The effect and mechanism of inhibiting miR-33 expression on pulmonary fibrosis in rats with acute respiratory distress syndrome

doi:10.11958/20220034

Abstract

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

CLC Number:

R563

Figures/Tables 7

References 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^＊＊