miR-204-3P对二氧化硅粉尘所致大鼠矽肺纤维化的干预作用

Intervention effect of miR-204-3P on silica dust-induced silicosis fibrosis in rats

[背景]长期暴露于游离二氧化硅颗粒会导致肺组织的纤维化,微小核糖核酸(miRNA)的异常表达可以影响纤维化的发生和进程。[目的]通过非暴露气管滴入二氧化硅粉尘悬液构建大鼠矽肺模型,通过尾静脉注射miR-204-3p过表达腺病毒观察其对二氧化硅粉尘所致矽肺纤维化的干预作用。[方法] 40只SD大鼠随机分为4组:对照组、矽肺模型组、miRNA-NC组和miR-204-3p干预组。在乙醚麻醉下,矽肺模型组、miRNA-NC组和miR-204-3p干预组的大鼠一次性气管非暴露注入游离二氧化硅粉尘悬液1 mL(50 mg·mL-1),而对照组则注入相同体积的生理盐水。染尘30 d时,miR-204-3p干预组尾静脉注射包装rno-mir-204的腺病毒载体过表达miR-204-3p,miRNA-NC组给予空病毒载体,并在正常饲养30 d后使用水合氯醛麻醉处死动物,取肺组织进行后续实验。采用实时荧光定量PCR(RT-qPCR)检测各组大鼠肺组织中miR-204-3p的相对表达水平,苏木精伊红染色、马森染色、天狼星红染色进行病理学观察,免疫组织化学法检测纤连蛋白(Fibronectin)、Ⅰ型胶原蛋白(Collagen Ⅰ)在各组大鼠肺组织中的表达情况,RTqPCR检测各组大鼠肺组织中纤维化标志物Fibronectin、波形蛋白(Vimentin)、Collagen Ⅰ、Ⅲ型胶原蛋白(Collagen Ⅲ)的mRNA相对表达水平,采用Western blot法检测各组大鼠肺组织中纤维化标志物Fibronectin、Vimentin、Collagen Ⅰ、Collagen Ⅲ的蛋白表达水平。[结果]肺组织大体解剖结果显示:正常肺组织呈粉红色,质地柔软表面光滑,而矽肺模型组织呈灰白色,质地较硬,表面可见瘢痕和灰白色的矽结节。与矽肺模型组相比miR-204-3p干预组肺组织的颜色变得红润,表面较为光滑,质地也变得柔软。染色结果显示:正常肺组织组织肺泡壁薄、肺泡内有少量的毛细血管、肺泡结构清晰完整;矽肺模型组肺组织中肺泡壁变厚、肺间隔部分断裂、肺泡结构缺损并伴随大量胶原纤维的沉积;miR-204-3p干预组的肺组织肺泡结构比较清晰且有少量胶原纤维的沉积。RT-qPCR结果显示:与对照组相比,矽肺模型组和miRNA-NC组肺组织中miR-204-3p的相对表达水平均降低(P <0.05),miR-204-3p干预组肺组织中miR-204-3p的相对表达水平明显升高(P <0.05)。免疫组织化学结果显示:与对照组相比矽肺模型组肺组织中Fibronectin和Collagen Ⅰ的表达水平均升高(P <0.05),与矽肺模型组相比,miR-204-3p干预组大鼠肺组织中Fibronectin和Collagen Ⅰ的相对表达水平明显降低(P <0.05)。RT-qPCR和Western blot结果显示:与对照组相比矽肺模型组大鼠肺组织中纤维化因子Fibronectin、Vimentin、Collagen Ⅰ、Collagen Ⅲ的基因相对表达水平和蛋白相对表达水平上升(P <0.05),与矽肺模型组相比,miR-204-3p干预组大鼠肺组织中纤维化因子Fibronectin、Vimentin、Collagen Ⅰ、Collagen Ⅲ的基因相对表达水平和蛋白相对表达水平均有所降低(P <0.05)。[结论]二氧化硅粉尘可引起大鼠肺组织纤维化,体内过表达miR-204-3P可以减轻二氧化硅粉尘引起的大鼠矽肺纤维化。

[Background]Long-term exposure to free silica particles will lead to fibrosis of lung tissue,and abnormal expression of microRNA(miRNA)may affect the occurrence and process of fibrosis.[Objective]To observed possible intervention effect of miR-204-3p overexpression adenovirus on silicosis fibrosis induced by silica dust using a silicosis rat model via non-exposed intratracheal instillation.[Methods]Forty SD rats were randomly divided into four groups:control group,silicosis model group,miRNA-NC group,and miR-204-3p intervention group.Under ether anesthesia,rats in the silicosis model group,miRNA-NC group,and miR-204-3p intervention group were injected with 1 mL(50 mg·mL−1)of free silica dust suspension into the trachea,while the control group was injected with the same volume of normal saline.After 30 d of dust exposure,the miR-204-3p intervention group was injected with rno-mir-204 adenovirus vector to overexpress miR-204-3p,and the miRNA-NC group was given empty virus vector.After 30 d of normal feeding,the animals were sacrificed by chloral hydrate anesthesia,and the lung tissue was taken for subsequent experiments.The relative expression level of miR-204-3p in lung tissue of rats in each group was detected by real-time fluorescence quantitative PCR(RT-qPCR).HE staining,Masson staining,and Sirius red staining were used for pathological observation.Immunohistochemistry was used to detect the expression of Fibronectin and Collagen I in lung tissue of rats in each group.RT-qPCR was used to detect the relative gene expression levels of fibrosis markers Fi-bronectin,Vimentin,Collagen I,and Collagen III in lung tissue of rats in each group.Western blot was used to detect the protein expression levels of fibrosis markers Fibronectin,Vimentin,Collagen I,and Collagen III in lung tissue of rats in each group.[Results]The anatomical features of lung tissue in the control group were pink lung tissue with soft texture and smooth surface,while those in the silicosis model were grayish white tissue with hard texture and scars and grayish white silicon nodules on the surface.Compared with the silicosis model group,the color of lung tissue in the miR-204-3p intervention group became ruddy,the surface was smooth,and the texture became soft.The staining results showed that the alveolar wall of the control group was thin,there were a small number of capillaries in the alveoli,and the alveolar structure was clear and complete.In the silicosis model group,the alveolar wall became thicker,the pulmonary septum was partially broken,the alveolar structure was defective,and a large amount of collagen fibers were deposited.The alveolar structure of the miR-204-3p intervention group was relatively clear and there was a small amount of collagen fiber deposi-tion.RT-qPCR results showed that compared with the control group,the relative expression levels of miR-204-3p in lung tissue of the silicosis model group and the miRNA-NC group were decreased(P<0.05),and the relative expression level of miR-204-3p in lung tissue of the miR-204-3p intervention group was increased(P<0.05).The results of immunohistochemistry showed that compared with the control group,the expression levels of Fibronectin and Collagen I in lung tissue of the silicosis model group were increased(P<0.05).Compared with the silicosis model group,the relative expression levels of Fibronectin and Collagen I in lung tissue of the rats in the miR-204-3p in-tervention group were significantly decreased(P<0.05).The results of RT-qPCR and Western blot showed that compared with the control group,the relative protein and gene expression levels of fibrosis factors Fibronectin,Vimentin,Collagen I,and Collagen III in lung tissue of the silicosis model group increased(P<0.05).Compared with the silicosis model group,the relative gene and protein expression levels of fibrosis factors Fibronectin,Vimentin,Collagen I,and Collagen III in lung tissue of rats in the miR-204-3p intervention group were decreased(P<0.05).[Conclusion]Silica dust can cause lung fibrosis in rats,and overexpression of miR-204-3P in vivo can reduce silicosis fibrosis in rats caused by silica dust.