SGLT2 inhibitor empagliflozin promotes revascularization in diabetic mouse hindlimb ischemia by inhibiting ferroptosis

Jing-xuan Han1,2, Lai-liu Luo1,2, Yi-cheng Wang1,2,3, Makoto Miyagishi4, Vivi Kasim1,2,3, Shou-rong Wu1,2,3
1 Key Laboratory for Biorheological Science and Technology of Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044, China
2 The 111 Project Laboratory of Biomechanics and Tissue Repair, College of Bioengineering, Chongqing University, Chongqing 400044, China
3 State and Local Joint Engineering Laboratory for Vascular Implants, College of Bioengineering, Chongqing University, Chongqing 400044, China
4 Molecular Composite Medicine Research Group, Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan
Correspondence to: Vivi Kasim:, Shou-rong Wu:,
DOI: 10.1038/s41401-022-01031-0
Received: 21 June 2022
Accepted: 13 November 2022
Advance online: 12 December 2022


Gliflozins are known as SGLT2 inhibitors, which are used to treat diabetic patients by inhibiting glucose reabsorption in kidney proximal tubules. Recent studies show that gliflozins may exert other effects independent of SGLT2 pathways. In this study we investigated their effects on skeletal muscle cell viability and paracrine function, which were crucial for promoting revascularization in diabetic hindlimb ischemia (HLI). We showed that treatment with empagliflozin (0.1−40 μM) dose-dependently increased high glucose (25 mM)-impaired viability of skeletal muscle C2C12 cells. Canagliflozin, dapagliflozin, ertugliflozin, ipragliflozin and tofogliflozin exerted similar protective effects on skeletal muscle cells cultured under the hyperglycemic condition. Transcriptomic analysis revealed an enrichment of pathways related to ferroptosis in empagliflozin-treated C2C12 cells. We further demonstrated that empagliflozin and other gliflozins (10 μM) restored GPX4 expression in high glucose-treated C2C12 cells, thereby suppressing ferroptosis and promoting cell viability. Empagliflozin (10 μM) also markedly enhanced the proliferation and migration of blood vessel-forming cells by promoting paracrine function of skeletal muscle C2C12 cells. In diabetic HLI mice, injection of empagliflozin into the gastrocnemius muscle of the left hindlimb (10 mg/kg, every 3 days for 21 days) significantly enhanced revascularization and blood perfusion recovery. Collectively, these results reveal a novel effect of empagliflozin, a clinical hypoglycemic gliflozin drug, in inhibiting ferroptosis and enhancing skeletal muscle cell survival and paracrine function under hyperglycemic condition via restoring the expression of GPX4. This study highlights the potential of intramuscular injection of empagliflozin for treating diabetic HLI.
Keywords: diabetic hindlimb ischemia; empagliflozin; therapeutic angiogenesis; revascularization; ferroptosis

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