SGLT2 inhibitor empagliflozin downregulates miRNA-34a-5p and targets GREM2 to inactivate hepatic stellate cells and ameliorate non-alcoholic fatty liver disease-associated fibrosis

Activation of hepatic stellate cells (HSCs), the central event of fibrosis, indicates the severe stage of non-alcoholic fatty liver disease (NAFLD). MicroRNAs (miRNAs) participate in this process. Treatment with a sodium-glucose cotransporter 2 inhibitor (SGLT2i) alleviates liver fibrosis in patients with type 2 diabetes and NAFLD; however, the role of SGLT2i in ameliorating liver fibrosis in NAFLD by regulating miRNAs remains unclear. We monitored the expression of NAFLD-associated miRNAs in the livers of two NAFLD models and observed high expression of miR-34a-5p. miR-34a-5p was highly expressed in mouse primary liver non-parenchymal cells and LX-2 HSCs, and this miRNA was positively correlated with alanine transaminase levels in NAFLD models. Overexpression of miR-34a-5p enhanced LX-2 activation, whereas its inhibition prevented HSCs activation by regulating the TGFβ signaling pathway. The SGLT2i empagliflozin significantly downregulated miR-34a-5p, inhibited the TGFβ signaling pathway, and ameliorated hepatic fibrosis in NAFLD models. Subsequently, GREM2 was identified as a direct target of miR-34a-5p through database prediction and a dual-luciferase reporter assay. In LX-2 HSCs, the miR-34a-5p mimic and inhibitor directly downregulated and upregulated GREM2, respectively. Overexpressing GREM2 inactivated the TGFβ pathway whereas GREM2 knockdown activated it. Additionally, empagliflozin upregulated Grem2 expression in NAFLD models. In methionine- and choline-deficient diet-fed ob/ob mice, a fibrosis model, empagliflozin downregulated miR-34a-5p and upregulated Grem2 to improve liver fibrosis. Empagliflozin ameliorates NAFLD-associated fibrosis by downregulating miR-34a-5p and targeting GREM2 to inhibit the TGFβ pathway in HSCs. In NAFLD-associated fibrosis, elevated miR-34a-5p levels inhibit GREM2 expression and activate the TGFβ signaling pathway to activate HSCs. SGLT2i empagliflozin downregulated miR-34a-5p expression and upregulated GREM2 expression to inhibit the TGFβ signaling pathway in HSCs to alleviate NAFLD-associated fibrosis. The graphical abstract was created with BioRender (www.BioRender.com). SGLT2, sodium-glucose cotransporter 2; HSC, hepatic stellate cell; ECM, extracellular matrix. [Display omitted] •miR-34a-5p was highly expressed in NAFLD models and hepatic stellate cells (HSCs).•GREM2, a target of miR-34a-5p, was downregulated by miR-34a-5p to activate the TGFβ signaling pathway in HSCs of NAFLD.•Empagliflozin downregulates miRNA-3