细胞间黏附分子-1在支气管哮喘儿童支气管黏膜中的表达及其对支气管上皮细胞凋亡的影响
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摘要
目的探讨细胞间黏附分子-1(ICAM-1)在支气管哮喘儿童支气管黏膜中的表达及其对支气管上皮细胞凋亡的影响。方法收集驻马店市中心医院2014年5月至2017年12月收治的28例哮喘儿童的支气管黏膜组织标本,同时选取28例支气管扩张非哮喘儿童的支气管黏膜组织作为对照;采用实时荧光定量聚合酶链反应(qRT-PCR)和Western blot法检测支气管哮喘儿童支气管黏膜组织中ICAM-1 mRNA和蛋白表达。将正常培养的支气管上皮细胞16HBE分为对照组、感染组、阴性组和干扰组。对照组细胞正常培养,不做任何处理;感染组细胞感染呼吸道合胞病毒(RSV),但不进行转染;阴性组细胞转染siRNA-control后感染RSV;干扰组细胞转染ICAM-1 siRNA后感染RSV。采用qRT-PCR和Western blot法检测各组细胞中ICAM-1 mRNA和蛋白表达,流式细胞术检测细胞凋亡情况,酶联免疫吸附试验检测细胞培养基上清液中白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)水平,Western blot法检测细胞中活化的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved Caspase-3)、核因子-κB p65(NF-κB p65)表达。结果哮喘支气管黏膜组织中ICAM-1 mRNA和蛋白表达显著高于正常支气管黏膜组织(P <0. 05)。感染组、阴性组、干扰组细胞中ICAM-1 mRNA和蛋白表达显著高于对照组(P <0. 05)。干扰组细胞中ICAM-1 mRNA和蛋白表达显著低于感染组和阴性组(P <0. 05)。感染组、阴性组、干扰组细胞凋亡率显著高于对照组(P <0. 05)。干扰组细胞凋亡率显著低于感染组和阴性组(P <0. 05)。感染组、阴性组、干扰组细胞培养基上清液中IL-1β、TNF-α水平显著高于对照组(P <0. 05)。干扰组细胞培养基上清液中IL-1β、TNF-α水平显著低于感染组和阴性组(P <0. 05)。感染组、阴性组、干扰组细胞中Cleaved Caspase-3、NF-κB p65蛋白水平显著高于对照组(P <0. 05)。干扰组细胞中Cleaved Caspase-3、NF-κB p65蛋白水平显著低于感染组和阴性组(P <0. 05)。结论 ICAM-1在哮喘儿童支气管黏膜组织中表达上调,抑制ICAM-1可以降低RSV诱导的支气管上皮细胞凋亡,降低支气管上皮细胞中Cleaved Caspase-3、NF-κB p65蛋白表达,减少细胞分泌炎性因子。
Objective To investigate the expression of intercellular adhesion molecule-1( ICAM-1) in bronchial mucosa of asthmatic children and its effect on the apoptosis of bronchial epithelial cells. Methods Twenty-eight bronchial mucosa of asthmatic children in the Central Hospital of Zhumadian City from May 2014 to December 2017,and twenty-eight bronchial mucosa of bronchiectasis without asthmatic children were selected as control. The expression of ICAM-1 mRNA and protein in bronchial mucosa of asthmatic children were detected by quantitative real-time polymerase chain reaction( qRTPCR) and Western blot. The bronchial epithelial cells who was cultured normally were divided into control group,infection group,negative group and interference group. The cells in the control group were cultured normally without any treatment. The cells in the infection group were infected with respiratory syncytial virus( RSV),but they were not transfected. The cells in the negative group were transfected with siRNA-control and then they were infected with RSV. The cells in the interference group were transfected with ICAM-1 siRNA and then they were infected with RSV. The expressions of ICAM-1 mRNA and protein in each group were determined by qRT-PCR and Western blot,and the cell apoptosis was measured by flow cytometry. The interleukin( IL)-1β and tumor necrosis factor( TNF)-α levels in supernatant of cell culture medium were detected by enzyme linked immunosorbent assay. The expressions of cleaved cysteinyl aspartate specific proteinase( Cleaved Caspase-3) and nuclear factor κB( NF-κB) p65 in cells were determined by Western blot. Results The expressions of ICAM-1 mRNA and protein in bronchial mucosa of asthmatic patients were significantly higher than those in normal bronchial mucosa( P < 0. 05).The expressions of ICAM-1 mRNA and protein in the infection group,negative group and interference group were significantly higher than those in the control group( P < 0. 05). The expressions of ICAM-1 mRNA and protein in the interference group were significantly lower than those in the infection group and the negative group( P < 0. 05). The cell apoptosis rate of the infection group,negative group and interference group was significantly higher than that of the control group( P < 0. 05). The cell apoptosis rate in the interference group was significantly lower than that in the infection group and the negative group( P <0. 05). The levels of IL-1β and TNF-α in supernatant of cell culture medium in the infection group,the negative group and the interference group were significantly higher than those in the control group( P < 0. 05). The levels of IL-1β and TNF-α in supernatant of cell culture medium in the interference group were significantly lower than those in the infection group and the negative group( P < 0. 05). The levels of Cleaved Caspase-3 and NF-κB p65 protein in the infection group,the negative group and the interference group were significantly higher than those in the control group( P < 0. 05). The levels of Cleaved Caspase-3 and NF-κB p65 protein in the interference group were significantly lower than those in the infection group and the negative group( P < 0. 05). Conclusion The ICAM-1 expression in bronchial mucosa of asthmatic children is up-regulated. Inhibiting ICAM-1 can reduce the level of apoptosis induced by RSV in bronchial epithelial cells,down-regulate the levels of Cleaved Caspase-3 and NF-κB p65 protein in bronchial epithelial cells,and reduce the secretion of inflammatory factors.
Objective To investigate the expression of intercellular adhesion molecule-1( ICAM-1) in bronchial mucosa of asthmatic children and its effect on the apoptosis of bronchial epithelial cells. Methods Twenty-eight bronchial mucosa of asthmatic children in the Central Hospital of Zhumadian City from May 2014 to December 2017,and twenty-eight bronchial mucosa of bronchiectasis without asthmatic children were selected as control. The expression of ICAM-1 mRNA and protein in bronchial mucosa of asthmatic children were detected by quantitative real-time polymerase chain reaction( qRTPCR) and Western blot. The bronchial epithelial cells who was cultured normally were divided into control group,infection group,negative group and interference group. The cells in the control group were cultured normally without any treatment. The cells in the infection group were infected with respiratory syncytial virus( RSV),but they were not transfected. The cells in the negative group were transfected with siRNA-control and then they were infected with RSV. The cells in the interference group were transfected with ICAM-1 siRNA and then they were infected with RSV. The expressions of ICAM-1 mRNA and protein in each group were determined by qRT-PCR and Western blot,and the cell apoptosis was measured by flow cytometry. The interleukin( IL)-1β and tumor necrosis factor( TNF)-α levels in supernatant of cell culture medium were detected by enzyme linked immunosorbent assay. The expressions of cleaved cysteinyl aspartate specific proteinase( Cleaved Caspase-3) and nuclear factor κB( NF-κB) p65 in cells were determined by Western blot. Results The expressions of ICAM-1 mRNA and protein in bronchial mucosa of asthmatic patients were significantly higher than those in normal bronchial mucosa( P < 0. 05).The expressions of ICAM-1 mRNA and protein in the infection group,negative group and interference group were significantly higher than those in the control group( P < 0. 05). The expressions of ICAM-1 mRNA and protein in the interference group were significantly lower than those in the infection group and the negative group( P < 0. 05). The cell apoptosis rate of the infection group,negative group and interference group was significantly higher than that of the control group( P < 0. 05). The cell apoptosis rate in the interference group was significantly lower than that in the infection group and the negative group( P <0. 05). The levels of IL-1β and TNF-α in supernatant of cell culture medium in the infection group,the negative group and the interference group were significantly higher than those in the control group( P < 0. 05). The levels of IL-1β and TNF-α in supernatant of cell culture medium in the interference group were significantly lower than those in the infection group and the negative group( P < 0. 05). The levels of Cleaved Caspase-3 and NF-κB p65 protein in the infection group,the negative group and the interference group were significantly higher than those in the control group( P < 0. 05). The levels of Cleaved Caspase-3 and NF-κB p65 protein in the interference group were significantly lower than those in the infection group and the negative group( P < 0. 05). Conclusion The ICAM-1 expression in bronchial mucosa of asthmatic children is up-regulated. Inhibiting ICAM-1 can reduce the level of apoptosis induced by RSV in bronchial epithelial cells,down-regulate the levels of Cleaved Caspase-3 and NF-κB p65 protein in bronchial epithelial cells,and reduce the secretion of inflammatory factors.
引文
[1] LIN Y L,CHEN S H,WANG J Y. Critical role of IL-6 in dendritic cell-induced allergic inflammation of asthma[J]. J Mol Med(Berl),2016,94(1):51-59.
[2]杨哲,宋欣,李硕,等.支气管哮喘控制患儿小气道功能状况及影响因素分析[J].中华实用儿科临床杂志,2017,32(16):1244-1247.
[3] SHIOBARA T,CHIBANA K,WATANABE T,et al. Dipeptidyl peptidase-4 is highly expressed in bronchial epithelial cells of untreated asthma and it increases cell proliferation along with fibronectin production in airway constitutive cells[J]. Respir Res,2016,17(1):1-11.
[4]冯媛元,李晓泳,宋春兰,等. ADRB2基因多态性与儿童支气管哮喘易感及耐药的相关性[J].中华实用儿科临床杂志,2018,33(16):1237-1241.
[5] VIGNOLA A M,CHIAPPARA G,SIENA L,et al. Proliferation and activation of bronchial epithelial cells in corticosteroid-dependent asthma[J]. J Allergy Clin Immunol,2001,108(5):738-746.
[6] BLUME C,REALE R,HELD M,et al. Cellular crosstalk between airway epithelial and endothelial cells regulates barrier functions during exposure to double-stranded RNA[J]. Immun Inflamm Dis,2016,5(1):45-56.
[7]杨丽华,张国成.呼吸道合胞病毒感染与支气管哮喘发病机制研究进展[J].中华实用儿科临床杂志,2016,31(21):1675-1677.
[8]邓艳凤,朱黎明. ICAM-1与支气管哮喘的研究进展[J].临床与病理杂志,2015,35(1):150-154.
[9]古力鲜·马合木提,李新,张慧,等. VEGF、ICAM-1、IL-13及IL-17在慢阻肺、支气管哮喘及慢阻肺合并哮喘患者中的表达及意义[J].临床肺科杂志,2017,22(9):1563-1566.
[10] GUO X,LIU T,SHI H,et al. Respiratory syncytial virus infection upregulates NLRC5 and MHC I expression through RIG-I induction in airway epithelial cells[J]. J Virol,2015,89(15):7636-7645.
[11] GOBEL J,GARTLAND M,GURLEY S H,et al. A phenotypic high-throughput screen with RSV-infected primary human small airway epithelial cells(SAECs)[J]. Screen J Biomol,2015,20(6):729-738.
[12]马玉梅,张建华.鼻病毒感染与支气管哮喘研究进展[J].中华实用儿科临床杂志,2016,31(21):1678-1680.
[13]李静,葛心,马建民,等.特发性眼眶炎性假瘤患者血清细胞间黏附分子-1水平及其临床意义[J].眼科新进展,2015,35(1):39-41.
[14] RELOVA A J,SHAHANA S,MAKEEVA N,et al. Effect of cytokines on ICAM-1 and ZO-1 expression on human airway epithelial cells[J]. Cell Biol Int,2013,29(9):768-777.
[15] CAO J,WONG C K,YIN Y,et al. Activation of human bronchial epithelial cells by inflammatory cytokines IL-27 and TNF-alpha:implications for immunopathophysiology of airway inflammation[J]. J Cell Physiol,2010,223(3):788-797.
[16] BOURDILLON M C,POSTON R N,COVACHO C,et al. ICAM-1deficiency reduces atherosclerotic lesions in double-knockout mice(ApoE-/-/ICAM-1-/-)fed a fat or a chow diet[J]. Arterioscler Thromb Vasc Biol,2000,144(12):2630-2635.
[17]唐力琼,李英,罗银河,等.咳喘宁对RSV诱发哮喘大鼠ICAM-1表达的影响[J].湖南中医药大学学报,2016,36(10):27-30.
[18]刘斌,刘鑫,罗晓青,等.气道上皮细胞在哮喘中的作用[J].现代生物医学进展,2013,13(8):1583-1585.
[19] HIROTA J A,KNIGHT D A. Human airway epithelial cell innate immunity:relevance to asthma[J]. Curr Opin Immunol,2012,24(6):740-746.
[20] YAMADA Y,MATSUMOTO K,HASHIMOTO N,et al. Effect of Th1/Th2 cytokine pretreatment on RSV-induced gene expression in airway epithelial cells[J]. Int Arch Allergy Immunol,2011,154(3):185-194.
[21] WHITE S R,FISCHER B M,MARROQUIN B A,et al. Interleukin-1beta mediates human airway epithelial cell migration via NFkappaB[J]. Am J Physiol Mol Physiol Cell Lung,2008,295(6):1018-1027.
[22] ZHANG T,YANG S,DU J,et al. Platycodin D attenuates airway inflammation in a mouse model of allergic asthma by regulation NF-κB pathway[J]. Inflammation,2015,38(3):1221-1228.
[23]王金磊,李承德,孙宏伟,等.黄芪多糖抑制NF-κB/MAPK信号通路和改善哮喘大鼠气道炎症的作用[J].中国药理学通报,2016,32(4):489-493.
[24]黄虎翔,张万明,王辉.白细胞介素-4、干扰素-γ及核因子-κB p65在解淀粉芽孢杆菌致BALB/c小鼠哮喘模型中的表达[J].新乡医学院学报,2017,34(1):18-21.
[25] MEURER S K,WEISKIRCHEN R. Primary peritoneal mast cells(PMC)reduce the fibrogenic response of hepatic stellate cells while inducing caspase 3 cleavage and activity[J]. Zeitschrift Für Gastroenterologie,2016,54(12):1343-1404.
[26] FANZO J C,REAVES S K,CUI L,et al. Zinc status affects p53,gadd45,and c-fos expression and caspase-3 activity in human bronchial epithelial cells[J]. Am J Physiol Cell Physiol,2001,281(3):751-757.
[2]杨哲,宋欣,李硕,等.支气管哮喘控制患儿小气道功能状况及影响因素分析[J].中华实用儿科临床杂志,2017,32(16):1244-1247.
[3] SHIOBARA T,CHIBANA K,WATANABE T,et al. Dipeptidyl peptidase-4 is highly expressed in bronchial epithelial cells of untreated asthma and it increases cell proliferation along with fibronectin production in airway constitutive cells[J]. Respir Res,2016,17(1):1-11.
[4]冯媛元,李晓泳,宋春兰,等. ADRB2基因多态性与儿童支气管哮喘易感及耐药的相关性[J].中华实用儿科临床杂志,2018,33(16):1237-1241.
[5] VIGNOLA A M,CHIAPPARA G,SIENA L,et al. Proliferation and activation of bronchial epithelial cells in corticosteroid-dependent asthma[J]. J Allergy Clin Immunol,2001,108(5):738-746.
[6] BLUME C,REALE R,HELD M,et al. Cellular crosstalk between airway epithelial and endothelial cells regulates barrier functions during exposure to double-stranded RNA[J]. Immun Inflamm Dis,2016,5(1):45-56.
[7]杨丽华,张国成.呼吸道合胞病毒感染与支气管哮喘发病机制研究进展[J].中华实用儿科临床杂志,2016,31(21):1675-1677.
[8]邓艳凤,朱黎明. ICAM-1与支气管哮喘的研究进展[J].临床与病理杂志,2015,35(1):150-154.
[9]古力鲜·马合木提,李新,张慧,等. VEGF、ICAM-1、IL-13及IL-17在慢阻肺、支气管哮喘及慢阻肺合并哮喘患者中的表达及意义[J].临床肺科杂志,2017,22(9):1563-1566.
[10] GUO X,LIU T,SHI H,et al. Respiratory syncytial virus infection upregulates NLRC5 and MHC I expression through RIG-I induction in airway epithelial cells[J]. J Virol,2015,89(15):7636-7645.
[11] GOBEL J,GARTLAND M,GURLEY S H,et al. A phenotypic high-throughput screen with RSV-infected primary human small airway epithelial cells(SAECs)[J]. Screen J Biomol,2015,20(6):729-738.
[12]马玉梅,张建华.鼻病毒感染与支气管哮喘研究进展[J].中华实用儿科临床杂志,2016,31(21):1678-1680.
[13]李静,葛心,马建民,等.特发性眼眶炎性假瘤患者血清细胞间黏附分子-1水平及其临床意义[J].眼科新进展,2015,35(1):39-41.
[14] RELOVA A J,SHAHANA S,MAKEEVA N,et al. Effect of cytokines on ICAM-1 and ZO-1 expression on human airway epithelial cells[J]. Cell Biol Int,2013,29(9):768-777.
[15] CAO J,WONG C K,YIN Y,et al. Activation of human bronchial epithelial cells by inflammatory cytokines IL-27 and TNF-alpha:implications for immunopathophysiology of airway inflammation[J]. J Cell Physiol,2010,223(3):788-797.
[16] BOURDILLON M C,POSTON R N,COVACHO C,et al. ICAM-1deficiency reduces atherosclerotic lesions in double-knockout mice(ApoE-/-/ICAM-1-/-)fed a fat or a chow diet[J]. Arterioscler Thromb Vasc Biol,2000,144(12):2630-2635.
[17]唐力琼,李英,罗银河,等.咳喘宁对RSV诱发哮喘大鼠ICAM-1表达的影响[J].湖南中医药大学学报,2016,36(10):27-30.
[18]刘斌,刘鑫,罗晓青,等.气道上皮细胞在哮喘中的作用[J].现代生物医学进展,2013,13(8):1583-1585.
[19] HIROTA J A,KNIGHT D A. Human airway epithelial cell innate immunity:relevance to asthma[J]. Curr Opin Immunol,2012,24(6):740-746.
[20] YAMADA Y,MATSUMOTO K,HASHIMOTO N,et al. Effect of Th1/Th2 cytokine pretreatment on RSV-induced gene expression in airway epithelial cells[J]. Int Arch Allergy Immunol,2011,154(3):185-194.
[21] WHITE S R,FISCHER B M,MARROQUIN B A,et al. Interleukin-1beta mediates human airway epithelial cell migration via NFkappaB[J]. Am J Physiol Mol Physiol Cell Lung,2008,295(6):1018-1027.
[22] ZHANG T,YANG S,DU J,et al. Platycodin D attenuates airway inflammation in a mouse model of allergic asthma by regulation NF-κB pathway[J]. Inflammation,2015,38(3):1221-1228.
[23]王金磊,李承德,孙宏伟,等.黄芪多糖抑制NF-κB/MAPK信号通路和改善哮喘大鼠气道炎症的作用[J].中国药理学通报,2016,32(4):489-493.
[24]黄虎翔,张万明,王辉.白细胞介素-4、干扰素-γ及核因子-κB p65在解淀粉芽孢杆菌致BALB/c小鼠哮喘模型中的表达[J].新乡医学院学报,2017,34(1):18-21.
[25] MEURER S K,WEISKIRCHEN R. Primary peritoneal mast cells(PMC)reduce the fibrogenic response of hepatic stellate cells while inducing caspase 3 cleavage and activity[J]. Zeitschrift Für Gastroenterologie,2016,54(12):1343-1404.
[26] FANZO J C,REAVES S K,CUI L,et al. Zinc status affects p53,gadd45,and c-fos expression and caspase-3 activity in human bronchial epithelial cells[J]. Am J Physiol Cell Physiol,2001,281(3):751-757.