Objective: To investigate the injury of cyanate on the pulmonary function and morphology of C57/BL6N mice.
Methods: Forty male C57/BL6N mice were randomly divided into two groups: normal control group (20 mice) and cyanate group (20 mice). Mice were exposed to 100 mmol/L cyanate feeding for 4 weeks, and pulmonary Raw (Resistance in Air Way) was measured at the beginning and end of the experiment. The mice were sacrificed at the end of the fourth week of the experiment, and the lung tissues were collected for pathological observation and molecular detection of E-Cadherin and Fibronectin. Well-growing A549 cells in logarithmic growth phase were treated with cyanate at the concentrations of 0, 0.25, 0.5 and 1 mmol/L for 24 h, and the cell viability was detected by CCK8 method; reactive oxygen species ROS fluorescent probe (DCFH-DA) was used to detect the changes of ROS levels, and expressions of E-Cadherin and Fibronectin in cells and pulmonary tissues were detected by Western blot.
Results: At the beginning of the experiment, the pulmonary airway resistance values of the mice in the normal control group and the cyanate group were (1.82±0.76)cmH2O/(L·s) and (1.85±0.78)cmH2O/(L·s), respectively, with no significant difference. Four weeks later, the pulmonary airway resistance value of mice in the cyanate group was increased to (4.86±0.87)cmH2O/(L·s) (P＜0.01). The HE staining showed that, compared with the normal control group, the injured alveolar structure, the thickened tracheal wall and the significantly proliferated pulmonary interstitial tissue were observed in the cyanate group. The Masson staining showed that elastic fibers were deposited around the trachea of mice in the cyanate group. The results of CCK8 assay for the viability of A549 cells showed that 0.5 mmol/L cyanate exposure could reduce the viability (P＜0.01). The immunofluorescence staining showed that cyanate could increase ROS level in A549 cells by producing green fluorescence in a concentration-dependent manner. The results of Western blotting showed that 0.5 mmol/L of cyanate treatment on A549 cells could reduce the expression of E-Cadherin (P＜0.01) with increasing concentration of cyanate. The expression level of Fibronectin in A549 cells was increased with the increasing cyanate concentration, and there was a significant difference (P＜0.01) on 1 mmol/L cyanate. Western blot results of lung showed the decreasing expression of E-Cadherin (P＜0.01) and increasing expression of Fibronectin (P＜0.01) in cyanate mice.
Conclusion: Pathological concentrations of cyanate can induce the proliferation of pulmonary interstitial tissue, fibrous deposition, and increased pulmonary airway resistance in mice, which may be related to damaged pulmonary epithelial cell viability, enhanced ROS production, and induced pathologic changes of extracellular matrix by cyanate.
目的: 观察氰酸盐对C57/BL6N小鼠肺气道阻力和肺组织结构的影响,以及对人肺A549细胞系细胞活力和蛋白表达水平的影响。方法: 选取40只雄性C57/BL6N小鼠随机分为两组:正常对照组(20只)、氰酸盐组(20只),适应性喂养一周后,氰酸盐组是在小鼠饮水中添加100 mmol/L氰酸盐喂养4周,并分别在实验开始与结束时测定小鼠肺气道阻力(Raw)。第4周实验结束时处死小鼠,取肺组织采用HE与Masson染色法进行病理观察。另取生长良好的对数生长期A549细胞经 0、0.25、0.5、1 mmol/L 浓度的氰酸盐处理24 h后,采用CCK8法检测细胞活力;活性氧ROS荧光探针(DCFH-DA)检测细胞ROS水平变化;蛋白免疫印迹法检测肺组织与细胞E-Cadherin与Fibronectin表达情况。结果: 实验开始前,正常对照组与氰酸盐组小鼠肺气道阻力值分别为(1.82±0.76) cm H2O/(L·s)与(1.85 ±0.78) cm H2O/(L·s),差异无显著性;实验结束后,氰酸盐组小鼠肺气道阻力值增高至(4.86±0.87) cm H2O/(L·s)(P＜0.01)。HE染色结果显示:与正常对照组相比,氰酸盐组小鼠肺泡结构破坏,气管壁增厚,肺间质组织增生明显。Masson染色结果显示:氰酸盐组小鼠气管周围弹力纤维沉积。CCK8法测定A549细胞活力结果显示:0.5 mmol/L及以上浓度的氰酸盐暴露可引起A549细胞活力下降。免疫荧光结果显示,0.25 mmol/L氰酸盐即可刺激A549细胞内ROS增加而出现绿色荧光,细胞内绿色荧光强度随氰酸盐浓度增加而增强。蛋白免疫印迹法结果显示: 0.5 mmol/L的氰酸盐处理即可使A549细胞中E-Cadherin的表达水平显著降低(P＜0.01),并随浓度增加降低更加显著。A549细胞中Fibronectin的表达水平随氰酸盐浓度增加而上升,在1 mmol/L浓度氰酸盐时其表达水平显著上升(P＜0.01)。小鼠肺组织蛋白免疫印迹结果显示,与正常对照组相比,氰酸盐组小鼠E-Cadherin表达水平显著减少(P＜0.01),Fibronectin的表达水平显著增加(P＜0.01)。结论: 病理浓度的氰酸盐可以引起小鼠肺间质组织增生、纤维沉积,导致肺气道阻力增加;其机制可能与氰酸盐损伤肺上皮细胞活力、促进ROS增加,诱导细胞外基质成分的病理改变有关。.
Keywords: C57/BL6N mice; cyanate; extracellular matrix; pulmonary epithelium.