平喘颗粒对哮喘小鼠肺泡灌洗液中外泌体的分泌及miR-23b介导的气道平滑肌细胞增殖的影响
|
摘要
目的:观察平喘颗粒对外泌体的分泌及miR-23b介导的气道平滑肌细胞增殖的影响。方法:通过构建哮喘小鼠模型并以空白小鼠为对照组,观察不同处理方式下小鼠肺泡灌洗液中外泌体的分泌及外泌体中miR-23b的表达;通过外泌体干预气道平滑肌细胞,分别检测平滑肌细胞的增殖、凋亡,及细胞中TβRⅡ蛋白的表达。结果:与空白组比较,模型组外泌体数量、标记蛋白Alix、TSG101、CD9、CD63的表达、平滑肌细胞的增殖及TβRⅡ蛋白的表达显著增加,miR-23b的表达和ASMCs的凋亡显著降低(P<0.01);与模型组比较,平喘颗粒显著降低外泌体数量、Alix、TSG101、CD9、CD63的表达、平滑肌细胞的增殖及TβRⅡ蛋白的表达,增加miR-23b的表达和ASMCs的凋亡(P<0.01)。结论:平喘颗粒能够促进外泌体中miR-23b的表达,进而下调ASMCs中TβRⅡ蛋白的表达,调控ASMCs增殖和凋亡,阻止哮喘气道重塑。
Objective: To study the effects of Pingchuan Granules on secretion of exosomes and airway smooth muscle cells proliferation by inhibiting miR-23 b. Methods: Asthmatic mice model was established and normal mice was used as control group. The secretion of exosomes in alveolar irrigating solution of asthmatic mice and the expression of miR-23 b in secretion of exosomes were observed under different treatments. Proliferation and apoptosis of airway smooth muscle cells were detected by CCK8 and flow cytometry, with the expression of TβRII protein was detect by Western Blot. Results: Compared with the control group, the number of exosomes, expression of Alix, TSG101, CD9, CD63 proteins, the proliferation of smooth muscle cells and the expression of TβRII protein in alveolar irrigating solution of the model group were significantly increased, and the expression of miR-23 b and the apoptosis of ASMCs were significantly decreased(P<0.01). Compared with the model group, the number of exosomes, the expression of Alix, TSG101, CD9, CD63 proteins, the proliferation of smooth muscle cells and the expression of TβRII protein in alveolar irrigating solution of the Pingchuang Granule group were significantly decreased, and the expression of miR-23 b and the apoptosis of ASMCs were significantly increased(P<0.01). Conclusion: Pingchuan Granules can promote the expression of miR-23 b in Exosome, further down-regulate the expression of TβRII protein in ASMCs, regulate proliferation and apoptosis of ASMCs, and prevent airway remodeling of asthma.
Objective: To study the effects of Pingchuan Granules on secretion of exosomes and airway smooth muscle cells proliferation by inhibiting miR-23 b. Methods: Asthmatic mice model was established and normal mice was used as control group. The secretion of exosomes in alveolar irrigating solution of asthmatic mice and the expression of miR-23 b in secretion of exosomes were observed under different treatments. Proliferation and apoptosis of airway smooth muscle cells were detected by CCK8 and flow cytometry, with the expression of TβRII protein was detect by Western Blot. Results: Compared with the control group, the number of exosomes, expression of Alix, TSG101, CD9, CD63 proteins, the proliferation of smooth muscle cells and the expression of TβRII protein in alveolar irrigating solution of the model group were significantly increased, and the expression of miR-23 b and the apoptosis of ASMCs were significantly decreased(P<0.01). Compared with the model group, the number of exosomes, the expression of Alix, TSG101, CD9, CD63 proteins, the proliferation of smooth muscle cells and the expression of TβRII protein in alveolar irrigating solution of the Pingchuang Granule group were significantly decreased, and the expression of miR-23 b and the apoptosis of ASMCs were significantly increased(P<0.01). Conclusion: Pingchuan Granules can promote the expression of miR-23 b in Exosome, further down-regulate the expression of TβRII protein in ASMCs, regulate proliferation and apoptosis of ASMCs, and prevent airway remodeling of asthma.
引文
[1]Lambrecht B N,Hammad H.The immunology of asthma.Nat Immunol,2014,16(1):45-56
[2]Martinez F D,Vercelli D.Asthma.Lancet,2013,382(9901):1360-1372
[3]李竹英,师留杰,高风丽.PM2.5与哮喘关系的中西医结合认识.中国中医急症,2016,25(12):2316-2318
[4]马晓燕,张志红.PM_(2.5)与哮喘关系的研究进展.环境与职业医学,2015,32(3):279-283
[5]李星,王雪慧,蒋鹏娜,等.平喘颗粒提取工艺的研究.中华中医药杂志,2017,32(7):3150-3153
[6]蒋鹏娜,李星,王雪慧,等.平喘颗粒质量标准研究.中华中医药杂志,2017,32(5):2132-2136
[7]毛文丽,刘建秋.平喘颗粒对哮喘慢性持续期mi R-21及嗜酸性粒细胞的影响.世界最新医学信息文摘,2016,16(36):237-239
[8]蒋鹏娜,刘建秋,李竹英,等.平喘颗粒对哮喘大鼠气道重塑后表皮生长因子蛋白表达的影响.长春中医药大学学报,2017,33(6):886-889
[9]李伟伟,李晓丰,侯萍,等.人脐带间充质干细胞外泌体可抑制Th22细胞的表达及功能.中国组织工程研究,2018,22(29):4657-4662
[10]Johnstone R M,Adam M,Hammond J R,et al.Vesicle formation duringreticulocytematuration.Associationofplasma membrane activities with released vesicles(exosomes).J Biol Chem,1987,262(19):9412-9420
[11]Harris D A,Patel S H,Gucek M,et al.Exosomes released from breast cancer carcinomas stimulate cell movement.Plos One,2015,10(3):e0117495
[12]白丰玺,潘珏.外泌体与肺疾病的研究现状.国际呼吸杂志,2015,35(8):629-632
[13]Biancone L,Bruno S,Deregibus M C,et al.Therapeutic potential of mesenchymal stem cell-derived microvesicles.Nephrol Dial Transplant,2012,27(8):3037-3042
[14]王湘云,陈乾,孙亚红,等.不同严重程度哮喘患者血清外泌体中微RNA-21的表达水平及其诊断价值.第二军医大学学报,2018,39(7):740-744
[15]贺争鸣.实验动物管理与使用指南.北京:科学出版社,2016:142-150
[16]赵龙,王宁,石晓岚,等.MicroRNA-34a对小鼠气道平滑肌细胞增殖和ORMDL3表达的影响.中国现代医学杂志,2018,28(8):6-12
[17]叶广亿,李书渊,陈艳芬,等.枇杷叶不同提取物的止咳化痰平喘作用比较研究.中药药理与临床,2013,29(2):100-102
[18]Busser H,Najar M,Raicevic G,et al.Isolation and characterization of human mesenchymal stromal cell subpopulations:Comparison of bone marrow and adipose tissue.Stem Cells Dev,2015,24(18):2142-2157
[19]Liga A,Vliegenthart A D,Oosthuyzen W,et al.Exosome isolation:A microfluidic road-map.Lab Chip,2015,15(11):2388-2394
[20]Elias J A.Airway remodeling in asthma.Unanswered questions.Am J Respir Crit Care Med,2000,161(2):168-171
[21]Davies D E,Wicks J,Powell R M,et al.Airway remodeling in asthma:New insights.J Allergy Clin Immunol,2003,111(2):215-225
[22]Hershenson M B,Brown M,Camoretti-Mercado B,et al.Airway smooth muscle in asthma.Annu Rev Pathol,2008,3:523-555
[23]Kariyawasam H H,Aizen M,Barkans J,et al.Remodeling and airway hyperresponsiveness but not cellular inflammation persist after allergen challenge in asthma.Am J Respir Crit Care Med,2007,175(9):896-904
[24]Folli C,Descalzi D,Scordamaglia F,et al.New insights into airway remodeling in asthma and its possible modulation.Curr Opin Allergy Clin Immunol,2008,8(5):367-375
[25]阮晓琳,周星星,彭来君,等.气阴双补中药对哮喘大鼠肺组织TGF-β1及Smad2/3表达的影响.中华中医药杂志,2017,32(3):1271-1275
[26]Budi E H,Xu J,Derynck R.Regulation of TGF-βReceptors.Methods Mol Biol,2016,1344:1-33
[27]Zhang L,Zhou F,Dinther M V,et al.Determining TGF-βReceptor Levels in the Cell Membrane.Methods Mol Biol,2016,1344:34-47
[2]Martinez F D,Vercelli D.Asthma.Lancet,2013,382(9901):1360-1372
[3]李竹英,师留杰,高风丽.PM2.5与哮喘关系的中西医结合认识.中国中医急症,2016,25(12):2316-2318
[4]马晓燕,张志红.PM_(2.5)与哮喘关系的研究进展.环境与职业医学,2015,32(3):279-283
[5]李星,王雪慧,蒋鹏娜,等.平喘颗粒提取工艺的研究.中华中医药杂志,2017,32(7):3150-3153
[6]蒋鹏娜,李星,王雪慧,等.平喘颗粒质量标准研究.中华中医药杂志,2017,32(5):2132-2136
[7]毛文丽,刘建秋.平喘颗粒对哮喘慢性持续期mi R-21及嗜酸性粒细胞的影响.世界最新医学信息文摘,2016,16(36):237-239
[8]蒋鹏娜,刘建秋,李竹英,等.平喘颗粒对哮喘大鼠气道重塑后表皮生长因子蛋白表达的影响.长春中医药大学学报,2017,33(6):886-889
[9]李伟伟,李晓丰,侯萍,等.人脐带间充质干细胞外泌体可抑制Th22细胞的表达及功能.中国组织工程研究,2018,22(29):4657-4662
[10]Johnstone R M,Adam M,Hammond J R,et al.Vesicle formation duringreticulocytematuration.Associationofplasma membrane activities with released vesicles(exosomes).J Biol Chem,1987,262(19):9412-9420
[11]Harris D A,Patel S H,Gucek M,et al.Exosomes released from breast cancer carcinomas stimulate cell movement.Plos One,2015,10(3):e0117495
[12]白丰玺,潘珏.外泌体与肺疾病的研究现状.国际呼吸杂志,2015,35(8):629-632
[13]Biancone L,Bruno S,Deregibus M C,et al.Therapeutic potential of mesenchymal stem cell-derived microvesicles.Nephrol Dial Transplant,2012,27(8):3037-3042
[14]王湘云,陈乾,孙亚红,等.不同严重程度哮喘患者血清外泌体中微RNA-21的表达水平及其诊断价值.第二军医大学学报,2018,39(7):740-744
[15]贺争鸣.实验动物管理与使用指南.北京:科学出版社,2016:142-150
[16]赵龙,王宁,石晓岚,等.MicroRNA-34a对小鼠气道平滑肌细胞增殖和ORMDL3表达的影响.中国现代医学杂志,2018,28(8):6-12
[17]叶广亿,李书渊,陈艳芬,等.枇杷叶不同提取物的止咳化痰平喘作用比较研究.中药药理与临床,2013,29(2):100-102
[18]Busser H,Najar M,Raicevic G,et al.Isolation and characterization of human mesenchymal stromal cell subpopulations:Comparison of bone marrow and adipose tissue.Stem Cells Dev,2015,24(18):2142-2157
[19]Liga A,Vliegenthart A D,Oosthuyzen W,et al.Exosome isolation:A microfluidic road-map.Lab Chip,2015,15(11):2388-2394
[20]Elias J A.Airway remodeling in asthma.Unanswered questions.Am J Respir Crit Care Med,2000,161(2):168-171
[21]Davies D E,Wicks J,Powell R M,et al.Airway remodeling in asthma:New insights.J Allergy Clin Immunol,2003,111(2):215-225
[22]Hershenson M B,Brown M,Camoretti-Mercado B,et al.Airway smooth muscle in asthma.Annu Rev Pathol,2008,3:523-555
[23]Kariyawasam H H,Aizen M,Barkans J,et al.Remodeling and airway hyperresponsiveness but not cellular inflammation persist after allergen challenge in asthma.Am J Respir Crit Care Med,2007,175(9):896-904
[24]Folli C,Descalzi D,Scordamaglia F,et al.New insights into airway remodeling in asthma and its possible modulation.Curr Opin Allergy Clin Immunol,2008,8(5):367-375
[25]阮晓琳,周星星,彭来君,等.气阴双补中药对哮喘大鼠肺组织TGF-β1及Smad2/3表达的影响.中华中医药杂志,2017,32(3):1271-1275
[26]Budi E H,Xu J,Derynck R.Regulation of TGF-βReceptors.Methods Mol Biol,2016,1344:1-33
[27]Zhang L,Zhou F,Dinther M V,et al.Determining TGF-βReceptor Levels in the Cell Membrane.Methods Mol Biol,2016,1344:34-47