTT colorimetric assay, and [3H] thymidine incorporation. Apoptosises of ASMC were detected by in situ end labeling and Annexin-V FITC PI double staining. The expressions of ERK mRNA, ERK protein, p-ERK1/2 protein and PCNA protein were observed by RT-PCR and Western blotting, respectively. Thus this study was designed to investigate the effect of ERK antisense oligodeoxynucleotides on proliferation and apoptosis of airway smooth muscle cells (ASMCs) from chronic asthmatic rats.
Results
1. The thickening of smooth muscle and structural remodeling in airway were observed in chronic asthmatic rats by image analysis. The enhanced expressions of ERK and PCNA appeared obviously increased in same lung tissue and the expressions of protein or mRNA of ERK and PCNA were significantly increased in ASM.
2. Compared with control group, the percentage of G0/G1 phase were significantly decreased and the percentage of S + G2/M phase were significantly increased, absorbance ( A490 ) value, DNA synthesis value,the expression of PCNA protein,the expression of cyclin D1 protein and the expression of CDK2 protein in ASMC from chronic asthmatic group were significantly increased. The expression of ERK mRNA, ERK1/2 protein, p-ERK1/2 protein and the activation ratio of ERK in ASMC from chronic asthmatic group were significantly increased compared to those in control group. After treatment with PD98059,the percentage of S + G2/M phase, A490 value, DNA synthesis value,the expression of PCNA protein,the expression of cyclin D1 protein and the expression of CDK2 protein in ASMC from chronic asthmatic group were significantly decreased,the expression of ERK mRNA, ERK1/2 protein, p-ERK1/2 protein and the activation ratio of ERK in ASMC from chronic asthmatic group were significantly decreased compared to those before treatment. After treatment with EGF, the percentage of S + G2/M phase, A490 value, DNA synthesis value,the expression of PCNA protein,the expression of cyclin D1 protein and the expression of CDK2 protein in ASMC from chronic asthmatic group were significantly increased compared to those before treatment. PD98059 inhibited markedly the effect of EGF.
3. Compared with control group, the apoptotic index and the percentage of the early apoptotic cells in ASMC from chronic asthmatic group were significantly decreased. The expression of ERK mRNA, ERK1/2 protein, p-ERK1/2 protein and the activation ratio of ERK in ASMC from chronic asthmatic group were significantly increased compared to those in control group. After treatment with PD98059,the apoptotic index, the percentage of the early apoptotic cells, the expression of bax and the expression of caspase-3 protein in ASMC from chronic asthmatic group were significantly increased compared to those before treatment, the expressions of ERK mRNA, ERK1/2 protein, p-ERK1/2 protein and the activation ratio of ERK were also significantly down-regulated. Compared with those before treatment, apoptotic index and the percentage of the early apoptotic cells were significantly increased in EGF-treated cells. PD98059 inhibited markedly the effect of EGF.
4. The percentage of G0/G1 phase were significantly increased, the percentage of S + G2/M phase, A490 value, DNA synthesis value, the expression of PCNA protein, the expression of ERK mRNA, ERK1/2 protein, p-ERK1/2 protein and the activation ratio of ERK in ASMC from antisense oligodeoxynucleotides group were significantly decreased compared to those of chronic asthmatic group. The apoptotic index and the percentage of the early apoptotic cells in ASMC from antisense oligodeoxynucleotides group were significantly increased compared to those of chronic asthmatic group. However, the sense and the mismatched did not have these effects.
Conclusions
1. The thickening of smooth muscle and structural remodeling in airway were observed in asthmatic rat model. It indicated the rat model of chronic asthma was established.
2. The enhanced expressions of ERK and PCNA appeared obviously in same lung tissue and the expressions of protein or mRNA of ERK and PCNA were significantly increased in ASM from asthmatic rat model. It indicated ERK signaling pathway might be an important pathway in regulating cell proliferation of ASM resulting in asthmatic airway remodeling.
3. The endogenous proliferation activity of ASMC from chronic asthmatic rats was significantly increased and the expressions of protein or mRNA of ERK and the activation ratio of ERK in chronic asthmatic rats were significantly increased compared with that from normal ones. ERK1/2 participate in this process by inducing enhanced expression of cyclinD1 and CDK2,promote asthmatic ASMC from G0/G1 into S phase, leading to cell proliferation.ERK signaling pathway might play an important role in regulating ASMC proliferation, leading to asthmatic airway remodeling.
4. Apoptotic activation is significantly decreased in ASMC from chronic asthmatic group while the endogenous proliferation activation is significantly increased. The regulation of cell apoptosis was possibly related to ERK signaling pathway. The bcl-2 family and caspase-3 may participate in the regulation mechanism of cell apoptosis by ERK signaling pathway in ASMC from chronic asthmatic group.
5. ERK antisense ODN could inhibit the proliferation and increase the apoptosis in cultured ASMC from chronic asthmatic rats.
So, our results suggest that ERK signaling pathway is crucial for the regulation of ASMC abnormal proliferation. It plays a pivotal role on regulation of the proliferation and apoptosis. ERK1/2 are involved in the regulation of asthmatic ASMC proliferation and apoptosis via cell cycle, bcl-2 family and caspace-3. The results should be helpful for further providing the novel clue for further elucidating the pathogenesis in asthma and finding the treatment in severe asthmatics.
引文
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[6]Lazaar AL, Panettieri RA Jr. Airway smooth muscle: a modulator of airway remodeling in asthma[J]. J Allergy Clin Immunol, 2005, 116: 488-95.
[7]Platanias LC. Map kinase signaling pathways and hematologic malignancies[J]. Blood, 2003, 101:4667-79.
[8]Hu Y, Dietrich H, Metzler B, et al. Hyperexpression and activation of extracellular signal-regulated kinase(ERK1/2)in atherosclerotic lesions of cholesterol-fed rabbits[J]. Arterioscler Thromb Vasc Biol,2000, 20: 18-26.
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[1]Kumar RK, Herbert C, Thomas PS,et al. Inhibition of inflammation and remodeling by roflumilast and dexamethasone in murine chronic asthma[J]. J Pharmacol Exp Ther,2003,307(1): 349-355.
[2]章晓初,姚婉贞,何其华,等.哮喘豚鼠气道重塑与气道反应性的图像分析[J].中华结核和呼吸杂志,2001,24(2): 87-89.
[3] Black JL, Johnson PR. Factors controlling smooth muscle proliferation and airway remodelling[J]. Curr Opin Allergy Clin Immunol, 2002, 2(1): 47-51
[4]Borger P, Tamm M, Black JL, et al. Asthma: is it due to an abnormal airway smooth muscle cell[J] ? Am J Respir Crit Care Med,2006,174(4): 367-372.
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