miR-204-3P对二氧化硅粉尘所致大鼠矽肺纤维化的干预作用

胡文选, 俞静, 陈芳, 皮洋阳, 焦子铭, 王发选

胡文选, 俞静, 陈芳, 皮洋阳, 焦子铭, 王发选. miR-204-3P对二氧化硅粉尘所致大鼠矽肺纤维化的干预作用[J]. 环境与职业医学, 2024, 41(4): 367-374. DOI: 10.11836/JEOM23341
引用本文: 胡文选, 俞静, 陈芳, 皮洋阳, 焦子铭, 王发选. miR-204-3P对二氧化硅粉尘所致大鼠矽肺纤维化的干预作用[J]. 环境与职业医学, 2024, 41(4): 367-374. DOI: 10.11836/JEOM23341
HU Wenxuan, YU Jing, CHEN Fang, PI Yangyang, JIAO Ziming, WANG Faxuan. Intervention effect of miR-204-3P on silica dust-induced silicosis fibrosis in rats[J]. Journal of Environmental and Occupational Medicine, 2024, 41(4): 367-374. DOI: 10.11836/JEOM23341
Citation: HU Wenxuan, YU Jing, CHEN Fang, PI Yangyang, JIAO Ziming, WANG Faxuan. Intervention effect of miR-204-3P on silica dust-induced silicosis fibrosis in rats[J]. Journal of Environmental and Occupational Medicine, 2024, 41(4): 367-374. DOI: 10.11836/JEOM23341

miR-204-3P对二氧化硅粉尘所致大鼠矽肺纤维化的干预作用

基金项目: 国家自然科学基金项目(82060584)
详细信息
    作者简介:

    胡文选(1997—),男,硕士生;E-mail:huwenxuan1208@163.com

    通讯作者:

    王发选,E-mail:faxuan203@163.com

  • 中图分类号: R114

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

Funds: This study was funded.
More Information
  • 摘要:
    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,RT-qPCR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。

     

    Abstract:
    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 Fibronectin, 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 deposition. 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 intervention 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.

     

  • 矽肺是一种由吸入游离二氧化硅粉尘引起的间质性肺部疾病,其特征是急性和慢性炎症伴弥漫性纤维化[1]。《2021年我国卫生健康事业发展统计公报》显示:全国2021年职业病新发病例共报告15407例,其中职业性尘肺病11809例(职业性尘肺病占职业病新发病例的76.6%),是我国发病率最高的职业病,其中矽肺病占了大约40%,是尘肺病中危害最严重的一种[23]。由于矽肺发病具有不可逆性,导致了较高的发病率和患病率,然而除肺移植外,迄今为止仍然没有有效的药物或治疗方法来逆转或阻止矽肺病纤维化过程[4]。因此,矽肺纤维化新的治疗靶点的发现对制定有效的矽肺治疗策略有重要意义。目前对于矽肺纤维化具有治疗潜力的方法主要包括抗纤维化药物、抗细胞因子治疗、抗氧化剂和微小核糖核酸(microRNA, miRNA)等[5]。miRNA是长度为20~24 nt的非编码单链RNA分子,主要通过转录抑制或降解信使RNA(messenger ribonucleic acid, mRNA)参与基因的转录和转录后调控,多项研究表明miRNA的失调在肺纤维化过程中发挥作用[67]。课题组前期建立了大鼠矽肺模型,通过miRNA微阵列分析对可能参与矽肺病纤维化的miRNA进行了大规模筛选,结果发现miR-204-3P在矽肺大鼠肺组织中表达降低[8],Li等[9]研究发现miR-204-3P可以靶向结合转化生长因子-β I型受体(transforming growth factor-beta receptor type I, TGF-βR I),从而调节晶状体上皮细胞的迁移和上皮间充质转化。那么miR-204-3P是否在二氧化硅所致矽肺纤维化过程发挥着同样的作用呢?为了验证miR-204-3P对二氧化硅粉尘所致大鼠矽肺纤维化的干预作用,本研究通过建立经典大鼠矽肺模型进行miR-204-3P的干预实验,对miR-204-3P在二氧化硅粉尘所致大鼠矽肺纤维化中的影响进行观察,进一步明确miR-204-3P在矽肺纤维化中的干预效果,为矽肺纤维化的干预提供可能的分子干预靶点。

    二氧化硅(0.5~10 μm,99%(美国Sigma公司);rno-mir-204腺病毒载体(锐博生物科技有限公司)、生理盐水、4%多聚甲醛、Triton X-100、牛血清白蛋白(bovine serum albumin, BSA)、Tween-20(北京索莱宝生物公司);RNAstore Reagent(北京天根生化公司);Trizol试剂聚偏二氟乙烯(PVDF)膜(美国Invitrogen公司);异丙醇(天津大茂化学试剂厂);cDNA反转录试剂盒(北京全式金生物公司);全蛋白提取试剂盒、二辛可酸(bicinchoninic acid, BCA)蛋白浓度检测试剂盒、Lysis buffer(南京凯基生物公司);5×loarding buffer上样缓冲液(北京康为世纪生物科技有限公司);三羟甲基氨基甲烷[tris(hydroxymethyl)methyl aminomethane, Tris]、甘氨酸(glycine, Gly)、十二烷基硫酸钠(sodium dodecyl sulfate, SDS)(北京博奥拓达科技有限公司);脱脂奶粉(美国BD公司);通用型抗体稀释液(苏州新赛美生物公司);甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase, GAPDH)单克隆抗体(60004-1-Ig)(美国Proteintech公司);I型胶原兔多克隆抗体(ab34710)、III型胶原兔多克隆抗体(ab7778)、纤连蛋白兔单克隆抗体(ab45688)、波形蛋白小鼠单克隆抗体(ab8978)(英国abcam公司);辣根酶标记山羊抗兔IgG(ZB-2301)、辣根酶标记山羊抗小鼠IgG(ZB-5305)(北京中杉金桥生物公司);Multiskan GO全波长酶标仪、超灵敏多功能成像仪、NanoDrop™ One/OneC微量紫外-可见分光光度计(美国Thermo公司);CFX96实时荧光定量PCR仪(美国Bio-Rad公司);CK倒置光学显微镜(德国Leica公司)。

    从宁夏医科大学实验动物中心购买6~8周龄(200 g±10 g)SPF级SD雄性大鼠40只,并由宁夏医科大学伦理委员会审查和批准(编号:IACUC-NYLAC-2021-191)。SD大鼠培养7~10 d。采用随机数表法将40只大鼠分为4组(每组n=10):对照组、矽肺模型组、miRNA-NC组、miR-204-3p干预组。矽肺模型组、miRNA-NC组、miR-204-3p干预组大鼠在乙醚麻醉下,一次性气管非暴露注入游离二氧化硅粉尘1 mL(50 mg·mL−1),对照组注入等体积无菌生理盐水。在染尘第30天,miR-204-3p干预组尾静脉注射包装rno-mir-204腺病毒载体,而miRNA-NC组给予空病毒载体,正常饲养30 d后水合氯醛麻醉处死动物。

    使用麻醉剂(10%水合氯醛300 mg·kg−1)使大鼠处于无痛苦状态,将其固定并在大鼠胸腔中央做切口,取出肺组织,取左肺上叶用RNAstore保存,用于mRNA表达水平的检测,右上肺用4%多聚甲醛进行固定用于组织病理学分析,收取右下肺速冻并储存在−80°C以进行蛋白表达水平的测定。

    HE染色、Masson染色和天狼星红染色:首先从固定液中取出组织进行脱水,包埋,并将包埋组织切成5 μm厚的切片,将切片固定在载玻片上放入乙醇中脱水,随后进行染色,染色10 min后进行脱水和透明处理,用显微镜观察染色结果。

    免疫组织化学:将组织从固定液中取出进行脱水处理。随后放入二甲苯、梯度稀释的乙醇中进行脱蜡,并将组织切片置于盛满乙二胺四乙酸(ethylene diamine tetraacetic acid,EDTA)抗原修复缓冲液的修复盒中,用微波炉加热进行抗原修复接着将切片放入3%过氧化氢溶液中消除内源性过氧化物酶的活性,并用PBS(pH=7.4)洗涤。用5% BSA进行封闭后加入一抗过夜孵育,使用PBS洗涤3次后使用二抗孵育并再次洗涤,然后滴加二氨基联苯胺(diaminobenzidine,DAB)显色液显色,最后进行苏木素复染、分化、返蓝和脱水透明处理,经乙醇梯度脱水后封片并在显微镜下拍片并观察实验结果,每组选取3个生物学样本进行分析。

    取RNAstore中保存的肺组织与离心管中(每组选取3个生物学样本),加入Trizol试剂后离心,将上清液转移到新管中并加入异丙醇混合使RNA沉淀,用75%乙醇洗涤RNA沉淀去除杂质,用焦碳酸二乙酯水溶解RNA沉淀得到纯化的RNA溶液。使用微量紫外-可见分光光度计测定所提取RNA的质量和浓度。使用所提取的RNA为模板用逆转录试剂盒配置逆转录体系进行cDNA的合成,以此为模板进行实时荧光定量,miRNA和mRNA分别以U6和GAPDH为内参,将实验所获得的各组内参和目的因子的定量循环(quantification cycle, CQ)值基于2−∆∆Ct法分别计算miR-204-3p、纤连蛋白(Fibronectin)、波形蛋白(Vimentin)I型胶原蛋白(Collagen I)、III型胶原蛋白(Collagen I)的mRNA相对表达水平,最后通过GraphPad Prism 10进行作图、SPSS 25软件进行实验数据的整理和分析。所需U6和miRNA的引物购买自广州锐博生物公司,其他mRNA引物由上海生工生物合成,引物序列见表1

    表  1  RT-qPCR引物序列
    Table  1.  RT-qPCR primer sequences
    基因名称
    (Gene name)
    引物序列(Primer sequence)
    正向反向
    FibronectinAGGCACAAGGTCCGAGAAGAGGCATGAGTCATCCGTAGGCTGGTTC
    VimentinGACCGCTTCGCCAACTACATCGCGCAACTCCCTCATCTCCTCCTC
    Collagen IGACAGGCGAACAAGGTGACAGAGTGCAGGACCAGAAGCTCCAGAG
    Collagen IIIAGTCGGAGGAATGGGTGGCTATCCAGGAGATCCAGGATGTCCAGAGG
    GAPDHCTGGAGAAACCTGCCAAGTATGGGTGGAAGAATGGGAGTTGCT
    下载: 导出CSV 
    | 显示表格

    将冻存的组织取出,分别取各组肺组织于离心管中加入裂解液和研磨珠充分研磨(每组选取3个生物学样本进行检测),经裂解、离心等步骤获得纯净的蛋白质溶液,用BCA定量法对所提取蛋白质溶液进行浓度检测,通过计算、稀释、高温变性将所提取各组蛋白质溶液配制为2 μg·μL−1的蛋白质溶液。将等体积的组织提取液在7.5%和10%十二烷基硫酸钠-聚丙烯酰胺凝胶上进行凝胶电泳分离(上层胶电压80 V,下层胶电压120 V),根据分子量大小将蛋白质分离成不同的条带,用湿转法将凝胶中的蛋白质转移到PVDF膜上形成蛋白质的印迹,条带在5%脱脂牛奶在室温下封闭2 h,用TBST缓冲盐溶液(Tris缓冲盐水与吐温20)洗膜3次,每次10 min,然后在4 ℃下与用抗体稀释液稀释的一抗(1∶1000)孵育过夜并用TBST缓冲盐溶液洗膜3次,将膜与相应的二抗孵育1 h后再次用TBST缓冲盐溶液洗涤3次,最后将蛋白质条带由Invitrogen iBright凝胶成像系统可视化,最后通过ImageJ1.51a进行灰度值分析。

    采用SPSS 22.0软件对实验数据进行统计分析,作图采用Graphpad 9.5软件,实验数据以均数±标准差表示,各组间基因和蛋白表达水平比较采用单因素方差分析,组间两两比较采用Tukey检验,检验水准为α=0.05。

    RT-qPCR结果如图所示(图1):与对照组相比,矽肺模型组和miRNA-NC组肺组织中miR-204-3p的相对表达水平降低(P<0.05);miR-204-3p干预组与矽肺模型组相比,肺组织中miR-204-3p的相对表达水平明显升高(P<0.05)。

    图  1  各组大鼠肺组织中miR-204-3p的相对表达水平
    a:对照组;b:矽肺模型组;c:miRNA-NC组;d:miR-204-3p干预组。数据以均数±标准差表示,n=3。**:与对照组相比,P<0.05;##:与矽肺模型组相比,P<0.05。
    Figure  1.  The relative expression level of miR-204-3 p in lung tissue of rats in each group
    a: Control group; b: Silicosis model group; c: miRNA-NC group; d: miR-204-3p intervention group. The data are expressed as mean ± standard deviation, n=3 for each group. **: Compared with the control group, P<0.05; ##: Compared with the silicosis model group, P<0.05.

    组织大体图(图2)显示:正常肺组织呈粉红色,质地柔软,表面光滑;矽肺模型组织呈灰白色,质地硬,表面出现疤痕和灰白色矽结节;经miR-204-3p干预后的肺组织色泽较红润,表面逐渐光滑,质地变得柔软。

    图  2  各组大鼠肺组织的大体图片
    a:对照组;b:矽肺模型组;c:miRNA-NC组;d:miR-204-3p干预组。图中黑色箭头所指为矽结节。
    Figure  2.  Anatomic presentation of lung tissue of rats in each group
    a: Control group; b: Silicosis model group; c: miRNA-NC group; d: miR-204-3p intervention group. The black arrow in the figure refers to silicon nodules.

    图3结果显示:正常肺组织HE染色结果显示肺泡结构完整,肺泡壁薄,间质内少量胶原纤维,细胞排列有序,没有明显的炎症细胞浸润和纤维化表现,Masson染色显示少量的胶原纤维分布在间质内,天狼星红染色呈现为较为均匀的纤维结构,颜色较浅。矽肺纤维化组的HE染色结果显示肺泡结构被破坏,肺泡壁增厚,间质内明显增多的胶原纤维,伴有炎症细胞浸润,肺泡腔内出现明显纤维化斑块,Masson染色显示大量的胶原纤维沉积在肺泡壁和间质内,形成纤维化斑块,呈现深蓝色,而天狼星红染色显示大量的胶原纤维呈现为深红色,形成纤维化区域。体内过表达miR-204-3p的肺组织HE染色结果显示肺泡结构得到一定程度的修复,肺泡壁变薄,间质内胶原纤维减少,炎症反应减轻,纤维化斑块减少,Masson染色和天狼星红染色结果显示胶原纤维的密度减少,纤维化斑块减少颜色较浅。表明体内过表达miR-204-3p对二氧化硅所致矽肺纤维化有一定缓解作用。

    图  3  各组大鼠肺组织HE染色(A)、Masson染色(B)、天狼星红染色(C)结果(×100)
    a:对照组;b:矽肺模型组;c:miRNA-NC组;d:miR-204-3p干预组。
    Figure  3.  Results of HE staining (A), Masson staining (B), and Sirius red staining (C) in lung tissue of rats in each group (×100)
    a: Control group; b: Silicosis model group; c: miRNA-NC group; d: miR-204-3p intervention group.

    免疫组织化学结果如图45所示:与对照组相比,矽肺模型组和miRNA-NC组肺组织中Fibronectin和Collagen I的表达水平升高(P<0.05);miR-204-3p干预组与矽肺模型组相比,肺组织中Fibronectin和Collagen I的表达水平明显降低(P<0.05)。

    图  4  各组大鼠肺组织Fibronectin(A)和Collagen I(B)免疫组织化学染色结果(×200)
    a:对照组;b:矽肺模型组;c:miRNA-NC组;d:miR-204-3p干预组。阳性表达呈棕黄色或褐色,着色程度代表阳性程度。
    Figure  4.  Immunohistochemical results of Fibronectin (A)and Collagen I (B)in lung tissue of rats in each group (×200 )
    a: Control group; b: Silicosis model group; c: miRNA-NC group; d: miR-204-3p intervention group. The positive expression is brownish yellow or brown, and the degree of staining represents the positive degree.
    图  5  各组大鼠肺组织Fibronectin和Collagen I平均光密度值
    数据以均数±标准差表示,n=3;a:对照组;b:矽肺模型组;c:miRNA-NC组;d:miR-204-3p干预组。**:与对照组相比,P<0.05;##:与矽肺模型组相比,P<0.05。
    Figure  5.  The average optical density values of Fibronectin and Collagen I in lung tissue of rats in each group
    The data are expressed as mean ± standard deviation, n=3 for each group; a: Control group; b: Silicosis model group; c: miRNA-NC group; d: miR-204-3p intervention group. **: Compared with the control group, P<0.05; ##: Compared with the silicosis model group, P<0.05.

    RT-qPCR结果如图6:与对照组相比,矽肺模型组和miRNA-NC组肺组织中Fibronectin1、Vimentin、Collagen I、Collagen III的mRNA相对表达水平均升高(P<0.05);miR-204-3p干预组与矽肺模型组相比,大鼠肺组织中Fibronectin1VimentinCollagen ICollagen III的mRNA相对表达水平显著降低(P<0.05)。

    图  6  各组大鼠肺组织中纤维化相关因子mRNA相对表达水平
    数据以均数±标准差表示,n=3;a:对照组;b:矽肺模型组;c:miRNA-NC组;d:miR-204-3p干预组。**:与对照组相比,P<0.05;##:与矽肺模型组相比,P<0.05。
    Figure  6.  The relative mRNA expression levels of fibrosis-related factors in lung tissue of rats in each group
    The data are expressed as mean ± standard deviation, n=3 for each group; a: Control group; b: Silicosis model group; c: miRNA-NC group; d: miR-204-3p intervention group; **: Compared with the control group, P<0.05; ##: Compared with the silicosis model group, P<0.05.

    Western blot结果如图7所示:与对照组相比,矽肺模型组和miRNA-NC组肺组织中Fibronectin、Vimentin、Collagen I、Collagen III的蛋白表达水平均升高(P<0.05);miR-204-3p干预组与矽肺模型组相比,大鼠肺组织中Fibronectin、Vimentin、Collagen I、Collagen III的蛋白表达水平明显降低(P<0.05)。

    图  7  各组大鼠肺组织中纤维化相关因子的蛋白相对表达水平
    图A:蛋白条带图;图B、C、D、E:Fibronectin、Vimentin、Collagen I、Collagen III蛋白相对表达量;a:对照组;b:矽肺模型组;c:miRNA-NC组;d:miR-204-3p干预组。**:与对照组相比,P<0.05;##:与矽肺模型组相比,P<0.05。
    Figure  7.  The relative protein expression levels of fibrosis-related factors in lung tissue of rats in each group
    A : Protein band; B, C, D, E: Relative protein expression levels of Fibronectin, Vimentin, Collagen I, and Collagen III; a: Control group; b: Silicosis model group; c: miRNA-NC group; d: miR-204-3p intervention group. **: Compared with the control group, P<0.05; ##: Compared with the silicosis model group, P<0.05.

    二氧化硅引起的矽肺纤维化仍然是一个严重的公共卫生问题。职业环境中二氧化硅暴露仍然是矽肺纤维化的主要原因[10]。当吸入含有二氧化硅粉尘的空气时,粉尘颗粒会沉积在肺组织中,最终导致炎症和肺纤维化[11]。本研究通过一次性气管非暴露注入二氧化硅粉尘悬液建立大鼠矽肺模型,在矽肺模型组中大鼠肺组织结构受损,部分肺泡间隔断裂,肺间质发生纤维化并可见灰白色矽结节,结节中心存在大量胶原纤维,说明矽肺模型建立成功。miR-204-3p干预组肺组织肺泡结构相对完整,与矽肺模型组相比胶原纤维的密度减少,纤维化斑块减少颜色较浅,初步验证了miR-204-3P的激活对二氧化硅所致大鼠矽肺纤维化具有一定的保护作用。

    miRNA是小的非编码RNA,能够通过抑制信使RNA(mRNA)翻译或促进mRNA降解来调节转录后水平的基因表达[6]。miRNA在许多生理和病理过程中起着重要的调节作用,包括肺腺症,心血管疾病,纤维化疾病等[1214]。miR-326、miR-29b和miR-34a等几种miRNA已成功应用于研究体外或体内矽肺纤维化,这些miRNA的异常表达可以减轻疾病纤维化程度[1517]。目前,已发现miR-204-3P可以通过调控多种靶标基因参与调控纤维化过程,包括影响细胞增殖、细胞凋亡、细胞迁移和细胞分化等,并在肺腺癌和动脉粥样硬化等疾病中有抑制作用[1819]。本研究通过建立大鼠矽肺模型体内过表达miR-204-3p对miR-204-3P在二氧化硅粉尘所致大鼠矽肺纤维化的干预作用进行验证,结果发现:矽肺模型组肺组织中miR-204-3p的相对表达水平降低,miR-204-3p干预组的miR-204-3p的相对表达水平升高,说明通过静脉注射包装rno-mir-204腺病毒载体可以在体内过表达miR-204-3p;通过免疫组织化学法对各组肺组织的Fibronectin、Collagen I在肺组织的表达进行检测,发现miR-204-3P的干预可以减少纤维化标志物Fibronectin、Collagen I在肺组织的表达,证实miR-204-3P对二氧化硅粉尘所致大鼠矽肺纤维化过程具有干预作用。

    矽肺发病机制包括初始免疫反应、肺泡细胞损伤、成纤维细胞扩增和活化,以及细胞外基质(extracellular matrix, ECM)的沉积[20]。ECM的成分主要为纤连蛋白、波形蛋白、I型胶原等,过多的ECM沉积是矽肺病的标志[21]。为了进一步研究miR-204-3P干预对肺组织纤维化的影响,检测了各组肺组织中纤维化标志物Fibronectin、Vimentin、Collagen I、Collagen III的表达情况,RT-qPCR结果与Western blot结果均证明:经二氧化硅刺激后肺组织中纤维化标志物Fibronectin1VimentinCollagen ICollagen III的mRNA和蛋白相对表达水平升高,体内过表达miR-204-3P后纤维化因子Fibronectin1VimentinCollagen ICollagen III的表达有所降低,证明miR-204-3P在二氧化硅所致大鼠矽肺纤维化的干预中发挥作用。结合课题组前期富集分析和靶点预测,miR-204-3P可能与TGF-βRⅠ的3'非翻译区结合形成一个miRNA-RISC复合物抑制蛋白质合成,通过阻断mRNA的翻译过程影响TGF-βRⅠ的功能而减少成纤维细胞活化和细胞外基质产生[89]。这种影响导致了纤维化标志物Fibronectin、Vimentin、Collagen I和Collagen III的表达减少,通过减少这些纤维化标志物的表达达到减轻矽肺纤维化程度的作用。

    综上,二氧化硅会导致矽肺纤维化的发生,miR-204-3P的过表达可以减缓肺纤维化进程,体内过表达miR-204-3P会降低纤维化标志物Fibronectin、Vimentin、Collagen I、Collagen III的表达,在矽肺纤维化过程中延缓纤维化进程。本研究通过建立大鼠矽肺模型模拟矽肺的发生发展进程,对miR-204-3P的过表达在二氧化硅粉尘所致大鼠矽肺纤维化过程的干预作用进行验证。实验结果表明,在体内过表达miR-204-3P可以在一定程度上延缓二氧化硅引起的矽肺纤维化进程,这些发现强调了miR-204-3P在肺纤维化进程中的重要性,并提示其具有作为矽肺纤维化治疗靶点的潜力。尽管miR-204-3P对二氧化硅粉尘引起的大鼠矽肺纤维化具有干预作用,但由于种属差异和矽肺发病的复杂性,推广miR-204-3P靶向药物治疗仍需要进行更深入的机制研究和大量的前期临床试验验证。

  • 图  1   各组大鼠肺组织中miR-204-3p的相对表达水平

    a:对照组;b:矽肺模型组;c:miRNA-NC组;d:miR-204-3p干预组。数据以均数±标准差表示,n=3。**:与对照组相比,P<0.05;##:与矽肺模型组相比,P<0.05。

    Figure  1.   The relative expression level of miR-204-3 p in lung tissue of rats in each group

    a: Control group; b: Silicosis model group; c: miRNA-NC group; d: miR-204-3p intervention group. The data are expressed as mean ± standard deviation, n=3 for each group. **: Compared with the control group, P<0.05; ##: Compared with the silicosis model group, P<0.05.

    图  2   各组大鼠肺组织的大体图片

    a:对照组;b:矽肺模型组;c:miRNA-NC组;d:miR-204-3p干预组。图中黑色箭头所指为矽结节。

    Figure  2.   Anatomic presentation of lung tissue of rats in each group

    a: Control group; b: Silicosis model group; c: miRNA-NC group; d: miR-204-3p intervention group. The black arrow in the figure refers to silicon nodules.

    图  3   各组大鼠肺组织HE染色(A)、Masson染色(B)、天狼星红染色(C)结果(×100)

    a:对照组;b:矽肺模型组;c:miRNA-NC组;d:miR-204-3p干预组。

    Figure  3.   Results of HE staining (A), Masson staining (B), and Sirius red staining (C) in lung tissue of rats in each group (×100)

    a: Control group; b: Silicosis model group; c: miRNA-NC group; d: miR-204-3p intervention group.

    图  4   各组大鼠肺组织Fibronectin(A)和Collagen I(B)免疫组织化学染色结果(×200)

    a:对照组;b:矽肺模型组;c:miRNA-NC组;d:miR-204-3p干预组。阳性表达呈棕黄色或褐色,着色程度代表阳性程度。

    Figure  4.   Immunohistochemical results of Fibronectin (A)and Collagen I (B)in lung tissue of rats in each group (×200 )

    a: Control group; b: Silicosis model group; c: miRNA-NC group; d: miR-204-3p intervention group. The positive expression is brownish yellow or brown, and the degree of staining represents the positive degree.

    图  5   各组大鼠肺组织Fibronectin和Collagen I平均光密度值

    数据以均数±标准差表示,n=3;a:对照组;b:矽肺模型组;c:miRNA-NC组;d:miR-204-3p干预组。**:与对照组相比,P<0.05;##:与矽肺模型组相比,P<0.05。

    Figure  5.   The average optical density values of Fibronectin and Collagen I in lung tissue of rats in each group

    The data are expressed as mean ± standard deviation, n=3 for each group; a: Control group; b: Silicosis model group; c: miRNA-NC group; d: miR-204-3p intervention group. **: Compared with the control group, P<0.05; ##: Compared with the silicosis model group, P<0.05.

    图  6   各组大鼠肺组织中纤维化相关因子mRNA相对表达水平

    数据以均数±标准差表示,n=3;a:对照组;b:矽肺模型组;c:miRNA-NC组;d:miR-204-3p干预组。**:与对照组相比,P<0.05;##:与矽肺模型组相比,P<0.05。

    Figure  6.   The relative mRNA expression levels of fibrosis-related factors in lung tissue of rats in each group

    The data are expressed as mean ± standard deviation, n=3 for each group; a: Control group; b: Silicosis model group; c: miRNA-NC group; d: miR-204-3p intervention group; **: Compared with the control group, P<0.05; ##: Compared with the silicosis model group, P<0.05.

    图  7   各组大鼠肺组织中纤维化相关因子的蛋白相对表达水平

    图A:蛋白条带图;图B、C、D、E:Fibronectin、Vimentin、Collagen I、Collagen III蛋白相对表达量;a:对照组;b:矽肺模型组;c:miRNA-NC组;d:miR-204-3p干预组。**:与对照组相比,P<0.05;##:与矽肺模型组相比,P<0.05。

    Figure  7.   The relative protein expression levels of fibrosis-related factors in lung tissue of rats in each group

    A : Protein band; B, C, D, E: Relative protein expression levels of Fibronectin, Vimentin, Collagen I, and Collagen III; a: Control group; b: Silicosis model group; c: miRNA-NC group; d: miR-204-3p intervention group. **: Compared with the control group, P<0.05; ##: Compared with the silicosis model group, P<0.05.

    表  1   RT-qPCR引物序列

    Table  1   RT-qPCR primer sequences

    基因名称
    (Gene name)
    引物序列(Primer sequence)
    正向反向
    FibronectinAGGCACAAGGTCCGAGAAGAGGCATGAGTCATCCGTAGGCTGGTTC
    VimentinGACCGCTTCGCCAACTACATCGCGCAACTCCCTCATCTCCTCCTC
    Collagen IGACAGGCGAACAAGGTGACAGAGTGCAGGACCAGAAGCTCCAGAG
    Collagen IIIAGTCGGAGGAATGGGTGGCTATCCAGGAGATCCAGGATGTCCAGAGG
    GAPDHCTGGAGAAACCTGCCAAGTATGGGTGGAAGAATGGGAGTTGCT
    下载: 导出CSV
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出版历程
  • 收稿日期:  2023-09-30
  • 录用日期:  2024-03-03
  • 网络出版日期:  2024-05-05
  • 刊出日期:  2024-04-24

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