抑制miR-193a-5p表达对急性呼吸窘迫综合征大鼠肺纤维化的改善作用及其机制

doi:10.13481/j.1671-587X.20240602

摘要/Abstract

摘要：

目的探讨抑制微小RNA（miR）-193a-5p表达对急性呼吸窘迫综合征（ARDS）大鼠肺纤维化的影响，并阐明相关作用机制。方法 60只雄性SD大鼠分为假手术组、模型组、miR-193a-5p拮抗剂组（Antagomir组）和阴性对照组（Antagomir-NC组），每组15只。采用暴露气管法滴注10 mg·kg^-1脂多糖（LPS）诱导构建ARDS大鼠模型，假手术组大鼠滴注等体积生理盐水，造模成功后Antagomir组和Antagomir-NC组大鼠给予miR-193a-5p Antagomir或Antagomir-NC尾静脉注射。测定各组大鼠动脉血氧分压（PaO₂）和氧合指数（OI），HE和Masson染色观察各组大鼠肺组织病理形态表现及肺组织中胶原纤维沉积情况，试剂盒检测各组大鼠肺组织中羟脯氨酸（Hyp）水平，酶联免疫吸附试验（ELISA）法检测各组大鼠肺泡灌洗液（BALF）中炎性因子肿瘤坏死因子α（TNF-α）、白细胞介素（IL）-1β和IL-6水平，实时荧光定量PCR（RT-qPCR）法检测各组大鼠肺组织中miR-193a-5p表达水平，Western blotting法检测各组大鼠肺组织中β连环蛋白（β-catenin）、蜗牛家族转录抑制因子1（Snail1）和α-平滑肌肌动蛋白（α-SMA）蛋白表达水平。结果与假手术组比较，模型组大鼠PaO₂和OI均明显降低（P<0.05）；与模型组比较，Antagomir组大鼠PaO₂和OI均明显升高（P<0.05）。HE染色观察，假手术组大鼠肺组织结构正常，未见明显的炎性改变；与假手术组比较，模型组和Antagomir-NC组大鼠肺组织结构轻度异常，肺泡萎缩塌陷，肺泡腔内发现有大量的淋巴细胞和少量的中性粒细胞浸润，肺泡间隙增宽；与模型组比较，Antagomir组大鼠肺组织的肺泡腔内淋巴细胞明显减少，中性粒细胞浸润较少，未见明显增生。Masson染色观察，假手术组大鼠肺组织无明显蓝色胶原纤维沉积；与假手术组比较，模型组和Antagomir-NC组大鼠肺组织中出现大量蓝色胶原纤维沉积，肺泡结构损坏严重，呈现明显的肺纤维化情况；与模型组比较，Antagomir组大鼠肺组织黏液中蓝色染色的胶原纤维沉积明显减少。与假手术组比较，模型组大鼠肺组织中Hyp水平明显升高（P<0.05）；与模型组比较，Antagomir组大鼠肺组织中Hyp水平明显降低（P<0.05）。ELISA法检测，与假手术组比较，模型组大鼠BALF中TNF-α、IL-1β和IL-6水平均明显升高（P<0.05）；与模型组比较，Antagomir组大鼠BALF中TNF-α、IL-1β和IL-6水平均明显降低（P<0.05）。RT-qPCR法检测，与假手术组比较，模型组大鼠肺组织中miR-193a-5p表达水平明显升高（P<0.05）；与模型组比较，Antagomir组大鼠肺组织中miR-193a-5p表达水平明显降低（P<0.05）。Western blotting法检测，与假手术组比较，模型组大鼠肺组织中β-catenin、Snail1和α-SMA蛋白表达水平均明显升高（P<0.05）；与模型组比较，Antagomir组大鼠肺组织中β-catenin、Snail1和α-SMA蛋白表达水平均明显降低（P<0.05）。结论抑制miR-193a-5p表达可通过降低肺组织炎症反应和下调β-catenin、Snail1及α-SMA等蛋白表达，改善ARDS大鼠肺功能和肺纤维化。

关键词: 微小RNA-193a-5p, 急性呼吸窘迫综合征, 肺纤维化, Wnt/β-catenin信号通路

Abstract:

Objective To discuss the effect of inhibiting microRNA（miR）-193a-5p expression on pulmonary fibrosis in the rats with acute respiratory distress syndrome（ARDS）， and to clarify the related mechanism. Methods A total of 60 male SD rats were divided into sham operation group， model group， miR-193a-5p antagonist group（Antagomir group）， and negative control group （Antagomir-NC group）， and there were 15 rats in each group. The ARDS animal model was induced by administering 10 mg·kg^-1 lipopolysaccharide（LPS） via tracheal instillation， while the rats in sham operation group received an equal volume of saline. After successful modeling， the rats in Antagomir group and Antagomir-NC group were treated with miR-193a-5p Antagomir or Antagomir-NC via tail vein injection. The arterial partial pressure of oxygen （PaO₂） and oxygenation index（OI） of the rats in various groups were measured； HE staining and Masson staining were used to observe the pathology and collagen fiber deposition in lung tissue of the rats； kit was used to detect the level of hydroxyproline（Hyp） in lung tissue of the rats in various groups； enzyme-linked immunosorbent assay（ELISA） method was used to detect the levels of inflammatory factors tumor necrosis factor α（TNF-α）， interleukin （IL）-1β， and IL-6 in bronchoalveolar lavage fluid（BALF） of the rats in various groups； real-time fluorescence quantitative PCR（RT-qPCR） method was used to detect the expression levels of miR-193a-5p in lung tissue of the rats in various groups； Western blotting method was used to detect the expression levels of β-catenin， Snail family transcriptional repressor 1（Snail1）， and α-smooth muscle actin（α-SMA） proteins in lung tissue of the rats in various groups. Results Compared with sham operation group， the PaO₂ and OI of the rats in model group were significantly decreased （P<0.05）； compared with model group， the PaO₂ and OI of the rats in Antagomir group were significantly increased（P<0.05）. The HE staining results showed that the lung tissue structure of the rats in sham operation group was normal， and there were no obvious inflammatory changes； compared with sham operation group， mild abnormalities in lung tissue structure， alveolar atrophy， and collapse were observed in the rats in model group and Antagomir-NC group， with a large number of lymphocytes and a small number of neutrophils infiltrating in the alveolar cavities， and widened alveolar spaces； compared with model group， the rats in Antagomir group showed a significant reduction in lymphocytes and neutrophil infiltration in the alveolar cavities and there were no obvious hyperplasia. The Masson staining results showed no obvious blue collagen fiber deposition in lung tissue of the rats in sham operation group； compared with sham operation group， significant blue collagen fiber deposition was observed in lung tissue of the rats in model group and Antagomir-NC group， with severe damage of the alveolar structure， indicating obvious pulmonary fibrosis； compared with model group， the deposition of blue-stained collagen fibers in lung tissue of the rats in Antagomir group was significantly reduced. Compared with sham operation group， the level of Hyp in lung tissue of the rats in model group was significantly increased（P<0.05）； compared with model group， the level of Hyp of the rats in Antagomir group was significantly decreased（P<0.05）. The ELISA results showed that compared with sham operation group， the levels of TNF-α， IL-1β， and IL-6 in BALF of the rats in model group were significantly increased（P<0.05）； compared with model group， the levels of TNF-α， IL-1β， and IL-6 of the rats in Antagomir group were significantly decreased（P<0.05）. The RT-qPCR results showed that compared with sham operation group， the expression level of miR-193a-5p in lung tissue of the rats in model group was significantly increased（P<0.05）； compared with model group， the expression level of miR-193a-5p of the rats in Antagomir group was significantly decreased（P<0.05）. The Western blotting results showed that compared with sham operation group， the expression levels of β-catenin， Snail1， and α-SMA proteins in lung tissue of the rats in model group were significantly increased（P<0.05）； compared with model group， the expression levels of β-catenin， Snail1， and α-SMA proteins in lung tissue of the rats in Antagomir group were significantly decreased（P<0.05）. Conclusion Inhibition of miR-193a-5p expression can improve the lung function and alleviate the pulmonary fibrosis in the ARDS rats by reducing the inflammatory responses and downregulating the expressions of β-catenin， Snail1， and α-SMA proteins.

Key words: Micro RNA-193a-5p, Acute respiratory distress syndrome, Pulmonary fibrosis, Wnt/β-catenin signaling pathway

中图分类号:

R563.9

龙光文,张谦,杨秀林,孙鸿鹏,吉春玲. 抑制miR-193a-5p表达对急性呼吸窘迫综合征大鼠肺纤维化的改善作用及其机制[J]. 吉林大学学报(医学版), 2024, 50(6): 1491-1498.

Guangwen LONG,Qian ZHANG,Xiulin YANG,Hongpeng SUN,Chunling JI. Improvement effect of inhibiting miR-193a-5p expression on pulmonary fibrosis in rats with acute respiratory distress syndrome and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2024, 50(6): 1491-1498.

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Gene	Primer sequence
MiR-193a-5p	F 5'-AATGCGGGGCTAGGGCTA-3'
	R 5'-GCCGCGTGAAATGTTTA GGACCAC-3'
U6	F 5'-CTCGCTTCGGCAGCACA-3'
	R 5'-AACGCTTCACGAATTTGCGT-3'

Group	PaO₂	OI
Sham operation	97.73±7.49	459.30±21.27
Model	68.11±1.99^*	158.94±18.48^*
Antagomir-NC	69.60±1.55	157.19±13.64
Antagomir	80.24±1.91^△	268.27±22.52^△

Group	Hyp[m_B/（μg·g^－1）]
Sham operation	412.04±31.62
Model	812.67±28.57^*
Antagomir-NC	810.63±38.81
Antagomir	537.68±29.14^△

Group	TNF-α	IL-6	IL-1β
Sham operation	24.42±1. 15	35.42±1.77	13.28±0.56
Model	78.36±5.24^*	128.96±9.33^*	45.83±3.53^*
Antagomir-NC	77.54±5.18	134.87±7.48	65.03±7.86
Antagomir	45.58±2.28^△	55.21±5.02^△	25.18±6.94^△

Group	MiR-193a-5p
Sham operation	1.03±0.09
Model	8.07±0.76^*
Antagomir-NC	8.05±0.46
Antagomir	3.28±0.24^△