摘要
目的:探讨孟鲁司特钠(Mon)对哮喘大鼠气道平滑肌细胞增殖和凋亡及炎症因子分泌的影响,阐明哮喘大鼠气道重塑及炎症反应的分子机制。方法:采用卵清蛋白致敏和激发构建急性哮喘大鼠模型,原代培养大鼠气道平滑肌细胞,取第5代细胞用于实验。实验分为对照组(正常气道平滑肌细胞)、模型组(哮喘模型大鼠气道平滑肌细胞)和治疗组(哮喘模型大鼠气道平滑肌细胞+Mon干预)。采用噻唑蓝(MTT)法检测各组细胞增殖活性,流式细胞术检测各组细胞凋亡率,酶联免疫吸附实验(ELISA)法检测各组细胞上清液中白细胞介素6(IL-6)和转化生长因子β1(TGF-β1)水平,Western blotting法检测各组细胞中增殖细胞核抗原(PCNA)、细胞周期蛋白D1(CyclinD1)、B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、Toll样受体4(TLR4)和核因子κB(NF-κB)p65蛋白表达水平,逆转录-聚合酶链反应(RT-PCR)检测各组细胞中TLR4和NF-κB p65 mRNA表达水平。结果:与对照组比较,模型组和治疗组细胞活性明显升高(P<0.05),细胞凋亡率明显降低(P<0.05),IL-6和TGF-β1水平升高(P<0.05),PCNA、CyclinD1、Bcl-2、TLR4和NF-κB p65蛋白表达水平及TLR4和NF-κB p65 mRNA表达水平均明显升高(P<0.05),而Bax蛋白表达水平明显降低(P<0.05);与模型组比较,治疗组细胞增殖活性明显降低(P<0.05),细胞凋亡率明显升高(P<0.05),IL-6和TGF-β1水平降低(P<0.05),PCNA、CyclinD1、Bcl-2、TLR4和NF-κB p65蛋白表达水平及TLR4和NF-κB p65 mRNA表达水平均明显降低(P<0.05),而Bax蛋白表达水平明显升高(P<0.05)。结论:Mon可抑制哮喘大鼠气道平滑肌细胞增殖,促进细胞凋亡,减轻哮喘气道炎症反应,其作用机制可能与抑制TLR4/NF-κB信号通路有关。
Objective:To investigate the effects of montelukast(Mon)on the proliferation,apoptosis and secretion of inflammatory factors of the airway smooth muscle cells of the asthmatic rats,and to elucidate the molecular mechanism of airway remodeling and inflammatory response in the asthmatic rats.Methods:The acute asthma rat models were established by ovalbumin sensitization and stimulation.The primary cultured airway smooth muscle cells at the fifth generation were carried out for the experiment.The experiment was divided into control group(normal airway smooth muscle cells),model group(asthmatic model rat airway smooth muscle cells)and treatment group(asthmatic model rat airway smooth muscle cells+Mon intervention).The proliferation activities of the cells in various groups were detected by MTT method,the apoptotic rates of the cells in various groups were measured by flow cytometry,and the levels of interleukin-6(IL-6)and transforming growth factor-β1(TGF-β1)in cell supernatant in various groups were tested by ELISA method.The expression levels of proliferating cell nuclear antigen(PCNA),CyclinD1,B-cell lymphoma-2(Bcl-2),Bcl-2 associated X protein(Bax),Toll-like receptor-4(TLR4)and nuclear factor-κB(NF-κB)p65 proteins in the cells in various groups were examined by Western blotting method,and the expression levels of TLR4 and NF-κB p65 mRNA in the cells in various groups were detected by RT-PCR method.Results:Compared with control group,the proliferation activities of the cells in model group and treatment group were increased significantly(P<0.05),the apoptotic rates were decreased significantly(P<0.05),and the levels of IL-6 and TGF-β1 were obviously increased(P<0.05);the expression levels of PCNA,CyclinD1,Bcl-2,TLR4 and NF-κB p65 proteins and the expression levels of TLR4 and NF-κB mRNA were remarkably increased(P<0.05),but the expression levels of Bax protein was distinctly decreased(P<0.05).Compared with model group,the proliferation activity of the cells in treatment group was decreased(P<0.05),the apoptotic rate was increased(P<0.05),and the levels of IL-6 and TGF-β1 were clearly decreased(P<0.05);the expression levels of PCNA,CyclinD1,Bcl-2,TLR4 and NF-κB p65 proteins and the expression levels of TLR4 and NF-κB mRNA were remarkably decreased(P<0.05),but the expression levels of Bax protein was obviously increased(P<0.05).Conclusion:Mon can inhibit the proliferation of airway smooth muscle cells,promote the apoptosis and alleviate the airway inflammation in the asthmatic rats,and its mechanism may be related to inhibition of TLR4/NF-κB signaling pathway.
作者
王亚洲
何鹏
王丹虹
WANG Yazhou;HE Peng;WANG Danhong(Department of Pediatrics,Hainan Woman and Children’s Medical Center,Haikou 570206,China;Department of Pediatrics,People’s Hospital of Guangxi Zhuang Autonomous Region,Nanning 530021,China;Department of Pediatrics,Affiliated Haikou Hospital,Xiangya Medical College,Central South University,Haikou 570208,China)
出处
《吉林大学学报（医学版）》
CAS
CSCD
北大核心
2020年第2期274-279,共6页
Journal of Jilin University：Medicine Edition
基金
海南省卫健委卫生计生行业科研项目资助课题(18A200042)。
关键词
哮喘
气道平滑肌细胞
孟鲁司特钠
细胞增殖
炎症
asthma
airway smooth muscle cells
montelukast
cell proliferation
inflammation
