蛋白酶激活受体-1 拮抗剂对矽肺大鼠肺纤维化的干预作用

Intervention effect of protease activated receptor-1 antagonist on pulmonary fibrosis in silicosis rats

关键词: 矽肺  纤维化  凝血级联反应  沃拉帕沙(Vorapaxar)  蛋白酶激活受体-1(PAR-1)

Keywords: silicosis  fibrosis  coagulation cascade reaction  Vorapaxar  protease activated receptor-1 (PAR-1)

摘 要: 目的 探讨矽肺大鼠凝血级联反应变化情况以及蛋白酶激活受体-1(PAR-1)拮抗剂对其肺纤维化的干预作用。方法 27 只 SPF 级健康雄性 Wistar 大鼠随机分为对照组、模型组( SiO₂ )及 Vorapaxar(PAR-1拮抗剂)组,采用口鼻吸入暴露法建立矽肺大鼠模型; SiO₂粉尘浓度 800mg/m³, 染尘 6h/d,连续吸入 28d;Vorapaxar 组动物于造模第 1 天开始给予 Vorapaxar(3.5mg/kg)灌胃。每组动物分别在染毒后第 14、28、56d 处死,收集肺组织及血清;采用 HE、Masson 染色观察各组大鼠肺组织病理和纤维化变化,免疫组化法检测肺组织转化生长因子(TGF)-β1 表达,酶联免疫吸附试验(ELISA)检测各组大鼠血清克拉拉蛋白(CC16)、组织因子(TF)、凝血酶-抗凝血酶复合物(TAT)和纤溶酶原激活物抑制因子(PAI-1)水平,蛋白免疫印迹试验(Western blot)检测各组大鼠肺组织胶原蛋白Ⅰ(Col-Ⅰ)、整合素(αvβ)5、αvβ6、PAR-1 和结缔组织生长因子(CTGF)的蛋白表达。结果 染尘 14、28、56d 时,血清中 TF、TAT 和 PAI-1 水平均较对照组明显升高(P<0.05),但CC16 水平显著降低(P<0.05);模型组大鼠染尘 56d 时肺内炎症和纤维化程度,肺组织 Col-Ⅰ、αvβ5、αvβ6、PAR-1、CTGF 的蛋白表达均高于对照组(P<0.05)。Vorapaxar 干预显著降低了各时间点血清 TAT、PAI-1 以及染尘 28、56d 时大鼠血清 TF 水平(P<0.05), 增高染尘 14、28d 大鼠血清 CC16 的水平(P<0.05),并使染尘 56d 大鼠肺组织炎症和纤维化程度减轻,肺组织 Col-Ⅰ、αvβ5、αvβ6、PAR-1 和 CTGF 蛋白表达水平降低(P<0.05)。结论 SiO₂ 吸入可导致大鼠肺上皮细胞损伤,激活凝血级联反应和增加肺组织 PAR-1 表达,诱发肺部炎症和纤维化;PAR-1 拮抗剂可显著抑制 SiO₂ 诱导的大鼠凝血级联反应, 减轻肺部炎症和纤维化。

Abstract: Objective To explore the changes in coagulation cascade of silicosis in rats and the therapeutic effects of protease activated receptor-1 (PAR-1) antagonist on the pulmonary fibrosis in rats.Methods Twenty-seven SPF grade healthy male Wistar rats were randomly divided into three groups: control,model (SiO₂),and Vorapaxar (PAR-1 antagonist) group.The silicosis rat model was established using intranasal inhalation of silica dust method (SiO₂ concentration was 800mg/m³),6 hours/day for 28 consecutive days;the rats in Vorapaxar group were given Vorapaxar by oral from the first day of modeling.Rats were euthanized on the 14th,28th,and 56th day after silica dust exposure,collect lung tissues and serum.Then,HE and Masson s trichrome staining were used to observe the pathological and fibrotic changes in lung tissue of the rats,immunohistochemistry was performed to detect the expression of TGF-β1 in lung tissues,enzyme linked immunosorbent assay (ELISA) was used to detect the serum levels of Clara cell secretory protein-16 (CC16),tissue factor (TF),thrombin antithrombin complex (TAT),and plasminogen activator inhibitor-1 (PAI-1) of the rats,Western blot analysis was used to assess the protein expression of collagen typeⅠ (Col-Ⅰ),integrins αvβ5 and αvβ6,PAR-1,and connective tissue growth factor (CTGF) in lung tissues of the rats.Results The results showed that the serum levels of TF,TAT,and PAI-1 in the rats of model group were significantly increased compared to the control rats on the days of 14th,28th,and 56th after silica dust exposure(P<0.05),while the serum level of CC16 level was decreased (P<0.05);meanwhile,the degrees of inflammation and fibrosis in lungs of the rats after 56 days exposure to silica dust and the expression levels of Col-Ⅰ,αvβ5,αvβ6,PAR-1,and CTGF proteins in lung tissues were all significantly higher than that in control rats (P<0.05).Vorapaxar intervention significantly reduced serum levels of TAT and PAI-1 levels at various time points,and serum TF level in rats exposed to silica dust for 28 and 56 days (P<0.05),and increased the serum CC16 level in rats exposed to silica dust for 14 and 28 days (P<0.05).Moreover, Vorapaxar might alleviate lung tissue inflammation and fibrosis,as well as might decrease the protein expression levels of Col-Ⅰ,αvβ5,αvβ6,PAR-1,and CTGF in rats exposed to dust for 56 days (P<0.05).Conclusion The results suggested that inhalation of SiO₂ dust might cause damage to epithelial cells,activate coagulation cascade reaction, and PAR-1 expression in lung tissue,leading to pulmonary inflammation and fibrosis;PAR-1 antagonist might significantly inhibit the SiO₂-induced coagulation cascade reaction,alleviates lung inflammation and fibrosis in silica dust exposed rats.