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.