miR-499a-5p attenuates lung injury in rats with acute respiratory distress syndrome by targeting MMP-16 via Nrf2 signaling pathway_Chinese Journal of Respiratory and Critical Care Medicine

Authors：

DUAN Lingxiao ¹ , LI Qin ² ,  WU Changxue ¹

1. Department of Critical Care Medicine, Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan 646000, P. R. China;
2. Department of Pharmacy, Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan 646000, P. R. China;

Corresponding?author：

WU Changxue, Email: wcxfywjk888@163.com

Keywords：

Acute respiratory distress syndrome; miR-499a-5p; Matrix metallopeptidase-16; Nrf2 signaling pathway

DOI：

10.7507/1671-6205.201907059

Video：

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Objective To investigate effects of high expression of miR-499a-5p on lung injury in rats with acute respiratory distress syndrome (ARDS) by targeting matrix metallopeptidase-16 (MMP-16).Methods The experiment set up sham operation group, model group, miR-499a-5p mimic group, MMP-16 group, miR-499a-5p mimic+MMP-16 group, D-ribofuranosylbenzimidazole (DRB, Nrf2 signaling pathway inhibitor) group, miR-499a-5p mimic+DRB group. A rat model of ARDS was constructed by cecal puncture. One hour before surgery, the transfection complex (50 μL) was injected into the trachea with a micro-syringe. DRB (5 mg/kg) was intraperitoneally injected 30 min before surgery. The expression levels of miR-499a-5p and MMP-16 in lung tissue were detected by RT-qPCR; Alveolar type Ⅱ epithelial cells of model group rats were separated and MMP-16 3 'UTR WT and MUT luciferase report plasmid were transfected into alveolar type Ⅱ epithelial cells with miR-499 respectively to verify the targeting relationship between miR-499 and MMP-16; the targeted relationship was verified by the dual luciferase reporter gene; lung injury was observed by hematoxylin-eosin staining; The level of inflammatory factors in bronchoalveolar lavage fluid (BALF) and the level of oxidative stress in lung tissue were detected by enzyme-linked immunosorbent assay; The expression levels of NAD(P)H: quinone oxidoreductase 1 (NQO1), heme oxygenase (HO)-1, and nuclear factor-erythroid 2-related factor 2 (Nrf2) proteins in lung tissues were analyzed by Western blotting.Results miR-499a-5p was down-regulated in the lungs of ARDS model rats (P<0.01), while MMP-16 was highly expressed (P<0.01); miR-499a-5p and MMP-16 3'UTR regions had binding sites, and miR-499a-5p directly targeted negative regulation of MMP-16 expression (P<0.01); overexpression of miR-499a-5p significantly reduced the right lung wet-to-dry weight ratio in the ARDS rats (P<0.05), reduced lung tissue damage (P<0.01), and reduced tumor necrosis factor α, interleukin (IL)-1β and IL-6 levels in BALF (P<0.01), decreased malondialdehyde and myeloperoxidase levels in lung tissue, increased total anti-oxidant capacity (P<0.01), and up-regulated NQO1, HO-1, Nrf2 protein expression in lung tissue (P<0.01). However, this phenomenon was significantly reversed after the addition of MMP-16 and DRB.Conclusion Overexpression of miR-499a-5p attenuates lung injury in rats with ARDS by targeting negative regulation of MMP-16 via activating the Nrf2 signaling pathway.

Citation： DUAN Lingxiao, LI Qin, WU Changxue. miR-499a-5p attenuates lung injury in rats with acute respiratory distress syndrome by targeting MMP-16 via Nrf2 signaling pathway. Chinese Journal of Respiratory and Critical Care Medicine, 2021, 20(7): 487-494. doi: 10.7507/1671-6205.201907059 Copy

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12.	劉琳, 李濤平. 急性呼吸窘迫綜合征大鼠肺泡Ⅱ型上皮細胞的超微結構觀察. 第一軍醫大學學報, 2003, 23(7): 690-691, 695.
13.	Umbrello M, Formenti P, Bolgiaghi L, et al. Current concepts of ARDS: a narrative review. Int J Mol Sci, 2017, 18(1): 64.
14.	宋婷, 張青苗, 歐陽為相, 等. 妊娠期糖尿病胎鼠肺發育與 mTOR 關系的研究. 醫學分子生物學雜志, 2018, 15(5): 289-294.
15.	D'Alessio FR. Mouse models of acute lung injury and ARDS. Methods Mol Biol, 2018, 1809(2): 341-350.
16.	Zeng Z, Gong H, Li Y, et al. Upregulation of mir-146a contributes to the suppression of inflammatory responses in LPS-induced acute lung injury. Exp Lung Res, 2013, 39(7): 275-282.
17.	Chen L, Li W, Qi D, et al. Lycium barbarum polysaccharide protects against LPS-induced ARDS by inhibiting apoptosis, oxidative stress, and inflammation in pulmonary endothelial cells. Free Radic Res, 2018, 52(4): 480-490.
18.	Zhang S, Wang P, Zhao P, et al. Pretreatment of ferulic acid attenuates inflammation and oxidative stress in a rat model of lipopolysaccharide-induced acute respiratory distress syndrome. Int J Immunopathol Pharmacol, 2018, 32(3): 518.
19.	Das A, Kole L, Wang L, et al. BALT development and augmentation of hyperoxic lung injury in mice deficient in NQO1 and NQO2. Free Radic Biol Med, 2006, 40(10): 1843-1856.
20.	Hsieh YH, Deng JS, Pan HP, et al. Sclareol ameliorate lipopolysaccharide-induced acute lung injury through inhibition of MAPK and induction of HO-1 signaling. Int Immunopharmacol, 2017, 44(2): 16-25.
21.	Gao D, Liu F, Li Z, et al. Isobavachalcone attenuates sephadex-induced lung injury via activation of A20 and NRF2/HO-1 in rats. Eur J Pharmacol, 2019, 848(5): 49-54.

1. Lee KY. Pneumonia, acute respiratory distress syndrome, and early immune-modulator therapy. Int J Mol Sci, 2017, 18(2): 388.
2. Seethala RR, Hou PC, Aisiku IP, et al. Early risk factors and the role of fluid administration in developing acute respiratory distress syndrome in septic patients. Ann Intensive Care, 2017, 7(1): 11.
3. Dinglas VD, Aronson Friedman L, Colantuoni E, et al. Muscle weakness and 5-year survival in acute respiratory distress syndrome survivors. Crit Care Med., 2017, 45(3): 446-453.
4. Fan E, Brodie D, Slutsky AS. Acute respiratory distress syndrome: advances in diagnosis and treatment. JAMA, 2018, 319(7): 698-710.
5. 林曉亮, 張建華. 卡介苗調控 OVA 誘導的哮喘小鼠 M-CSF、IL-12 表達的研究. 醫學分子生物學雜志, 2019, 16(2): 169-173.
6. Li M, Zhang S, Wu N, et al. Overexpression of mir-499-5p inhibits non-small cell lung cancer proliferation and metastasis by targeting vav3. Sci Rep, 2016, 6(2): 3100.
7. Li Y, Lu J, Bao X, et al. Mir-499-5p protects cardiomyocytes against ischaemic injury via anti-apoptosis by targeting pdcd4. Oncotarget, 2016, 7(24): 35607-35617.
8. 施凱, 劉景全, 馮月娟, 等. 微小 RNA-320 檢測在體外循環所致急性呼吸窘迫綜合征中的臨床價值及其機制研究. 中華危重癥醫學雜志, 2018, 11(1): 22-28.
9. Chao W, Deng JS, Huang SS, et al. 3,4-dihydroxybenzalacetone attenuates lipopolysaccharide-induced inflammation in acute lung injury via down-regulation of MMP-2 and MMP-9 activities through suppressing ROS-mediated MAPK and PI3K/AKT signaling pathways. Int Immunopharmacol, 2017, 50(2): 77-86.
10. Zhu G, Xin X, Liu Y, et al. Geraniin attenuates LPS-induced acute lung injury via inhibiting NF-κB and activating Nrf2 signaling pathways. Oncotarget, 2017, 8(14): 22835-22841.
11. Yun L, Zheng-Zhao L, Jian-Feng Z, et al. Microrna-494 inhibition alleviates acute lung injury through Nrf2 signaling pathway via nqo1 in sepsis-associated acute respiratory distress syndrome. Life Sci, 2018, 210(1): 1-8.
12. 劉琳, 李濤平. 急性呼吸窘迫綜合征大鼠肺泡Ⅱ型上皮細胞的超微結構觀察. 第一軍醫大學學報, 2003, 23(7): 690-691, 695.
13. Umbrello M, Formenti P, Bolgiaghi L, et al. Current concepts of ARDS: a narrative review. Int J Mol Sci, 2017, 18(1): 64.
14. 宋婷, 張青苗, 歐陽為相, 等. 妊娠期糖尿病胎鼠肺發育與 mTOR 關系的研究. 醫學分子生物學雜志, 2018, 15(5): 289-294.
15. D'Alessio FR. Mouse models of acute lung injury and ARDS. Methods Mol Biol, 2018, 1809(2): 341-350.
16. Zeng Z, Gong H, Li Y, et al. Upregulation of mir-146a contributes to the suppression of inflammatory responses in LPS-induced acute lung injury. Exp Lung Res, 2013, 39(7): 275-282.
17. Chen L, Li W, Qi D, et al. Lycium barbarum polysaccharide protects against LPS-induced ARDS by inhibiting apoptosis, oxidative stress, and inflammation in pulmonary endothelial cells. Free Radic Res, 2018, 52(4): 480-490.
18. Zhang S, Wang P, Zhao P, et al. Pretreatment of ferulic acid attenuates inflammation and oxidative stress in a rat model of lipopolysaccharide-induced acute respiratory distress syndrome. Int J Immunopathol Pharmacol, 2018, 32(3): 518.
19. Das A, Kole L, Wang L, et al. BALT development and augmentation of hyperoxic lung injury in mice deficient in NQO1 and NQO2. Free Radic Biol Med, 2006, 40(10): 1843-1856.
20. Hsieh YH, Deng JS, Pan HP, et al. Sclareol ameliorate lipopolysaccharide-induced acute lung injury through inhibition of MAPK and induction of HO-1 signaling. Int Immunopharmacol, 2017, 44(2): 16-25.
21. Gao D, Liu F, Li Z, et al. Isobavachalcone attenuates sephadex-induced lung injury via activation of A20 and NRF2/HO-1 in rats. Eur J Pharmacol, 2019, 848(5): 49-54.

Chinese Journal of Respiratory and Critical Care Medicine

miR-499a-5p attenuates lung injury in rats with acute respiratory distress syndrome by targeting MMP-16 via Nrf2 signaling pathway

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