The protective effects of etomidate against interleukin-1β (IL-1β)-induced oxidative stress, extracellular matrix alteration and cellular senescence in chondrocytes

Abstract

Osteoarthritis (OA) is a common chronic inflammatory disease associated with aging. Etomidate is an intravenous anesthetic with profound antioxidant and anti-inflammatory effects. We speculated that etomidate might exert a beneficial effect on OA. Herein, we explored the effects of etomidate on interleukin-1β (IL-1β)- induced chondrocytes. Our results prove that etomidate ameliorated the IL-1β-induced oxidative stress in C28/12 chondrocytes by decreasing and increasing the reactive oxygen species (ROS) and glutathione peroxidase (GPx) levels, respectively. Etomidate prevented the IL-1β-induced increase in the expressions of matrix metalloproteinase-3 (MMP-3) and matrix metalloproteinase-13 (MMP-13) in C28/I2 chondrocytes at both mRNA and protein levels. It also caused a significant reduction in the percentage of senescence-associated-β-galactosidase (SA-β-Gal)-stained chondrocytes, while inducing elevated telomerase activity in IL-1β-treated C28/I2 chondrocytes. The expression levels of senescence regulators, plasminogen activator inhibitor-1 (PAI-1) and p16, were also inhibited by etomidate in IL-1β-treated C28/I2 chondrocytes. In addition, etomidate caused the activation of Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK), along with upregulated expression levels of phosphorylated AMPKα and phosphorylated acetyl-Co A carboxylase (ACC). Moreover, blockage of AMPK using compound C abolished the protective effects of etomidate on IL-1β-challenged C28/I2 chondrocytes. Taken together, these results demonstrate that etomidate protected C28/I2 chondrocytes from IL-1β-induced oxidative stress, ECM degradation, and cellular senescence via activating AMPK signaling.

Keywords: AMP-activated protein kinase (AMPK); Osteoarthritis (OA); aging; chondrocytes; etomidate.

Figure 1.

Cytotoxicity of etomidate in human C28/I2 chondrocytes. Cells were stimulated with etomidate at the concentrations of 0, 0.3, 0.6, 3, 6, 30, 60 μM for 24 hours. (a). Molecular structure of etomidate; (b). LDH release (^{#, ##}P < 0.05, 0.01 vs. vehicle group).

Figure 2.

Etomidate ameliorates IL-1β-induced oxidative stress. Cells were incubated with IL-1β (10 ng/mL) in the presence or absence of 3 and 6 μM etomidate for 24 hours. (a). Intracellular ROS; (b). The level of GPx (^###P < 0.005 vs. vehicle group; *^, **P < 0.05, 0.01 vs. IL-1β group).

Figure 3.

Etomidate prevents IL-1β-induced increase in the expression of MMP-3 and MMP-13. Cells were incubated with IL-1β (10 ng/mL) in the presence or absence of 3 and 6 μM etomidate for 24 hours. (a). mRNA of MMP-3 and MMP-13 as measured by real-time PCR; (b). Protein of MMP-3 and MMP-13 (^###P < 0.005 vs. vehicle group; *^, **P < 0.05, 0.01 vs. IL-1β group).

Figure 4.

Etomidate mitigates IL-1β-induced cellular senescence. Cells were incubated with IL-1β (10 ng/mL) in the presence or absence of 3 and 6 μM etomidate for 7 days. Cellular senescence was assayed with senescence-associated β- galactosidase (SA-β-Gal) staining (^###P < 0.005 vs. vehicle group; *^, **P < 0.05, 0.01 vs. IL-1β group).

Figure 5.

Etomidate attenuates IL-1β-induced reduction in telomerase activity. Cells were incubated with IL-1β (10 ng/mL) in the presence or absence of 3 and 6 μM etomidate for 7 days. Telomerase activity was measured using a commercial kit (^###P < 0.005 vs. vehicle group; *^, **P < 0.05, 0.01 vs. IL-1β group).

Figure 6.

Etomidate inhibits the expression of PAI-1 and p16 against IL-1β. Cells were incubated with IL-1β (10 ng/mL) in the presence or absence of 3 and 6 μM etomidate. (a). mRNA of PAI-1 and p16; (b). Western blot of PAI-1 and p16 (^###P < 0.005 vs. vehicle group; *^, **P < 0.05, 0.01 vs. IL-1β group).

Figure 7.

Etomidate ameliorates IL-1β- induced inactivation of AMPKα against IL-1β. Cells were incubated with IL-1β (10 ng/mL) in the presence or absence of 3 and 6 μM etomidate. Phosphorylated AMPKα and phosphorylated ACC were measured using Western blot analysis (^###P < 0.005 vs. vehicle group; *^, **P < 0.05, 0.01 vs. IL-1β group).

Figure 8.

Blockage of AMPK abolishes the protective effects of Etomidate against IL-1β- induced cellular senescence. Cells were incubated with IL-1β (10 ng/mL) in the presence or absence of 6 μM Etomidate or the AMPK inhibitor compound C (10 μM). (a). mRNA levels of PAI-1 and p16; (b). Telomerase activity; (c). SA-β-Gal staining (^###P < 0.005 vs. vehicle group; *^, **P < 0.05, 0.01 vs. IL-1β group).