祛风宣肺方对神经生长因子介导的人支气管上皮细胞神经源性炎症的干预作用及机制
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摘要
目的探讨祛风宣肺方对神经生长因子(NGF)介导的人支气管上皮细胞(HBEC)神经源性炎症的干预作用及机制。方法人支气管上皮细胞随机分为正常组,模型组,阿斯美组,祛风宣肺方高、中、低剂量组,利用NGF制备体外神经源性炎症细胞模型,造模后给予含药血清干预。采用四甲基偶氮唑蓝(MTT)法检测各组细胞的增殖率,使用流式细胞仪检测各组细胞凋亡率,并用RT-PCR及蛋白质印迹(Western blot)法检测各组细胞Pan-Ras、c-fos、神经激肽1受体(NK-1R) mRNA及蛋白表达情况。结果与正常组相比,模型组细胞增殖率、凋亡率、Pan-Ras、c-fos、NK-1R mRNA及蛋白的表达均升高,差异有统计学意义(P<0.05)。与模型组相比,阿斯美组、祛风宣肺方高、中、低剂量组细胞增殖率、凋亡率、Pan-Ras、c-fos mRNA及蛋白的表达均降低,差异有统计学意义(P<0.05);祛风宣肺方高、中剂量组均能够明显降低NK-1R mRNA及蛋白的表达,差异具有统计学意义(P<0.05)。结论祛风宣肺方通过下调Ras-Raf-MAPK信号通路相关基因和蛋白的表达,从而减轻NGF介导的气道神经源性炎症,降低咳嗽敏感性。
Objective To investigate the interventional effect and mechanism of Qufeng Xuanfei Fang(Wind-expelling Lung-dispersing Decoction, QFXF Decoction) to neurogenic inflammation of human bronchial epithelial cells(HBEC) mediated by nerve growth factor(NGF). Methods HBEC were randomly divided into normal group, model group, Asmeton group, and high-dose, mid-dose and low-dose QFXF Decoction groups. The model of in vitro neurogenic inflammation was established by using NGF. The normal group was given normal serum, model group, 100 ng/mL of NGF and normal serum, Asmeton group, 100 ng/mL of NGF and Asmeton medicated serum, and high-dose, mid-dose and low-dose QFXF Decoction groups, QFXF Decoction medicated serum in different doses(100 ng/mL of NGF and high-dose, mid-dose or low-dose QFXF Decoction). The proliferation rate of HBEC was detected by using MTT, apoptosis rate was detected by using flow cytometry, and mRNA and protein expressions of Pan-Ras, c-fos and NK-1 R were detected by byusing RT-PCR and Western blotting assay. Results The proliferation rate and apoptosis rate of HBEC, and mRNA and protein expressions of Pan-Ras, c-fos and NK-1 R increased all in model group compared with normal group(P<0.05). The proliferation rate and apoptosis rate of HBEC, and mRNA and protein expressions of Pan-Ras and c-fos decreased in Asmeton group, and high-dose, mid-dose and low-dose QFXF Decoction groups compared with model group(P<0.05). The mRNA and protein expressions of NK-1 R decreased significantly in high-dose and mid-dose QFXF Decoction groups(P<0.05), while they had no changes in Asmeton group and low-dose QFXF Decoction group(P>0.05). Conclusion QFXF Decoction can relieve HBEC neurogenic inflammation mediated by NGF and reduce cough sensitivity through down-regulating the expressions of genes and protein related to Ras-Raf-MAPK signaling pathway.
Objective To investigate the interventional effect and mechanism of Qufeng Xuanfei Fang(Wind-expelling Lung-dispersing Decoction, QFXF Decoction) to neurogenic inflammation of human bronchial epithelial cells(HBEC) mediated by nerve growth factor(NGF). Methods HBEC were randomly divided into normal group, model group, Asmeton group, and high-dose, mid-dose and low-dose QFXF Decoction groups. The model of in vitro neurogenic inflammation was established by using NGF. The normal group was given normal serum, model group, 100 ng/mL of NGF and normal serum, Asmeton group, 100 ng/mL of NGF and Asmeton medicated serum, and high-dose, mid-dose and low-dose QFXF Decoction groups, QFXF Decoction medicated serum in different doses(100 ng/mL of NGF and high-dose, mid-dose or low-dose QFXF Decoction). The proliferation rate of HBEC was detected by using MTT, apoptosis rate was detected by using flow cytometry, and mRNA and protein expressions of Pan-Ras, c-fos and NK-1 R were detected by byusing RT-PCR and Western blotting assay. Results The proliferation rate and apoptosis rate of HBEC, and mRNA and protein expressions of Pan-Ras, c-fos and NK-1 R increased all in model group compared with normal group(P<0.05). The proliferation rate and apoptosis rate of HBEC, and mRNA and protein expressions of Pan-Ras and c-fos decreased in Asmeton group, and high-dose, mid-dose and low-dose QFXF Decoction groups compared with model group(P<0.05). The mRNA and protein expressions of NK-1 R decreased significantly in high-dose and mid-dose QFXF Decoction groups(P<0.05), while they had no changes in Asmeton group and low-dose QFXF Decoction group(P>0.05). Conclusion QFXF Decoction can relieve HBEC neurogenic inflammation mediated by NGF and reduce cough sensitivity through down-regulating the expressions of genes and protein related to Ras-Raf-MAPK signaling pathway.
引文
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[7] 季坤,马建岭,闫妍,等.祛风宣肺方对豚鼠咳嗽敏感性增高模型的肺组织病理及磷酸化p38丝裂原活化蛋白激酶的影响[J].现代中医临床,2017,24(5):20-24.Ji K,Ma JL,Yan Y,et al.The effect of Qufeng Xuanfei formula on lung tissue pathology and the protein expression of phosphorylated p38 mitogen-activated protein kinase in cough sensitivity increases guinea pigs model[J].Modern Chinese Clinical Medicine,2017,24(5):20-24.
[8] Rhodes J,Lutka FA,Jordan-sciutto KL,et al.Altered expression and distribution of FAC1 during NGF-induced neurite outgrowth of PC12 cells[J].Neuroreport,2003,14(3):449-452.
[9] 季坤.祛风宣肺方治疗感染后咳嗽的疗效观察及对气道神经源性炎症的作用研究[D].北京:北京中医药大学,2013.Ji K.Observation on curative effect of Qufeng Xuanfei Fang on cough after infection and its effect on airway neurogenic inflammation[D].Beijing:Beijing University of Chinese Medicine,2013.
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[11] Santarpia L,Lippman SM,El-Naggar AK.Targeting the MAPK-RAS- RAF signaling pathway in cancer therapy[J].Expert Opin Ther Targets,2012,16(1):103-119.
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[15] 林彬,史利卿,马建岭,等.慢性咳嗽伏邪病机认识探讨[J].现代中医临床,2017,24(5):12-15.Lin B,Shi LQ,Ma JL,et al.Pathogenesis study of hidden pathogen in chronic cough[J].Modern Chinese Clinical Medicine,2017,24(5):12-15.
[16] 沈金鳌.杂病源流犀烛[M].上海:上海科学技术出版社,1962:32-33.Shen JA.Za Bing Yuan Liu Xi Zhu[M].Shanghai:Shanghai Scientific and Technical Publishers,1962:32-33.
[17] 王月娇,马健.叶天士从脾胃辨治慢性咳嗽探析[J].北京中医药大学学报,2016,39(7):545-546.Wang YJ,Ma J.Differentiation and treatment of chronic cough from spleen and stomach By Ye Tianshi[J].Journal of Beijing University of Traditional Chinese Medicine,2016,39(7):545-546.
[18] 史利卿,刘志国,席宁,等.宣肺止咳方治疗呼吸道病毒感染后咳嗽临床观察[J].北京中医药大学学报(中医临床版),2004,11(3):9-10.ShiLQ,Liu ZG,Xi N,et al.Clinical observation on Xuanfei Zhike Fang for treatment of cough after respiratory virus infection[J].Journal of Beijing University of Traditional Chinese Medicine (Clinical Medicine),2004,11 (3):9-10.
[19] 马建岭,史利卿,席宁.祛风宣肺颗粒治疗感染后咳嗽60例临床观察[J].北京中医药大学学报,2007,30(1):67-69.Ma JL,Shi LQ,Xi N.Analysis and study on 60 cases of postinfectious cough treated with eliminating wind and releasing lung medicines[J].Journal of Beijing University of Traditional Chinese Medicine,2007,30(1):67-69.
[20] 史利卿,席宁,逯勇,等.祛风宣肺方对豚鼠咳嗽模型气道反应性影响的实验研究[J].北京中医药大学学报,2013,36(10):660-664.Shi LQ,Xi N,Lu Y,et al.Influence of Qufeng Xuanfei Fang on airway responsiveness in guinea pigs with cough[J].Journal of Beijing University of Traditional Chinese Medicine,2013,36(10):660-664.
[21] 马建岭,温绍惠,席宁,等.祛风宣肺方对气道敏感性增高豚鼠组胺、白三烯B4及嗜中性粒细胞弹力蛋白酶水平的影响[J].中华中医药杂志,2013,28(9):2588-2591.Ma JL,Wen SH,Xi N,et al.Effects of Eliminating Wind and Dispersing Lung Formula on HIS,LTB4 and NE in plasma of the guinea pigs with airway hypersensitivity[J].China Journal of Traditional Chinese Medicine and Pharmacy,2013,28 (9):2588-2591.
[2] 王岚,余莉,邱志宏,等.上气道咳嗽综合征的咳嗽敏感性改变及其意义[J].同济大学学报(医学版),2012,33(3):81-84.Wang L,Yu L,Qiu ZH,et al.Changes and implications of cough sensitivity in upper airway cough syndrome[J].Journal of Tongji University (Medical Science),2012,33(3):81-84.
[3] 赖克方,方章福,姚红梅.咳嗽高敏感综合征:不明原因慢性咳嗽的新概念[J].解放军医学杂志,2014,39(5):343-349.Lai KF,Fang ZF,Yao HMi.Cough hypersensitivity syndrome:a new concept for chronic idiopathic cough[J].Medical J Chinese PLA,2014,39(5):343-349.
[4] 刘春丽,赖克方,钟南山.气道神经源性炎症与慢性咳嗽的发病机制[J].国外医学(呼吸系统分册),2004,24(4):237-239.Liu CL,Lai KF,Zhong NS.Pathogenesis of airway neurogenic inflammation and chronic cough[J].Sect Respir Sys Foreign Med Sci,2004,24 (4):237-239.
[5] 韩桂玲,张纾难.基于病证结合的慢性咳嗽的中医诊治策略[J].现代中医临床,2017,24(5):16-19.Han GL,Zhang SN.The diagnostic and therapeutic strategy of traditional Chinese medicine about chronic cough based on combination of disease and syndrome[J].Modern Chinese Clinical Medicine,2017,24(5):16-19.
[6] 史利卿,马建岭,杨凯,等.慢性咳嗽中医病因病机及证候学规律的认识与探讨[J].现代中医临床,2017,24(5):1-5.Shi LQ,Ma JL,Yang K,et al.The study and discussion on traditional Chinese medicine etiology,pathogenesis and syndrome rule of chronic cough[J].Modern Chinese Clinical Medicine,2017,24(5):1-5.
[7] 季坤,马建岭,闫妍,等.祛风宣肺方对豚鼠咳嗽敏感性增高模型的肺组织病理及磷酸化p38丝裂原活化蛋白激酶的影响[J].现代中医临床,2017,24(5):20-24.Ji K,Ma JL,Yan Y,et al.The effect of Qufeng Xuanfei formula on lung tissue pathology and the protein expression of phosphorylated p38 mitogen-activated protein kinase in cough sensitivity increases guinea pigs model[J].Modern Chinese Clinical Medicine,2017,24(5):20-24.
[8] Rhodes J,Lutka FA,Jordan-sciutto KL,et al.Altered expression and distribution of FAC1 during NGF-induced neurite outgrowth of PC12 cells[J].Neuroreport,2003,14(3):449-452.
[9] 季坤.祛风宣肺方治疗感染后咳嗽的疗效观察及对气道神经源性炎症的作用研究[D].北京:北京中医药大学,2013.Ji K.Observation on curative effect of Qufeng Xuanfei Fang on cough after infection and its effect on airway neurogenic inflammation[D].Beijing:Beijing University of Chinese Medicine,2013.
[10] Ma L,Li K,Guo YX,et al.Ras-Raf-MAPK signaling promotes nuclear localization of FOXA transcription factor SGF1 via Ser91 phosphorylation[J].BBA Mol Cell Res,2018,865(4):560-571.
[11] Santarpia L,Lippman SM,El-Naggar AK.Targeting the MAPK-RAS- RAF signaling pathway in cancer therapy[J].Expert Opin Ther Targets,2012,16(1):103-119.
[12] Lee JH,Min DS,Lee CW,et al.Ginsenosides from Korean Red Ginseng ameliorate lung inflammatory responses:inhibition of the MAPKs/NF-κB/c-Fos pathways[J].J Ginseng Res,2018,42(4):476-484.
[13] Terashima T,Xu Q,Yamaguchi S,et al.Intrathecal P/Q-and R-type calcium channel blockade of spinal substance P release and c-Fos expression[J].Neuropharmacology,2013,75:1-8.
[14] 赵丹,张洪春,王辛秋,等.风咳源流与临床新释义[J].北京中医药大学学报,2011,34(5):337-338.Zhao D,Zhang HC,Wang XQ,et al.Origin and new clinical definition of wind cough[J].Journal of Beijing University of Traditional Chinese Medicine,2011,34(5):337-338.
[15] 林彬,史利卿,马建岭,等.慢性咳嗽伏邪病机认识探讨[J].现代中医临床,2017,24(5):12-15.Lin B,Shi LQ,Ma JL,et al.Pathogenesis study of hidden pathogen in chronic cough[J].Modern Chinese Clinical Medicine,2017,24(5):12-15.
[16] 沈金鳌.杂病源流犀烛[M].上海:上海科学技术出版社,1962:32-33.Shen JA.Za Bing Yuan Liu Xi Zhu[M].Shanghai:Shanghai Scientific and Technical Publishers,1962:32-33.
[17] 王月娇,马健.叶天士从脾胃辨治慢性咳嗽探析[J].北京中医药大学学报,2016,39(7):545-546.Wang YJ,Ma J.Differentiation and treatment of chronic cough from spleen and stomach By Ye Tianshi[J].Journal of Beijing University of Traditional Chinese Medicine,2016,39(7):545-546.
[18] 史利卿,刘志国,席宁,等.宣肺止咳方治疗呼吸道病毒感染后咳嗽临床观察[J].北京中医药大学学报(中医临床版),2004,11(3):9-10.ShiLQ,Liu ZG,Xi N,et al.Clinical observation on Xuanfei Zhike Fang for treatment of cough after respiratory virus infection[J].Journal of Beijing University of Traditional Chinese Medicine (Clinical Medicine),2004,11 (3):9-10.
[19] 马建岭,史利卿,席宁.祛风宣肺颗粒治疗感染后咳嗽60例临床观察[J].北京中医药大学学报,2007,30(1):67-69.Ma JL,Shi LQ,Xi N.Analysis and study on 60 cases of postinfectious cough treated with eliminating wind and releasing lung medicines[J].Journal of Beijing University of Traditional Chinese Medicine,2007,30(1):67-69.
[20] 史利卿,席宁,逯勇,等.祛风宣肺方对豚鼠咳嗽模型气道反应性影响的实验研究[J].北京中医药大学学报,2013,36(10):660-664.Shi LQ,Xi N,Lu Y,et al.Influence of Qufeng Xuanfei Fang on airway responsiveness in guinea pigs with cough[J].Journal of Beijing University of Traditional Chinese Medicine,2013,36(10):660-664.
[21] 马建岭,温绍惠,席宁,等.祛风宣肺方对气道敏感性增高豚鼠组胺、白三烯B4及嗜中性粒细胞弹力蛋白酶水平的影响[J].中华中医药杂志,2013,28(9):2588-2591.Ma JL,Wen SH,Xi N,et al.Effects of Eliminating Wind and Dispersing Lung Formula on HIS,LTB4 and NE in plasma of the guinea pigs with airway hypersensitivity[J].China Journal of Traditional Chinese Medicine and Pharmacy,2013,28 (9):2588-2591.