| Objective: To investigate the important role and potential molecular mechanism of Dapagliflozin(Dapa)in down-regulating autophagy and improving endothelial(HUVECs)disorders of human umbilical vein endothelial cells induced by high glucose and high fat(HG/HF).Materials and methods: 1.Thoracic aortic tissue samples from patients undergoing aortic dissection were collected and the expression of SGLT-2 was detected by immunohistochemistry and immunofluorescence;2.HUVECs were transfected with overexpressed plasmid SLC5A2 to overexpress SGLT-2protein,and the changes in autophagy and cell function of HUVECs were detected after overexpression of SGLT-2;3.The HUVECs overexpressing SGLT-2 were pretreated with autophagy inhibitor 3-MA(5 m M)to observe the changes in endothelial function,determined the relationship between SGLT-2-induced autophagy and endothelial cell dysfunction;4.HUVECs were used as in vitro experimental subjects.CCK-8 was used to detect the proliferation of HUVECs after 24 hours of cell stimulation by different treatment factors,and the optimal experimental induction group and drug action group were selected;5.The experiment was divided into normal group(5.5m M),control group(16.5m M mannitol + 10% bovine serum albumin+ 0.1M sodium hydroxide),HG/PA group(22m M glucose,0.125 m M palmitic acid)and HG /PA+ DAPA group(5μM).The expression of SGLT-2 protein on HUVECs was detected by immunoimprinting and immunofluorescence techniques to understand the changes of SGLT-2 protein.In addition,endothelial cell function was evaluated by cell proliferation,migration,tubular formation,and apoptosis;The level of autophagy in endothelial cells was evaluated by immunoimprinting and the number of autophagosomes under electron microscopy.Results: 1.The expression of SGLT-2 was found in human thoracic aorta tissues and HUVECs.Immunohistochemistry,immunofluorescence and western blotting showed that the expression of SGLT-2 was up-regulated in atherosclerotic of human thoracic aorta tissue and HG/PA induced HUVECs;2.After the overexpression of SGLT-2 in HUVECs,the expression of autophagy proteins LC3-II,Beclin1 and Atg5 was up-regulated,the expression of P62 was down-regulated,the proliferation,migration and tubule formation ability of HUVECs were inhibited,apoptosis was increased,and apoptotic related proteins were up-regulated(P<0.05);3.After preconditioning with the autophagy pathway inhibitor 3-MA,the overexpression of SGLT-2-induced migration and tubule dysfunction were restored(P<0.05),and the cell apoptosis was improved(P<0.05);4.Different concentrations of HG/PA(11/0.125,22/0.25,33/0.5,44/1.0,55/2.0m M)stimulated HUVECs for24 hours,HG/PA reduced the proliferation of HUVECs in a concentration-dependent manner(P<0.05),inhibited the migration of HUVECs,decreased the tubule formation of HUVECs,significantly increased the fluorescence spots of Hoechst,and increased the contents of apoptotic proteins Caspase-3 and Bax,and the expression of Bcl 2 was down-regulated;After the addition of Sodium-glucose co-transporters 2 Inhibitor Dapagliflozin at the optimal treatment concentration,the proliferation of HUVECs inhibited by HG/PA was improved(P<0.05),and the tubule formation and migration ability of cells inhibited by HG/PA were significantly restored;Hoechst fluorescent spots were reduced,the expression of Caspase-3 and Bax were down-regulated,and the expression of Bcl 2 was increased(P<0.05);5.The contents of LC3-II,Beclin1,ATG5,P62 and autophagosomes were significantly increased 24 hours after HG/PA(22/0.25 m M)stimulation of HUVECs(P<0.05),and the addition of Dapagliflozin significantly reduced the number of autophagosomes and the number of autophagosomes in HG/PA induced HUVECs(P<0.05).Conclusions: 1.The expression of SGLT-2 was found in both human thoracic aorta tissues and HUVECs,and the expression of SGLT-2 was upregulated in atherosclerotic of human thoracic aorta tissue and HG/PA induced HUVECs;2.By targeting SGLT-2,Dapagliflozin down-regulates HG/PA induced autophagy and apoptosis of HUVECs,and improves HUVECs dysfunction. |
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