Fig. 6.

Curcumol induces mitochondrial ROS generation and depolarization in HSCs via a RIPK3-dependent mechanism. Human HSC-LX2 cells were treated with DMSO (0.02%, w/v), or curcumol at indicated concentrations for 24 h. Following the treatment, (A) ROS production was assessed by DCFH-DA staining, Scale bar, 50 µm. (C) Mitochondrial superoxide was detected by immunofluorescence using MitoSox Red staining. Scale bar, 50 µm. (F) The JC-1 fluorescence ratio evaluated mitochondrial membrane potential. Scale bar, 50 µm. (B) HSC-LX2 cells were pretreated with NAC (10 mM) for 1 h and then were treated with the indicated concentration of curcumol for 24 h. Cell viability was evaluated by Cell Counting Kit-8 analysis. HSC-LX2 cells were steadily transfected with RIPK3 siRNA, and then treated with the indicated concentration of curcumol for 24 h. Following the treatment, (D) MitoSOX Red immunofluorescence staining evaluated mitochondrial superoxide. Scale bar, 50 µm. (E) MDA levels, SOD cellular viability, and the GSH/GSSG ratio were measured by corresponding test kit. (G) Mitochondrial membrane potential was detected by the JC-1 fluorescence ratio. Scale bar, 50 µm. Data are expressed as mean ± SD (n = 3); *P < 0.05 versus DMSO, **P < 0.01 versus DMSO, and ***P < 0.001 versus DMSO. ^&& P < 0.01 versus control. ^#P < 0.05 versus curcumol (30 μM), ^##P < 0.01 versus curcumol (30 μM), ^###P < 0.001 versus curcumol (30 μM).