吸入激素对哮喘大鼠基质金属蛋白酶-9及其抑制剂的表达调控

目的探讨基质金属蛋白酶9(MMP9)及其组织抑制物(TIMP1)在支气管哮喘发病中的作用,评价吸入糖皮质激素对其调节作用。方法建立哮喘大鼠模型,用ELISA、免疫组织化学、westernblot及RTPCR技术,对激素治疗前后哮喘大鼠肺组织中MMP9及TIMP1表达进行研究。结果(1)通过大鼠行为学改变、呼吸功能曲线、IgE水平以及肺组织切片证实哮喘模型成立。(2)哮喘组肺组织细小支气管壁及伴行动脉周围炎性细胞浸润,黏膜断裂,杯状细胞增生,平滑肌增厚;21天组气道壁基膜增厚和平滑肌增生更加明显。(3)大鼠肺组织MMP9mRNA表达:哮喘7天组为(2.71±0.37),21天组为(1.76±0.27),激素治疗组为(0.88±0.18),正常对照组为(0.52±0.10),组间比较差异有统计学意义(F=151.52,P<0.01)。大鼠肺组织TIMP1mRNA表达:哮喘7天组为(1.13±0.19),21天组为(1.55±0.24),激素治疗组为(0.77±0.15),正常对照组为(0.47±0.08),组间比较差异有统计学意义(F=69.46,P<0.01)。(4)免疫组织化学及蛋白质表达结果与RTPCR结果一致。结论从蛋白质水平及mRNA水平证实哮喘大鼠模型肺组织MMP9表达升高,TIMP1表达亦增强;激素下调MMP9及TIMP1表达,可能是其抑制气道炎症和气道重塑的机制之一。

Regulatory effects of inhaled steroids on expression of matrix metalloproteinase-9 and its inhibitor in asthmatic rats

OBJECTIVE: To investigate the role of matrix metalloproteinase-9 (MMP-9) and its tissue inhibitor (TIMP-1) in the pathogenesis of bronchial asthma and assess the effect of steroid treatment on MMP-9 and TIMP-1 levels. Matrix metalloproteinases are a family of zinc and calcium-dependent endopeptidases. Many MMPs such as MMP-1, MMP-2, MMP-3 are associated with asthma, in which MMP-9 is the key factor in asthma. Tissue inhibitor-1 of metalloproteinases is a specific inhibitor of MMP-9; the MMP-9 and TIMP-1 imbalance could lead to airway inflammation and remodeling in lung disease such as asthma. METHODS: Forty Wistar rats were divided into 4 groups randomly: control, asthma model 7 days (7-day group), asthma model 21 days (21-day group) and steroid treatment groups. Asthma model of rats were established by ovalbumin (OVA) sensitization and challenge with mist inhalation. The expression of MMP-9 and TIMP-1 in lung tissues was detected by immunocytochemistry, RT-PCR and Western blotting. RESULTS: (1) By observing the changes of action, tracing respiratory curves, detecting level of serum IgE level and observing the lung tissues sections, the authors demonstrated that the rat asthmatic models were successfully established. (2) The lung tissue sections of the asthma groups stained with hematoxiline and eosin (HE) showed many inflammatory cell infiltrations around the bronchioli and accompanying arterioles, hyperplasia of caliciform cells, broken bronchial mucous membrane and thickening of submucosal layer. The hyperplasia of airway smooth muscle and basement membrane were more significant in asthma model 21-day group than that in 7-day group. These changes were improved after treatment. (3) The expression of MMP-9 in rat's lung tissues: the expression was 2.71 +/- 0.37 in 7-day group, 1.76 +/- 0.27 in 21-day group, 0.88 +/- 0.18 in the treatment group and 0.52 +/- 0.10 in the control group (F = 151.52, P < 0.01). The expression of TIMP-1 in rat's lung tissues was 1.13 +/- 0.19 in the 7-day group, 1.55 +/- 0.24 in 21-day group, 0.77 +/- 0.15 in the treatment group and 0.47 +/- 0.08 in the control group (F = 69.46, P < 0.01). (4) The results of immunocytochemistry and protein expression were consistent with those of RT-PCR. CONCLUSION: The protein and mRNA expression level of MMP-9 and TIMP-1 was high in asthmatic rat's lung tissues. Down-regulation of the expression of MMP-9 and TIMP-1 by steroids may be one of the mechanisms by which airway inflammation and remodeling are inhibited in asthma.