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This Article Full Text (PDF) All Versions of this Article: 16/14/1988 02-0406fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by KANG, K. W. Articles by KIM, S. G. Search for Related Content PubMed PubMed Citation Articles by KANG, K. W. Articles by KIM, S. G.

Oltipraz regenerates cirrhotic liver through CCAAT/enhancer binding protein-mediated stellate cell inactivation ¹

KEON WOOK KANG^*,, YOON GYOON KIM^*,, MIN KYONG CHO^*,, SOO KYUNG BAE, CHOON WON KIM, MYUNG GULL LEE and SANG GEON KIM^*,2

^* National Research Laboratory,
College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul; and
Department of Clinical Pathology, Hanyang University Medical School, Seoul, Korea

²Correspondence: College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Sillim-dong, Kwanak-gu, Seoul 151–742, Korea. E-mail: sgk{at}snu.ac.kr

SPECIFIC AIMS

No effective therapeutic agent is available for liver cirrhosis (LC), a chronic disease with a high mortality rate. We were challenged to study the effectiveness of oltipraz, which has been used clinically and studied extensively as a cancer chemopreventive agent in regeneration of cirrhotic liver with the hypothesis that activation of CCAAT/enhancer binding protein (C/EBP) inhibits transforming growth factor ß1 (TGF-ß1) gene expression in stellate cells.

PRINCIPAL FINDINGS

1. Oltipraz increases survival rates of cirrhotic rats as a result of improvement of liver function and regeneration of cirrhotic liver
We induced LC in rats by injecting multiple doses of DMN for 4 wk and determined whether the survival rate of cirrhotic rats was improved during the next 4 wk of oltipraz treatment. A large fraction of vehicle-treated cirrhotic rats died within the first 3 wk. Oltipraz treatment (30 mg/kg, three times per week, orally for 4 wk) improved the survival rate (P<0.05; ² test) of these rats to 82% on day 28 compared with 47% in vehicle-treated cirrhotic animals. Rats treated with oltipraz had significantly greater body weight gain on day 28 than that of vehicle-treated ones. Fluid accumulation in the peritoneal cavity was indexed after treatment of cirrhotic rats with oltipraz. The ascitic index of cirrhotic rats was 1.6 ± 0.4; oltipraz treatment (30 mg/kg) significantly reduced it to 0.3 ± 0.2. Decreased synthesis of albumin in the liver accompanies edema and ascites formation. Plasma albumin, which was significantly decreased to 76% of control in cirrhotic rats, was restored by oltipraz to 90% of healthy control animals. To determine whether oltipraz affected liver regeneration, weights of the major organs excised after drug treatment were measured. In contrast to no or minimal changes in brain and kidney, liver weight in cirrhotic rats was decreased to 55% of that in healthy control. The liver-to-brain weight ratio in oltipraz-treated rats increased up to 93% of that in control. Proliferating cell nuclear antigen (PCNA) is expressed in dividing cells in late G1 and S phases and used as a marker for cell proliferation. Western blot analysis showed an increase in the band intensity of 36 kDa PCNA in the liver homogenates of cirrhotic rats treated with oltipraz vs. healthy control or vehicle-treated cirrhotic animals. The increase in PCNA in conjunction with the increase in the liver-to-brain weight ratio supports the notion that oltipraz regenerates cirrhotic liver.

2. Oltipraz reduces the intensity of liver fibrotic and cirrhotic nodules and eliminates accumulated extracellular matrix
We next histopathologically examined cirrhotic nodules, extent of liver fibrosis, intralobular hepatocyte degeneration, and portal inflammation of surviving cirrhotic rats after 4 wk of vehicle or drug treatment. Healthy control rats showed no pathological changes (Fig. 1 A-a). Masson’s trichrome staining revealed that extracellular matrix was heavily accumulated around and within thick multiple fibrotic nodules, particularly in proximity to portal spaces in the liver of cirrhotic rats (Fig. 1A -b). Treatment of cirrhotic rats with 15 mg/kg of oltipraz notably decreased the intensities of liver fibrotic nodules (Fig. 1A -c). Furthermore, liver fibrotic nodules completely disappeared after 30 mg/kg of oltipraz treatment. Only marginal fibrous bands were detected (Fig. 1A -d). In cirrhotic animals treated with 30 mg/kg oltipraz, fibrosis scores significantly decreased (Fig. 1B , left). By multiple analyses, we obtained Knodell scores, which were also significantly decreased by oltipraz treatment (Fig. 1B , right). Type I collagen that accumulated in LC disappeared after oltipraz treatment (Fig. 1C ).

View larger version (38K): [in this window] [in a new window]   Figure 1. Oltipraz dissolves the fibers accumulated in cirrhotic liver. A) Masson’s trichrome stainings. Liver sections from healthy control rats (a), cirrhotic rats treated with vehicle (b), 15 mg/kg oltipraz (c), or 30 mg/kg oltipraz (d) (3 times/wk for 4 wk) were stained with Masson’s trichrome (100x). Microphotographs show low power views of the liver sections: a) no pathological changes, central vein (CV), and portal space (PS); b) nodular appearance surrounded by thick fibrous bands (), piecemeal necrosis (), bridging necrosis (); c) piecemeal necrosis (), bridging necrosis (), intralobular acidophilic bodies (#), and portal inflammation (*); d) piecemeal necrosis () and fibrous portal expansion (). B) Fibrosis and Knodell scores. Fibrosis and Knodell scores were evaluated in liver sections stained with Masson’s trichrome by a certified pathologist in a blinded fashion. Extent of fibrosis was graded as 0 = no increase; 1 = slight increase; 2 = moderate increase; 3 = distinct increase; 4 = severe increase. Extent of periportal bridging (severe=10), intralobular degeneration (severe=4), portal inflammation (severe=4), and fibrosis (severe=4) was also graded according to Knodell’s scoring method. The score represents a sum of each severity score (significant compared with cirrhotic rats, *P<0.05, **P<0.01; n=8–10). C) Representative immunohistochemistry of type I collagen. The extent of type I collagen accumulated was immunohistochemically assessed (400x). Data were confirmed by repeated experiments.

3. Oltipraz inactivates stellate cells in cirrhotic liver
Activated hepatic stellate cells produce collagens in cirrhotic livers. As a second approach, we sought to determine the number of activated stellate cells in cirrhotic liver by immunostaining of -smooth muscle actin (-SMA), a definitive marker. No -SMA-positive cells were detected in the liver of healthy control rats. As expected, -SMA-positive cells multiplied and stained as single cells or clusters lined within or in the vicinity of accumulated fibers. In cirrhotic rats treated with oltipraz (30 mg/kg), -SMA-positive cells were rarely detected. Expression of -SMA in cirrhotic rats substantially increased whereas oltipraz treatment decreased protein expression.

4. The pharmacological molecular targets for the inactivation of activated stellate cells include CCAAT/enhancer binding protein (C/EBP)
To determine whether oltipraz inhibited trans-activation of hepatic stellate cells and promoted stellate cell inactivation, the expression of -SMA and TGF-ß1 was measured in cultured stellate cells exposed to oltipraz in vitro. Induction of -SMA was notably inhibited by oltipraz at 3–10 µM for 1 or 3 day(s) (Fig. 2 A). TGF-ß1 as a key fibrogenic mediator causes synthesis of extracellular matrix proteins and inhibits collagenase activity in the liver. We next determined whether oltipraz directly inhibits TGF-ß1 expression in activated stellate cells. The TGF-ß1 mRNA level was assessed by RT-PCR analysis. The level of TGF-ß1 mRNA was decreased by the presence of 3–30 µM oltipraz but that of glyceraldehyde-3-phosphate dehydrogenase mRNA did not change (Fig. 2B ). Consistent with the inhibition of TGF-ß1, oltipraz decreased the expression of procollagen 1(III) in activated stellate cells (Fig. 2B ). Putative C/EBP response elements are present in the 5'-flanking region of the rat TGF-ß1 gene. Given the activation of C/EBPß by oltipraz and the presence of C/EBP response element in the TGF-ß1 gene, we assessed the role of C/EBPß in TGF-ß1 gene expression in stellate cells. Immunocytochemistry revealed that oltipraz (30 µM) allowed cytosolic C/EBPß to translocate into the nucleus in activated stellate cells (Fig. 2C ), which was confirmed by Western blot analysis (data not shown). We used C/EBP-specific decoy oligodeoxynucleotides (dODN) to determine the role of C/EBPß in suppression of the TGF-ß1 gene. RT-PCR (Fig. 2D , upper) and real-time RT-PCR (Fig. 2D , lower) analyses revealed that oltipraz inhibited TGF-ß1 expression in cells treated with mutant C/EBP dODN. In the present study, C/EBP dODN enhanced the expression of TGF-ß1 in control cells as a result of blocking the negative regulation of TGF-ß1 expression by C/EBP. In stellate cells treated with oltipraz, C/EBP dODN reversed the inhibition of TGF-ß1 mRNA by oltipraz (Fig. 2D , upper and lower).

View larger version (49K): [in this window] [in a new window]   Figure 2. Oltipraz inactivates activated stellate cells. A) Inhibition of -SMA expression by oltipraz in activated stellate cells. The level of -SMA protein was measured in the lysates of stellate cells treated with 3–10 µM oltipraz for 1 or 3 day(s). B) Inhibition of TGF-ß1 and procollagen 1(III) [1(III)] expression by oltipraz in activated stellate cells. Total RNA prepared from stellate cells treated with oltipraz (3–30 µM) for 12 h was used for cDNA synthesis and TGF-ß1 or procollagen 1(III) mRNA expression was assessed by RT-PCR. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was amplified by RT-PCR as control. C) Immunocytochemistry of C/EBPß in activated stellate cells. Nuclear translocation of cytosolic C/EBPß was monitored in stellate cells incubated with vehicle (dimethylsulfoxide) or 30 µM oltipraz for 1 h. The same fields were counterstained with propidium iodide. D) Role of C/EBP in the suppression of TGF-ß1 expression by oltipraz. Activated stellate cells were preincubated with 10 µM of mutant C/EBP (mC/EBP) dODN or C/EBP dODN for 1 h and further incubated with or without 30 µM oltipraz for 12 h. Levels of TGF-ß1 and GAPDH mRNA were assessed by RT-PCR analysis (upper). SYBR Green real-time RT-PCR analysis was used to confirm changes in the TGF-ß1 mRNA in stellate cells (lower). The amplification curve was right-shifted by the presence of oltipraz with the increase of CT value in cells exposed to mutant C/EBP dODN. In contrast, oltipraz failed to increase CT value in cells pretreated with C/EBP dODN. Results were confirmed by repeated experiments.

CONCLUSIONS AND SIGNIFICANCE

In the U.S. and Western world as well as oriental countries, LC is the leading cause of death by disease. Therapeutic management of LC is primarily symptomatic, directed at clinical manifestations such as ascites, esophageal varices, and hepatic encephalopathy. Because no agent is currently available for regeneration of cirrhotic liver, a few drugs (including ursodeoxycholic acid, silymarin, and lamivudine) have been used for cirrhotic patients. The efficacy of these drugs is uncertain.

Oltipraz, structurally related to the 1,2-dithiolthiones naturally found in cruciferous vegetables, has been extensively studied as a cancer chemopreventive agent. In animal models, oltipraz significantly decreased tumor incidence and multiplicity in liver, colon, skin, and lung. Oltipraz was originally used as a potential schistosomicidal agent in humans at a daily dose of 30 mg/kg. In the present study, oltipraz at the daily equivalent dose of 13 mg/kg or less was highly efficacious as a treatment for LC. Oltipraz is the first therapeutic agent that regenerates cirrhotic liver, and its clinical use will fill an important need for patients with LC.

We conclude that the inhibition of extracellular matrix accumulation by oltipraz in the liver of cirrhotic rats may result from C/EBPß-mediated TGF-ß1 suppression in stellate cells. TGF-ß1 stimulates collagen synthesis in stellate cells, and Smad proteins are transcriptional activators for the collagen genes inducible by TGF-ß1 superfamily members. Activation of C/EBP negatively regulates the expression of type I collagen gene. Smad proteins suppress C/EBPß-mediated transcriptional activation. Hence, activation of C/EBPß by oltipraz may antagonize Smad-mediated induction of collagen and activate collagenase for the resolution of fibers accumulated in the liver. Inhibition of TGF-ß1 expression in stellate cells via activation of C/EBP provides a pharmacological target for the treatment of LC (Fig. 3 ).

View larger version (25K): [in this window] [in a new window]   Figure 3. Schematic diagram illustrating the possible mechanism by which oltipraz regenerates cirrhotic liver. The activation of CCAAT/enhancer binding protein (C/EBP) by oltipraz inhibits transforming growth factor ß1 (TGF-ß1) gene expression in stellate cells, which provides a molecular target for pharmacological treatment of LC. Oltipraz within the clinical dose range regenerates cirrhotic liver in the established LC rats as a result of inactivation of stellate cells, thereby reducing the intensities of cirrhotic nodules and improving survival rate.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0406fje; to cite this article, use FASEB J. (October 18, 2002) 10.1096/fj.02-0406fje

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This Article Full Text (PDF) All Versions of this Article: 16/14/1988 02-0406fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by KANG, K. W. Articles by KIM, S. G. Search for Related Content PubMed PubMed Citation Articles by KANG, K. W. Articles by KIM, S. G.