De novo synthesis of glutathione is a prerequisite for curcumin to inhibit hepatic stellate cell (HSC) activation

Abstract

On liver injury, quiescent hepatic stellate cells (HSC), the most relevant cell type for hepatic fibrogenesis, become active, characterized by enhanced cell growth and overproduction of extracellular matrix (ECM). Oxidative stress facilitates HSC activation and the pathogenesis of hepatic fibrosis. Glutathione (GSH) is the most important intracellular antioxidant. We previously showed that curcumin, the yellow pigment in curry from turmeric, significantly inhibited HSC activation. The aim of this study is to elucidate the underlying mechanisms. It is hypothesized that curcumin might inhibit HSC activation mainly by its antioxidant capacity. Results from this study demonstrate that curcumin dose and time dependently attenuates oxidative stress in passaged HSC demonstrated by scavenging reactive oxygen species and reducing lipid peroxidation. Curcumin elevates the level of cellular GSH and induces de novo synthesis of GSH in HSC by stimulating the activity and gene expression of glutamate-cysteine ligase (GCL), a key rate-limiting enzyme in GSH synthesis. Depletion of cellular GSH by the inhibition of GCL activity using L-buthionine sulfoximine evidently eliminates the inhibitory effects of curcumin on HSC activation. Taken together, our results demonstrate, for the first time, that the antioxidant property of curcumin mainly results from increasing the level of cellular GSH by inducing the activity and gene expression of GCL in activated HSC in vitro. De novo synthesis of GSH is a prerequisite for curcumin to inhibit HSC activation. These results provide novel insights into the mechanisms of curcumin as an antifibrogenic candidate in the prevention and treatment of hepatic fibrosis.

Figure 1. Curcumin time-, and dose-dependently reduces the level of ROS and LPO in activated HSC in vitro

Passaged HSC were either treated with curcumin at 20μM for various hours (A), or with curcumin at various concentrations (B & C) for 24 hr. The levels of intracellular ROS (A & B) and LPO (C) were assessed as described in Methods. The levels of cellular ROS (A & B), or LPO (C), were expressed as fold changes compared to that in the control either at the time 0 (A), or without curcumin treatment (B & C). Values were presented as means ± S.D. (n≥3). *p<0.05, versus the control (the 1^st point, or column, on the left).

Figure 2. Curcumin causes a time- and dose-dependent increase in the content of cellular GSH in activated HSC in vitro

Passaged HSC were either treated with curcumin at 20μM for various hours (A), or with curcumin at various concentrations (B & C) for 24 hr. The levels of total cellular GSH (A & B) and the ratio of GSH/GSSG (C) were determined as described in Methods. The level of total cellular GSH was expressed as nmol/μg protein. Values were presented as means ± S.D. (n≥3). *p<0.05, versus the control (the 1^st point, or column, on the left).

Figure 3. The pretreatment with BSO reduces cellular GSH content in activated HSC

Passaged HSC were either treated with BSO at various concentrations for 24 hr (A), or with BSO at 0.25 mM for various hours (B). The level of total cellular GSH was expressed as nmol/μg protein. The number of percentage indicated the reduction in the level of cellular GSH in cells treated with BSO at 0.25 mM, compared to that in the control cells with no treatment (the 1^st column on the left). Values were presented as means ± S.D. (n≥3). *p<0.05, versus the control (the 1^st column, or point, on the left).

Figure 4. The antioxidant capability of curcumin mainly results from de novo synthesis of GSH

Passaged HSC were either maintained in DMEM with 10% of FBS, or treated for 24 hr with BSO (0.25mM), or curcumin (20μM), or NAC (5mM) with or without the pre-exposure to BSO (0.25mM) for 1 hr. The levels of total cellular GSH (A), ROS (B) and LPO (C) were, respectively, determined and expressed as fold changes compared to that in the control with no treatment. The number of percentage in (A) indicated the increase in the level of cellular GSH in cells treated with curcumin or NAC compared to that in the control cells with no treatment. Values were means ± S.D. (n≥3). *p<0.05, versus the control with no treatment (the 1^st column on the left); ‡ p<0.05, versus cells treated with curcumin or NAC (the 2^nd, or 3^rd column respectively, on the left).

Figure 5. Curcumin dose-dependently increases the activity of GCL in passaged HSC

HSC were treated with curcumin at indicated concentrations for 24 hr. The activity of GCL was determined as described in Methods. Values were expressed as means ± S.D. (n≥6). The number of percentage indicated the increase in GCL activity in cells treated with curcumin at 20 μM, compared to that in control cells with no treatment (0). *p<0.05, versus control cells with no treatment (the 1^st column on the left).

Figure 6. Curcumin induces gene expression of the two GCL subunits in passaged HSC

HSC were treated with curcumin at indicated concentrations for 24 hr. (A). Luciferase assays of cells transfected with the plasmid pGCLc-Luc, containing −1758bp of Gclc gene promoter. Luciferase activities were normalized with ß-galactosidase activities (n≥6). *p<0.05, versus control cells with no treatment (the 1^st column on the left). The floating schema denotes the pGCLc-Luc luciferase reporter construct in use and the application of curcumin to the system. (B). Real-time PCR assays of Gclc and Gclm. GAPDH was used as an invariant control for calculating mRNA fold changes (n=3). Values were means ± S.D. *p<0.05, versus cells with no treatment (the 1^st columns on the left). (C). Western blotting analyses of GCLc and GCLm. ß-actin was used as an internal control. Representatives were shown from three independent experiments.

Figure 7. de novo synthesis of GSH plays a critical role in the inhibitory effect of curcumin on HSC growth

Passaged HSC were either maintained in DMEM with 10% of FBS, or treated for 24 hr with BSO (0.25mM), or curcumin (20μM), or NAC (5mM) with or without the pre-exposure to BSO (0.25mM) for 1 hr. (A). Western blotting analyses of proteins relevant to cell growth. ß-actin was used as an invariant control for equal loading. Representatives of three independent experiments were shown; (B). Cell growth was determined by MTS assays. Values were expressed as means ± S.D (n=3). *p<0.05, versus control cells with no treatment (the 1^st column on the left); ‡ p<0.05, versus cells treated with curcumin, or NAC (the 2^nd or 3^rd column, respectively, on the left). (C). Flow cytometric analyses of apoptosis (n=3). *p<0.05, versus control cells with no treatment (the 1^st column on the left); ** p<0.05, versus cells treated with curcumin (the 2^nd column on the left).

Figure 8. de novo synthesis of GSH is a necessity for curcumin to regulate expression of genes relevant to fibrogenesis in activated HSC in vitro

Passaged HSC were either maintained in DMEM with 10% of FBS, or treated for 24 hr with BSO (0.25mM), or curcumin (20μM), or NAC (5mM) with or without the pre-exposure to BSO (0.25mM) for 1 hr. (A). Real-time PCR analyses. GAPDH was used as an invariant control for calculating mRNA fold changes. Values were expressed as means ± S.D (n=3). *p<0.05, versus control cells (the 1^st column on the left); ‡ p<0.05, versus cells treated with curcumin or NAC (the 2^nd or 3^rd column on the left). (B). Western blotting analyses. ß-actin was used as an invariant control for equal loading. Representatives of three independent experiments were shown.

Figure 9. A Schema of the role of the antioxidant capability of curcumin in the inhibition of HSC activation

Curcumin increases the level of intracellular GSH by inducing expression of GCL genes and stimulating the activity of GCL, leading to the attenuation of oxidative stress in activated HSC. These actions, together with others, reduce cell growth and fibrogenesis, resulting in the inhibition of HSC activation.