射干麻黄汤对慢性哮喘小鼠气道炎症和气道重塑的影响
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摘要
目的:观察射干麻黄汤对哮喘小鼠PPAR-γ、DC及TGF-β1表达的影响,探讨其在气道炎症和气道重塑两方面治疗哮喘的作用机制,为临床应用提供实验依据。
方法:健康清洁级雌性BALB/c小鼠60只,随机分组,每组10只。分组如下:A组(正常对照组),B组(哮喘模型组),C组(地塞米松组),D组(低剂量中药组),E组(中剂量中药组),F组(高剂量中药组)。B、C、D、E、F组建立慢性哮喘模型。D、E、F组小鼠予不同剂量的射干麻黄汤灌胃;C组予地塞米松腹腔注射。取材后,对各组小鼠采用双抗体夹心ABC-ELISA法检测BALF中TGF-β1的含量;半定量评定支气管周围炎症细胞浸润程度、杯状细胞比例;图像分析测量血管管壁厚度、气管平滑肌层厚度、气道旁和血管旁胶原纤维含量;透射电镜观察DC形态;免疫组化染色检测肺组织DC、TGF-β1及PPAR-γ表达情况;RT-PCR技术检测肺组织PPAR-γmRNA的表达水平;Western blot技术检测肺组织PPAR-γ在核蛋白中表达强度;实时定量PCR技术检测肺组织TGF-β1mRNA表达水平。
结果: 1、与正常组比较,模型组炎症细胞浸润程度明显加重,肺组织杯状细胞评分及BALF中的白细胞总数、EOS、淋巴细胞等均明显增高,均有显著性差异(P<0.05);低、中、高剂量射干麻黄汤组较模型组炎症细胞浸润程度、肺组织杯状细胞评分及BALF中的白细胞总数、EOS、淋巴细胞均降低,有显著性差异(P<0.05);中、高剂量组与地塞米松组效果相近,无显著性差异(P>0.05);中、高剂量组效果相近,无显著性差异(P>0.05)。
2、与正常组比较,模型组哮喘发作程度明显加重,肺组织血管管壁厚度、气管平滑肌层厚度、气道旁和血管旁胶原纤维含量明显增加,均有显著性差异(P<0.05);中、高剂量组较模型组哮喘发作程度减轻,肺组织血管管壁厚度、气管平滑肌层厚度、气道旁和血管旁胶原纤维含量均明显降低,有显著性差异(P<0.05);中、高剂量组与地塞米松组比较,无显著性差异(P>0.05);中、高剂量组效果相近,无显著性差异(P>0.05)。
3、与正常组比较,模型组DC数量明显增加,有显著性差异(P<0.05);低、中、高剂量射干麻黄汤组较模型组DC数量降低,有显著性差异(P<0.05);中、高剂量与地塞米松组效果相近,无显著性差异(P>0.05);中、高剂量组效果相近,无显著性差异(P>0.05)。电镜下,A组和B组小鼠肺组织DC呈典型表现,C、D、E、F组小鼠DC呈不典型表现。
4、与正常组比较,模型组肺组织PPAR-γ数量、PPAR-γmRNA及PPAR-γ在核蛋白中表达均明显增加,有显著性差异(P<0.05);地塞米松组较模型组PPAR-γ数量、PPAR-γmRNA及PPAR-γ在核蛋白中表达明显减少,而中、高剂量射干麻黄汤组PPAR-γ数量、PPAR-γmRNA及PPAR-γ在核蛋白中表达明显增强,均有显著性差异(P<0.05);中、高剂量组效果相近,无显著性差异(P>0.05)。
5、与正常组比较,模型组BALF中TGF-β1含量、肺组织TGF-β1数量、TGF-β1mRNA表达强度明显增加,有显著性差异(P<0.05);低、中、高剂量射干麻黄汤BALF中TGF-β1含量、肺组织TGF-β1数量及TGF-β1mRNA表达较模型组均明显降低,有显著性差异(P<0.05);中、高剂量组较地塞米松组效果相当,无显著性差异(P>0.05);中、高剂量组效果相近,无显著性差异(P>0.05)。
结论:1.射干麻黄汤可降低哮喘小鼠肺组织DC数量。2.射干麻黄汤可降低哮喘小鼠BALF中TGF-β1含量、肺组织TGF-β1数量及TGF-β1mRNA表达强度。3.射干麻黄汤可增加哮喘小鼠肺组织PPAR-γ数量,增强PPAR-γmRNA表达及PPAR-γ在核蛋白中的表达强度。4射干麻黄汤可通过影响DC、TGF-β1及PPAR-γ表达情况,抑制哮喘气道炎症和重塑,从而达到治疗哮喘的目的。
Objective: Through investigate the effection of She Gan Ma Huang Tang (SGMHT) on the expressions of PPAR-γ、DC and TGF-β1 in chronic asthma mice,to explore the mechanisms of SGMHT treating on asthma,and to provide experimental basis for its clinical application.
Methods: 60 clean female BALB/c mice were divided averagely into six groups according to the random number table,as follows:A(control group),B( asthma group),C(dexamethasone group),D(low dose SGMHT group),E(middle dose SGMHT group),F(high dose SGMHT group).GroupB,C,D,E,F were esta- blished model of chronic asthma.Group D,E,F were gastric irrigated with different doses of SGMHT;group C was intraperitoneal injection of dexamet- hasone.After drawn the materials in each group,detected TGF-β1 content in BALF by ABC-ELISA ;assessed the bronchial inflammatory cell infiltration and goblet cell ratio by semi-quantitative;measured the thickness of vascular wall, thickness of airway smooth muscle,collagen content of around airway vessels and perivascular by pathological image;observed DC morphology by electron microscopy;measured the numbers of DC, TGF-β1 and PPAR-γin the lung tissue by immunohistochemical staining;measured the expression of PPAR-γmRNA in lung tissue by RT-PCR;measured the expression of PPAR-γin nuclear protein by Western blot;measured the expression of TGF-β1mRNA in lung tissue by real-time PCR.
Results:1.Compared with group A,group B's inflammatory cell infiltrati- on,goblet cell scores and theleukocytes,EOS,lymphocytes in BALF increased, there were significant differences(P<0.05);compared with group B,these indi- cators decreased in group D,E,F,there were significant differences(P<0.05); compared with group C,these indicators were similar in groupE and F(P>0.05 );group E and F were similar(P>0.05).
2.Compared with group A,asthma attacks,thickness of vascular wall and airway smooth muscler,collagen content of around airway perivascular and vessels increased in group B,there were significant difference(P<0.05);comp- ared with group B,these indicators decreased in group E and F, there were significant differences(P<0.05);compared with group C,these indicators were similar in group E and F(P>0.05);group E and F were similare(P>0.05).
3.Compared with group A,group B's the number of DC increased,there was significant difference(P<0.05);in group D,E,F,the number of DC is lower than group B(P<0.05);compared with groupC,groupE and F were similar(P> 0.05);groupE and F was similar(P>0.05).Observe DC morphology in lung tissue by electron microscopy, groupA and B were typical show, group C,D,E, F were untypical show.
4.Compared with group A,the number of PPAR-γ,the expression of PPA- R-γmRNA and PPAR-γin the nuclear protein increased in group B,there were significant difference(P<0.05);compared with groupB,groupC's indicators are decreased,group E andF's are increased,there were significant difference(P< 0.05);group E and F were similar(P>0.05).
5.Compared with group A,the content of TGF-β1 in the BALF,the number of TGF-β1 and the expression of TGF-β1mRNA in lung increased in groupB(P <0.05);in group D,E and F,these indicators are lower than group B(P<0.05); compared with group C,group E and F's indicators were similar(P>0.05); groupE and F were similar,there were no significant difference(P>0.05).
Conclusion: 1.SGMHT could reduce the number of DC in lung tissue of asthmatic mice.2.SGMHT could reduce the content of TGF-β1 in BALF,the expressions of TGF-β1 and TGF-β1mRNA in lung tissue of asthmatic mice.3. SGMHT could increase PPAR-γnumber of lung,enhance the expression of PPAR-γmRNA and PPAR-γof nuclear protein in lung tissue of asthmatic mice. 4.This experiment prompted SGMHT could influence the expressions of DC, TGF-β1 and PPAR-γ,inhibit airway inflammation and remodelling, so as to achieve the purpose of treatment of asthma.
方法:健康清洁级雌性BALB/c小鼠60只,随机分组,每组10只。分组如下:A组(正常对照组),B组(哮喘模型组),C组(地塞米松组),D组(低剂量中药组),E组(中剂量中药组),F组(高剂量中药组)。B、C、D、E、F组建立慢性哮喘模型。D、E、F组小鼠予不同剂量的射干麻黄汤灌胃;C组予地塞米松腹腔注射。取材后,对各组小鼠采用双抗体夹心ABC-ELISA法检测BALF中TGF-β1的含量;半定量评定支气管周围炎症细胞浸润程度、杯状细胞比例;图像分析测量血管管壁厚度、气管平滑肌层厚度、气道旁和血管旁胶原纤维含量;透射电镜观察DC形态;免疫组化染色检测肺组织DC、TGF-β1及PPAR-γ表达情况;RT-PCR技术检测肺组织PPAR-γmRNA的表达水平;Western blot技术检测肺组织PPAR-γ在核蛋白中表达强度;实时定量PCR技术检测肺组织TGF-β1mRNA表达水平。
结果: 1、与正常组比较,模型组炎症细胞浸润程度明显加重,肺组织杯状细胞评分及BALF中的白细胞总数、EOS、淋巴细胞等均明显增高,均有显著性差异(P<0.05);低、中、高剂量射干麻黄汤组较模型组炎症细胞浸润程度、肺组织杯状细胞评分及BALF中的白细胞总数、EOS、淋巴细胞均降低,有显著性差异(P<0.05);中、高剂量组与地塞米松组效果相近,无显著性差异(P>0.05);中、高剂量组效果相近,无显著性差异(P>0.05)。
2、与正常组比较,模型组哮喘发作程度明显加重,肺组织血管管壁厚度、气管平滑肌层厚度、气道旁和血管旁胶原纤维含量明显增加,均有显著性差异(P<0.05);中、高剂量组较模型组哮喘发作程度减轻,肺组织血管管壁厚度、气管平滑肌层厚度、气道旁和血管旁胶原纤维含量均明显降低,有显著性差异(P<0.05);中、高剂量组与地塞米松组比较,无显著性差异(P>0.05);中、高剂量组效果相近,无显著性差异(P>0.05)。
3、与正常组比较,模型组DC数量明显增加,有显著性差异(P<0.05);低、中、高剂量射干麻黄汤组较模型组DC数量降低,有显著性差异(P<0.05);中、高剂量与地塞米松组效果相近,无显著性差异(P>0.05);中、高剂量组效果相近,无显著性差异(P>0.05)。电镜下,A组和B组小鼠肺组织DC呈典型表现,C、D、E、F组小鼠DC呈不典型表现。
4、与正常组比较,模型组肺组织PPAR-γ数量、PPAR-γmRNA及PPAR-γ在核蛋白中表达均明显增加,有显著性差异(P<0.05);地塞米松组较模型组PPAR-γ数量、PPAR-γmRNA及PPAR-γ在核蛋白中表达明显减少,而中、高剂量射干麻黄汤组PPAR-γ数量、PPAR-γmRNA及PPAR-γ在核蛋白中表达明显增强,均有显著性差异(P<0.05);中、高剂量组效果相近,无显著性差异(P>0.05)。
5、与正常组比较,模型组BALF中TGF-β1含量、肺组织TGF-β1数量、TGF-β1mRNA表达强度明显增加,有显著性差异(P<0.05);低、中、高剂量射干麻黄汤BALF中TGF-β1含量、肺组织TGF-β1数量及TGF-β1mRNA表达较模型组均明显降低,有显著性差异(P<0.05);中、高剂量组较地塞米松组效果相当,无显著性差异(P>0.05);中、高剂量组效果相近,无显著性差异(P>0.05)。
结论:1.射干麻黄汤可降低哮喘小鼠肺组织DC数量。2.射干麻黄汤可降低哮喘小鼠BALF中TGF-β1含量、肺组织TGF-β1数量及TGF-β1mRNA表达强度。3.射干麻黄汤可增加哮喘小鼠肺组织PPAR-γ数量,增强PPAR-γmRNA表达及PPAR-γ在核蛋白中的表达强度。4射干麻黄汤可通过影响DC、TGF-β1及PPAR-γ表达情况,抑制哮喘气道炎症和重塑,从而达到治疗哮喘的目的。
Objective: Through investigate the effection of She Gan Ma Huang Tang (SGMHT) on the expressions of PPAR-γ、DC and TGF-β1 in chronic asthma mice,to explore the mechanisms of SGMHT treating on asthma,and to provide experimental basis for its clinical application.
Methods: 60 clean female BALB/c mice were divided averagely into six groups according to the random number table,as follows:A(control group),B( asthma group),C(dexamethasone group),D(low dose SGMHT group),E(middle dose SGMHT group),F(high dose SGMHT group).GroupB,C,D,E,F were esta- blished model of chronic asthma.Group D,E,F were gastric irrigated with different doses of SGMHT;group C was intraperitoneal injection of dexamet- hasone.After drawn the materials in each group,detected TGF-β1 content in BALF by ABC-ELISA ;assessed the bronchial inflammatory cell infiltration and goblet cell ratio by semi-quantitative;measured the thickness of vascular wall, thickness of airway smooth muscle,collagen content of around airway vessels and perivascular by pathological image;observed DC morphology by electron microscopy;measured the numbers of DC, TGF-β1 and PPAR-γin the lung tissue by immunohistochemical staining;measured the expression of PPAR-γmRNA in lung tissue by RT-PCR;measured the expression of PPAR-γin nuclear protein by Western blot;measured the expression of TGF-β1mRNA in lung tissue by real-time PCR.
Results:1.Compared with group A,group B's inflammatory cell infiltrati- on,goblet cell scores and theleukocytes,EOS,lymphocytes in BALF increased, there were significant differences(P<0.05);compared with group B,these indi- cators decreased in group D,E,F,there were significant differences(P<0.05); compared with group C,these indicators were similar in groupE and F(P>0.05 );group E and F were similar(P>0.05).
2.Compared with group A,asthma attacks,thickness of vascular wall and airway smooth muscler,collagen content of around airway perivascular and vessels increased in group B,there were significant difference(P<0.05);comp- ared with group B,these indicators decreased in group E and F, there were significant differences(P<0.05);compared with group C,these indicators were similar in group E and F(P>0.05);group E and F were similare(P>0.05).
3.Compared with group A,group B's the number of DC increased,there was significant difference(P<0.05);in group D,E,F,the number of DC is lower than group B(P<0.05);compared with groupC,groupE and F were similar(P> 0.05);groupE and F was similar(P>0.05).Observe DC morphology in lung tissue by electron microscopy, groupA and B were typical show, group C,D,E, F were untypical show.
4.Compared with group A,the number of PPAR-γ,the expression of PPA- R-γmRNA and PPAR-γin the nuclear protein increased in group B,there were significant difference(P<0.05);compared with groupB,groupC's indicators are decreased,group E andF's are increased,there were significant difference(P< 0.05);group E and F were similar(P>0.05).
5.Compared with group A,the content of TGF-β1 in the BALF,the number of TGF-β1 and the expression of TGF-β1mRNA in lung increased in groupB(P <0.05);in group D,E and F,these indicators are lower than group B(P<0.05); compared with group C,group E and F's indicators were similar(P>0.05); groupE and F were similar,there were no significant difference(P>0.05).
Conclusion: 1.SGMHT could reduce the number of DC in lung tissue of asthmatic mice.2.SGMHT could reduce the content of TGF-β1 in BALF,the expressions of TGF-β1 and TGF-β1mRNA in lung tissue of asthmatic mice.3. SGMHT could increase PPAR-γnumber of lung,enhance the expression of PPAR-γmRNA and PPAR-γof nuclear protein in lung tissue of asthmatic mice. 4.This experiment prompted SGMHT could influence the expressions of DC, TGF-β1 and PPAR-γ,inhibit airway inflammation and remodelling, so as to achieve the purpose of treatment of asthma.
引文
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