二陈汤加味对COPD大鼠转化生长因子-β_1,组蛋白去乙酰化酶2基因表达的影响
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摘要
目的:观察二陈汤加味对慢性阻塞性肺疾病(COPD)大鼠肺组织中转化生长因子-β_1(TGF-β_1)及其受体(TGF-βRII)基因表达,外周血单个核细胞(PBMCs)中组蛋白去乙酰化酶2(HDAC2)基因表达及活性的影响。方法:采用烟熏加脂多糖方法制备COPD大鼠模型。随机分为正常组、模型组、二陈汤加味高、中、低剂量组(20,10,5 g·kg~(-1))。正常组、模型组灌胃生理盐水(10 g·kg~(-1)),二陈汤加味高、中、低剂量组分别灌胃,连续14 d。荧光酶联免疫法测定PBMCs中HDAC2活性,酶联免疫吸附测定(ELISA)法测定肺组织匀浆中TGF-β_1及PBMCs中HDAC2含量;实时荧光定量PCR(Real-time PCR)检测肺组织匀浆中TGF-β_1mRNA及PBMCs中HDAC2 mRNA表达;免疫组化检测TGF-β_1与TGF-βRⅡ蛋白在肺组织的原位表达,光镜观察组织结构。结果:与正常组比较,模型组大鼠肺组织匀浆中TGF-β_1含量升高(P<0.05);肺组织中TGF-β_1mRNA,TGF-β_1与TGF-βRⅡ蛋白表达均显著增强(P<0.01);模型组大鼠PBMCs中HDAC2 mRNA表达、含量及其活性均明显减低(P<0.05,P<0.01),差异均有统计学意义。与模型组比较,二陈汤加味高、中剂量组肺组织匀浆中TGF-β_1mRNA表达(P<0.01)和TGF-β_1蛋白含量均显著降低(P<0.05,P<0.01),免疫组化检测肺组织中TGF-β_1与TGF-βRⅡ蛋白表达均显著弱于模型组(P<0.01),PBMCs中HDAC2 mRNA表达、含量及其活性均升高(P<0.05,P<0.01),肺组织结构明显改善。结论:二陈汤加味对COPD有抗炎作用。其机制可能与增强PBMCs中HDAC2基因表达,提高HDAC2活性,抑制TGF-β_1及其受体基因的表达有关。
Objective: To observe the effect of modified Erchentang on gene expression of transforming growth factor-β_1(TGF-β_1) and its receptor TGF-βRⅡ in lung tissue,the expression of mRNA and activity of histone deacetylase 2(HDAC2) in peripheral blood mononuclerar cells(PBMCs) of rats with chronic obstructive pulmonary disease(COPD). Method: The method of smoke combined with lipopolysaccharide(LPS) was used to prepare COPD model in rats. The experimental animals were randomly divided into normal control group,model group,high,middle and low-dose modified Erchentang groups(20,10,5 g·kg~(-1)). Normal group and model group were orally given normal saline(10 g·kg~(-1)),and high,middle and low-dose modified Erchentang groups were orally given the drug for consecutively 14 days. The activity of HDAC2 was determinate by Enzyme-linked immune fluorescence. Euzyme-linked immunosorbent assay was used to determine the levels of TGF-β_1in lung homogenates and histone deacetylase 2(HDAC2) in peripheral blood mononuclerarcells(PBMCs) of all groups before and after treatment. Real-time fluorescence quantitative PCR(Real-time PCR) method was used to detect the expression of TGF-β_1mRNA and HDAC2 mRNA. Immunohistochemical(IHC) detection was performed for the expression of TGF-β_1and TGF-βRⅡ protein in lung tissues. Result: Compared with the normal group,the levels of TGF-β_1was significantly increased(P<0. 05),expression of TGF-β_1mRNA,and TGF-β_1and TGF-βR Ⅱprotein expressions in lung homogenates were significantly higher(P<0.01),while the expression of mRNA,content and activity of HDAC2 in PBMCs were decreased significantly in model group,with statistically significant differences. Compared with the model group,expressions of TGF-β_1mRNA(P<0.01) and TGF-βRⅡ protein in lung tissue of the rats treated with high and middle-dose modified Erchentang(20,10 g·kg~(-1)) were decreased significantly(P<0. 05,P<0.01),TGF-β_1and TGF-βRⅡ protein expressions were lower than model group(P <0.01),the expression of HDAC2 mRNA,content and activity of HDAC2 were higher significantly(P<0. 05,P <0.01),with statistical significance and obvious improvement in lung tissue structures. Conclusion: Modified Erchentang has an anti-inflammatory effect on COPD. Its mechanism may be correlated with inhibition of expression of TGF-β_1and TGF-β receptor Ⅱ genes,improvement of the expression and activity of HDAC2 in PBMCs,and inhibition of the inflammatory response.
Objective: To observe the effect of modified Erchentang on gene expression of transforming growth factor-β_1(TGF-β_1) and its receptor TGF-βRⅡ in lung tissue,the expression of mRNA and activity of histone deacetylase 2(HDAC2) in peripheral blood mononuclerar cells(PBMCs) of rats with chronic obstructive pulmonary disease(COPD). Method: The method of smoke combined with lipopolysaccharide(LPS) was used to prepare COPD model in rats. The experimental animals were randomly divided into normal control group,model group,high,middle and low-dose modified Erchentang groups(20,10,5 g·kg~(-1)). Normal group and model group were orally given normal saline(10 g·kg~(-1)),and high,middle and low-dose modified Erchentang groups were orally given the drug for consecutively 14 days. The activity of HDAC2 was determinate by Enzyme-linked immune fluorescence. Euzyme-linked immunosorbent assay was used to determine the levels of TGF-β_1in lung homogenates and histone deacetylase 2(HDAC2) in peripheral blood mononuclerarcells(PBMCs) of all groups before and after treatment. Real-time fluorescence quantitative PCR(Real-time PCR) method was used to detect the expression of TGF-β_1mRNA and HDAC2 mRNA. Immunohistochemical(IHC) detection was performed for the expression of TGF-β_1and TGF-βRⅡ protein in lung tissues. Result: Compared with the normal group,the levels of TGF-β_1was significantly increased(P<0. 05),expression of TGF-β_1mRNA,and TGF-β_1and TGF-βR Ⅱprotein expressions in lung homogenates were significantly higher(P<0.01),while the expression of mRNA,content and activity of HDAC2 in PBMCs were decreased significantly in model group,with statistically significant differences. Compared with the model group,expressions of TGF-β_1mRNA(P<0.01) and TGF-βRⅡ protein in lung tissue of the rats treated with high and middle-dose modified Erchentang(20,10 g·kg~(-1)) were decreased significantly(P<0. 05,P<0.01),TGF-β_1and TGF-βRⅡ protein expressions were lower than model group(P <0.01),the expression of HDAC2 mRNA,content and activity of HDAC2 were higher significantly(P<0. 05,P <0.01),with statistical significance and obvious improvement in lung tissue structures. Conclusion: Modified Erchentang has an anti-inflammatory effect on COPD. Its mechanism may be correlated with inhibition of expression of TGF-β_1and TGF-β receptor Ⅱ genes,improvement of the expression and activity of HDAC2 in PBMCs,and inhibition of the inflammatory response.
引文
[1]钟南山.慢性阻塞性肺疾病在中国[J].中国实用内科杂志,2011,31(5):321-323.
[2]葛均波,徐永健.内科学[M].8版.北京:人民卫生出版社,2013:21-25.
[3]中华中医药学会内科分会肺系病专业委员会.慢性阻塞性肺疾病中医诊疗指南(2011版)[J].中医杂志,2012,53(2):177-178.
[4]Fisman E Z,Motro M,Tenenbaum A.Cardiovascular diabetology in the core of a novel interleukins class infication the bad the good and the aloof Cardiovase[J].Diabetoly,2003,2(1):11-15.
[5]Utsugi M,Dobashi K,Kaga Y,et al.Glntathione redox regulates lipolysaccharideinduced IL-12 production through p38 mitogen activated protein kinase activation in human monocytes role of glutathi one redox in IFNgamma prining of IL-12 productioy[J].Leukoo Biol,2002,71:339-347.
[6]刘经义,李德光,李素萍.COPD急性发作期和缓解期血清细胞因子水平的变化[J].山东医药,2010,50(33):87-88.
[7]陈延伟,艾文,李一禄,等.1-磷酸鞘氨醇在慢性阻塞性肺疾病大鼠肺部炎症作用的研究[J].武汉大学学报:医学版,2015,36(1):11-14.
[8]尚立芝,王峰,谢文英,等.爱罗咳喘宁对慢性阻塞性肺疾病大鼠可溶性细胞间黏附分子-1、肿瘤坏死因子-α、白细胞介素-6及及炎细胞的影响[J].中国老年学杂志,2015,35(17):4762-4763.
[9]尚立芝,谢文英,张良芝,等.爱罗咳喘宁对COPD大鼠p38MAPK、TNF-α及AQP5基因表达的影响[J].中国实验方剂学杂志,2014,20(23):174-179.
[10]尚立芝,谢文英,张良芝,等.爱罗咳喘宁对COPD大鼠肺组织炎症因子及氧化应激的影响[J].中国实验方剂学杂志,2014,20(24):168-171.
[11]尚立芝,谢文英,张良芝,等.爱罗咳喘宁对慢性阻塞性肺疾病大鼠白三烯B4、白细胞介素-6及肺组织病理形态的影响[J].中国实验方剂学杂志,2014,20(12):170-173.
[12]尚立芝,谢文英,张良芝,等.爱罗咳喘宁对COPD大鼠气道水通道蛋白5和黏蛋白5AC基因表达的影响[J].中国实验方剂学杂志,2014,20(22):127-132.
[13]尚立芝,张紫娟,谢文英,等.爱罗咳喘宁对慢性阻塞性肺疾病大鼠肿瘤坏死因子-α、巨噬细胞炎性蛋白-2、髓过氧化物酶及肺组织病理变化的影响[J].中国老年学杂志,2015,35(20):5705-5708.
[14]Mahadeva R,Shapiro S D.Chronic obstructive pulmonary disease:experimental animalmodels of ulmonary emphysema[J].Thorax,2002,57(10):908-914.
[15]宋小莲,王昌惠,白冲.脂多糖结合熏烟法和单纯熏烟法建立慢性阻塞性肺病大鼠模型的比较[J].第二军医大学学报,2010,31(3):246-249.
[16]宋一平,崔德健,茅培英,等.慢性阻塞性肺疾病大鼠模型的建立及药物干预的影响[J].中华内科杂志,2000,39(8):556-557.
[17]尚立芝,谢文英,张良芝,等.爱罗咳喘宁对COPD大鼠气道水通道蛋白5和黏蛋白5AC基因表达的影响[J].中国实验方剂学杂,2014,20(22):127-133.
[18]尚立芝,张紫娟,谢文英,等.爱罗咳喘宁对慢性阻塞性肺疾病大鼠肿瘤坏死因子-α、巨噬细胞炎性蛋白-2、髓过氧化物酶、炎细胞及肺组织病理变化的影响[J].中国老年学杂,2015,35(20):5705-5708.
[19]黄建华,李理,袁伟锋,等.肺表面活性物质对LPS诱导的急性肺损伤小鼠的肺保护作用及可能机制[J].免疫学杂志,2013,29(7):583-587.
[20]李亚清,严建平,许武林,等.核因子κB和p38丝裂原活化蛋白激酶调节脂多糖诱导的气道上皮细胞白细胞介素8的表达[J].中国药理学通报,2011,27(2):182-186.
[21]彭红星,杨荣时,曾玉兰,等.吸烟诱导的慢性支气管炎大鼠模型巨噬细胞炎性蛋白-2及髓氧化物酶研究[J].中国现代医学杂志,2012,22(12):10-13.
[22]王今越,丁树哲,王小虹,等.p38,NF-κB,IL-6在长期大强度运动诱导大鼠骨骼肌发生中的作用[J].体育科学,2010,30(2):75-82.
[23]Schiffer M,von Gersdorff G,Bitzer M,et al.Smad proteins and transforming growth factor-beta signaling[J].Kidney Int Suppl,2000,77(2):45-52.
[24]Ten Dijke P,Hill C S.New insights into TGF-beta-Smad signaling[J].Trends Biochem Sci,2004,29(5):265-273.
[25]Adcock I M.Glucocorticoid-regulated transcription factors[J].Pulm Pharmacol Ther,2001,14(3):211-219.
[26]王洁,张心怡,郝彩丽,等.组蛋白去乙酰化酶抑制剂的研究进展.现代生物医学进展,2014,14(14):2783-2785.
[27]Ropero S,Esteller M.The role of histone deacetylases(HDACs)in human cancer[J].Mol Oncol,2007,1(1):19-25.
[28]ZHOU L,Ruvolo V R,Mc Queen T.HDAC inhibition by SNDX-275(Entinostat)restores expression of silenced leukemia-associated transcription factors Nur77 and Nor1and of key pro-apoptotic pro-teins in AML[J].Leukemia,2013,27(6):1358-1368.
[29]WANG X,SONG Y,Jennifer J L,et al.Inhibition of histone deacetylases antagonized FGF2 and IL-1βeffects on MMP expression in human articular chondrocytes[J].Growth Factors,2009,27(1):40-49.
[30]Espallergues J,Teegarden S L,Veerakumar A,et al.HDAC6 regu-lates glucocorticoid receptor signaling in serotonin pathways with critical impact on stress resilience[J].J Neurosci,2012,32(13):4400-4416.
[31]To M,Takagi D,Akashi K,et al.Sputum plasminogen activator inhibitor-1 elevation by oxidative stressdependent nuclear factor-κB activation in COPD[J].Chest,2013,144(2):515-521.
[32]Ito K,Ito M,Elliott W M.Deacetylase activity in chronic obstructive pulmonary disease[J].N Engl J Med,2005,352(19):1967-1976.
[33]Barnes P J.Histone deacetylase-2 and airway disease[J].Ther Adv Respir Dis,2009,3(5):235-243.
[34]Pace E,Ferraro M,Di Vincenzo S,et al.Comparative cytoprotective effects of carbocysteine and fluticasone propionate in cigarette smoke extract-stimulated bronchial epithelial cells[J].Cell Stress Chaperones,2013,18(6):733-743.
[35]王玉梅,黄琼,罗鹏,等.香烟对大鼠肺白介素-8、谷胱甘肽及组蛋白去乙酰化酶2的影响[J].中国医学创新,2014,11(22):18-23.
[36]Cosio B G,Tsaprouni L,Ito K,et al.Theophylline restores histone deacetylase activity and steroid responses in COPD macrophages[J].J Exp Med,2004,200(5):689-695.
[37]Barnes P J.Role of HDAC2 in the pathophysiology of COPD[J].Annu Rev Physiol,2009,71(3):451-464.
[2]葛均波,徐永健.内科学[M].8版.北京:人民卫生出版社,2013:21-25.
[3]中华中医药学会内科分会肺系病专业委员会.慢性阻塞性肺疾病中医诊疗指南(2011版)[J].中医杂志,2012,53(2):177-178.
[4]Fisman E Z,Motro M,Tenenbaum A.Cardiovascular diabetology in the core of a novel interleukins class infication the bad the good and the aloof Cardiovase[J].Diabetoly,2003,2(1):11-15.
[5]Utsugi M,Dobashi K,Kaga Y,et al.Glntathione redox regulates lipolysaccharideinduced IL-12 production through p38 mitogen activated protein kinase activation in human monocytes role of glutathi one redox in IFNgamma prining of IL-12 productioy[J].Leukoo Biol,2002,71:339-347.
[6]刘经义,李德光,李素萍.COPD急性发作期和缓解期血清细胞因子水平的变化[J].山东医药,2010,50(33):87-88.
[7]陈延伟,艾文,李一禄,等.1-磷酸鞘氨醇在慢性阻塞性肺疾病大鼠肺部炎症作用的研究[J].武汉大学学报:医学版,2015,36(1):11-14.
[8]尚立芝,王峰,谢文英,等.爱罗咳喘宁对慢性阻塞性肺疾病大鼠可溶性细胞间黏附分子-1、肿瘤坏死因子-α、白细胞介素-6及及炎细胞的影响[J].中国老年学杂志,2015,35(17):4762-4763.
[9]尚立芝,谢文英,张良芝,等.爱罗咳喘宁对COPD大鼠p38MAPK、TNF-α及AQP5基因表达的影响[J].中国实验方剂学杂志,2014,20(23):174-179.
[10]尚立芝,谢文英,张良芝,等.爱罗咳喘宁对COPD大鼠肺组织炎症因子及氧化应激的影响[J].中国实验方剂学杂志,2014,20(24):168-171.
[11]尚立芝,谢文英,张良芝,等.爱罗咳喘宁对慢性阻塞性肺疾病大鼠白三烯B4、白细胞介素-6及肺组织病理形态的影响[J].中国实验方剂学杂志,2014,20(12):170-173.
[12]尚立芝,谢文英,张良芝,等.爱罗咳喘宁对COPD大鼠气道水通道蛋白5和黏蛋白5AC基因表达的影响[J].中国实验方剂学杂志,2014,20(22):127-132.
[13]尚立芝,张紫娟,谢文英,等.爱罗咳喘宁对慢性阻塞性肺疾病大鼠肿瘤坏死因子-α、巨噬细胞炎性蛋白-2、髓过氧化物酶及肺组织病理变化的影响[J].中国老年学杂志,2015,35(20):5705-5708.
[14]Mahadeva R,Shapiro S D.Chronic obstructive pulmonary disease:experimental animalmodels of ulmonary emphysema[J].Thorax,2002,57(10):908-914.
[15]宋小莲,王昌惠,白冲.脂多糖结合熏烟法和单纯熏烟法建立慢性阻塞性肺病大鼠模型的比较[J].第二军医大学学报,2010,31(3):246-249.
[16]宋一平,崔德健,茅培英,等.慢性阻塞性肺疾病大鼠模型的建立及药物干预的影响[J].中华内科杂志,2000,39(8):556-557.
[17]尚立芝,谢文英,张良芝,等.爱罗咳喘宁对COPD大鼠气道水通道蛋白5和黏蛋白5AC基因表达的影响[J].中国实验方剂学杂,2014,20(22):127-133.
[18]尚立芝,张紫娟,谢文英,等.爱罗咳喘宁对慢性阻塞性肺疾病大鼠肿瘤坏死因子-α、巨噬细胞炎性蛋白-2、髓过氧化物酶、炎细胞及肺组织病理变化的影响[J].中国老年学杂,2015,35(20):5705-5708.
[19]黄建华,李理,袁伟锋,等.肺表面活性物质对LPS诱导的急性肺损伤小鼠的肺保护作用及可能机制[J].免疫学杂志,2013,29(7):583-587.
[20]李亚清,严建平,许武林,等.核因子κB和p38丝裂原活化蛋白激酶调节脂多糖诱导的气道上皮细胞白细胞介素8的表达[J].中国药理学通报,2011,27(2):182-186.
[21]彭红星,杨荣时,曾玉兰,等.吸烟诱导的慢性支气管炎大鼠模型巨噬细胞炎性蛋白-2及髓氧化物酶研究[J].中国现代医学杂志,2012,22(12):10-13.
[22]王今越,丁树哲,王小虹,等.p38,NF-κB,IL-6在长期大强度运动诱导大鼠骨骼肌发生中的作用[J].体育科学,2010,30(2):75-82.
[23]Schiffer M,von Gersdorff G,Bitzer M,et al.Smad proteins and transforming growth factor-beta signaling[J].Kidney Int Suppl,2000,77(2):45-52.
[24]Ten Dijke P,Hill C S.New insights into TGF-beta-Smad signaling[J].Trends Biochem Sci,2004,29(5):265-273.
[25]Adcock I M.Glucocorticoid-regulated transcription factors[J].Pulm Pharmacol Ther,2001,14(3):211-219.
[26]王洁,张心怡,郝彩丽,等.组蛋白去乙酰化酶抑制剂的研究进展.现代生物医学进展,2014,14(14):2783-2785.
[27]Ropero S,Esteller M.The role of histone deacetylases(HDACs)in human cancer[J].Mol Oncol,2007,1(1):19-25.
[28]ZHOU L,Ruvolo V R,Mc Queen T.HDAC inhibition by SNDX-275(Entinostat)restores expression of silenced leukemia-associated transcription factors Nur77 and Nor1and of key pro-apoptotic pro-teins in AML[J].Leukemia,2013,27(6):1358-1368.
[29]WANG X,SONG Y,Jennifer J L,et al.Inhibition of histone deacetylases antagonized FGF2 and IL-1βeffects on MMP expression in human articular chondrocytes[J].Growth Factors,2009,27(1):40-49.
[30]Espallergues J,Teegarden S L,Veerakumar A,et al.HDAC6 regu-lates glucocorticoid receptor signaling in serotonin pathways with critical impact on stress resilience[J].J Neurosci,2012,32(13):4400-4416.
[31]To M,Takagi D,Akashi K,et al.Sputum plasminogen activator inhibitor-1 elevation by oxidative stressdependent nuclear factor-κB activation in COPD[J].Chest,2013,144(2):515-521.
[32]Ito K,Ito M,Elliott W M.Deacetylase activity in chronic obstructive pulmonary disease[J].N Engl J Med,2005,352(19):1967-1976.
[33]Barnes P J.Histone deacetylase-2 and airway disease[J].Ther Adv Respir Dis,2009,3(5):235-243.
[34]Pace E,Ferraro M,Di Vincenzo S,et al.Comparative cytoprotective effects of carbocysteine and fluticasone propionate in cigarette smoke extract-stimulated bronchial epithelial cells[J].Cell Stress Chaperones,2013,18(6):733-743.
[35]王玉梅,黄琼,罗鹏,等.香烟对大鼠肺白介素-8、谷胱甘肽及组蛋白去乙酰化酶2的影响[J].中国医学创新,2014,11(22):18-23.
[36]Cosio B G,Tsaprouni L,Ito K,et al.Theophylline restores histone deacetylase activity and steroid responses in COPD macrophages[J].J Exp Med,2004,200(5):689-695.
[37]Barnes P J.Role of HDAC2 in the pathophysiology of COPD[J].Annu Rev Physiol,2009,71(3):451-464.