Fig 1.
Suppressive effects of edaravone on ROS in primary HCEpiCs exposed to hyperosmotic media.
ROS was evaluated via DCFH-DA staining (ANOVA: *, P<0.001 vs. untreated control; #, P<0.001 vs. 450 mOsM treated group).
Fig 2.
Suppressive effects of edaravone on mitochondrial function at 10 or 20 μM.
ROS in primary HCEpiCs exposed to hyperosmotic media (400–450 mOsM), evaluated via MitoSox Red staining (ANOVA: *, P<0.001 vs. untreated control; #, P<0.001 vs. 450 mOsM treated group).
Fig 3.
Effects of edaravone on mitochondrial function in primary HCEpiCs exposed to hyperosmotic media.
(A) Representative fluorescence photos of mitochondrial membrane potential (MMP) show reduced intracellular MMP in primary HCEpiCs, which was ameliorated by edaravone in a dose-dependent manner; (B) Quantitative results show that exposure to hyperosmotic media (400–450 mOsM) reduced intracellular ATP in HCEpiCs, which was attenuated by edaravone in a dose-dependent manner (ANOVA: *, P<0.001 vs. untreated control; #, P<0.001 vs. 450 mOsM treated group).
Fig 4.
Edaravone prevents cell death induced by exposure to hyperosmotic media (400–450 mOsM).
(A) Cell viability was measured by MTT assay; (B) Cell death was measured by LDH release assay. All experiments were repeated at least three times (ANOVA: *, P<0.001 vs. untreated control; #, P<0.001 vs. 450 mOsM treated group).
Fig 5.
Effects of edaravone on apoptosis in primary HCEpiCs exposed to hyperosmotic media (400–450 mOsM).
Apoptotic cells were stained using a TUNEL Assay Kit. Nuclear DNA was stained with DAPI.
Fig 6.
Effects of edaravone on cytosol cytochrome C release as measured by western blot analysis.
Results indicate that edaravone treatment attenuates hyperosmotic exposure (400–450 mOsM)-induced release of cytochrome C. (A) Cytosolic fractions; (B) Mitochondrial fractions (ANOVA:*, P<0.001 vs. non-treatment control; #, P<0.001 vs. 450 mOsM treated group).
Fig 7.
Effects of edaravone on cleaved caspase-3 as measured by western blot analysis.
Edaravone treatment inhibits the increase in cleaved caspase-3 induced by hyperosmotic exposure (400–450 mOsM) (ANOVA:*, P<0.001 vs. non-treatment control; #, P<0.001 vs. 450 mOsM treated group).
Fig 8.
Effects of edaravone on the expression of Nrf2.
Levels of Nrf2 in whole cell extracts were determined by the western blot analysis (ANOVA:*, P<0.001 vs. non-treatment control; #, P<0.001 vs. 450 mOsM treated group).
Fig 9.
Effects of edaravone on Nrf2 target gene expression in primary HCEpiCs.
Cells were treated with edaravone for 24 h. Gene expression was determined by real-time PCR analysis. (A) HO-1 mRNA; (B) GPx-1 mRNA; (C) GCLC mRNA; (D) GSH levels (ANOVA:*, P<0.001 vs. non-treatment control; #, P<0.001 vs. 450 mOsM treated group).