五味子乙素对香烟烟雾提取物和脂多糖损伤的肺上皮细胞干预作用研究
|
摘要
目的:观察五味子乙素(Sch B)对慢性阻塞性肺疾病(COPD)急性加重期细胞模型的影响,并初步探讨其作用机制。方法:用香烟烟雾提取物(CSE)联合脂多糖(LPS)作用于肺上皮细胞株A549,制备COPD急性加重期体外模型,并给予相应浓度的五味子乙素进行干预,通过MTT法测定各组细胞存活率、RT-PCR法测定细胞炎症因子白细胞介素8(IL-8)、环氧合酶2(COX-2)的mRNA水平; ELISA法测定细胞上清中IL-8、COX-2水平; Western blot法测定细胞磷酸化NF-κB蛋白水平。结果:4%CSE联合0. 1μg/ml LPS作用24 h能够促进肺上皮细胞增殖,炎症因子表达明显上升,给予Sch B能够明显抑制CSE联合LPS对细胞的增殖作用,改善细胞炎症状态;降低损伤细胞IL-8、COX-2 mRNA表达水平;减少细胞上清中IL-8、COX-2含量;降低细胞中磷酸化NF-κB的蛋白表达水平。结论:Sch B可改善CSE联合LPS引起的肺上皮细胞炎症,其作用机制可能与抑制肺上皮细胞增殖,抑制炎症因子表达,发挥抗炎作用有关。该研究为Sch B应用于COPD急性加重期的临床治疗提供实验依据。
Objective: To explore the effect of Schisandrin B( Sch B) on cigarette smoke extract( CSE) combined with lipopolysaccharide( LPS) injury in lung epithelial cells of chronic obstructive pulmonary disease( COPD) and to explore its mechanism. Methods: By using CSE combined with LPS induced injury of pulmonary epithelial cell line A549 as acute exacerbation model of COPD in vitro; and proper concentration of Sch B was given to prevent the injury of CSE and LPS,then the cell viability was determined by MTT assay,mRNA levels of interleukin-8( IL-8) and cyclooxygenase-2( COX-2) were measured by RT-PCR,the levels of IL-8 and COX-2 in supernatant were measured by ELISA,the level of phosphorylated NF-κB protein was measured by Western blot. Results: Co-cultured with 4% CSE combined with 0. 1 μg/ml LPS for 24 h,lung epithelial cell proliferation was promoted,inflammatory factor significantly increased,Sch B could significantly inhibit the proliferation of lung epithelial cells and improve the inflammation induced by CSE combined with LPS,reduce the expression of IL-8 and COX-2 mRNA in injured cells,decrease IL-8 and COX-2 levels in the supernatant,reduce the level of phosphorylated NF-κB protein expression in cells. Conclusion: Sch B can inhibit the proliferation of lung epithelial cells,and the inflammatory factor expression induced by CSE combined with LPS,which provides experimental evidence for the clinical application of Sch B in the treatment of acute exacerbation of COPD.
Objective: To explore the effect of Schisandrin B( Sch B) on cigarette smoke extract( CSE) combined with lipopolysaccharide( LPS) injury in lung epithelial cells of chronic obstructive pulmonary disease( COPD) and to explore its mechanism. Methods: By using CSE combined with LPS induced injury of pulmonary epithelial cell line A549 as acute exacerbation model of COPD in vitro; and proper concentration of Sch B was given to prevent the injury of CSE and LPS,then the cell viability was determined by MTT assay,mRNA levels of interleukin-8( IL-8) and cyclooxygenase-2( COX-2) were measured by RT-PCR,the levels of IL-8 and COX-2 in supernatant were measured by ELISA,the level of phosphorylated NF-κB protein was measured by Western blot. Results: Co-cultured with 4% CSE combined with 0. 1 μg/ml LPS for 24 h,lung epithelial cell proliferation was promoted,inflammatory factor significantly increased,Sch B could significantly inhibit the proliferation of lung epithelial cells and improve the inflammation induced by CSE combined with LPS,reduce the expression of IL-8 and COX-2 mRNA in injured cells,decrease IL-8 and COX-2 levels in the supernatant,reduce the level of phosphorylated NF-κB protein expression in cells. Conclusion: Sch B can inhibit the proliferation of lung epithelial cells,and the inflammatory factor expression induced by CSE combined with LPS,which provides experimental evidence for the clinical application of Sch B in the treatment of acute exacerbation of COPD.
引文
[1]Cai Z,Liu J,Bian H,et al.Suppression of P2X7/NF-κB pathways by Schisandrin B contributes to attenuation of lipopolysaccharideinduced inflammatory responses in acute lung injury[J].Arch Pharm Res,2016,39(4):1-9.
[2]Liu N,Zheng JX,Zhuang YS,et al.Anti-inflammatory effects of schisandrin B on LPS-stimulated BV2 microglia via,activating PPAR-γ[J].Inflammation,2017:1-6.
[3]Victoni T,Gleonnec F,Lanzetti M,et al.Roflumilast N-Oxide prevents cytokine secretion induced by cigarette smoke combined with LPS through JAK/STAT and ERK1/2 inhibition in airway epithelial cells[J].PLo S One,2014,9(1):e85243.
[4]Wang H,Yang T,Shen Y,et al.Ghrelin inhibits interleukin-6 production induced by cigarette smoke extract in the bronchial epithelial cell via NF-κB pathway[J].Inflammation,2016,39(1):190-198.
[5]Liu N,Zheng JX,Zhuang YS,et al.Anti-Inflammatory Effects of Schisandrin B on LPS-Stimulated BV2 Microglia via Activating PPAR-[J].Inflammation,2017,40(3):1-6.
[6]Guo CX,Deng S,Yin JY,et al.Schisandrin A and B induce organic anion transporting polypeptide 1B1 transporter activity[J].Pharmazie,2015,70(1):29-32.
[7]沈晓君,刘玉翠,曲晓波,等.HPLC法同时测定不同产地五味子中4种木脂素类成分的含量[J].中国药房,2017,28(3):387-390.Xiao JS,Yu CL,Xiao BQ,et al.Simultaneous determination of four lignin components in schisandra from different habitats by HPLC[J].Chinese Pharmacy,2017,28(3):387-390.
[8]梁婧,侯海燕,兰晓霞,等.五味子乙素的药理作用及其分子机制的研究进展[J].中国现代应用药学,2014,31(4):506-510.Jing L,Hai YH,Xiao XL,et al.Advances in research on the pharmacological effects and molecular mechanism of schisandrin B[J].China Modern Applied Pharmacy,2014,31(4):506-510.
[9]Yang X,Shuai W,Mu Y,et al.Schisandrin B inhibits cell proliferation and induces apoptosis in human cholangiocarcinoma cells[J].Oncol Rep,2016,36(4):1799-1806.
[10]Wu J,Jia J,Liu L,et al.Schisandrin B displays a protective role against primary pulmonary hypertension by targeting transforming growth factor[J].J Am Chem Soc,2017,11(3):148-157.
[11]Lv XJ,Zhao LJ,Hao YQ,et al.Schisandrin B inhibits the proliferation of human lung adenocarcinoma A549 cells by inducing cycle arrest and apoptosis[J].Int J Clin Exp Med,2015,8(5):6926-6936.
[12]Sun YP,Bae YS,Min JK,et al.Comparison of anti-inflammatory potential of four different dibenzocyclooctadiene lignans in microglia;action via activation of PKA and Nrf-2 signaling and inhibition of MAPK/STAT/NF-κB pathways[J].Mol Nutr Food Res,2014,58(4):738-748.
[13]Neder JA,Arbex FF,Alencar MC,et al.Exercise ventilatory inefficiency in mild to end-stage COPD[J].Eur Respir J,2014,45(2):377-387.
[14]Nie Y,Huang C,Zhong S,et al.Cigarette smoke extract(CSE)induces transient receptor potential ankyrin 1(TRPA1)expression via activation of HIF1 in A549 cells[J].Free Radical Bio Med,2016,99:498-507.
[15]Victoni T,Gleonnec F,Lanzetti M,et al.Roflumilast N-oxide prevents cytokine secretion induced by cigarette smoke combined with LPS through JAK/STAT and ERK1/2 inhibition in airway epithelial cells[J].PLo S One,2013,9(1):e85243.
[16]张晓慧.炎性细胞因子水平检测对慢性阻塞性肺疾病的临床意义[J].海南医学院学报,2016,22(3):240-242.Xiao HZ.Clinical significance of measurement of inflammatory cytokine levels in chronic obstructive pulmonary disease[J].JHainan Med Univ,2016,22(3):240-242.
[17]Chen B,You WJ,Xue S,et al.Overexpression of farnesoid X receptor in small airways contributes to epithelial to mesenchymal transition and COX-2 expression in chronic obstructive pulmonary disease[J].J Thorac Dis,2016,8(11):3063-3074.
[18]袁彬凡,吴东,何若云,等.哮喘-COPD重叠综合征机械通气患者肺泡灌洗液SP-D及IL-8意义分析[J].中国免疫学杂志,2016,32(7):1035-1038.Bin FY,Dong W,Ruo YH,et al.Analysis of the significance of SP-D and IL-8 in bronchoalveolar lavage fluid in patients with mechanical ventilation with asthma-COPD overlap syndrome[J].Chin J Immunol,2016,32(7):1035-1038.
[19]Zhong S,Nie YC,Gan ZY,et al.Effects of Schisandra chinensis,extracts on cough and pulmonary inflammation in a cough hypersensitivity guinea pig model induced by cigarette smoke exposure[J].J Ethnopharmacol,2015,165:73-82.
[20]Wang H,Yang T,Shen Y,et al.Ghrelin inhibits interleukin-6production induced by cigarette smoke extract in the bronchial epithelial cell via NF-κB pathway[J].Inflammation,2016,39(1):190-198.
[21]Cai Z,Liu J,Bian H,et al.Suppression of P2X7/NF-κB pathways by Schisandrin B contributes to attenuation of lipopolysaccharideinduced inflammatory responses in acute lung injury[J].Arch Pharm Res,2016,39(4):499-507.
[22]Kuan LP,Shan WH,Chen J,et al.Differential action between schisandrin A and schisandrin B in eliciting an anti-inflammatory action:the depletion of reduced glutathione and the induction of an antioxidant response[J].PLo S One,2016,11(5):e0155879.
[2]Liu N,Zheng JX,Zhuang YS,et al.Anti-inflammatory effects of schisandrin B on LPS-stimulated BV2 microglia via,activating PPAR-γ[J].Inflammation,2017:1-6.
[3]Victoni T,Gleonnec F,Lanzetti M,et al.Roflumilast N-Oxide prevents cytokine secretion induced by cigarette smoke combined with LPS through JAK/STAT and ERK1/2 inhibition in airway epithelial cells[J].PLo S One,2014,9(1):e85243.
[4]Wang H,Yang T,Shen Y,et al.Ghrelin inhibits interleukin-6 production induced by cigarette smoke extract in the bronchial epithelial cell via NF-κB pathway[J].Inflammation,2016,39(1):190-198.
[5]Liu N,Zheng JX,Zhuang YS,et al.Anti-Inflammatory Effects of Schisandrin B on LPS-Stimulated BV2 Microglia via Activating PPAR-[J].Inflammation,2017,40(3):1-6.
[6]Guo CX,Deng S,Yin JY,et al.Schisandrin A and B induce organic anion transporting polypeptide 1B1 transporter activity[J].Pharmazie,2015,70(1):29-32.
[7]沈晓君,刘玉翠,曲晓波,等.HPLC法同时测定不同产地五味子中4种木脂素类成分的含量[J].中国药房,2017,28(3):387-390.Xiao JS,Yu CL,Xiao BQ,et al.Simultaneous determination of four lignin components in schisandra from different habitats by HPLC[J].Chinese Pharmacy,2017,28(3):387-390.
[8]梁婧,侯海燕,兰晓霞,等.五味子乙素的药理作用及其分子机制的研究进展[J].中国现代应用药学,2014,31(4):506-510.Jing L,Hai YH,Xiao XL,et al.Advances in research on the pharmacological effects and molecular mechanism of schisandrin B[J].China Modern Applied Pharmacy,2014,31(4):506-510.
[9]Yang X,Shuai W,Mu Y,et al.Schisandrin B inhibits cell proliferation and induces apoptosis in human cholangiocarcinoma cells[J].Oncol Rep,2016,36(4):1799-1806.
[10]Wu J,Jia J,Liu L,et al.Schisandrin B displays a protective role against primary pulmonary hypertension by targeting transforming growth factor[J].J Am Chem Soc,2017,11(3):148-157.
[11]Lv XJ,Zhao LJ,Hao YQ,et al.Schisandrin B inhibits the proliferation of human lung adenocarcinoma A549 cells by inducing cycle arrest and apoptosis[J].Int J Clin Exp Med,2015,8(5):6926-6936.
[12]Sun YP,Bae YS,Min JK,et al.Comparison of anti-inflammatory potential of four different dibenzocyclooctadiene lignans in microglia;action via activation of PKA and Nrf-2 signaling and inhibition of MAPK/STAT/NF-κB pathways[J].Mol Nutr Food Res,2014,58(4):738-748.
[13]Neder JA,Arbex FF,Alencar MC,et al.Exercise ventilatory inefficiency in mild to end-stage COPD[J].Eur Respir J,2014,45(2):377-387.
[14]Nie Y,Huang C,Zhong S,et al.Cigarette smoke extract(CSE)induces transient receptor potential ankyrin 1(TRPA1)expression via activation of HIF1 in A549 cells[J].Free Radical Bio Med,2016,99:498-507.
[15]Victoni T,Gleonnec F,Lanzetti M,et al.Roflumilast N-oxide prevents cytokine secretion induced by cigarette smoke combined with LPS through JAK/STAT and ERK1/2 inhibition in airway epithelial cells[J].PLo S One,2013,9(1):e85243.
[16]张晓慧.炎性细胞因子水平检测对慢性阻塞性肺疾病的临床意义[J].海南医学院学报,2016,22(3):240-242.Xiao HZ.Clinical significance of measurement of inflammatory cytokine levels in chronic obstructive pulmonary disease[J].JHainan Med Univ,2016,22(3):240-242.
[17]Chen B,You WJ,Xue S,et al.Overexpression of farnesoid X receptor in small airways contributes to epithelial to mesenchymal transition and COX-2 expression in chronic obstructive pulmonary disease[J].J Thorac Dis,2016,8(11):3063-3074.
[18]袁彬凡,吴东,何若云,等.哮喘-COPD重叠综合征机械通气患者肺泡灌洗液SP-D及IL-8意义分析[J].中国免疫学杂志,2016,32(7):1035-1038.Bin FY,Dong W,Ruo YH,et al.Analysis of the significance of SP-D and IL-8 in bronchoalveolar lavage fluid in patients with mechanical ventilation with asthma-COPD overlap syndrome[J].Chin J Immunol,2016,32(7):1035-1038.
[19]Zhong S,Nie YC,Gan ZY,et al.Effects of Schisandra chinensis,extracts on cough and pulmonary inflammation in a cough hypersensitivity guinea pig model induced by cigarette smoke exposure[J].J Ethnopharmacol,2015,165:73-82.
[20]Wang H,Yang T,Shen Y,et al.Ghrelin inhibits interleukin-6production induced by cigarette smoke extract in the bronchial epithelial cell via NF-κB pathway[J].Inflammation,2016,39(1):190-198.
[21]Cai Z,Liu J,Bian H,et al.Suppression of P2X7/NF-κB pathways by Schisandrin B contributes to attenuation of lipopolysaccharideinduced inflammatory responses in acute lung injury[J].Arch Pharm Res,2016,39(4):499-507.
[22]Kuan LP,Shan WH,Chen J,et al.Differential action between schisandrin A and schisandrin B in eliciting an anti-inflammatory action:the depletion of reduced glutathione and the induction of an antioxidant response[J].PLo S One,2016,11(5):e0155879.