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

Xiao-xue Li1, Zheng-dong Chen1, Xue-jiao Sun1, Yi-qing Yang1, Hong Jin1, Nai-feng Liu1
1 Department of Cardiology, Zhongda Hospital, Southeast University School of Medicine, Nanjing 210009, China
Correspondence to: Nai-feng Liu:,
DOI: 10.1038/s41401-023-01217-0
Received: 20 September 2023
Accepted: 14 December 2023
Advance online: 3 January 2024


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.

Keywords: type 2 diabetes mellitus; vascular calcification; empagliflozin; NLRP3 inflammasome; Bhlhe40

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