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. 2009 Jul;150(7):3011-20.
doi: 10.1210/en.2008-1601. Epub 2009 Mar 19.

Curcumin eliminates leptin's effects on hepatic stellate cell activation via interrupting leptin signaling

Youcai Tang  1 , Shizhong Zheng, Anping Chen
Affiliations

Curcumin eliminates leptin's effects on hepatic stellate cell activation via interrupting leptin signaling

Youcai Tang et al. Endocrinology. 2009 Jul.

Abstract

Nonalcoholic steatohepatitis (NASH) is commonly found in patients with obesity and is often accompanied with abnormally elevated levels of plasma leptin, i.e. hyperleptinemia. A relatively high population of NASH patients develops hepatic fibrosis, even cirrhosis. Hepatic stellate cells (HSCs) are the major effector cells during liver fibrogenesis and could be activated by leptin. The antioxidant curcumin, a phytochemical from turmeric, has been shown to suppress HSC activation in vitro and in vivo. This project is to evaluate the effect of curcumin on leptin-induced HSC activation and to elucidate the underlying mechanisms. We hypothesize that curcumin abrogates the stimulatory effect of leptin on HSC activation by interrupting leptin signaling and attenuating leptin-induced oxidative stress. Curcumin eliminates the stimulatory effects of leptin on regulating expression of genes closely relevant to HSC activation. Curcumin interrupts leptin signaling by reducing phosphorylation levels of leptin receptor (Ob-R) and its downstream intermediators. In addition, curcumin suppresses gene expression of Ob-R in HSCs, which requires the activation of endogenous peroxisome proliferator-activated receptor-gamma and de novo synthesis of glutathione. In conclusion, our results demonstrate that curcumin abrogates the stimulatory effect of leptin on HSC activation in vitro by reducing the phosphorylation level of Ob-R, stimulating peroxisome proliferator-activated receptor-gamma activity, and attenuating oxidative stress, leading to the suppression of Ob-R gene expression and interruption of leptin signaling. These results provide novel insights into therapeutic mechanisms of curcumin in inhibiting HSC activation and intervening liver fibrogenesis associated with hyperleptinemia in NASH patients.

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Figures

Figure 1
Figure 1
Curcumin (Cur) eliminates the effects of leptin on regulating expression of genes relevant to HSC activation. Serum-starved passaged HSCs were treated with or without leptin at 100 ng/ml plus curcumin at various concentrations in serum-depleted media for 24 h. Total RNA and whole cell extracts were prepared. A, Real-time PCR assays of mRNA of representative genes relevant to HSC activation. Values were presented as mean ± sd (n = 3). *, P < 0.05 vs. the untreated control (corresponding first column on the left); ‡, P < 0.05 vs. cells treated with leptin only (corresponding second column). B, Western blotting analyses of proteins of genes relevant to HSC activation. β-Actin was used as an invariant control for equal loading. Representatives were from three independent experiments. Italic numbers were fold changes in densities of the bands compared with the control with no treatment (first well) after normalization (n = 3). Because of the limited space, sd values were not presented. Tβ-RI, TGF-βRI; Tβ-RII, TGF-βRII.
Figure 2
Figure 2
A and B, Curcumin (Cur) interrupts leptin signaling pathways in activated HSCs by reducing phosphorylation levels of Ob-R and its downstream intermediators, and suppressing gene expression of leptin and its receptors. Serum-starved HSCs were pretreated with curcumin at indicated concentrations for 1 h before the stimulation with leptin at 100 ng/ml for an additional 30 min. Whole cell extracts were prepared. Representatives were from three independent experiments. Italic numbers were fold changes in densities of the bands compared with the control with no treatment (corresponding first well) after normalization (n = 3). A, Western blotting analyses of phosphorylated (p)-Ob-R from immunoprecipitated total Ob-R. Nonspecifically recognized heavy chain of IgG was used for normalization. Molecular weights of standards (Std) were presented on the left. B, Western blotting analyses of phosphorylated (p)-ERK1/2, JNK-p46, AKTser473, JAK2, and STAT3. Corresponding total proteins were used as internal controls for normalization. C–E, Passaged HSCs were treated with curcumin at indicated concentrations for 24 h. Total RNA and whole cell extracts were prepared. Representatives from three independent Western blotting analyses were presented. Italic numbers beneath blots were fold changes in densities of the bands compared with the control without treatment in the blot (n = 3), after normalization. C, Real-time PCR assays of mRNA of leptin and three isoforms of Ob-Rs. Values were presented as mean ± sd (n = 3). *, P < 0.05 vs. the untreated control (the corresponding first column on the left). D, Western blotting analyses of leptin. β-Actin was used as an invariant control for equal loading and normalization. E, Western blotting analyses of immunoprecipitated Ob-R. Nonspecifically recognized heavy chain of IgG was used for normalization.
Figure 3
Figure 3
Curcumin (Cur) eliminates the effects of leptin on the induction of oxidative stress by reducing the levels of cellular ROS and LPO, increasing the contents of cellular GSH and improving the ratio of GSH to GSSG in cultured HSCs. Serum-starved HSCs were stimulated with or without leptin at 100 ng/ml plus curcumin at indicated concentrations in serum-depleted media for 24 h. Cell extracts were prepared for assays. Values were presented as fold changes (mean ± sd, n = 3), compared with the untreated control (corresponding first column on the left). *, P < 0.05 vs. the untreated control (first column); ‡, P < 0.05 vs. cells treated with leptin only (second column). A, Analyses of ROS levels. B, Analyses of LPO levels. C, Analyses of GSH contents. D, Determination of the ratio of GSH to GSSG.
Figure 4
Figure 4
Curcumin (Cur) increases GCL activities in activated HSCs in vitro by inducing gene expression of GCLc and GCLm. Serum-starved HSCs were stimulated with or without leptin at 100 ng/ml plus curcumin at indicated concentrations in serum-depleted media for 24 h. Assays were performed. *, P < 0.05 vs. the untreated control (corresponding first column); ‡, P < 0.05 vs. cells treated with leptin only (corresponding second column). A, Analyses of GCL activities. Values were presented as fold changes (mean ± sd, n = 3), compared with the untreated control (first column on the left). B, Real-time PCR analyses of GCLc and GCLm mRNA (n = 3). C, Western blotting analyses of GCLc and GCLm proteins. β-Actin was used as an invariant control for equal loading. Representatives were from three independent experiments. Italic numbers beneath blots were fold changes in densities of the bands compared with the control without treatment in the blot (n = 3), after normalization.
Figure 5
Figure 5
The depletion of cellular GSH by BSO eliminates the suppressive effect of curcumin (Cur) on gene expression of Ob-R in activated HSCs in vitro. Serum-starved HSCs were divided into two groups. In one group, cells were stimulated with or without leptin (100 ng/ml) plus curcumin (20 μm) or NAC (5 mm) in serum-free media for 24 h. In the other group, cells were pretreated with BSO (0.25 mm) for 1 h before the addition of leptin (100 ng/ml) plus curcumin (20 μm) or NAC (5 mm) in serum-free media for an additional 24 h. Total RNA or whole cell extracts were prepared. A, Real-time PCR analyses of Ob-R mRNA (n = 3). *, P < 0.05 vs. the untreated control (first column); **, P < 0.05 vs. cells treated with leptin only (second column); ‡, P < 0.05, vs. cells treated with leptin plus NAC or curcumin (third or fourth column, respectively). B, Western blotting analyses of immunoprecipitated Ob-R. Representatives were from three independent experiments. Italic numbers were fold changes in densities of the bands compared with the untreated cells (first well) after normalization with nonspecifically recognized heavy chain of IgG (n = 3).
Figure 6
Figure 6
The activation of PPARγ by curcumin (Cur) mediates the effect of the phytochemical on the suppression of gene expression of Ob-R in activated HSCs in vitro. A and B, Semi-confluent HSCs were pretreated with or without PD68235 (PD) (20 μm) for 30 min before the exposure to curcumin (20 μm) for an additional 24 h. Total RNA or whole cell extracts were prepared. A, Real-time PCR assays of Ob-R mRNA (n = 3). *, P < 0.05 vs. the untreated control (first column); **, P < 0.05 vs. cells treated with leptin only (second column). B, Western blotting analyses of immunoprecipitated Ob-R. Italic numbers were fold changes in densities of the bands compared with the control with no treatment (first well) after normalization with nonspecifically recognized heavy chain of IgG (n = 3). C and D, HSCs were transiently transfected with indicated plasmids. After recovery, cells were treated with curcumin (20 μm) in DMEM with FBS (10%), or with PGJ2 (5 μm) in serum-depleted media, for 24 h. Luciferase activities were expressed as relative units after β-galactosidase normalization (means ± sd, n ≥ 6). The floating schema denotes plasmid(s) in use and the application of curcumin or PGJ2 to the system. C, HSCs were transfected with pOb-R-Luc. *, P < 0.05 vs. the untreated control (corresponding first column of each group). D, HSCs were cotransfected with pOb-R-Luc and pPPARγcDNA at indicated doses. *, P < 0.05 vs. the untreated control (second column).
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