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The SGLT2 inhibitor empagliflozin attenuates atherosclerosis progression by inducing autophagy

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Abstract

Cardiovascular disease due to atherosclerosis is one of the leading causes of death worldwide; however, the underlying mechanism has yet to be defined. The sodium-dependent glucose transporter 2 inhibitor (SGLT2i) empagliflozin is a new type of hypoglycemic drug. Recent studies have shown that empagliflozin not only reduces high glucose levels but also exerts cardiovascular-protective effects and slows the process of atherosclerosis. The purpose of this study was to elucidate the mechanism by which empagliflozin ameliorates atherosclerosis. Male apolipoprotein E-deficient (ApoE−/−) mice were fed a high-fat Western diet to establish an atherosclerosis model. The area and size of atherosclerotic lesions in ApoE−/− mice were then assessed by performing hematoxylin–eosin (HE) staining after empagliflozin treatment. Concurrently, oxidized low-density lipoprotein (oxLDL) was used to mimic atherosclerosis in three different types of cells. Then, following empagliflozin treatment of macrophage cells (RAW264.7), human aortic smooth muscle cells (HASMCs), and human umbilical vein endothelial cells (HUVECs), western blotting was applied to measure the levels of autophagy-related proteins and proinflammatory cytokines, and green fluorescent protein (GFP)-light chain 3 (LC3) puncta were detected using confocal microscopy to confirm autophagosome formation. Oil Red O staining was performed to detect the foaming of macrophages and HASMCs, and flow cytometry was used for the cell cycle analysis. 5-ethynyl-2′-deoxyuridine (EdU), cell counting kit-8 (CCK-8), and scratch assays were also performed to examine the proliferation and migration of HASMCs. Empagliflozin suppressed the progression of atherosclerotic lesions in ApoE−/− mice. Empagliflozin also induced autophagy in RAW246.7 cells, HASMCs, and HUVECs via the adenosine monophosphate–activated protein kinase (AMPK) signaling pathway, and it significantly increased the levels of the Beclin1 protein, the LC3B-II/I ratio, and p-AMPK protein. In addition, empagliflozin decreased the expression of P62 and the protein levels of inflammatory cytokines, and it inhibited the foaming of RAW246.7 cells and HASMCs, as well as the expression of inflammatory factors by inducing autophagy. Empagliflozin activated autophagy through the AMPK signaling pathway to delay the progression of atherosclerosis. Furthermore, the results of flow cytometry, EdU assays, CCK-8 cell viability assays, and scratch assays indicated that empagliflozin blocked HASMCs proliferation and migration. Empagliflozin activates autophagy through the AMPK signaling pathway to delay the evolution of atherosclerosis, indicating that it may represent a new and effective drug for the clinical treatment of atherosclerosis.

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Abbreviations

AS :

Atherosclerosis

EMPA :

Empagliflozin

SGLT2i :

Sodium-dependent glucose transporter 2 inhibitor

HUVECs :

Human umbilical vein endothelial cells

HUSMCs :

Human aortic smooth muscle cells

RAW 264.7 :

A mouse macrophage line

GFP-LC3 :

Green fluorescent protein-light chain 3

oxLDL :

Human oxidized low-density lipoprotein

AMPK :

Adenosine monophosphate-activated protein kinase

T2DM :

Type 2 diabetes mellitus

TNF-α :

Tumor necrosis factor-α

IL-6 :

Interleukin-6

mTORC1 :

Mammalian target of rapamycin complex 1

ASCVD :

Atherosclerotic cardiovascular disease

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Funding

This study was financially supported by the Scientific and Technological Department of Hubei Province (NO.2022CFA036), the National Natural Science Foundation of China (NO. 81771522), Hubei Provincial Health Commission Project (NO. WJ2021M258) and Cultivating Project for Young Scholars at Hubei University of Medicine (No. 2020QDJZR026).

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Authors and Affiliations

  1. Postgraduate Training Basement of Jinzhou Medical University, Taihe Hospital, Hubei University of Medicine, Shiyan, 442000, China

    Hualin Xu & Zheng Cao

  2. Department of Cardiology, Taihe Hospital, Hubei University of Medicine, Shiyan, 442000, Hubei, China

    Hualin Xu, Jie Fu, Qiang Tu, Qingyun Shuai & Zheng Cao

  3. School of Basic Medical Sciences, Hubei University of Medicine, Shiyan, 442000, Hubei, China

    Yizhi Chen & Fuyun Wu

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  1. Hualin Xu
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  2. Jie Fu
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  4. Qingyun Shuai
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  5. Yizhi Chen
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  6. Fuyun Wu
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Contributions

The authors declare that all data were generated in-house and that no paper mill was used. The authors’ responsibilities were as follows: HLX performed the experiments, analyzed the data, prepared the figures and wrote the manuscript; JF, QT, QYS and YZC provided support for experimental techniques and analyzed the data; ZC and FYW conceived the study, designed the experiments and helped with data analysis. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Fuyun Wu or Zheng Cao.

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Ethical approval and consent to participate

This experiment was approved by the Animal Management Committee of Hubei University of Medicine in accordance with the Animal Laboratory Animal Guidelines of Hubei University of Medicine and strictly abided by the rules of the Experimental Animal Ethics Committee of Hubei Medical College.

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Key Points

• Empagliflozin reduces the area of atherosclerotic plaques in ApoE−/− mice.

• Empagliflozin induces autophagy in RAW246.7 cells, HASMCs and HUVECs.

• Empagliflozin may slow the progression of atherosclerosis by inducing autophagy.

• Empagliflozin may induce autophagy by activating the AMPK signaling pathway.

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Xu, H., Fu, J., Tu, Q. et al. The SGLT2 inhibitor empagliflozin attenuates atherosclerosis progression by inducing autophagy. J Physiol Biochem 80, 27–39 (2024). https://doi.org/10.1007/s13105-023-00974-0

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