抑制miR-33表达对急性呼吸窘迫综合征大鼠肺纤维化的影响及机制研究

doi:10.11958/20220034

摘要/Abstract

摘要：

目的探讨抑制miR-33表达对急性呼吸窘迫综合征（ARDS）大鼠肺纤维化的影响及其机制。方法将60只SD大鼠按随机数字表法分成假手术组（Sham组）、ARDS模型组（Model组）、antagomir阴性对照组（antagomir-NC组）和miR-33 antagomir组（antagomir组）,每组15只。除Sham组外,其他各组大鼠均通过颈部气管滴注脂多糖（LPS）建立ARDS模型。造模成功后给予miR-33 antagomir或antagomir-NC尾静脉注射。测定动脉血氧分压[p（O₂）]及氧合指数（OI）;HE和Masson染色观察肺组织病理学变化;碱性水解法检测肺组织中羟脯氨酸（Hyp）含量;酶联免疫吸附试验（ELISA）检测肺泡灌洗液中炎性因子白细胞介素（IL）-1β、IL-6和肿瘤坏死因子（TNF）-α水平;实时荧光定量PCR检测肺组织中miR-33表达水平及转化生长因子β1（TGF-β1）、胶原蛋白（Collagen）Ⅰ和CollagenⅢ mRNA表达水平;Western blot检测肺组织中TGF-β1、Smad2、p-Smad2、Smad3、p-Smad3和α-平滑肌肌动蛋白（α-SMA）蛋白表达水平。结果与Sham组比较,Model组大鼠中p（O₂）和OI均降低（P<0.05）,肺组织损伤严重,且有明显的胶原纤维沉积,肺组织中Hyp含量及肺泡灌洗液中IL-1β、IL-6和TNF-α水平均升高（P<0.05）,肺组织中miR-33和TGF-β1、CollagenⅠ、CollagenⅢ mRNA表达水平及TGF-β1、p-Smad2/Smad2、p-Smad3/Smad3和α-SMA蛋白表达水平均增加（P<0.05）。与Model组和antagomir-NC组比较,antagomir组大鼠中p（O₂）和OI均升高（P<0.05）,肺组织损伤明显改善,胶原纤维沉积减少,肺组织中Hyp含量及肺泡灌洗液中IL-1β、IL-6和TNF-α水平均降低（P<0.05）,miR-33和TGF-β1、CollagenⅠ、CollagenⅢ mRNA表达水平及TGF-β1、p-Smad2/Smad2、p-Smad3/Smad3、α-SMA蛋白表达水平均降低（P<0.05）。结论抑制miR-33表达可能通过抑制TGF-β1/Smad信号通路从而改善ARDS大鼠肺纤维化。

关键词: 转化生长因子β, Smad蛋白质类, 肺纤维化, 呼吸窘迫综合征,成人, miR-33

Abstract:

Objective To investigate the effect and mechanism of inhibiting miR-33 expression on pulmonary fibrosis in rats with acute respiratory distress syndrome (ARDS). Methods A total of 60 SD rats were divided into the Sham group, the ARDS model group (Model group), the antagomir negative control group (antagomir-NC group) and the miR-33 antagomir group (antagomir group) according to random number table method, with 15 rats in each group. Except for the Sham group, ARDS model was established by endotracheal drop of lipopolysaccharide (LPS) in other groups. After the model was successfully established, miR-33 antagomir or antagomir-NC were injected through tail vein. Arterial partial pressure of oxygen [p(O₂) ] and oxygenation index (OI) were measured. HE staining and Masson staining were used to observe the histopathological changes and the degree of pulmonary fibrosis of lung. The content of hydroxyproline (Hyp) in lung tissue was detected by alkaline hydrolysis method. The levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) in alveolar lavage fluid were detected by ELISA. The mRNA expression levels of miR-33 and TGF-β1, Collagen Ⅰ and Collagen Ⅲ in lung tissues were detected by qPCR. The protein expression levels of TGF-β1, Smad2, p-Smad2, Smad3, p-Smad3 and α-smooth muscle actin (α-SMA) were detected by Western blot assay. Results Compared with the Sham group, the level of p(O₂) and OI were significantly decreased in the Model group (P<0.05). Lung tissue was severely damaged and there were obvious collagen fiber deposition. The Hyp content in lung tissue and levels of IL-1β, IL-6 and TNF-α in alveolar lavage fluid were significantly increased in the Model group (P<0.05). The expression levels of miR-33, the mRNA expression levels of TGF-β1, Collagen Ⅰ and Collagen Ⅲ and the protein expression levels of TGF-β1, p-Smad2/Smad2, p-Smad3/Smad3 and α-SMA in lung tissue were significantly increased (P<0.05). Compared with the Model group and the antagomir-NC group, the levels of p（O₂） and OI were significantly increased in the antagomir group (P<0.05), lung tissue damage was significantly improved, and collagen fiber deposition was significantly decreased. The Hyp content in lung tissue and levels of IL-1β, IL-6 and TNF-α in alveolar lavage fluid were significantly decreased (P<0.05). The expression levels of miR-33, the mRNA expression levels of TGF-β1, Collagen Ⅰ, Collagen Ⅲ and the protein expression levels of TGF-β1, p-Smad2/Smad2, p-Smad3/Smad3 and α-SMA were significantly decreased (P<0.05). Conclusion The inhibition of miR-33 expression may improve pulmonary fibrosis in ARDS rats by inhibiting TGF-β1/Smad signaling pathway.

Key words: transforming growth factor beta, Smad proteins, pulmonary fibrosis, respiratory distress syndrome,adult, miR-33

中图分类号:

R563

图/表 7

参考文献 23

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基因名称	引物序列（5'→3'）	产物大小（bp）
miR-33	上游：GATCCTCAGTGCATTGTAGTTGC	69
miR-33	下游：CTCTGTCTCTCGTCTTGTTGGTAT	69
U6	上游：CCTGCTTCGGCAG CACA	96
U6	下游：AACGCTTCACGAATTTGCGT	96
TGF-β1	上游：GTGGAAATCAACGGGATCAG	188
TGF-β1	下游：CGCACACAGCAGTTCTTCTC	188
CollagenⅠ	上游：TTGCTTCCCAGATGTCCTATG	108
CollagenⅠ	下游：CTTCCCCATCATCTCCATTCT	108
CollagenⅢ	上游：CAGACGGGA GTTTCTCCTCGGACGT	210
CollagenⅢ	下游：GACCAGGAGGACCAGGAAGTCCACGT	210
GAPDH	上游：AACTGCTTAGCACCCCTGGC	92
GAPDH	下游：ATGACCTTGCCCA CACAGCCTT	92

基因名称	引物序列（5'→3'）	产物大小（bp）
miR-33	上游：GATCCTCAGTGCATTGTAGTTGC	69
miR-33	下游：CTCTGTCTCTCGTCTTGTTGGTAT	69
U6	上游：CCTGCTTCGGCAG CACA	96
U6	下游：AACGCTTCACGAATTTGCGT	96
TGF-β1	上游：GTGGAAATCAACGGGATCAG	188
TGF-β1	下游：CGCACACAGCAGTTCTTCTC	188
CollagenⅠ	上游：TTGCTTCCCAGATGTCCTATG	108
CollagenⅠ	下游：CTTCCCCATCATCTCCATTCT	108
CollagenⅢ	上游：CAGACGGGA GTTTCTCCTCGGACGT	210
CollagenⅢ	下游：GACCAGGAGGACCAGGAAGTCCACGT	210
GAPDH	上游：AACTGCTTAGCACCCCTGGC	92
GAPDH	下游：ATGACCTTGCCCA CACAGCCTT	92

组别	p（O₂）	OI
Sham组	97.73±7.49	459.30±21.27
Model组	68.11±1.99^a	158.94±18.48^a
antagomir-NC组	69.60±1.55^a	157.19±13.64^a
antagomir组	80.24±1.91^abc	268.27±22.52^abc
F	33.845^＊＊	162.902^＊＊

组别	p（O₂）	OI
Sham组	97.73±7.49	459.30±21.27
Model组	68.11±1.99^a	158.94±18.48^a
antagomir-NC组	69.60±1.55^a	157.19±13.64^a
antagomir组	80.24±1.91^abc	268.27±22.52^abc
F	33.845^＊＊	162.902^＊＊

组别	IL-1β	IL-6	TNF-α
Sham组	28.70±8.30	41.84±10.71	107.51±19.57
Model组	61.56±11.72^a	158.18±24.13^a	431.92±23.58^a
antagomir-NC组	60.79±8.85^a	157.15±35.72^a	430.37±16.17^a
antagomir组	39.83±2.23^bc	88.64±12.92^abc	288.11±23.74^abc
F	10.859^＊＊	18.017^＊＊	160.420^＊＊