Activation of PPARgamma is required for curcumin to induce apoptosis and to inhibit the expression of extracellular matrix genes in hepatic stellate cells in vitro

Abstract

During liver fibrogenesis, quiescent HSC (hepatic stellate cells) become active, a transformation that is associated with enhanced cell proliferation and overproduction of ECM (extracellular matrix). Inhibition of cell proliferation and induction of apoptosis are potential strategies to block the activation of HSC for the prevention and treatment of liver fibrosis. Levels of PPARgamma (peroxisome proliferator-activated receptor gamma) are dramatically diminished in parallel with HSC activation. Stimulation of PPARgamma by its agonists inhibits HSC activation in vitro and in vivo. We demonstrated recently that curcumin, the yellow pigment in curry, inhibited HSC activation in vitro, reducing cell proliferation, inducing apoptosis and inhibiting ECM gene expression. Further studies indicated that curcumin induced the gene expression of PPARgamma and stimulated its activity in activated HSC in vitro, which was required for curcumin to inhibit HSC proliferation. The aims of the present study were to evaluate the roles of PPARgamma activation in the induction of apoptosis and suppression of ECM gene expression by curcumin in activated HSC, and to elucidate the underlying mechanisms. Our results demonstrated that blocking PPARgamma activation abrogated the effects of curcumin on the induction of apoptosis and inhibition of the expression of ECM genes in activated HSC in vitro. Further experiments demonstrated that curcumin suppressed the gene expression of TGF-beta (transforming growth factor-beta) receptors and interrupted the TGF-beta signalling pathway in activated HSC, which was mediated by PPARgamma activation. Taken together, our results demonstrate that curcumin stimulated PPARgamma activity in activated HSC in vitro, which was required for curcumin to reduce cell proliferation, induce apoptosis and suppress ECM gene expression. These results provide novel insight into the mechanisms responsible for the inhibition of HSC activation by curcumin. The characteristics of curcumin, which has no adverse health effects, make it a potential candidate for prevention and treatment of hepatic fibrosis.

Figure 1. Activation of PPARγ mediates curcumin suppression of cyclin D1 expression in passaged HSC, which, in turn, facilitates the activation of PPARγ

Passaged HSC were pretreated with or without the PPARγ antagonist PD 68235 (20 μM) for 30 min prior to the addition of curcumin (Cur.) at 20 μM for an additional 24 h. Total RNA and protein extracts were prepared for real-time PCR (A) (n=3) and Western blotting analyses (B) (n=3) respectively. Significance: *P<0.05 compared with cells without curcumin; **P<0.05 compared with cells with curcumin. (C) To assess the effects of cyclin D1 (ClnD1) on PPARγ activity, HSC in 6-well culture plates were co-transfected with a total of 4.5 μg of plasmid DNA per well, including 2 μg of pPPRE-TK-Luc, 0.5 μg of pSV-β gal, pCMV-cyclinD1 at the indicated doses and an empty vector. The amount of DNA in pCMV-cyclinD1 plus the empty vector was 2 μg. After recovery, cells were treated with or without curcumin at 20 μM for 36 h. Luciferase activities were expressed as relative units after β-galactosidase normalization (n=6). Significance: *P<0.05 compared with cells without curcumin; †P<0.05 compared with cells transfected with no pCMV-cyclinD1, with curcumin treatment (second column).

Figure 2. Curcumin dose-dependently increases the abundance of PPARγ and pro-apoptotic Bax and decreases that of anti-apoptotic Bcl-2 in passaged HSC

Passaged HSC were treated with curcumin at indicated concentrations for 24 h. Whole-cell protein extracts were prepared for Western blotting analyses (n=3). β-Actin was an internal control for equal loading.

Figure 3. Blocking PPARγ activation abrogates the pro-apoptotic effects of curcumin on activated HSC in vitro

Passaged HSC were pretreated or not with the specific PPARγ antagonist PD 68235 (10 or 20 μM) for 30 min prior to the addition of curcumin (Cur.; 20 μM) for an additional 24 h. Total RNA or whole-cell protein extracts were prepared. (A) Real-time PCR. GAPDH was used as an invariant control for calculating fold changes in mRNA levels (n=3). Values are expressed as means±S.D. Significance: *P<0.05 compared with cells with no treatment; †P<0.05 compared with cells treated only with curcumin. (B) Western blotting analyses. β-Actin was an internal control for equal loading (n=3). (C) Caspase 3 activity assays. Values are expressed as means±S.D. (n=3 independent experiments). Significance: *P<0.05 compared with cells with no treatment; ‡P<0.05 compared with cells treated only with curcumin.

Figure 4. PPARγ activation by curcumin is required for induction of HSC apoptosis

Passaged HSC were pretreated or not with the specific PPARγ antagonist PD 68235 for 30 min prior to the addition of curcumin (Cur.; 20 μM) for an additional 24 h. HSC (≥1×10⁶ cells/sample) were fixed with ethanol and then labelled with propidium iodide (PI) for flow cytometric analyses (n=3). Results representative of three independent experiments are shown. Values are expressed as means±S.D. Significance: *P<0.05 compared with cells with no treatment; **P<0.05 compared with cells treated only with curcumin.

Figure 5. PPARγ activation mediates the inhibitory effects of curcumin on the gene expression of α1(I) collagen, fibronectin and α-SMA

Passaged HSC were pretreated or not with the specific PPARγ antagonist PD 68235 (10 or 20 μM) for 30 min prior to the addition of curcumin (20 μM) for an additional 24 h. Total RNA was prepared for real-time PCR (n=3). Fold changes in mRNA levels were calculated by using GAPDH as an invariant control. Values are expressed as means±S.D. Significance: *P<0.05 compared with cells with no treatment; †P<0.05 compared with cells treated only with curcumin.

Figure 6. Activation of PPARγ by curcumin blocks TGF-β signalling in activated HSC

(A) Passaged HSC were transiently transfected with the TGF-β-inducible luciferase reporter plasmid p3TP-Lux. Cells were then pretreated with or without PD 68235 (20 μM) prior to the addition of curcumin (Cur.) at the indicated concentrations for an additional 36 h. Luciferase activities are expressed in relative units after β-galactosidase normalization (n=6). Significance: *P<0.05 compared with cells without curcumin; **P<0.05 compared with cells treated with 20 μM curcumin. (B) HSC in 6-well culture plates were co-transfected with a total of 4.5 μg of plasmid DNA per well, including 2 μg of p3TP-Lux, 0.5 μg of pSV-β gal, pPPARγcDNA at the indicated doses and an empty vector. The amount of DNA in pPPARγcDNA plus the empty vector was 2 μg. Cells were then treated with or without 20 μM curcumin for 36 h. Luciferase activities are expressed in relative units after β-galactosidase normalization (n=6). Significance: *P<0.05 compared with cells transfected with no pPPARγ cDNA and not treated with curcumin.

Figure 7. Curcumin has no effect on the release and/or activation of latent TGF-β1 in activated HSC

(A) Passaged HSC were treated with curcumin at the indicated concentrations for 24 h. Conditioned media were collected and analysed for the amounts of total TGF-β1 and the active form of TGF-β1 by the TGF-β1 Emax ImmunoAssay System (ELISA). Values are expressed as means±S.D. (n=6). (B) Passaged HSC were transfected with the TGF-β-inducible plasmid p3TP-Lux. Cells were then pretreated with 20 μM curcumin for 24 h prior to the addition of exogenous active TGF-β1 for an additional 24 h. Luciferase activities are expressed as relative units after β-galactosidase normalization (n=6). Significance: *P<0.05 compared with cells with no treatment; **P<0.05 compared with cells treated with curcumin but without exogenous TGF-β1.

Figure 8. Curcumin inhibits the expression of TGF-β receptor genes in passaged HSC, which requires the activation of PPARγ

Passaged HSC were pretreated with or without the specific PPARγ antagonist PD 68235 (10 or 20 μM) for 30 min prior to the addition of curcumin (Cur.; 20 μM) for an additional 24 h. Total RNA and whole-cell protein extracts were prepared. (A) Real-time PCR of the TGF-β receptors Tβ-RI and Tβ-RII. GAPDH was used as an invariant control for calculating fold changes in mRNA levels (n=3). Values are expressed as means±S.D. Significance: *P<0.05 compared with cells with no treatment; †P<0.05 compared with cells treated only with curcumin. (B) Western blotting analyses of Tβ-RI and Tβ-RII. β-Actin was an internal control for equal loading (n=3).

Figure 9. Forced expression of PPARγ mimics the inhibitory effect of curcumin on the expression of TGF-β receptor genes in passaged HSC

Passaged HSC were co-transfected with the pPPARγcDNA and a TGF-β receptor luciferase reporter plasmid (pTβ-RI or pTβ-RII). HSC in 6-well culture plates were transfected with a total of 4.5 μg of plasmid DNA per well, including 2 μg of the TGF-β receptor luciferase reporter pTβ-RI or pTβ-RII, 0.5 μg of pSV-β gal, pPPARγcDNA at the indicated doses and an empty vector. The amount of DNA in pPPARγcDNA plus the empty vector was 2 μg. Cells were then treated with or without 20 μM curcumin (Cur) for 36 h. Luciferase activities are expressed as relative units after β-galactosidase normalization (n=6). Significance: *P<0.05 compared with cells not transfected with pPPARγ cDNA and not treated with curcumin.

Figure 10. Possible mechanisms underlying the inhibition of HSC activation by curcumin

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