养清尘肺方对矽肺模型大鼠上皮细胞间质转化的影响

Effect of Yangqing Chenfei Formula on epithelial-mesenchymal transition in silicon dioxide-induced silicosis rats

目的探讨养清尘肺方(YCF)对矽肺模型大鼠肺组织上皮细胞间质转化的影响。方法选择无特定病原体级雄性SD大鼠,随机分为对照组、模型组、汉防己甲素组和YCF组,每组8只。后3组大鼠均一次性气管内注入l.00 mL质量浓度为50.0 g/L的二氧化硅混悬液,对照组大鼠予等体积0.9%氯化钠溶液;于造模后第15天,汉防己甲素组大鼠予汉防己甲素27.0 mg/kg体质量,YCF组大鼠予YCF 8.91 g/kg体质量,对照组和模型组大鼠均予2.00 mL的0.9%氯化钠溶液;每日早、晚共灌胃2次,连续灌胃14 d。实验第29天检测各组大鼠潮气量、功能残气量(FRC)和肺活量后,分离大鼠肺组织,采用苏木素-伊红染色法和Masson染色观察肺组织病理情况并进行纤维化评分,采用比色法检测羟脯氧酸水平,采用免疫组织化学法检测Ⅰ型胶原(COL-Ⅰ)、Ⅲ型胶原(COL-Ⅲ)、E-钙黏蛋白(E-Cad)、N-钙黏蛋白(N-Cad)和α-平滑肌肌动蛋白(α-SMA)表达,采用免疫荧光法检测上皮细胞黏附分子(EpCAM)和成纤维细胞特异性蛋白1(FSP-1)表达。结果对照组大鼠肺组织结构完整,肺泡结构基本正常;模型组大鼠肺泡结构破坏,肺泡壁增厚,可见细胞性结节;汉防己甲素组和YCF组大鼠肺组织病变均较模型组减轻,且该两组间病变程度无差异。与对照组比较,模型组大鼠潮气量、FRC和肺活量均降低(P值均<0.05),肺组织中E-Cad蛋白相对表达水平降低(P<0.05),纤维化评分和肺组织中羟脯氨酸、COL-Ⅰ、COL-Ⅲ、N-Cad和α-SMA蛋白相对表达水平均增加(P值均<0.05),EpCAM蛋白荧光强度降低,而FSP-1蛋白荧光强度增加。与模型组比较,汉防己甲素组和YCF组大鼠潮气量、FRC和肺活量均增加(P值均<0.05),肺组织纤维化评分、羟脯氨酸水平和COL-Ⅰ、N-Cad、α-SMA蛋白相对表达水平均降低(P值均<0.05),E-Cad蛋白相对表达水平均增加(P值均<0.05),EpCAM蛋白荧光强度均增加,FSP-1蛋白荧光强度均降低。与汉防己甲素组比较,YCF组大鼠仅肺组织中N-Cad相对表达水平降低(P<0.05)。结论YCF能够改善二氧化硅诱导的矽肺模型大鼠肺功能和胶原沉积,抑制肺组织上皮细胞间质转化,从而改善矽肺纤维化进程。

Objective To investigate the effect of Yangqing Chenfei Formula(YCF)on epithelial-mesenchymal transition(EMT)in lung tissues of silicosis model rats.Methods Specific pathogen free adult male SD rats were randomly divided into control group,model group,tetrandrine group and YCF group,with eight rats in each group.The rats in the model group,tetrandrine group and YCF group were intratracheally injected with 1.00 mL of silica suspension with a mass concentration of 50.0 g/L,and the rats in the control group were given an equal volume of 0.9%sodium chloride solution.On the 15th day after modeling,the tetrandrine group was given tetrandrine at a dose of 27.0 mg/kg body weight,the YCF group was given YCF with a dose of 8.91 g/kg body weight,while both the control group and model group were given 2.00 mL 0.9%sodium chloride solution.Gavage was performed twice a day in the morning and evening for 14 days.On day 29 of the experiment,after evaluating the tidal volume,functional residual volume(FRC)and vital capacity of rats in each group,lung tissues were collected,and hematoxylin-eosin staining and Masson staining were performed to examine the histopathological changes,and the fibrosis score was evaluated.Hydroxyproline level was detected by colorimetry.The expression of typeⅠcollagen(COL-Ⅰ),typeⅢcollagen(COL-Ⅲ),E-cadherin(E-Cad),N-cadherin(N-Cad)andα-smooth muscle actin(α-SMA)protein was detected by immunohistochemistry.The expression of epithelial cell adhesion molecule(EpCAM)and fibroblast specific protein 1(FSP-1)was detected by immunofluorescence.Results The lung structure was intact and the alveolar structure was normal in the control group.The alveolar structure was destroyed,the alveolar wall was thickened,and cellular nodules were observed/n the model group.The lung tissue lesions of rats in the tetrandrine group and YCF group were reduced compared with that in the model group,and there was no difference in the degree of lesions between the two groups.The tidal volume,FRC and vital capacity of rats in model group decreased(all P<0.05),the relative expression of E-Cad protein in lung tissue decreased(P<0.05),the fibrosis score and the level of hydroxyproline,the protein relative expression of COL-Ⅰ,COL-Ⅲ,N-Cad andα-SMA in lung tissue increased(all P<0.05),while the fluorescence intensity of EpCAM protein decreased,and that of FSP-1 protein increased compared with the control group.The tidal volume,FRC and vital capacity of rats in tetrandrine and YCF groups increased(all P<0.05),the fibrosis score and the level of hydroxyproline,the protein relative expression of COL-Ⅰ,N-Cad andα-SMA in lung tissue decreased(all P<0.05),the relative expression of E-Cad protein in lung tissues increased(P<0.05),while the EpCAM protein fluorescence intensity increased and FSP-1 protein fluorescence intensity decreased compared with the model group.The relative expression of N-Cad protein in lung tissues of YCF group was lower than that of the tetrandrine group(P<0.05).Conclusion YCF can improve the lung function,alleviate collagen deposition in lung tissues,and inhibit the epithelial-mesenchymal transition in silicosis model rats,and then attenuates the progression of silicotic fibrosis.