Intracellular glutathione depletion by oridonin leads to apoptosis in hepatic stellate cells

Abstract

Proliferation of hepatic stellate cells (HSCs) plays a key role in the pathogenesis of liver fibrosis. Induction of HSC apoptosis by natural products is considered an effective strategy for treating liver fibrosis. Herein, the apoptotic effects of 7,20-epoxy-ent-kaurane (oridonin), a diterpenoid isolated from Rabdosia rubescens, and its underlying mechanisms were investigated in rat HSC cell line, HSC-T6. We found that oridonin inhibited cell viability of HSC-T6 in a concentration-dependent manner. Oridonin induced a reduction in mitochondrial membrane potential and increases in caspase 3 activation, subG1 phase, and DNA fragmentation. These apoptotic effects of oridonin were completely reversed by thiol antioxidants, N-acetylcysteine (NAC) and glutathione monoethyl ester. Moreover, oridonin increased production of reactive oxygen species (ROS), which was also inhibited by NAC. Significantly, oridonin reduced intracellular glutathione (GSH) level in a concentration- and time-dependent fashion. Additionally, oridonin induced phosphorylations of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK). NAC prevented the activation of MAPKs in oridonin-induced cells. However, selective inhibitors of MAPKs failed to alter oridonin-induced cell death. In summary, these results demonstrate that induction of apoptosis in HSC-T6 by oridonin is associated with a decrease in cellular GSH level and increase in ROS production.

Figure 1

Oridonin decreased viability of HSC-T6. Cells were treated with DMSO (control) or indicated concentrations of oridonin for 24 h. (A) Chemical structure of 7,20-epoxy-ent-kaurane (oridonin). (B) After the incubation period, cell viability was determined using WST-1 assay. (C) Morphological changes in HSC-T6 were observed at 0 (control), 24, and 48 h. Bar = 10 μM. All data are presented as the mean ± SEM. (n = 6). *** p < 0.001 compared to control.

Figure 2

Oridonin induced apoptosis of HSC-T6. (A) Time-dependent changes in the subG1 phase population were determined after oridonin (30 μM) treatment or not (control). (B) Representative subG1 populations calculated from FACS histograms are shown (n = 4). (C) Changes in nuclear morphology by DMSO (control) or oridonin at 24 h were visualized using TUNEL staining. Bar = 10 μM. All data are presented as the mean ± SEM. *** p < 0.001 compared to control.

Figure 3

Oridonin increased caspase 3 activity and expression. HSC-T6 cells were treated with different concentrations of oridonin for 24 h. (A) Caspase 3 activity was measured using Caspase 3/CPP32 colorimetric assay kits. (n = 6). *** p < 0.001 compared to control. (B) The expressions of pro-caspase and cleaved caspase 3 were detected using western blotting analysis. β-Actin was used as a loading control. All data are presented as the mean ± SEM.

Figure 4

Oridonin induced ROS production. HSC-T6 cells were treated with oridonin for the indicated times. The levels of intracellular ROS were determined using DCF-DA, and the fluorescence was detected using FACS Calibur analysis. (A) Right shifts in fluorescence represented a change induced by oridonin (30 μM) or not (0 h, control). (B) The mean fluorescence intensity of DMSO (control) or oridonin is shown. (n = 6–8). All data are presented as the mean ± SEM. *** p < 0.001 compared to control.

Figure 5

NAC inhibited ROS production and cell viability in oridonin-treated HSC-T6. (A) Cells were pretreated with NAC (5 mM) for 1 h and then treated with DMSO (control) or oridonin (30 μM) for 6 h. The levels of intracellular ROS were determined using DCF-DA, and the fluorescence was detected using FACS Calibur analysis. (B) The mean fluorescence intensity is shown. (C) NAC reversed the oridonin-induced cell death. NAC (0.1, 1, and 5 mM) was preincubated for 1 h before the addition of oridonin (15 and 30 μM) for 24 h. Cell viability was detected using the WST-1 assay (n = 4). All data are presented as the mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001 compared to the corresponding oridonin alone.

Figure 6

Oridonin caused intracellular GSH depletion. (A) Cells were treated with DMSO or oridonin (15 or 30 μM) for the indicate times. Intracellular GSH levels were determined using mBCl. (n = 6). (B) GSH-MEE reversed the cell death caused by oridonin. GSH-MEE (0.05, 0.1, 0.5, and 1 mM) was preincubated for 1 h before the addition of oridonin (30 μM) for 24 h. Cell viability was detected using the WST-1 assay (n = 4). All data are presented as the mean ± SEM. *** p < 0.001 compared to DMSO; ^### p < 0.001 compared to oridonin alone.

Figure 7

Oridonin induced phosphorylation of MAPKs. (A) Oridonin induced the phosphorylation of MAPKs in a time-dependent manner. Cells were treated with oridonin (30 μM) for the indicated times. Phosphorylation of ERK, p38 MAPK, and JNK was analyzed using immunoblotting analysis with antibodies against phosphorylated and total protein. (B) NAC reversed oridonin-induced phosphorylation of MAPKs. NAC (5 mM) was preincubated for 1 h before the addition of oridonin (30 μM). Phosphorylation of ERK, p38 MAPK, and JNK was analyzed using immunoblotting analysis with antibodies against the phosphorylated and total protein.

Figure 8

Pharmacological inhibitors of MAPKs failed to alter oridonin-induced cell death. (A) PD98059, (B) SB203580, and (C) SP600125 did not reverse the cell death caused by oridonin. Cells were pretreated with various concentrations of inhibitors for 1 h before the addition of oridonin (30 μM). Cell viability was detected using the WST-1 assay (n = 3).

Figure 9

NAC and Z-DEVD-fmk reversed oridonin-induced mitochondrial dysfunction, caspase 3 activation, and DNA fragmentation. (A) NAC reversed the oridonin-induced loss of mitochondrial membrane potential. NAC (5 mM) was preincubated for 1 h before the addition of oridonin (30 μM) for 24 h. The mitochondrial membrane potential was determined using rhodamine 123, and the fluorescence was detected using FACS Calibur analysis. The mean fluorescence intensity is shown in the upper right corner (n = 4). (B) NAC and Z-DEVD-fmk reversed oridonin-induced caspase 3 activity. NAC (5 mM) or Z-DEVD-fmk (20 μM) was preincubated for 1 h before the addition of oridonin (30 μM) for 24 h. The caspase 3 activity was determined using Caspase 3/CPP32 colorimetric assay kits. (C) NAC and Z-DEVD-fmk suppressed oridonin-induced DNA fragmentation. NAC (5 mM) or Z-DEVD-fmk (20 μM) was preincubated for 1 h before the addition of oridonin (30 μM) for 24 h. Changes in the subG1 phase ratio were determined using propidium iodide (PI) staining at the indicated time points using FACS Calibur analysis. (D) Representative subG1 populations calculated from FACSalibur histograms are shown (n = 6). All data were presented as the mean ± SEM. ** p < 0.01 compared to oridonin alone.