Empagliflozin Reduces the Progression of Hepatic Fibrosis in a Mouse Model and Inhibits the Activation of Hepatic Stellate Cells via the Hippo Signalling Pathway

Abstract

Hepatic fibrosis is the excessive production and deposition of the extracellular matrix, resulting in the activation of the fibrogenic phenotype of hepatic stellate cells (HSCs). The Hippo/Yes-associated protein (YAP) signalling pathway is a highly conserved kinase cascade that is critical in regulating cell proliferation, differentiation, and survival, and controls stellate cell activation. Empagliflozin, a sodium-glucose cotransporter type-2 inhibitor, is an antidiabetic drug that may prevent fibrotic progression by reducing hepatic steatosis and inflammation. However, little is known about its mechanism of action in liver fibrosis. In this study, we used male C57 BL/6 J mice fed a choline-deficient, l-amino acid-defined, high-fat diet (CDAHFD) as a model for hepatic fibrosis. For 5 weeks, the mice received either a vehicle or empagliflozin based on their assigned group. Empagliflozin attenuated CDAHFD-induced liver fibrosis. Thereafter, we identified the Hippo pathway, along with its effector, YAP, as a key pathway in the mouse liver. Hippo signalling is inactivated in the fibrotic liver, but empagliflozin treatment activated Hippo signalling and decreased YAP activity. In addition, empagliflozin downregulated the expression of pro-fibrogenic genes and activated Hippo signalling in HSCs. We identified a mechanism by which empagliflozin ameliorates liver fibrosis.

Keywords: Hippo signalling pathway; empagliflozin; hepatic fibrosis; hepatic stellate cells.

Conflict of interest statement

All authors have no conflict of interest to declare.

Figures

Figure 1

Effects of empagliflozin on hepatic fibrosis in CDAHFD-induced mice. The mice were treated with the control chow diet (CD), choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD), or CDAHFD with empagliflozin (EMPA) treatment. (a) The liver histology was studied via macroscopic morphological examination, H&E staining, and Sirius Red staining. α-SMA signals were visualised via immunohistochemistry. (b) Fibrosis grades in the different groups. (c) α-SMA, fibronectin, and connective tissue growth factor (cTGF) signals were examined and quantified using Western blot. β-actin served as the loading control. (d) Relative expression of genes related to fibrosis was determined via RT-PCR. * p < 0.05, ** p < 0.01, and *** p < 0.001 in the comparisons of the CD-fed mice with the CDAHFD-fed mice. † p < 0.05, †† p < 0.01, and ††† p < 0.001 in the comparisons of the CDAHFD-fed mice with the EMPA treatment groups. PV: portal vein; CV: central vein.

Figure 2

Empagliflozin downregulates pro-fibrogenic markers and decreases proliferation in LX-2 cells. (a) Collagen 1 α1, α-SMA, fibronectin, and cTGF were assayed by Western blotting using specific antibodies. The maximum control phosphoprotein intensity was set to 100%, and relative test intensities were calculated. (b) The relative expression of α-SMA, Collagen 1 α1, and TGF-β was determined via real-time polymerase chain reaction (RT-PCR). (c) The cell proliferation marker, proliferating cell nuclear antigen (PCNA), was determined via RT-PCR. Data are expressed as means ± standard deviation of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to the control.

Figure 3

Effect of EMPA on Hippo signalling pathway in CDAHFD-induced mice. (a) Western blot of mice liver protein extracts with the indicated antibodies. (b) Quantitation and normalisation of phospho-specific signal to total protein or total protein to β-actin from a. (c) The relative expression of cyr61, cTGF, and AREG was determined via real-time PCR. ** p < 0.01, and *** p < 0.001 in the comparisons of the CD-fed mice with the CDAHFD-fed mice. † p < 0.05, †† p < 0.01, and ††† p < 0.001 in the comparisons of the CDAHFD-fed mice with the EMPA treatment groups.

Figure 4

Empagliflozin decreased Yes-associated protein (YAP) expression in LX-2 cells. (a) The representative protein expression of YAP and phospho-YAP was determined using Western blot. (b) Quantitation and normalisation of phospho-YAP to total YAP or total YAP to β-actin from a. (c) The relative expression of cyr61, cTGF, and AREG was determined via RT-PCR. Data are expressed as means ± standard deviation of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to the control.

Figure 5

Empagliflozin regulates Hippo signalling in LX-2 cells. (a) Following different doses of EMPA treatment in LX-2 cells for 12 h, the protein levels of MST1, MST2, phospho-MST1/2, MOB1, phospho-MOB1, LATS1, and phospho-LATS1 were evaluated via Western blot. (b) Statistical analysis on the relative expression of the Hippo pathway in LX-2 cells. Data are expressed as means ± standard deviation of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to the control.