Antifibrotic effect of silymarin in rat secondary biliary fibrosis is mediated by downregulation of procollagen alpha1(I) and TIMP-1

Department of Gastroenterology and Hepatology, Friedrich-Alexander University, Erlangen-Nuernberg, Erlangen, Germany.
Journal of Hepatology (Impact Factor: 10.4). 09/2001; 35(3):392-8.
Source: PubMed

ABSTRACT Silymarin reduces hepatic collagen accumulation by 35% in rats with secondary biliary cirrhosis. The aim of the present study was to explore its antifibrotic mechanism.
Thirty female adult Wistar rats were allocated to (1) bile duct occlusion, (2) bile duct occlusion and oral silymarin at 50 mg/kg per day, and (3) sham operation and oral silymarin at 50 mg/kg per day. Steady-state mRNA levels for procollagen alpha1(I), tissue inhibitor of metalloproteinases-1 (TIMP-1), and transforming growth factor (TGF) beta1 were determined by multi-probe ribonuclease protection assay.
After 6 weeks of bile duct occlusion, liver collagen content was increased 12-fold, when compared with the sham-operated controls. These animals displayed 17-, 6.5- and 16-fold higher transcript levels for procollagen alpha1(I), TIMP-1 and TGFbeta1 (P < 0.01). Silymarin downregulated elevated procollagen alpha1(I), TIMP-1 and TGFbeta1 mRNA levels by 40-60% (P < 0.01). These lowered hepatic profibrogenic transcript levels correlated with decreased serum levels of the aminoterminal propeptide of procollagen type III.
Silymarin suppresses expression of profibrogenic procollagen alpha1(I) and TIMP-1 most likely via downregulation of TGFbeta1 mRNA in rats with biliary fibrosis. The serum procollagen type III propeptide level mirrors profibrogenic mRNA expression in the liver.

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    • "Liver transplantation is currently the only therapeutic option for liver cirrhosis or tumor, but such method is risky and painful for the patient. Antifibrotic agents, such as silymarin, are widely accepted for the treatment of liver diseases and can downregulate TIMP-1 and TGF-␤1 expression, as well as suppress collagen synthesis (Jia et al., 2001); however, the treatment effect of silymarin is not outstanding. Therefore, the search for more effective antifibrotic agents with fewer side effects remains an active area of research. "
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    ABSTRACT: Fibrosis results from excessive accumulation of extracellular matrix (ECM), and hepatic stellate cell (HSC) plays a central role in hepatic fibrosis. Thus, removal of excess ECM and promotion of apoptosis in HSC are two antifibrotic approaches. Ethyl acetate fraction (EF) of Terminalia bellirica fruit has long been used in liver diseases, but little scientific evidence exists for EF mechanisms in such illnesses. EF has demonstrated both antiproliferative and proapoptotic activities in HSC-T6. The present study investigated the effects of EF on cellular proliferation, ECM removal, cellular signaling pathways, and apoptosis of a rat HSC line (HSC-T6). HSC-T6 cells were incubated with EF, and their proliferation was assessed by MTT assay. Expression of Smad2, platelet-derived growth factor receptor (PDGFR), alpha-smooth muscle actin (␣-SMA), matrix metalloproteinase-2 (MMP-2) was determined by real-time polymerase chain reaction (PCR) and western blot analysis. Expression of tissue inhibitor of metalloproteinase (TIMP)-1, and TIMP-2 mRNAs was determined by real-time PCR. Collagen I, collagen III, TGF-␤1, and hydroxyproline levels were assessed by ELISA. Apoptosis was measured and confirmed by Annexin V-PI staining using fluorescence microscopy and flow cytometry. Apoptotic pathways involving Fas/FasL expression and Bcl-2/Bax family were investigated by real-time PCR. Results showed that 31.25–250 ␮g/mL EF exhibited cytotoxic and antiproliferative effects on HSC-T6 cells. EF at 50 ␮g/mL significantly decreased the levels of collagen I, collagen III, TGF-␤1, and hydroxyproline. EF suppressed the gene expression of Smad2, PDGFR, ␣-SMA, TIMP-1, and TIMP-2 but elevated that of MMP-2. These events consequently facilitated ECM removal and fibrosis resolution. The highest potency of EF promoting HSC-T6 apoptosis was detected at 50 ␮g/mL concentration. This finding was associated with upregulation of Fas/FasL and Bax, as well as downregulation of Bcl-2 in HSC-T6. These results indicate that EF demonstrates antifibrotic activity, and its mechanism of action can be ascribed to inhibition of collagen synthesis, cytokine secretion, and TGF-␤1/Smad pathway. Furthermore, EF facilitates apoptosis in HSCs.
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    • "The primary active constituent of milk thistle is silymarin that is composed of four isomers: silybin, isosilybin, silychristin, and silydianin (El-Kamary et al. 2009). Silymarin medical properties are associated with antioxidant, anti-inflammatory, anti-carcinogenic (Kaur and Agarwal 2007; Ramasamy and Agarwal 2008), and anti-fibrotic properties (Jia et al. 2001) as well as with increased cellular-reduced glutathione (GSH) content, cellular protein biosynthesis potency, stimulation of regeneration ability, and increased stability of the cellular membrane in liver (Flora et al. 1998; Basiglio et al. 2009). However, there is a lack of reports on the side effect of silymarin in laboratory models (S ˇ eršeň et al. 2006) such as mice, rat, rabbit, and dog, and there is no information about oral administration of silymarin in fishes. "
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    • "Journal of the Neurological Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n s in vitro and is known to be involved in hepatic tissue repair [15] [16]. The fibrotic effect of TGF-β1 in lung fibroblasts results in an increase in the secretion and deposition of total ECM and collagens because of a decrease in MMP-1 secretion and an increase in TIMP-1 expression [14]. "
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    ABSTRACT: To investigate the role of tissue inhibitors of metalloproteinases (TIMPs) in muscular dystrophy, we examined the expression of TIMP-1 using plasma and biopsied muscle from patients with various muscular dystrophies by ELISA, immunohistochemistry, and Western blot analysis. TIMP-1 immunolocalization was also studied in mouse models of muscular dystrophy. Plasma TIMP-1 was elevated and correlated with TGF-β1 in Duchenne muscular dystrophy (DMD) and congenital muscular dystrophy (CMD), but not in Becker muscular dystrophy. In dystrophic human muscles, TIMP-1 was immunopositive in the regenerating and non-regenerating muscle fibers, and interstitial cells that consist of activated fibroblasts and macrophages. TIMP-1 immunoreactivity was also closely associated with TGF-β1. Western blot analysis showed elevated TIMP-1 protein in muscles in DMD. The semiquantitative analysis of TIMP-1 staining intensity and tissue fibrosis showed that TIMP-1 immunoreactivity is closely associated with the extent of tissue fibrosis in human and mouse dystrophic muscles. In conclusion, the present study implied that the TGF-β1-TIMP-1 pathway is activated in dystrophic muscles and the overexpression of TIMP-1 may result in increased deposition of extracellular matrix leading to tissue fibrosis.
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