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

作　　者：张强 罗婷 袁德政 付义[4] 袁嘉丽 ZHANG Qiang;LUO Ting;YUAN Dezheng;FU Yi;YUAN Jiali(School of Basic Medicine,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;School Of Basic Medicine,Yunnan University of Traditional Chinese Medicine,Kunming 650500,Yunnan,China;School of Clinical Medicine,Yunnan University of Traditional Chinese Medicine,Kunming 650500,Yunnan,China;Department of Pulmonary Diseases,Kunming Hospital of Traditional Chinese Medicine,Kunming 650500,Yunnan,China)

机构地区：[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

出　　处：《上海中医药大学学报》2022年第5期60-68,共9页Academic Journal of Shanghai University of Traditional Chinese Medicine

基　　金：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)。

摘　　要：目的: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: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:(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Objective:To observe the intervention effect and possible mechanism of Qilongtian on bleomycin induced pulmonary fibrosis in mice.Methods: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:(1)Compared 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).

关 键 词：七龙天 肺纤维化 转录组 IL-17/p38MAPK 

分 类 号：R285.5[医药卫生—中药学]