孟鲁司特钠通过抑制TLR4/NF-κB信号通路影响哮喘大鼠气道平滑肌细胞增殖和凋亡

doi:10.13481/j.1671-587x.20200211

摘要/Abstract

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。

关键词: 哮喘, 气道平滑肌细胞, 孟鲁司特钠, 细胞增殖, 炎症

Abstract: 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.

Key words: asthma, airway smooth muscle cells, montelukast, cell proliferation, inflammation

中图分类号:

R562.25

王亚洲, 何鹏, 王丹虹. 孟鲁司特钠通过抑制TLR4/NF-κB信号通路影响哮喘大鼠气道平滑肌细胞增殖和凋亡[J]. 吉林大学学报(医学版), 2020, 46(02): 274-279.

WANG Yazhou, HE Peng, WANG Danhong. Effects of montelukast on proliferation and apoptosis of airway smooth muscle cells in asthmatic rats by inhibiting TLR4/NF-κB signaling pathway[J]. Journal of Jilin University(Medicine Edition), 2020, 46(02): 274-279.

参考文献

[1] HOSHINO M, OHTAWA J, AKITSU K, et al. Effect of the addition of montelukast on airway inflammation and remodeling in symptomatic asthma[J].Respirology, 2017,50(61):PA4680.
[2] 李莲梅, 余博文, 毕建朋, 等. TLR4对肺炎链球菌诱导的A549细胞凋亡的影响[J]. 郑州大学学报(医学版), 2019, 54(1):73-77.
[3] DAI J P, WANG Q W, SU Y, et al. Oxymatrine inhibits influenza a virus replication and inflammation via TLR4, p38 MAPK and NF-κB Pathways.[J]. Int J Mol Sci, 2018,19(4):E965.
[4] 鲍晓, 何成松. 沉默TLR4对类风湿关节炎滑膜成纤维细胞增殖和侵袭的影响[J]. 郑州大学学报(医学版), 2019, 54(2):44-47.
[5] CHEN G, SUN W, YAN L, et al. Maternal diabetes modulates offspring cell proliferation and apoptosis during odontogenesis via the TLR4/NF-κB signalling pathway[J].Cell Prolif, 2017, 50(3):e12324.
[6] 鲍璐璐,崔立红.IBS-D大鼠结肠TLR4、NF-κB的表达变化及机制[J].解放军医学杂志,2019,44(8):648-651.
[7] 韦江红, 莫碧文, 黄剑伟. TLR4/NF-κB对哮喘大鼠气道平滑肌细胞增殖、凋亡的影响[J]. 中国现代医学杂志, 2011, 21(25):3110-3115.
[8] 余建玮, 薛汉荣, 付向春. 小青龙汤干预哮喘大鼠气道平滑肌细胞增殖作用的研究[J]. 四川中医, 2011,29(10):23-25.
[9] YE K. Observation on clinical effects of tuina plus montelukast sodium tablets for children with cough variant asthma[J]. J Acupunct Tuina Sci, 2017, 15(5):349-353.
[10] WEI Y, LI D, LIU J, et al. Therapeutic effect and safety of montelukast sodium combined with budesonide in children with cough variant asthma:a Meta analysis[J]. Chin J Contemp Pediatr, 2016, 18(11):1100.
[11] 吴玲玲. 布地奈德吸入联合孟鲁司特钠对小儿哮喘疗效及炎症因子的影响[J]. 中国妇幼保健, 2017, 32(1):191-194.
[12] LIN B S. Effect of glucocorticoid inhalation combined with oral montelukast on airway function and serum inflammatory cytokines in patients with bronchial asthma[J]. J Hainan Med Univ, 2017, 23(3):54-57.
[13] 王兮, 母前途, 冯思嘉. 基于TLR4/MyD88/NF-κB通路探讨乌司他丁减轻脂多糖诱导的大鼠急性肺损伤的作用[J]. 河北医学, 2019, 25(8):1299-1304.
[14] FENG Y, CUI Y, GAO J L, et al. Resveratrol attenuates neuronal autophagy and inflammatory injury by inhibiting the TLR4/NF-κB signaling pathway in experimental traumatic brain injury[J]. Int J Mol Med, 2016, 37(4):921-930.
[15] HAN B, LU Y, ZHAO H, et al. Electroacupuncture modulated the inflammatory reaction in MCAO rats via inhibiting the TLR4/NF-κB signaling pathway in microglia[J]. Int J Clin Exp Pathol, 2015, 8(9):11199-11205.
[16] LI Y, XIE G, LI L, et al. The effect of TLR4/MyD88/NF-κB signaling pathway on proliferation and apoptosis in human nasopharyngeal carcinoma 5-8F cells induced by LPS[J]. J Chin Otorhinolaryngol Head Neck Surg, 2015, 29(11):1012-1015.
[17] LI L, LOU C Y, LI M, et al. Effect of montelukast sodium intervention on airway remodeling and percentage of Th17 cells/CD4+ CD25+ regulatory T cells in asthmatic mice[J]. Chin J Comtemp Pediatr, 2016, 18(11):1174.
[18] 周洁, 黄星原, 魏丽,等. 孟鲁司特钠对柯萨奇B3病毒感染小鼠心肌的保护作用[J]. 现代生物医学进展, 2017, 17(12):2209-2213.
[19] 王力宁, 黄小琪, 李志峰,等. 中医分期序贯治疗对哮喘气道平滑肌细胞增殖周期调控的影响[J]. 广西中医药, 2015, 38(4):65-68.
[20] 闫新星, 王晶, 张巍,等. Toll样受体4在淋巴瘤细胞株中的表达及其对细胞增殖和耐药的影响[J]. 肿瘤防治研究, 2016, 43(6):437-441.
[21] 魏蔚, 刘玉华, 刘守燕. Toll样受体4特异性拮抗剂TAK-242对人卵巢癌细胞株SKOV-3增殖、凋亡的影响及其机制[J]. 山东医药, 2017, 57(19):37-39.
[22] CHEN W, HU Y Q, JIANG L F, et al. Mechanism of action of Zhuyu Annao pill in mice with cerebral intrahemorrhage based on TLR4[J]. Asian Pac J Trop Med, 2016, 9(11):1095-1100.
[23] LI S F, ZHOU Y, ZHENG X D, et al. Sphk1 promotes breast epithelial cell proliferation via NF-κB-p65-mediated cyclin D1 expression[J]. Oncotarget, 2016, 7(49):80579-80585.
[24] LI L, WU W J, HUANG W J, et al. NF-κB RNAi decreases the Bax/Bcl-2 ratio and inhibits;TNF-α-induced apoptosis in human alveolar epithelial cells[J]. Inflamm Res, 2013, 62(4):387-397.
[25] 孙婷婷,李京涛,魏海梁,等.TNFα/NF-κB信号通路调控非酒精性脂肪性肝病的研究现状[J].临床肝胆病杂志,2019,35(9):2095-2098.
[26] SHI L, LI L I, YUANG W F, et al. Inhibition of NF-κB p65 expression by RNAi affects production of inflammatory mediators in LPS-activated mouse marcophages[J]. Chin J Pathophysiol, 2011, 27(7):1264-1269.
[27] SHIMIZU H, BOLATI D, ADIJIANG A, et al. NF-κB plays an important role in indoxyl sulfate-induced cellular senescence, fibrotic gene expression, and inhibition of proliferation in proximal tubular cells[J]. Am J Physiol-Cell Physiol, 2011, 301(5):C1201-C1212.