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Empagliflozin ameliorates vascular calcification in diabetic mice through inhibiting Bhlhe40-dependent NLRP3 inflammasome activation

Acta Pharmacologica Sinica volume 45, pages 751–764 (2024)Cite this article

Abstract

Type 2 diabetes mellitus (T2DM) patients exhibit greater susceptibility to vascular calcification (VC), which has a higher risk of death and disability. However, there is no specific drug for VC therapy. NLRP3 inflammasome activation as a hallmark event of medial calcification leads to arterial stiffness, causing vasoconstrictive dysfunction in T2DM. Empagliflozin (EMPA), a sodium-glucose co-transporter 2 inhibitor (SGLT2i), restrains hyperglycemia with definite cardiovascular benefits. Given the anti-inflammatory activity of EMPA, herein we investigated whether EMPA protected against VC in the aorta of T2DM mice by inhibiting NLRP3 inflammasome activation. Since db/db mice receiving a normal diet developed VC at the age of about 20 weeks, we administered EMPA (5, 10, 20 mg·kg−1·d−1, i.g) to 8 week-old db/db mice for 12 weeks. We showed that EMPA intervention dose-dependently ameliorated the calcium deposition, accompanied by reduced expression of RUNX2 and BMP2 proteins in the aortas. We found that EMPA (10 mg·kg−1·d−1 for 6 weeks) also protected against VC in vitamin D3-overloaded mice, suggesting the protective effects independent of metabolism. We showed that EMPA (10 mg·kg−1·d−1) inhibited the abnormal activation of NLRP3 inflammasome in aortic smooth muscle layer of db/db mice. Knockout (KO) of NLRP3 significantly alleviated VC in STZ-induced diabetic mice. The protective effects of EMPA were verified in high glucose (HG)-treated mouse aortic smooth muscle cells (MOVASs). In HG-treated NLRP3 KO MOVASs, EMPA (1 μM) did not cause further improvement. Bioinformatics and Western blot analysis revealed that EMPA significantly increased the expression levels of basic helix-loop-helix family transcription factor e40 (Bhlhe40) in HG-treated MOVASs, which served as a negative transcription factor directly binding to the promotor of Nlrp3. We conclude that EMPA ameliorates VC by inhibiting Bhlhe40-dpendent NLRP3 inflammasome activation. These results might provide potential significance for EMPA in VC therapy of T2DM patients.

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Fig. 1: EMPA inhibited VC and alleviated vascular dysfunction in db/db mice.
Fig. 2: EMPA inhibited NLRP3 inflammasome activation in the arterial smooth muscle layer of db/db mice.
Fig. 3: EMPA attenuated HG-induced calcification in MOVASs by inhibiting NLRP3 inflammasome.
Fig. 4: EMPA inhibited NLRP3 inflammasome by reversing HG-induced Bhlhe40 suppression in MOVASs.
Fig. 5: EMPA attenuated HG-induced osteogenic differentiation and calcium deposition by increasing Bhlhe40 in MOVASs.
Fig. 6: EMPA reversed Bhlhe40 diminution in the arterial smooth muscle layer of db/db mice.

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Acknowledgements

This work was supported by National Natural Science Foundation of China (No. 81970381), the Fundamental Research Funds for the Central Universities, the Postgraduate Research & Practice Innovation Program of Jiangsu Province (No.5024002205).

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  1. Department of Cardiology, Zhongda Hospital, Southeast University School of Medicine, Nanjing, 210009, China

    Xiao-xue Li, Zheng-dong Chen, Xue-jiao Sun, Yi-qing Yang, Hong Jin & Nai-feng Liu

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XXL investigated, designed and performed the experiments; ZDC, XJS and YQY participated in part of the experiments and formal analysis; HJ investigated and modified the format; NFL conceived and supervised the study; XXL analyzed the data and wrote the manuscript.

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Li, Xx., Chen, Zd., Sun, Xj. et al. Empagliflozin ameliorates vascular calcification in diabetic mice through inhibiting Bhlhe40-dependent NLRP3 inflammasome activation. Acta Pharmacol Sin 45, 751–764 (2024). https://doi.org/10.1038/s41401-023-01217-0

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