螺内酯对实验性矽肺小鼠的干预研究

Effect of spirolactone on mouse model of experimental silicosis

目的：探讨螺内酯（spirolactone,SPI）在实验性小鼠矽肺病理进展中的作用及机制。方法：将120只雄性C57 BL/6小鼠（18~20 g）随机分为生理盐水组（NS组）、石英组（quartz组）、石英＋生理盐水组（quartz＋NS组）和石英＋螺内酯组（quartz＋SPI组）,经口咽吸入石英混悬液建立实验性小鼠矽肺模型;quartz＋SPI组及quartz＋NS组每天分别经灌胃给予螺内酯10 mg/kg或等体积的生理盐水。术后3 d、14 d和28 d处死小鼠,行常规支气管肺泡灌洗术,回收支气管肺泡灌洗液（bronchoalveolar lavage fluid,BALF）进行细胞分类和计数;采用ELISA法检测BALF上清中转化生长因子β1（TGF-β1）的含量;采用HE染色、Masson染色和免疫荧光染色观察肺组织病理学变化及纤维化程度;采用RT-q PCR检测各组肺组织中胶原Ⅰ（Col Ⅰ）、胶原Ⅲ（Col Ⅲ）及TGF-β1的mRNA表达水平。结果：经口咽吸入石英后,小鼠肺组织经历了典型的急性炎症期和纤维化期改变,给予SPI干预后的小鼠肺组织炎症反应程度相比于quartz＋NS组减轻（P〈0.05）。给予螺内酯干预后,在第3天小鼠肺组织TGF-β1的mRNA表达量显著下调（P〈0.01）,BALF中TGF-β1的含量也显著减少（P〈0.01）。到第28天,quartz＋SPI组小鼠肺组织Col Ⅰ、Col Ⅲ的mRNA表达量明显低于quartz＋NS组（P〈0.05）。结论：SPI可能通过调控TGF-β1的mRNA表达,减少TGF-β1蛋白的分泌,从而改善石英导致的早期急性炎症反应,并在纤维化期减轻了Col Ⅰ和Col Ⅲ的mRNA表达,抑制了纤维化的进展。

AIM： To investigate the role of spirolactone（ SPI） in the progression of quartz-induced experimental silicosis mouse model. METHODS： Male C57 BL /6 mice（ n = 120,18 ~ 20 g） were randomly divided into normal saline（ NS） group,quartz group,quartz ＋ SPI group and quartz ＋ NS group. Oropharyngeal aspiration of quartz suspension was performed to make model. The mice in quartz ＋ SPI group and quartz ＋ NS group were treated with SPI（ 10 mg / kg） or the same volume of NS daily by oral gavage throughout the experiment. The mice were sacrificed following anesthesia on days 3,14 and 28 after treatment. The total number and different types of bronchoalveolar lavage fluid（ BALF）-derived cells were routinely counted. HE staining,Masson＇s trichrome staining and immunofluorescent staining were used to conduct histopathologic examination. The mRNA expression levels of collagen Ⅰ（ Col Ⅰ）,collagen Ⅲ（ Col Ⅲ） and transforming growth factor-β1（ TGF-β1） were examined by real-time PCR. The content of TGF-β1 in BALF was measured by ELISA. RESULTS： The classical pathological stages of quartz-induced lung injury,including acute inflammation phase（ 3d）,progressive fibrosis phase（ 14 d） and late fibrosis phase（ 28 d）,were observed. Compared with quartz ＋ NS group,the inflammatory responses of the lungs in quartz ＋ SPI group were attenuated in the acute inflammation phase,and the degree of fibrosis was significantly reduced at 28 d after the inspiration of quartz. After the treatment with SPI,the mRNA expression of Col Ⅰ,Col Ⅲ and TGF-β1 in the lung tissues was effectively down-regulated. Meanwhile,the content of TGF-β1 in BALF was reduced. CONCLUSION： Treatment with SPI may significantly attenuates quartz-induced early inflammatory responses of the lung tissues by regulating the mRNA expression of TGF-β1 and secretion of TGF-β1 protein,and reduces the mRNA expression of Col Ⅰ and Col Ⅲ at fibrotic phase,resulting in the inhibition of the progression of fibrosis.