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.