1.Department of Children’s Heart Cardiovascular, the Second Afffliated Hospital & Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou 325027, China; 2.Department of Oncology, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325015, China
WU Chunhui,WU Huiyang,WU Mao, et al. Empagliflozin regulating lysosomal autophagy through transcription factor EB to alleviate human umbilical vein endothelial cells injury[J]. JOURNAL OF WEZHOU MEDICAL UNIVERSITY, 2024, 54(7): 517-527.
Abstract：Objective: To investigate the mechanism of how Empagliflozin (EMPA) improves endothelial injury in human primary umbilical vein endothelial cells (HUVECs). Methods: HUVECs were stimulated with were stimulated with TNFα, and divided into four groups: ①Control group: treated with PBS; ②TNFα group:stimulated with TNFα; ③EMPA group: TNFα+EMPA; ④BAF group: Bafilomycin A1 (Baf A1)+TNFα+EMPA;⑤siTFEB group: transcription factor EB (TFEB) was pre-interfered before cell modeling, and the rest processing was the same as the EMPA group. After 24 hours of modeling, cells were collected. Western blot (detecting the expression of proteins such as lysosome-associated membrane protein 1 (LAMP1), P62, LC3, VE-cadherin,TFEB, pTFEB, etc.), cell immunofluorescence (VE-cadherin, LAMP1, TFEB, etc.), monocyte adhesion, cell leakage, RNA sequencing, adenovirus transfection GFP-LC3 (detecting the amount of LC3 accumulation in cells),lentivirus transfection RFP-GFP-LC3 (detecting autophagy flow in cells) were performed. Results: Western blot results showed that, compared with the control group, the TNFα group significantly increased levels of P62,LC3, and pTFEB (P<0.01), while LAMP1 and TFEB levels decreased significantly (P<0.01). Compared with the TNFα group, the EMPA group showed decreased P62, LC3, and pTFEB levels (P<0.05) but increased LAMP1 and TFEB levels (P<0.05). The BAF group demonstrated higher P62 and LC3 expression than the EMPA group (P<0.01). After TFEB interference, the siTFEB group had increased P62 and LC3 expression compared with the EMPA group (P<0.01), with LAMP1 level decreased (P<0.01). Immunofluorescence results indicated that TNFα group had increased TFEB accumulation in the cytoplasm, compared with the control group (P<0.01), and the EMPA group had less TFEB cytoplasmic accumulation than the TNFα group (P<0.01). RNA measurements suggested that EMPA activated the autophagy pathway. The RFP-GFP-LC3 dual fluorescence results showed that the EMPA group had more autolysosome formation than the TNFα group (P<0.01). Conclusion: Empagliflozin enhances lysosomal autophagy through TFEB, thereby improving endothelial damage in HUVECs.
