Figure Captions
Figure 1 Empagliflozin improved glucose metabolism disorder in HFD/STZ-induced T2DM mice. (A) Flow chart of animal experiment design. All mice were randomly assigned to different groups: NCD+ citrate buffer+ normal saline (Control group), HFD+ STZ + normal saline (T2DM group), HFD+ STZ + Empagliflozin (T2DM+ Empagliflozin group). (B) The weight change curve of mice during 6-week Empagliflozin intervention. (C) Changes of random blood glucose in mice during 6-week Empagliflozin intervention. (D) Fasting blood glucose levels of mice in each group after 6-week Empagliflozin intervention. (E) Blood glucose at intervals of 0, 15, 30, 60, 120 minutes and AUC0~120min in OGTT. (F) Blood glucose at intervals of 0, 15, 30, 60, 120 minutes and AUC0~120min in IPGTT. Chow diet vs. High-fat diet or Control vs. T2DM: *P<0.05; ** P<0.01; ***P<0.001; T2DM vs. T2DM+ Empagliflozin, #P<0.05; ##P<0.01; ###P<0.001.
Figure 2 Empagliflozin ameliorated HFD/STZ-induced liver fibrosis in T2DM mice. (A) HE, Sirius red staining, and Masson’s trichrome staining of mouse liver. Sirius red staining was applied to assess the degree of liver fibrosis in mice (n = 5 per group, magnification, ×200), one-way ANOVA with Tukey post hoc test. (B) Western blot was applied to detect the protein expression levels of α-SMA and TGF-β1 in mice liver tissues. Each value is expressed as mean ± SD (n = 3 per group). (C) Relative mRNA expression of α-SMA, TGF-β1, Col1, Vimentin was detected using RT-qPCR. Each value is expressed as mean ± SD (n = 3 or 4 per group). (D) Representative graph of IHC staining for Col1, Col3 and Vimentin in liver tissues. Data are presented as mean ± SD (n=3 per group) and analyzed using one-way ANOVA with Tukey post hoc test.
Figure 3 Empagliflozin treatment rebalanced the gut dysbiosis in HFD/STZ-induced T2DM and liver fibrosis mice. (A) The barplots illustrated the taxonomic construction of different groups and their counterpart. (B) Venn diagram of gut microbiota showing the common genera. (C) Chao 1, Faith_pd, Shannon index and Observed_species were measured to evaluate alpha diversity. (D) PCoA plot showed the distribution of whole gut microbiota population. (E) Hierarchical cluster analysis of whole gut microbiota population. (F) Genus-level analysis of the relative abundances. (G) Relative abundance histogram of 3 representative strains at the genus level. Data are presented as mean ± SD (n=3 per group) and analyzed using one-way ANOVA with Tukey post hoc test. (H) The heatmap delineated the spearman correlation analysis between cecal microbial composition and fibrosis score or indicators related to glucose metabolism. Control vs. T2DM: *P<0.05; ** P<0.01; ***P<0.001; T2DM vs. T2DM+ Empagliflozin, #P<0.05; ##P<0.01; ###P<0.001.
Supplementary Figure 1 The weekly weight change curve of mice during HFD/STZ-induced T2DM and liver fibrosis.
Supplementary Figure 2 Changes in random blood glucose of mice after intraperitoneal injection of STZ at the week 8.
Supplementary Figure 3 Changes of food intake in mice during 6-week Empagliflozin intervention.
Supplementary Figure 4 Graphical abstract of the whole study.