基质金属蛋白酶对大鼠慢性阻塞性肺病模型气道细胞外基质重塑的作用及影响因素

The role of matrix metalloproteinases in airway remodelling in COPD rat models

目的:研究基质金属蛋白酶(MMPs)及其组织抑制剂(TIMP-1)在慢性阻塞性肺病(COPD)大鼠模型气道细胞外基质重塑中的作用及 N-乙酰半胱氨酸(NAC)、蛋白激酶 C 抑制剂 H7及转移生长因子(TGF-β)单抗干预的影响。方法:Wistar 大鼠随机分为(1)对照组;(2)COPD 模型组,采用熏香烟加气管注小量内毒素法复制 COPD 模型;(3)NAC 干预组(NAC 组);(4)H7干预组(H7组);(5)TGF-β单抗干预组。观察各组病理形态学改变;生化法测定支气管肺组织羟脯氨酸含量;免疫组化法和 RT-PCR 法测支气管肺组织 MMP-9、2及 TIMP-1,以及 TGF-βⅠ、Ⅱ受体的表达;SDA-PAGE 明胶酶谱学测定支气管肺组织 MMPs 酶活性。结果:模型组气道壁以Ⅰ型胶原为主的细胞外基质及羟脯氨酸含量较对照组显著增多,NAC 组及 TGF-β单抗组较模型组显著减少,H7组羟脯氨酸含量较模型组显著增多。模型组 MMP-9、2及 TIMP-1,以及 TGF-βⅠ、Ⅱ受体在气道上皮、肺泡巨噬细胞、肺血管内皮细胞等细胞中蛋白和(或)mRNA 表达显著增强,72 kdMMP-2及92 kdMMP-9酶原活性显著增高,三个干预组 MMP-2、9及 TIMP-1的蛋白及 mRNA 表达较模型组显著减弱,72 kdMMP-2及92 kdMMP-9酶原活性显著减低,三个干预组TGF-βⅠ、Ⅱ受体亦较模型组有不同程度减弱。结论:以Ⅰ型胶原为主的细胞外基质显著增生是 COPD 气道重塑的重要病理改变。COPD 气道细胞外基质降解及合成代谢异常活跃与气道细胞外基质重塑密切相关;NAC 可对 MMP-9/TIMP-1的失衡起调节作用,其表达及功能在一定程度上依赖 PKC 信号传导途径。抑制 TGF-β传导通路可能使胶原等细胞外基质降解及合成减少。

Objective;To study the role of metalloproteinasea (MMPs) in extracellular matrix remodelling of air- way in COPD rat models,and to observe the intervening effect of N-acetylcysteine (NAC),inhibitor of protein ki- nase C (H7) and TGF-βmonocolonal antibody in this process.Methods;53 Wistar rats were randomly divided into 5 groups,normal control group,COPD rat model group,NAC group,H7 group and TGF-βmonocolonal antibody group.Pathologic examinations of the airway and lung tissues were performed.Hydroxyproline contents of bron- chial lung tissue homogenates were determined with biochemistry method.Location of MMP-9,2,TIMP-1 was verified by immunohistological staining.MMP-9/2 and TIMP-1 mRNA espressions in bronchial lung tissue were determined with reverse transcription-PCR analysis.The gelatinolytic activities of MMPs in bronchial and lung tis- sues were performed with gelatin zymographic analysis.Results;The collagen deposition in the airway was signifi- cantly increased in COPD model than the control group,and it was decreased in NAC group and TGF-βmonoclonal antibody group compared with model group.Collagen in the H7 group was not decreased.Significant positive im- munoreactivities of TGF-βⅠ、Ⅱ receptor,MMP-2,MMP-9,and TIMP-1 were found in the model group com- pared with those in the control group.In treatment groups the expressions of above items were decreased,but there were no changes in TGF-βI receptor in NAC group and in MMP-9 of bronchus in TGF-βmonoclonal antibody group compared with model group.The mRNA expressions of MMP-2,MMP-9 and TIMP-1 in model group were significantly increased than the control group.The mRNA of MMP-9 and TIMP-1 were decreased in the treatment groups compared with the model group.Weak enzyme activities of MMP-2 (72kd),MMP-9 (92kd) were found in the control group,while they were strong in the model group.The activities were also weak in the other groups. Conclusion;The main features of extraceUular matrix remodeling in COPD was diffuse collagen deposition within the airway wall.The results showed that MMP-2,MMP-9 and TIMP-1 play an important role in extracellular ma- trix remodeling.The balance among MMP-2,MMP-9,TIMP-1 and TGF-βⅠ,Ⅱ receptor was regulated by PKC pathway,TGF-βand oxygen free radicals.H7,TGF-βmonoelonal antibody and NAC could downregulate their expression.TGF-β monoclonal antibody and NAC could degrade and decrease airway extracellular matrix.