Dapagliflozin Inhibits Myocardial Fibrosis By Promoting Lymphangiogenesis Through TGF-β/VEGFC Signaling Pathway

Objective:To investigate the pro-lymphatic neonatal effects of daglitazone(DAPA),a sodium-glucose co-transport protein 2 inhibitor(SGLT2 inhibitor),in angiotensin II(Ang II)-induced cardiac hypertrophic cells and mouse models and its mechanism.Methods:Eighteen C57BL/6 male mice aged 4-6 weeks and weighing 20-25 g were randomly divided into three groups: control group,angiotensin II group(Ang II,n=6)and angiotensin II combined with dapagliflozin group(Ang II-DAPA,n=6),and myocardial hypertrophy model was constructed by intraperitoneal injection of DAPA and subcutaneous injection of Ang II.The mice were weighed after 8 weeks of simultaneous feeding,and the cardiac function studies,including EF and FS,were evaluated using echocardiography after anesthesia in each group.After that,mice were executed and heart tissues were collected and weighed,and hematoxylin-eosin staining was used to observe the pathological changes of mouse hearts,while the expression of TGF-β1,type I collagen,type III collagen,α-SMA,VEGFC and VEGFR3 were observed using immunohistochemical staining,and protein expression in mouse heart tissues was quantified by Western blot.Then,human lymphatic vascular endothelial cell line HLECs were randomly divided into control group,Ang II-induced group and DAPA+Ang II intervention group,and different concentrations of Ang II and DAPA were given to induce myocardial hypertrophy model for 24 h.The migration ability of lymphatic vascular endothelial cells was observed by migration invasion assay,the expression of VEGFC and TGF-β in each group was quantified by Western blot,and the important role of TGF-β pathway in inhibiting myocardial fibrosis and promoting lymphatic vascular neogenesis was verified by rescue assay.Results:(1)Results of animal experiments: compared with the control group,mice in the angiotensin II group administered for 8 weeks showed significant myocardial hypertrophy,and the hearts of mice in the angiotensin II combined with dapagliflozin group were significantly reduced compared with those in the angiotensin II group alone,and myocardial hypertrophy was effectively inhibited.The hematoxylin-eosin staining results showed that the cardiac cell volumes of mice in both the angiotensin II and angiotensin II combined with dapagliflozin groups were higher than those in the conventional group,but the cardiac cell volumes of mice in the angiotensin combined with dapagliflozin group were smaller than those in the angiotensin II group.Masson staining results showed that mice in the angiotensin II group exhibited significant myocardial fibrosis compared with the control group.The angiotensin-induced myocardial fibrosis was attenuated by the dapagliflozin intervention.Immunohistochemical staining showed that the expression of VEGFC and VEGFR3 decreased and the expression of α-SMA,Collagen I and Collagen III increased in the heart tissue of mice in the angiotensin group,and dapagliflozin reversed the expression of these proteins.The expression of ANP,α-SMA and TGF-β were significantly increased in the angiotensin group(P<0.05),while the expression of VEGFC was significantly decreased,and again,dapagliflozin could reverse the angiotensin II-induced changes in the expression of these proteins.(2)Cellular assay results: The cell migration assay showed that angiotensin II intervention led to a decrease in the migration ability of lymphatic endothelial cells compared with the control group,while dapagliflozin enhanced the migration ability of lymphatic endothelial cells.western blot results showed that dapagliflozin inhibited myocardial fibrosis and promoted cardiac lymphatic vascular neogenesis by inhibiting the TGF-β signaling pathway.Rescue assay further demonstrated that the inhibition of myocardial fibrosis and promotion of lymphatic vascular neoplasia by dapagliflozin were dependent on the TGF-β signaling pathway.Conclusion:Dapagliflozin ameliorates angiotensin II-induced myocardial fibrosis and promotes cardiac lymphatic vascular neogenesis through the TGF-β/VEGFC signaling pathway in mice hearts.