Empagliflozin alleviates atherosclerotic calcification by inhibiting osteogenic differentiation of vascular smooth muscle cells

SGLT-2 inhibitors, such as empagliflozin, have been shown to reduce the occurrence of cardiovascular events and delay the progression of atherosclerosis. However, its role in atherosclerotic calcification remains unclear. In this research, ApoE-/- mice were fed with western diet and empagliflozin was added to the drinking water for 24 weeks. Empagliflozin treatment significantly alleviated arterial calcification assessed by alizarin red and von kossa staining in aortic roots and reduced the lipid levels, while had little effect on body weight and blood glucose levels in ApoE-/- mice. In vitro studies, empagliflozin significantly inhibits calcification of primary vascular smooth muscle cells (VSMCs) and aortic rings induced by osteogenic media (OM) or inorganic phosphorus (Pi). RNA sequencing of VSMCs cultured in OM with or without empagliflozin showed that empagliflozin negatively regulated the osteogenic differentiation of VSMCs. And further studies confirmed that empagliflozin significantly inhibited osteogenic differentiation of VSMCs via qRT-PCR. Our study demonstrates that empagliflozin alleviates atherosclerotic calcification by inhibiting osteogenic differentiation of VSMCs, which addressed a critical need for the discovery of a drug-based therapeutic approach in the treatment of atherosclerotic calcification.SGLT-2抑制剂（SGLT-2 inhibitors）如恩格列净（empagliflozin），已被证实可降低心血管事件发生率并延缓动脉粥样硬化进展。然而其在动脉粥样硬化钙化中的作用仍不明确。本研究中，ApoE-/-小鼠（ApoE-/- mice）被喂食西式饮食，并在饮用水中添加恩格列净，持续干预24周。经茜素红（alizarin red）与冯库萨（von kossa）染色评估，恩格列净治疗可显著减轻小鼠主动脉根部的动脉钙化程度，同时降低体内脂质水平，且对ApoE-/-小鼠的体重与血糖水平几乎无影响。体外实验方面，恩格列净可显著抑制成骨培养基（osteogenic media, OM）或无机磷（inorganic phosphorus, Pi）诱导的原代血管平滑肌细胞（vascular smooth muscle cells, VSMCs）及主动脉环的钙化反应。对经或未经恩格列净处理、于成骨培养基中培养的VSMCs进行RNA测序（RNA sequencing），结果显示恩格列净可负向调控VSMCs的成骨分化。后续通过实时定量聚合酶链反应（qRT-PCR）开展的验证实验，进一步证实恩格列净可显著抑制VSMCs的成骨分化。本研究证实，恩格列净可通过抑制VSMCs的成骨分化减轻动脉粥样硬化钙化，为动脉粥样硬化钙化的药物治疗策略研发解决了一项关键需求。