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4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol) alleviates lung injury by inhibiting SIRT6-HIF-1α signaling pathway activation through the upregulation of miR-212-5p expression

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Abstract

Objective

Obstructive sleep apnea is closely related to oxidative stress. 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol) can scavenge reactive oxygen species (ROS) and ameliorate oxidative damage in the body. The mechanism by which Tempol alleviates chronic intermittent hypoxia-induced lung injury has rarely been reported. This study aimed to confirm the molecular mechanism by which Tempol alleviates lung injury.

Methods

The levels of miR-212-5p and Sirtuin 6 (SIRT6) in injured lungs were analyzed using bioinformatics. In vitro, intermittent hypoxia (IH) treatment induced hypoxia in BEAS-2B cells and we established a model of chronic intermittent hypoxia (CIH) in mouse using a programmed hypoxia chamber. We used HE staining to observe the morphology of lung tissue, and the changes in lung fibers were observed by Masson staining. The levels of inflammatory factors in mouse serum were detected by ELISA, and the levels of the oxidative stress indicators GSH, MDA, SOD and ROS were detected using commercially available kits. Moreover, a real-time qPCR assay was used to detect miR-212-5p expression, and Western blotting was used to detect the levels of SIRT6, HIF-1α and apoptosis-related proteins. CCK-8 was used to detect cell proliferation. Subsequently, we used flow cytometry to detect cell apoptosis. Dual-luciferase gene reporters determine the on-target binding relationship of miR-212-5p and SIRT6.

Results

SIRT6 was highly expressed in CIH-induced lung injury, as shown by bioinformatics analysis; however, miR-212-5p expression was decreased. Tempol promoted miR-212-5p expression, and the levels of SIRT6 and HIF-1α were inhibited. In BEAS-2B cells, Tempol also increased proliferation, inhibited apoptosis and inhibited oxidative stress in BEAS-2B cells under IH conditions. In BEAS-2B cells, these effects of Tempol were reversed after transfection with an miR-212-5p inhibitor. miR-212-5p targeted and negatively regulated the level of SIRT6 and overexpression of SIRT6 effectively reversed the enhanced influence of the miR-212-5p mimic on Tempol’s antioxidant activity. Tempol effectively ameliorated lung injury in CIH mice and inhibited collagen deposition and inflammatory cell infiltration. Likewise, the therapeutic effect of Tempol could be effectively reversed by interference with the miR-212-5p inhibitor.

Conclusion

Inhibition of the SIRT6-HIF-1α signaling pathway could promote the effect of Tempol by upregulating the level of miR-212-5p, thereby alleviating the occurrence of lung injury and providing a new underlying target for the treatment of lung injury.

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Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

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Acknowledgements

Not applicable.

Funding

This research is supported by the National Natural Science Foundation of China (Project Number: 81960025).

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Authors and Affiliations

  1. Department of Respiratory and Critical Care Medicine, The Second Affiliated Hospital of Kunming Medical University, No 374 Dianmian Road, Kunming, 650101, Yunnan, China

    Li Ai, Ran Li, Yu Cao, Zhijuan Liu, Xiaoqun Niu & Yongxia Li

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  1. Li Ai
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  2. Ran Li
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  3. Yu Cao
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  4. Zhijuan Liu
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  5. Xiaoqun Niu
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Contributions

Conceptualization, LA and LR; methodology, LA, YL and YC; software, YC and ZL; validation, XN; formal analysis, RL, YC, ZL and XN; investigation, LA and YL; resources, LA and YL; data curation, RL and YC; writing—original draft preparation, LA and YL; writing—review and editing, LA and YL; visualization, LR, YC, ZL and XN; supervision, LA and YL; funding acquisition, LA and YL. All authors have read and agreed to the published version of the manuscript.

Corresponding author

Correspondence to Yongxia Li.

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The authors declare that they have no competing interests.

Ethical approval

All animal experiments were approved by the Experimental Animal Ethics Committee of Kunming Medical University (kmmu20211432). All authors confirm that all the methods were in accordance with the ARRIVE guidelines.

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Ai, L., Li, R., Cao, Y. et al. 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol) alleviates lung injury by inhibiting SIRT6-HIF-1α signaling pathway activation through the upregulation of miR-212-5p expression. Mol Biol Rep 51, 129 (2024). https://doi.org/10.1007/s11033-023-09039-3

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