3.1 Effects of empagliflozin and IL-1β on cell viability and
chondrocyte phenotype maintenance
The effects of empagliflozin and IL-1β on cell viability and chondrocyte
phenotype maintenance were evaluated by CCK-8 assay and the safranine O
staining. The results demonstrated that empagliflozin had no
cytotoxicity on mouse chondrocyte viability with concentration ≤5 μM at
24 h. Besides, chondrocytes treated with 1 and 5 μM concentrations of
empagliflozin could significantly improve the down-regulated cell
viability caused by IL-1β (Figure 1A). Similarly, the results of
safranine O staining revealed that chondrocytes were apparently less
stained after 24 h IL-1β treatment, but the mouse chondrocytes treated
with 1 and 5 μM of empagliflozin were observably reverse the IL-1β
caused safranine O stain loss (Figure 1B). Thus, 1 and 5 μM
concentrations of empagliflozin were used for the following in vitro
tests, and a concentration of 5 μM was used for the following in vivo
tests.
3.2 Effects of
empagliflozin on IL-1β-induced ECM degradation in mouse chondrocytes
Here the expression levels of catabolic markers of ECM were detected by
ELISA, RT-PCR and western blot to evaluate the effects of empagliflozin
on IL-1β-induced ECM degradation in mouse chondrocytes. As shown in
Figure 2, administration of empagliflozin could counteract the
increments of MMP9 and MMP13 secretion level in the cell medium caused
by IL-1β. In addition, pretreatment with empagliflozin significantly
decreased IL-1β-induced upregulation of matrix-degrading genes (MMP9,
MMP13) in a dose-dependent manner at both mRNA level and protein level.
Based on the results above, empagliflozin could protect mouse
chondrocytes from ECM degradation by inhibiting the MMPs.
3.3 Effects ofempagliflozin on
IL-1β-induced inflammation in mouse chondrocytes
To discover the anti-inflammation effects of empagliflozin on
mouse chondrocytes, the
expression of several inflammatory mediators in mouse chondrocytes was
assessed by ELISA, RT-PCR and western blot. As illustrated in Figure 3A,
the production and secretion of proinflammatory factors (NO, PGE2, and
IL-6) was increased under IL-1β treatment, and empagliflozin
pretreatment could dose-dependently offset these increments. Moreover,
the results also showed that empagliflozin significantly decreased
IL-1β-induced upregulation of other inflammatory genes (COX2 and INOS)
in both mRNA and protein expression levels (Figure 3B-C). These data
demonstrated that empagliflozin could significantly inhibited the
production of IL-1β-induced inflammatory mediators in mouse
chondrocytes.