七龙天干预博来霉素模型小鼠肺纤维化的作用机制研究

张强; 罗婷; 袁德政; 付义; 袁嘉丽

doi:10.16306/j.1008-861x.2022.05.011

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七龙天干预博来霉素模型小鼠肺纤维化的作用机制研究

实验研究 | 更新时间：2022-11-01

- 七龙天干预博来霉素模型小鼠肺纤维化的作用机制研究
- Mechanism study of Qilongtian on pulmonary fibrosis in bleomycin model mice
- 上海中医药大学学报 2022年36卷第5期页码：60-68
- 作者机构：
  
  1.上海中医药大学基础医学院（上海　201203）
  2.云南中医药大学基础医学院（云南　昆明　650500）
  3.云南中医药大学临床医学院（云南　昆明　650500）
  4.昆明市中医医院肺病科（云南　昆明　650500）
- 作者简介：
  
  张强，女，在读博士生，主要从事中西医结合防治重大疾病的基础研究
  袁嘉丽，教授，博士生导师；E-mail：2748132800@qq.com
- 基金信息：
  
  云南省科技中医联合重点项目［2017FF117（-007）］;云南省中医药学分子生物学重点实验室（云南中医药大学）建设项目(2019DG016)
- DOI：10.16306/j.1008-861x.2022.05.011
  中图分类号：
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张强,罗婷,袁德政等.七龙天干预博来霉素模型小鼠肺纤维化的作用机制研究[J].上海中医药大学学报,2022,36(05):60-68.

ZHANG Qiang,LUO Ting,YUAN Dezheng,et al.Mechanism study of Qilongtian on pulmonary fibrosis in bleomycin model mice[J].Academic Journal of Shanghai University of Traditional Chinese Medicine,2022,36(05):60-68.
张强,罗婷,袁德政等.七龙天干预博来霉素模型小鼠肺纤维化的作用机制研究[J].上海中医药大学学报,2022,36(05):60-68. DOI： 10.16306/j.1008-861x.2022.05.011.

ZHANG Qiang,LUO Ting,YUAN Dezheng,et al.Mechanism study of Qilongtian on pulmonary fibrosis in bleomycin model mice[J].Academic Journal of Shanghai University of Traditional Chinese Medicine,2022,36(05):60-68. DOI： 10.16306/j.1008-861x.2022.05.011.

摘要

目的,2,观察七龙天对博来霉素致小鼠肺纤维化的干预作用及可能的作用机制。,方法,2,将36只C57BL/6J小鼠随机分为空白组、模型组、七龙天低、中、高剂量组、吡非尼酮组。建立小鼠肺纤维化模型后，空白组和模型组以0.9% NaCl溶液灌胃，七龙天组和吡非尼酮组分别以七龙天和吡非尼酮灌胃。给药21 d后，采用HE染色和Masson染色检测小鼠肺组织形态和胶原沉积情况，碱水解法检测羟脯氨酸（HYP）含量，qRT-PCR法检测,TGF-β、α-SMA、COL-Ⅰ、COL-Ⅲ、TNF-α、CCL2、FOSL1、MMP9、CXCL5、AREG ,mRNA表达水平，Western blot、免疫荧光双标法检测TGF-β、α-SMA、COL-Ⅰ、COL-Ⅲ、TNF-α、IL-17R、p38MAPK、p-p38MAPK蛋白表达水平，并对空白组和模型组小鼠肺组织进行转录组测序。,结果,2,①与空白组比较，模型组小鼠肺组织呈现炎症细胞浸润和肺泡间隙扩大现象，胶原沉积较多（,P,<,0.01）；与模型组比较，发现七龙天各剂量组可抑制炎症细胞浸润和肺泡间隙扩大，且吡非尼酮组和七龙天各剂量组肺部胶原沉积明显减少（,P,<,0.05，,P,<,0.01）。②与空白组比较，模型组的HYP含量升高（,P,<,0.01）；与模型组比较，吡非尼酮组、七龙天各剂量组的HYP含量均降低（,P,<,0.05，,P,<,0.01）。③与空白组比较，模型组COL-Ⅰ、COL-Ⅲ、α-SMA、TGF-β、TNF-α的蛋白和mRNA表达水平升高（,P,<,0.01）；与模型组比较，吡非尼酮组和七龙天各剂量组的COL-Ⅰ、COL-Ⅲ、α-SMA、TGF-β、TNF-α蛋白和mRNA表达水平降低（,P,<,0.05，,P,<,0.01）。④通过对空白组和模型组小鼠肺组织的转录组测序分析可知，差异基因显著富集于IL-17/p38MAPK信号通路。⑤与空白组比较，模型组,CCL2、FOSL1、MMP9、CXCL5、AREG, mRNA和IL-17R、p-p38MAPK蛋白表达水平升高（,P,<,0.01）；与模型组比较，吡非尼酮组和七龙天各剂量组的,CCL2、FOSL1、MMP9、CXCL5、AREG ,mRNA和IL-17R、p-p38MAPK蛋白表达水平降低（,P,<,0.05，,P,<,0.01）。同时，七龙天对上述mRNA和蛋白表达的影响呈剂量依赖性，其中高剂量的作用更有效。,结论,2,七龙天可以通过下调炎症与胶原基因和蛋白的表达，减轻肺部炎症反应，降低肺部胶原过度沉积，发挥干预肺纤维化的作用，其作用机制可能为通过抑制IL-17/p38MAPK信号通路发挥作用。

Abstract

Objective： To observe the intervention effect and possible mechanism of Qilongtian on bleomycin induced pulmonary fibrosis in mice.,Methods,2,Thirty-six C57BL/6J mice were randomly divided into control group， model group， Qilongtian low， medium and high dose group， pirfenidone group. After the establishment of mouse pulmonary fibrosis model， the control group and model group were administrated with 0.9% NaCl solution by gavage， and the Qilongtian group and pirfenidone group were administrated with Qilongtian and pirfenidone by gavage respectively. After 21 d of administration， the morphology and collagen deposition of mice lung were measured by HE and Masson staining. The content of hydroxyproline （HYP） was detected by alkali hydrolysis method. The expression levels of ,TGF-β， α-SMA,， ,COL-Ⅰ， COL-Ⅲ,， ,TNF-α， CCL2， FOSL1， MMP9， CXCL5， AREG ,mRNA were detected by qRT-PCR. The expression levels of TGF-β， α-SMA， COL-Ⅰ， COL-Ⅲ， TNF-α， IL-17R， p38MAPK， and p-p38MAPK protein were detected by Western blot and immunofluorescence double labeling method.,Results,2,①Compared with the control group， the lung tissues of mice in model group showed inflammatory cell infiltration and alveolar space expansion， and the collagen deposition was more （,P,<,0.01）. Compared with the model group， Qilongtian each dose group inhibit the infiltration of inflammatory cells and the expansion of alveolar space， and collagen deposition was significantly reduced in pirfenidone group and Qilongtian each dose group （,P,<,0.05， ,P,<,0.01）. ② Compared with the control group， the content of HYP in the model group increased （,P,<,0.01）. Compared with the model group， the content of HYP in pirfenidone group and Qilongtian each dose group decreased （,P,<,0.05， ,P,<,0.01）. ③ Compared with the control group， the protein and mRNA expression level of COL-Ⅰ， COL-Ⅲ， α-SMA， TGF-β， TNF-α in the model group increased （,P,<,0.01）. Compared with the model group， the protein and mRNA expression levels of COL-Ⅰ， COL-Ⅲ， α-SMA， TGF-β， TNF-α in pirfenidone group and Qilongtian each dose group decreased （,P,<,0.05， ,P,<,0.01）. ④Transcriptome sequencing analysis of mice lung tissues in the blank group and model group showed that differential genes were significantly enriched in IL-17/p38MAPK signaling pathway. ⑤Compared with the control group， the expression levels of ,CCL2， FOSL1， MMP9， CXCL5， AREG ,mRNA and IL-17R， p-p38MAPK protein increased in the model group （,P,<,0.01）. Compared with the model group， the expression levels of ,CCL2， FOSL1， MMP9， CXCL5， AREG ,mRNA and IL-17R， p-p38MAPK protein decreased in pirfenidone group and Qilongtian each dose group （,P,<,0.05， ,P,<,0.01）. Meanwhile， the effects of Qilongtian on the above mRNA and protein expression were dose-dependent， and high dose was more effective.,Conclusion,2,Qilongtian can intervene pulmonary fibrosis by down regulating the expression of inflammation， collagen genes and proteins， alleviate pulmonary inflammatory response， reducing excessive deposition of pulmonary collagen， and its mechanism may be through inhibition of IL-17/p38MAPK signal pathway.

关键词

七龙天肺纤维化转录组IL-17/p38MAPK

Keywords

Qilongtianpulmonary fibrosistranscriptomeIL-17/p38MAPK

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