南葶苈子中的指标成分槲皮素-3-O-β-D-葡萄糖基-7-O-β-D-龙胆双糖苷对慢性阻塞性肺疾病大鼠肺部免疫功能调节机制研究
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摘要
目的研究南葶苈子中的指标成分槲皮素-3-O-β-D-葡萄糖基-7-O-β-D-龙胆双糖苷(QGG)对慢性阻塞性肺疾病(COPD)大鼠肺部免疫功能调节机制,阐释南葶苈子止咳平喘功效的物质基础与科学内涵。方法 SD大鼠随机分为对照组、模型组及QGG高(QGGH)、中(QGGM)、低(QGGL)剂量(0.20、0.15、0.10 g/kg)组和阳性对照氨茶碱(0.09 g/kg)组,连续8周采用香烟熏暴露联合细菌感染的方法制备COPD稳定期大鼠模型。各给药组大鼠于烟熏5周后开始ig给药,连续28 d。采用荧光定量PCR(q RT-PCR)法检测大鼠肺组织中Th17和Treg细胞特异性转录因子RORγt和FOXP3 mRNA表达水平;流式细胞仪检测Th17/Treg细胞比例;TUNEL法观察QGG对大鼠肺组织细胞凋亡的影响;Western blotting法检测大鼠肺组织蛋白提取液中凋亡相关蛋白(cytoC、Caspase-9、Caspase-3、Bax、Bcl-2)及胞质转录因子-κBp65(NF-κBp65)和胞核NF-κBp65的表达。结果对照组大鼠体质量增长率随周龄增长而明显增加,模型组大鼠体质量在第1～8周增长率变化缓慢。与模型组比较,QGGH和氨茶碱组大鼠体质量增长率从第5周开始呈增长趋势,而QGGM、QGGL组大鼠体质量增长缓慢。q RT-PCR检测结果显示,与对照组比较,模型组大鼠肺组织中Treg和Th17细胞相关转录因子FOXP3和RORγt的表达水平显著增高(P<0.05),FOXP3与RORγt比值显著升高。与模型组比较,QGGH组和阳性对照组大鼠FOXP3和RORγt的表达均显著降低(P<0.05),且QGGH组效果优于氨茶碱组。流式细胞仪检测结果表明,与模型组比较,QGGH组大鼠外周血中CD4~+IL-17~+/CD4~+和CD4~+FOXP3+/CD4~+的表达均显著下调(P<0.05);CD4~+IL-17~+/CD4~+FOXP3+显著降低(P<0.05)。氨茶碱组大鼠外周血中Th17和Treg细胞相关的表达与QGGH组相近,但是CD4~+IL-17~+/CD4~+FOXP3+无明显差异。与对照组比较,模型组大鼠肺组织细胞凋亡显著增加。与模型组比较,QGGH组和氨茶碱组大鼠肺组织细胞凋亡显著减少(P<0.05),QGGM、QGGL组改变不明显。与对照组比较,模型组大鼠肺组织Bax、cytoC、Caspase-9、Caspase-3、胞核NF-κBp65表达水平显著升高(P<0.05),而Bcl-2、胞质NF-κBp65表达水平显著降低(P<0.05)。与模型组比较,QGGH组和氨茶碱组大鼠肺组织Bax、cytoC、Caspase-9、Caspase-3、胞核NF-κBp65表达水平显著降低(P<0.05),而Bcl-2、胞质NF-κBp65表达水平显著升高(P<0.05)。结论 QGG可以显著改善COPD模型大鼠的生存状态、体质量,降低外周血CD4~+IL-17~+/FOXP3+CD4~+,调控Treg和Th17特征性转录因子FOXP3和RORγt的基因表达,平衡外周血中Th17与Treg比例,抑制肺组织细胞凋亡,修复损伤的组织,维持器官功能的完整性。
Objective To explore the mechanism of QGG from Descurainia sophia on the airway inflammation and remodeling in COPD rats. Methods SD Rats were randomly divided into six groups, including control, model, QGG high(QGGH, 0.20 g/kg), medium(QGGM 0.15 g/kg), low(QGGL 0.10 g/kg) groups and aminophylline group(0.09 g/kg). Rats with COPD in the stable phase were established by exposure to tobacco smoking combined with persistent bacterial infections for eight weeks. Rat in each group were ig administrated for twenty-eight days successive five weeks after smoking. Quantitative RT-PCR was applied to determine the mRNA expression of Th17 and Treg specific transcription factors RORγt and FOXP3. Ratio of Th17/Treg was tested by flow cytometry.Apoptosis cells of lung tissue were assayed by TUNEL. The expression of apoptotic related proteins cyctoC, Caspase-9, Caspase-3,bax, bcl-2, nucleus NF-κBp65, and cytoplasm NF-κBp65 in protein extracts of lung tissue were determined by Western blotting.Results The growth rate of control group increased obviously with weeks, however, that of model group increased slowly in COPD establishment stage and the following smoking stage. The growth rate in QGGH and aminophylline groups increased quickly from the fifth week but in QGGL and QGGM groups increased slowly. The expression of RORγt and FOXP3 was increased in model group(P <0.05). Compared with the model group, the expression of RORγt and FOXP3 was decreased in the QGGH and positive groups(P <0.05). The expression of CD4~+IL-17+/CD4~+, FOXP3+CD4~+/CD4 and the ratio of CD4~+IL-17+/FOXP3+CD4~+ in peripheral blood was significantly increased in the model group(P < 0.05). Compared with that of model, the expression of CD4~+IL-17+/CD4~+,FOXP3+CD4~+/CD4 and the ratio of CD4~+IL-17+/FOXP3+CD4~+ were decreased in the QGGH group(P < 0.05). The expression of CD4~+IL-17+/CD4~+, FOXP3+CD4~+/CD4 was decreased in the positive group, but the ratio of CD4~+IL-17+/FOXP3+CD4~+ was not changed. Compared with the control group, the apoptosis of lung tissues in the model group was significantly increased. Compared with the model group, apoptosis in the QGGH and positive groups was decreased(P < 0.05). The expression of bax, cytoC, Caspase-9, Caspase-3,and nucleus NF-κBp65 were significantly increased in the lung tissues of model rats, while that of bcl-2 and cytoplasm NF-κBp65 were decreased(P < 0.05). Compared with the model group, the expression of bax, cytoC, Caspase-9, Caspase-3, and nucleus NF-κBp65 in QGGH and positive groups were obviously decreased(P < 0.05), but that of bcl-2 and cytoplasm NF-κBp65 increased(P < 0.05).Conclusion QGG can significantly improve the survival conditions, growth ratio and pulmonary functions of rats. QGG can reduce the ratio of CD4~+IL-17+/FOXP3+CD4~+ in peripheral blood. It can modulate Treg and Th17 specific transcriptional factors FOXP3 and RORγt,balance the ratio of Th17/Treg. QGG can reduce the apoptosis of lung tissues, repair damaged tissue, and maintain the integrity of organ.
Objective To explore the mechanism of QGG from Descurainia sophia on the airway inflammation and remodeling in COPD rats. Methods SD Rats were randomly divided into six groups, including control, model, QGG high(QGGH, 0.20 g/kg), medium(QGGM 0.15 g/kg), low(QGGL 0.10 g/kg) groups and aminophylline group(0.09 g/kg). Rats with COPD in the stable phase were established by exposure to tobacco smoking combined with persistent bacterial infections for eight weeks. Rat in each group were ig administrated for twenty-eight days successive five weeks after smoking. Quantitative RT-PCR was applied to determine the mRNA expression of Th17 and Treg specific transcription factors RORγt and FOXP3. Ratio of Th17/Treg was tested by flow cytometry.Apoptosis cells of lung tissue were assayed by TUNEL. The expression of apoptotic related proteins cyctoC, Caspase-9, Caspase-3,bax, bcl-2, nucleus NF-κBp65, and cytoplasm NF-κBp65 in protein extracts of lung tissue were determined by Western blotting.Results The growth rate of control group increased obviously with weeks, however, that of model group increased slowly in COPD establishment stage and the following smoking stage. The growth rate in QGGH and aminophylline groups increased quickly from the fifth week but in QGGL and QGGM groups increased slowly. The expression of RORγt and FOXP3 was increased in model group(P <0.05). Compared with the model group, the expression of RORγt and FOXP3 was decreased in the QGGH and positive groups(P <0.05). The expression of CD4~+IL-17+/CD4~+, FOXP3+CD4~+/CD4 and the ratio of CD4~+IL-17+/FOXP3+CD4~+ in peripheral blood was significantly increased in the model group(P < 0.05). Compared with that of model, the expression of CD4~+IL-17+/CD4~+,FOXP3+CD4~+/CD4 and the ratio of CD4~+IL-17+/FOXP3+CD4~+ were decreased in the QGGH group(P < 0.05). The expression of CD4~+IL-17+/CD4~+, FOXP3+CD4~+/CD4 was decreased in the positive group, but the ratio of CD4~+IL-17+/FOXP3+CD4~+ was not changed. Compared with the control group, the apoptosis of lung tissues in the model group was significantly increased. Compared with the model group, apoptosis in the QGGH and positive groups was decreased(P < 0.05). The expression of bax, cytoC, Caspase-9, Caspase-3,and nucleus NF-κBp65 were significantly increased in the lung tissues of model rats, while that of bcl-2 and cytoplasm NF-κBp65 were decreased(P < 0.05). Compared with the model group, the expression of bax, cytoC, Caspase-9, Caspase-3, and nucleus NF-κBp65 in QGGH and positive groups were obviously decreased(P < 0.05), but that of bcl-2 and cytoplasm NF-κBp65 increased(P < 0.05).Conclusion QGG can significantly improve the survival conditions, growth ratio and pulmonary functions of rats. QGG can reduce the ratio of CD4~+IL-17+/FOXP3+CD4~+ in peripheral blood. It can modulate Treg and Th17 specific transcriptional factors FOXP3 and RORγt,balance the ratio of Th17/Treg. QGG can reduce the apoptosis of lung tissues, repair damaged tissue, and maintain the integrity of organ.
引文
[1]魏伟超,吴伟,王创畅.邓铁涛五脏相关理论在慢性阻塞性肺疾病治疗中的应用[J].中医杂志,2017,58(23):2068-2070.
[2]中国药典[S].一部.2015.
[3]Nie Y C,Wu H,Li P B,et al.Characteristic comparison of three rat models induced by cigarette smoke or combined with LPS:To establish a suitable model for study of airway mucus hypersecretion in chronic obstructive pulmonary disease[J].Pulm Pharmacol Ther,2012,25(5):349-356.
[4]朱闽,徐楠,何清湖,等.湿热消腰部热敷疗法对EAP小鼠模型前列腺Thl7/Treg相关特异性因子表达的影响[J].中华男科学杂志,2017,23(3):243-250.
[5]杨霞,邱诗林,李煜梅,等.烟草烟雾暴露小鼠中CD4+Foxp3+调节性T细胞亚型与Th1/Th17变化的实验研究[J].广西医科大学学报,2017,34(4):514-517.
[6]许和平,姚津剑,吴多益,等.细胞色素C氧化酶-2/前列腺素E2信号通路对老年慢性阻塞性肺疾病患者肺血管内皮细胞凋亡的作用[J].中国老年学杂志,2017,37(14):3432-3433.
[7]童祥丽,朱洁,杨程,等.益气化痰散瘀方含药血清对低氧大鼠肺动脉平滑肌细胞凋亡的影响[J].北京中医药大学学报,2017,40(3):200-207.
[8]Pauwels R A,Buist A S,Calverley P M,et al.Global strategy for the diagnosis,management,and prevention of chronic obstructive pulmonary disease.NHLBI/WHOglobal initiative for chronic obstructive lung disease(GOLD)workshop summary[J].Am J Respir Crit Care Med,2001,163(5):1256-1276.
[9]杨群,万丽玲,丁兆辉,等.调补肺肾膏调治慢性阻塞性肺疾病稳定期肺肾两虚证的临床研究[J].中医临床研究,2016,8(27):108-110.
[10]李连怀,张淑华,常广敬,等.南葶苈子和北葶苈子的电镜扫描与理化鉴别[J].河北医学院学报,1989,1(3):1-3.
[11]宋一平,崔德健,茅培英,等.慢性阻塞性肺疾病大鼠模型气道重塑及生长因子的研究[J].中华结核和呼吸杂志,2001,24(5):283-287.
[12]Vila J,Isaacs J D,Anderson A E.Regulatory T cells and autoimmunity[J].Curr Opin Hematol,2009,16(4):274-279.
[13]Yssel H,Lecart S,Pene J.Regulatory T cells and allergic asthma[J].Microb Infect,2001,3(11):899-904.
[14]Chatila T A.Role of regulatory T cells in human diseases[J].J Allergy Clin Immunol,2005,116(5):949-959.
[2]中国药典[S].一部.2015.
[3]Nie Y C,Wu H,Li P B,et al.Characteristic comparison of three rat models induced by cigarette smoke or combined with LPS:To establish a suitable model for study of airway mucus hypersecretion in chronic obstructive pulmonary disease[J].Pulm Pharmacol Ther,2012,25(5):349-356.
[4]朱闽,徐楠,何清湖,等.湿热消腰部热敷疗法对EAP小鼠模型前列腺Thl7/Treg相关特异性因子表达的影响[J].中华男科学杂志,2017,23(3):243-250.
[5]杨霞,邱诗林,李煜梅,等.烟草烟雾暴露小鼠中CD4+Foxp3+调节性T细胞亚型与Th1/Th17变化的实验研究[J].广西医科大学学报,2017,34(4):514-517.
[6]许和平,姚津剑,吴多益,等.细胞色素C氧化酶-2/前列腺素E2信号通路对老年慢性阻塞性肺疾病患者肺血管内皮细胞凋亡的作用[J].中国老年学杂志,2017,37(14):3432-3433.
[7]童祥丽,朱洁,杨程,等.益气化痰散瘀方含药血清对低氧大鼠肺动脉平滑肌细胞凋亡的影响[J].北京中医药大学学报,2017,40(3):200-207.
[8]Pauwels R A,Buist A S,Calverley P M,et al.Global strategy for the diagnosis,management,and prevention of chronic obstructive pulmonary disease.NHLBI/WHOglobal initiative for chronic obstructive lung disease(GOLD)workshop summary[J].Am J Respir Crit Care Med,2001,163(5):1256-1276.
[9]杨群,万丽玲,丁兆辉,等.调补肺肾膏调治慢性阻塞性肺疾病稳定期肺肾两虚证的临床研究[J].中医临床研究,2016,8(27):108-110.
[10]李连怀,张淑华,常广敬,等.南葶苈子和北葶苈子的电镜扫描与理化鉴别[J].河北医学院学报,1989,1(3):1-3.
[11]宋一平,崔德健,茅培英,等.慢性阻塞性肺疾病大鼠模型气道重塑及生长因子的研究[J].中华结核和呼吸杂志,2001,24(5):283-287.
[12]Vila J,Isaacs J D,Anderson A E.Regulatory T cells and autoimmunity[J].Curr Opin Hematol,2009,16(4):274-279.
[13]Yssel H,Lecart S,Pene J.Regulatory T cells and allergic asthma[J].Microb Infect,2001,3(11):899-904.
[14]Chatila T A.Role of regulatory T cells in human diseases[J].J Allergy Clin Immunol,2005,116(5):949-959.