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Stearic acid attenuates cholestasis-induced liver injury
Biochemical and Biophysical Research Communications 391 (2010) 1537–1542
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Stearic acid attenuates cholestasis-induced liver injury Pin-Ho Pan a,1, Shih-Yi Lin b,1, Yen-Chuan Ou c, Wen-Ying Chen d, Yu-Han Chuang b, Yu-Ju Yen d, Su-Lan Liao d, Shue-Ling Raung d, Chun-Jung Chen d,e,f,g,* a
Department of Pediatrics, Tung’s Taichung MetroHarbor Hospital, Taichung, Taiwan Division of Endocrinology and Metabolism, Taichung Veterans General Hospital, Taichung, Taiwan c Division of Urology, Taichung Veterans General Hospital, Taichung, Taiwan d Department of Education and Research, Taichung Veterans General Hospital, Taichung, Taiwan e Center for General Education, Tunghai University, Taichung, Taiwan f Institute of Medical and Molecular Toxicology, Chung-Shan Medical University, Taichung, Taiwan g Institute of Biomedical Sciences, National Chung-Hsing University, Taichung, Taiwan b
a r t i c l e
i n f o
Article history: Received 12 December 2009 Available online 28 December 2009 Keywords: Cholestasis Hepatotoxicity Inflammation Oxidative stress Stearic acid
a b s t r a c t Inflammation is involved in cholestasis-induced hepatic damage. Stearic acid has been shown to possess anti-inflammatory potential. We assessed whether stearic acid has protective effects against cholestasisrelated liver damage. Cholestasis was produced by bile duct ligation (BDL) in male Sprague–Dawley rats for 3 weeks. Daily administration of stearic acid was started 2 weeks before injury and lasted for 5 weeks. In comparison with the control group, the BDL group showed hepatic damage as evidenced by elevation in serum biochemicals, ductular reaction, fibrosis, and inflammation. These pathophysiological changes were attenuated by chronic stearic acid supplementation. The anti-fibrotic effect of stearic acid was accompanied by reductions in a-smooth muscle actin-positive matrix-producing cells and critical fibrogenic cytokine transforming growth factor beta-1 production. Stearic acid also attenuated BDL-induced leukocyte accumulation and NF-jB activation. The data indicate that stearic acid attenuates BDL-induced cholestatic liver injury. The hepatoprotective effect of stearic acid is associated with anti-inflammatory potential. Ó 2009 Elsevier Inc. All rights reserved.
Introduction Cholestasis is characterized by an abnormal accumulation of bile acids, which is caused by defects in the process of bile acid transport. Chronic cholestasis is a key histopathological change that occurs in biliary atresia, primary biliary cirrhosis, and primary sclerosing cholangitis and contributes to the later development of hepatocellular injury, progressive hepatic fibrogenesis, cirrhosis, and death from liver failure [1]. Bile duct proliferation is a hallmark of cholestatic disorders. Histologically, the increase in hepatic bile ductular structures is generally accompanied by hepatocyte death, inflammatory response, and periductular fibrosis [2–5]. Although mechanisms of liver damage in cholestasis are multifactorial, retained bile acids, oxidative stress, and inflammation have been implicated as important factors in cholestatic liver injury [2–8]. Therefore, bile acids, oxidative stress, and inflammation are consid-
* Corresponding author. Address: Department of Education and Research, Taichung Veterans General Hospital, No. 160, Sec. 3, Taichung-Kang Rd., Taichung 407, Taiwan. Fax: +886 4 23592705. E-mail address: [email protected] (C.-J. Chen). 1 These authors contributed equally to this work. 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.12.119
ered to be potential targets for therapeutic intervention in treating cholestatic liver disorders. Polyunsaturated fatty acids have been implicated in the prevention of various human diseases, including inflammation-associated diseases [9]. However, only a few articles have reported the effects of saturated fatty acids on human health. Saturated fatty acids are usually regarded as unhealthy, but nutritionists believe that some types of saturated fatty acids may be beneficial. Several lines of investigation indicate that dietary fat can modulate the severity of liver injury. In experimental animals, 3 weeks of polyunsaturated fatty acid supplementation reduced cholestatic liver injury [2]. Dietary saturated fatty acids have been shown to protect against alcohol-induced liver injury [10,11]. Among the saturated fatty acids commonly found in the food supply, stearic acid (18:0) is unique because, unlike palmitic, myristic, and lauric acid, its hypercholesterolemic effect is negligible [12,13]. A recent study showed that stearic acid may function as a natural ligand of the peroxisome proliferator-activated receptor and protect cells from oxidative damage [14]. Elevated production of anti-inflammatory cytokine interleukin-10 (IL-10) has been detected in stearic acidtreated hepatocytes [15]. Although the consequences of stearic acid supplementation vary, these findings imply that stearic acid is capable of alleviation of oxidative stress and inflammation.
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Cholestasis is a clinically important primary event that contributes to hepatic damage. Currently, little information is available about the effect of stearic acid on chronic cholestatic liver injury. Common bile duct ligation (BDL) and scission in animals represents a classical experimental model for the analysis of cholestasis and consequent hepatic damage. BDL produces cholestasis, triggers free radical generation and inflammation, induces progressive portal fibrosis, causes secondary biliary cirrhosis, and finally leads to liver failure [4]. The aims of the present study were to assay the preventive potential of stearic acid on rat hepatic damage induced by BDL and to elicit the role of the anti-inflammatory effect. Materials and methods Animals. Male Sprague–Dawley rats were randomly divided into six experimental groups. The control (n = 30) and BDL group (n = 30) rats underwent sham and BDL operation, respectively. Intraperitoneal administration of saline and stearic acid (250 and 1000 nmol/kg/day, respectively) was carried out and modified in accordance with the method used in our previous reports [9]. The rats in both groups received saline and stearic acid (10 animals/each group) for 5 weeks starting from 2 weeks before operation. All animals were sacrificed 3 weeks after surgery under pentobarbital anesthesia. The animal study was approved by the Animal Care and Use Committee of Taichung Veterans General Hospital. BDL operation. Rats (200–250 g) were anesthetized with pentobarbital (50 mg/kg) and the common bile duct was exposed and ligated by double ligatures with 3–0 silk. The first ligature was made below the junction of the hepatic ducts and the second ligature was made above the entrance of the pancreatic ducts. The common bile duct was then cut between the double ligatures. Thus, animals had total, permanent biliary obstruction [4]. In sham-operated rats, an abdominal incision was made without a ligation. Biochemical analysis. Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), c-glutamyl transpeptidase (GGT), total bilirubin, and albumin were measured by automated standardized procedures (Roche Hitachi 917/747, Mannheim, Germany). The serum level of transforming growth factor beta-1 (TGF-b1) was measured by enzyme-linked immunosorbent assay (ELISA). Histological and immunohistochemical examination. Excised liver specimens were fixed in 10% formalin and embedded with paraffin. Hematoxylin and eosin (H&E) staining and Sirius Red staining were performed according to standard procedures. For immunohistochemical examination, deparaffinized sections were incubated with a-smooth muscle actin (a-SMA) and CD68 primary antibody and biotinylated secondary antibody, followed by the avidin–biotin–peroxidase complex. The immunoreactive signal was developed by color deposition using diaminobenzidine as substrate. Tissue preparation and Western blot. The resected liver tissues were extracted with lysis buffer (1% Triton X-100; 50 mmol/l Tris–HCl, pH 7.6; 150 mmol/l NaCl; and 1% protease inhibitor cocktail). For Western blot, proteins were separated by SDS–PAGE and electrophoretically transferred to polyvinylidene difluoride membranes. After blocking, the membranes were incubated for 1 h at room temperature with the indicated antibodies including aSMA (Calbiochem Biotechnology), CD68 (BioSource), and GAPDH (Santa Cruz Biotechnology). Then, a 1:10,000 dilution of horseradish peroxidase-labeled IgG was added at room temperature for 1 h. The blots were developed using ECL Western blotting reagents and quantified by optical densitometry. The intensity of each signal was corrected by the values obtained from the immunodetection of GAPDH and the relative protein intensity was expressed as folds of the content in the control group.
Myeloperoxidase activity (MPO) assay. Proteins (10 lg/100 ll) obtained from liver tissues were mixed with 2.9 ml of the assay solution. The optical absorbance was determined at 470 nm for 1 min with a spectrophotometer [9]. MPO activity was calculated from a standard MPO activity curve. The assay solution consisted of: H2O, 26.9 ml; 0.1 M sodium phosphate buffer (pH 7.0), 3.0 ml; 0.1 M H2O2, 0.1 ml; guaiacol, 0.048 ml. Collagen measurement. Proteins (50 lg) extracted from liver tissues were subjected to ELISA with antibody against collagen IV (Sigma Chemical) for the determination of collagen [16]. Preparation of nuclear extracts and electrophoretic mobility shift assay (EMSA). Nuclear proteins were extracted from liver tissues. The isolation of nuclear extract and EMSA was conducted as described previously [9]. The oligonucleotides specific for NF-jB (50 -AGTTGAGGGGACTTTCCCAGGC) were synthesized and labeled with biotin. Nuclear extract (5 lg) was used for EMSA. The binding reaction mixture included 1 lg of poly(dI-dC), 0.1 lg of poly-L-lysine, and 100 fmol of biotin-labeled DNA probe in a 20 ll binding buffer (10 mM HEPES, pH 7.6; 50 mM NaCl; 0.5 mM MgCl2; 0.5 mM EDTA; 1 mM dithiothreitol; 5% glycerol). The DNA/protein complex was analyzed on 6% native polyacrylamide gels. Statistical analysis. All data are presented as mean ± standard deviation. For comparison, the statistical significance between means was determined using one-way analysis of variance (ANOVA) followed by Dunnett’s t-test. A p value of less than 0.05 was considered significant. Results Stearic acid attenuated BDL-induced liver injury Grossly, body mass and the average food intake of BDL rats were lower than those of sham control rats. There were no significant differences in body mass and average food intake between salineand stearic acid-supplemented animals (data not shown). Liver damage was first analyzed by histological examination (Fig. 1). No morphological abnormalities were observed in saline- and stearic acid-supplemented sham-operated control rats showing a regular morphology of liver parenchyma with intact hepatocytes, sinusoids, and portal tract. Diffuse severe/high bile duct hyperplasia, portal edema, and mild portal infiltrates, all features of extrahepatic cholestasis, were present in BDL rats. BDL rats also showed a loss of hepatic structure in periportal areas. Serum levels of AST, ALT, Alk-P, GGT, and total bilirubin, common biochemical indexes of hepatocellular injury, were significantly elevated in BDL rats (Table 1). BDL rats also had decreased serum level of albumin (Table 1). The incidence of BDL-induced histopathological changes was not affected by stearic acid supplementation (Table 2). However, the histopathological changes (Fig. 1) and histopathological scores for bile duct hyperplasia (Table 2) were improved by stearic acid supplementation. In addition, serum biochemical analysis (Table 1) showed moderate reduction in these pathophysiological changes except Alk-P and total bilirubin in BDL rats supplemented with stearic acid. These results indicate that stearic acid supplementation attenuates BDL-induced liver damage. Stearic acid attenuated BDL-induced fibrosis Biliary fibrosis, such as the type observed in conditions of chronic obstruction of the biliary tree, is characterized by accumulation of extracellular matrix at portal tracts, surrounding newly formed bile ducts emerging during the course of ductular reaction and originating from proliferation of pre-existing bile duct epithelial cells [17]. The histopathological scores for bile duct hyperplasia (Table 2) and serum activity measurements of GGT (Table 1) re-
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Fig. 1. Stearic acid attenuated BDL-induced liver injury. The obtained liver sections were subjected to histological examination. Representative photomicrographs of H&E staining are shown. Original magnification: 40.
Table 1 Biochemical parameters. SA (nmol/kg)
AST (U/l) ALT (U/l) Alk-P (U/l) GGT (U/l) Total bilirubin (mg/dl) Albumin (g/dl) TGF-b1 (pg/ml)
Sham 0
BDL 0
Sham 250
BDL 250
Sham 1000
BDL 1000
178 ± 32 44 ± 9 199 ± 88 1.8 ± 1.2 0.6 ± 0.3 3.8 ± 0.2 328 ± 144
924 ± 201** 199 ± 69** 599 ± 86** 53.3 ± 20.1** 8.4 ± 1.9** 2.3 ± 0.4** 1498 ± 395**
193 ± 30 49 ± 5 242 ± 106 1.7 ± 1.0 0.7 ± 0.4 3.8 ± 0.2 415 ± 168
783 ± 233** 168 ± 44** 545 ± 118** 45.2 ± 21.8** 8.1 ± 1.3** 2.8 ± 0.5** 1300 ± 379**
184 ± 32 47 ± 14 225 ± 80 1.5 ± 0.9 0.4 ± 0.3 3.8 ± 0.1 223 ± 122
646 ± 197**,# 134 ± 26**,# 538 ± 154** 32.7 ± 6.1**,# 7.9 ± 2.4** 3.1 ± 0.6*,# 835 ± 357**,#
Data are expressed as mean ± SD *p < 0.05 and **p < 0.01 vs. vehicle sham control group and #p < 0.05 vs. BDL control group, n = 10. AST, aspartate aminotransferase; ALT, alanine aminotransferase; Alk-P, alkaline phosphatase; GGT, c-glutamyl transpeptidase.
Table 2 Scores of bile duct hyperplasia. Histopathological findings
Hyperplasia, bile duct, focal, moderate to severe/higha Histopathology score of bile duct hyperplasiab
Group (SA (nmol/kg)) Sham 0
BDL 0
Sham 250
BDL 250
Sham 1000
BDL 1000
0/10
10/10
0/10
10/10
0/10
10/10
0
4.4 ± 0.5
0
4.0 ± 0.7
0
3.4 ± 0.7*
Data are expressed as mean ± SD. * p < 0.05 vs. BDL control group. a Incidence: affected rats/total examined rats. b Score of bile duct hyperplasia. Severity of lesions was graded according to the methods described by Shackelford et al. [31]. Degree of lesions was graded from one to five depending on severity: 1, minimal (<1%); 2, slight (1–25%); 3, moderate (26– 50%); 4, moderate/severe (51–75%); 5, severe/high (76–100%).
vealed that stearic acid alleviated BDL-induced ductular reaction. The next experiment was conducted to determine whether stearic acid has a role in BDL-induced fibrosis. Fibrosis was assessed by a classical histopathological technique using Sirius Red staining. In BDL rats, significant hepatic collagen deposition/accumulation was present in periportal and portal tracts. The presence of this deposition/accumulation and the bridging fibrosis were reduced in the stearic acid-treated groups (Fig. 2). The extent of liver fibrosis was also measured biochemically as hepatic collagen content.
In parallel to the observed improvement of liver histology, stearic acid reduced BDL-induced elevation in collagen content (Fig. 3A). These results indicate that stearic acid has an attenuating effect against BDL-induced liver fibrosis. Immunohistochemical results showed that BDL caused an elevation in a-SMA immunopositive signals in portal and periportal areas and the immunoreactive signals were reduced by stearic acid treatment (Supplementary Fig. 1). Western blot study showed that stearic acid caused a reduction of a-SMA protein (Fig. 3B). These results indicate that stearic acid decreases the activation of a-SMA-positive matrix-producing cells in hepatic tissues after BDL injury. Numerous studies indicate that TGF-b1 plays an important role in liver fibrogenesis through acting on matrix-producing cells [4,18]. The elevation of serum level of TGF-b1 in BDL rats was reduced by stearic acid in this study (Table 1). These data suggest that the anti-fibrotic effect of stearic acid is associated with reduction of TGF-b1 signaling. Stearic acid attenuated BDL-induced inflammation Prolonged biliary obstruction is associated with inflammatory cell infiltration, NF-jB activation, and cytokine over-production. Inhibition of inflammatory responses prevents cholestatic liver damage [3,5,19–21]. Immunohistochemical staining (Supplementary Fig. 2) and Western blotting (Fig. 4B) revealed an accumulation of monocytes/macrophages in the liver tissues of BDL rats. An increase in neutrophil infiltration in liver tissues assessed by measuring hepatic MPO activity occurred in BDL rats (Fig. 4A). The recruitment/accumulation of inflammatory cells was attenuated by stearic acid. The activation of NF-jB occurs in cholestatic
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Fig. 2. Stearic acid attenuated BDL-induced liver fibrosis. The obtained liver sections were subjected to histological examination. Representative photomicrographs of Sirius Red staining are shown. Original magnification: 40.
Fig. 3. Stearic acid attenuated BDL-induced liver fibrotic changes. (A) Proteins were isolated from livers and were subjected to ELISA for the measurement of collagen. The absorbance was measured at 450 nm using a spectrophotometer. (B) Proteins were isolated from livers and were subjected to Western blot with antibodies against a-SMA and GAPDH. Representative blots and the quantitative data are shown. **p < 0.01 vs. the sham control group and #p < 0.05 vs. the BDL control group.
liver damage and contributes to inflammatory responses [19]. EMSA revealed an increase of NF-jB activity in the liver tissues of BDL rats. BDL-induced NF-jB DNA binding activity was attenuated by stearic acid (Fig. 4C). These data show that stearic acid can attenuate BDL-induced inflammation by suppressing inflammatory cell recruitment/accumulation and NF-jB activation. Discussion Saturated fatty acids such as stearic acid have been proposed to possess several beneficial and detrimental biological activities. Increasing evidence indicates that stearic acid shows antioxidant and anti-inflammatory potential [10,11,14,15]. Previous studies revealed that chronic supplementation with saturated fatty acids prevented alcohol-induced liver damage [10,11]. Using a rat BDL model, we showed that stearic acid supplementation had a benefi-
cial effect against cholestatic liver injury, as evidenced by the results of biochemical analysis and pathohistological examination, decreases in ductular reaction, fibrosis, and inflammatory cell accumulation as well as suppression of NF-jB activity. Our findings suggest that stearic acid could serve as a hepatoprotective agent against cholestasis-related hepatic damage. Obstruction of the biliary tree by BDL causes cholestasis. Cholestasis results in poor bile secretion and, subsequently, impairment in lipid digestion and absorption of fat-soluble nutrients. In addition, the associated bacterial translocation, portal bacteremia, and endotoxemia might alter microflora populations [2,13]. Persistent cholestasis induces bile duct proliferation and dilation, attracts low-grade inflammatory infiltration, activates matrixproducing cells, and then leads to periportal and perineoductular fibrosis [17]. In this study, all these cholestatic changes were moderately attenuated by stearic acid supplementation. In most cases,
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Fig. 4. Stearic acid attenuated BDL-induced inflammation. (A) Proteins were isolated from livers and were subjected to enzymatic assay for the measurement of MPO activity. (B) Proteins were isolated from livers and were subjected to Western blot with antibodies against CD68 and GAPDH. Representative blots and the quantitative data are shown. (C) Nuclear proteins were isolated from livers and were subjected to EMSA for the measurement of NF-jB DNA binding activity. Representative blots and the quantitative data are shown. **p < 0.01 vs. the sham control group and #p < 0.05 vs. the BDL control group.
primary damage to the biliary epithelium leads to the development of cholestasis. Long-standing cholestasis causes an ordered bile duct proliferation or typical ductular reaction [17]. GGT is known to be selectively expressed by bile duct cells [22]. Stearic acid supplementation was accompanied by the alleviation of bile duct proliferation and ductular reaction in this animal model. Since the proliferation of bile ducts is an early event in cholestasis-related changes, the attenuation of hepatic injury and fibrosis in BDL rats by stearic acid might be associated with alleviation of ductular reaction. It is hypothesized that the bile flow, bile acid composition, and bile acid’s effects might be the potential targets for the beneficial effects of stearic acid against ductular reaction and hepatic damage. Hydrophobic bile acids possess biologically active potential. The retained hydrophobic bile acids are able to generate free radicals, trigger inflammatory reaction, cause hepatocyte damage, and stimulate cholangiocyte proliferation [17,23,24]. BDL-induced Alk-P and total bilirubin elevation was not altered by stearic acid, indicating the protective effect of stearic acid was not global. Total bilirubin level is highly reflective of early and acute cholestatic changes [2,5]. Evidence showed that the hepatoprotective effects of omega-3 fatty acids and simvastatin against cholestatic liver damage were independent of reduction of total bilirubin [2,5]. Dietary stearic acid has little effect on bile acid excretion but can alter bile acid composition resulting in lower hydrophobicity [12,13]. Therefore, it is hypothesized that the alteration of bile acid composition might be one of the actions targeted by stearic acid to lower the stimulating effect of bile acid. However, further studies are needed to elicit the action targets of stearic acid. Hepatic fibrosis is the result of the accumulation of matrix proteins in the liver due to the imbalance between fibrogenesis and fibrolysis. Matrix-producing cells play an important role in the positive regulation of fibrogenesis. Among fibrogenic mesenchymal cells in the liver, the most extensively studied populations include the hepatic stellate cells and portal myofibroblasts, in particular the former. In this study, the activated matrix-producing cells proliferated, migrated, and produced collagen. BDL injury caused acti-
vation of a-SMA-positive matrix-producing cells. Stearic acid inhibited the development of hepatic fibrosis and suppressed aSMA-positive cells. These findings indicate that stearic acid exerted a negative regulatory effect on fibrogenesis by suppression of aSMA-positive matrix-producing cells. Evidence indicates that mitogenic and fibrogenic factors are critical for the activation of matrixproducing cells. Tumor necrosis factor alpha, TGF-b1, and interleukin-6 are the most extensively studied mitogenic and fibrogenic factors. Bile duct epithelial cells, Kupffer cells, and other infiltrates are important cell sources for the synthesis of these factors. Among them, TGF-b1 as a key fibrogenic mediator can enhance extracellular matrix deposition by activating stellate cells and inhibiting collagenase activity [18,25]. This study demonstrated that stearic acid down-regulated BDL-induced TGF-b1 production. Taken together, these results indicate that the anti-fibrotic effect of stearic acid is associated with the blockade of TGF-b1-associated mitogenic and/or fibrogenic signaling. The most novel and relevant finding was that stearic acid supplementation was accompanied by the suppression of inflammatory cell recruitment/accumulation and NF-jB activation. Massive hepatocyte cell death and necrosis, inflammation, excessive repair and fibrosis occur in liver diseases induced by different stimuli [4,10,11]. Bile duct epithelial cells, hepatocytes, Kupffer cells, and recruited leukocytes are important cell sources for inflammatory responses. The consequences of inflammatory reaction contribute to regenerative processes and/or augmented liver damage. In these processes, NF-jB plays an important role in cholangiocyte survival/damage, hepatocyte survival/damage, stellate cell and inflammatory cell activation, and inflammatory cytokine production [19]. Chronic stearic acid supplementation attenuated BDL-induced leukocyte infiltration, cytokine production, and NFjB activation in this study. Dietary saturated fatty acids down-regulate inflammatory reactions in the alcohol-injured liver [10,11]. Anti-inflammatory cytokine IL-10 possesses a hepatoprotective effect [26]. Evidence suggests that stearic acid was able to stimulate IL-10 production in hepatocytes [15]. Free radicals are involved in
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the regulation of cell proliferation and death as well as gene expression such as TGF-b1 [27,28]. Stearic acid possesses the potential to neutralize oxidative stress [14]. Thus, the anti-inflammatory effect of stearic acid might be attributable to its antioxidant and/or anti-inflammatory cytokine producing effects. On the other hand, portal endotoxemia also plays an important role in cholestasis-induced inflammation [29]. Stearic acid can alter microflora populations and reduce endotoxemia [13]. Therefore, the effect of stearic acid on the severity of endotoxemia might be another reason to alleviate inflammation. It should be noted that stearic acid also possesses a pro-inflammatory effect by stimulating macrophages to release pro-inflammatory cytokines [30]. However, further study is needed to elucidate the detailed anti-inflammatory mechanisms of stearic acid. In conclusion, our findings suggest that stearic acid could serve as a hepatoprotective agent, and chronic stearic acid supplementation could attenuate cholestasis-related hepatic damage. However, the effect of stearic acid post-treatment against cholestatic liver damage was not investigated in the current study. Acknowledgment This study was supported by Grants (DOH96-TD-F-113-026 and NSC96-2314-B-075A-003-MY3) from the Department of Health and National Science Council, Taiwan. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2009.12.119. References [1] M.E. Guicciardi, G.J. Gores, Bile acid-mediated hepatocyte apoptosis and cholestatic liver disease, Dig. Liver Dis. 34 (2002) 387–392. [2] S. Lee, S. Kim, H.D. Le, J. Meisel, R.A.M. Strijbosch, V. Nose, M. Puder, Reduction of hepatocellular injury after common bile duct ligation using omega-3 fatty acids, J. Pediatr. Surg. 43 (2008) 2010–2015. [3] K. Reyes-Gordillo, J. Segovia, M. Shibayama, V. Tsutsumi, P. Vergara, M.G. Moreno, P. Muriel, Curcumin prevents and reverses cirrhosis induced by bile duct obstruction or CCl4 in rat: role of TGF-b modulation and oxidative stress, Fundam. Clin. Pharmacol. 22 (2008) 417–427. [4] W.Y. Chen, C.J. Chen, J.W. Liao, F.C. Mao, Chromium attenuates hepatic damage in a rat model of chronic cholestasis, Life Sci. 84 (2009) 606–614. [5] S. Dold, M.W. Laschke, S. Lavasani, M.D. Menger, B. Jeppsson, H. Thorlacius, Simvastatin protects against cholestasis-induced liver injury, Br. J. Pharmacol. 156 (2009) 466–474. [6] H. Greim, D. Trulzsch, J. Roboz, K. Dressler, P. Czygan, F. Hutterer, F. Schaffner, H. Popper, Mechanism of cholestasis. Bile acids in normal rat livers and in those after bile duct ligation, Gastroenterology 63 (1972) 837–845. [7] G.S. Baroni, L. D’Ambrosio, G. Ferretti, A. Casini, A.D. Sario, R. Salzano, F. Ridolfi, S. Saccomanno, A.M. Jezequel, A. Benedetti, Fibrogenic effect of oxidative stress on rat hepatic stellate cells, Hepatology 27 (1998) 720–726. [8] T.Z. Liu, K.T. Lee, C.L. Chern, J.T. Cheng, A. Stern, L.Y. Tsai, Free radical-triggered hepatic injury of experimental obstructive jaundice of rats involves overproduction of proinflammatory cytokines and enhanced activation of nuclear factor kappaB, Ann. Clin. Lab. Sci. 31 (2001) 383–390.
[9] H.C. Pan, T.K. Kao, Y.C. Ou, D.Y. Yang, Y.J. Yen, C.C. Wang, Y.H. Chuang, S.L. Liao, S.L. Raung, C.W. Wu, A.N. Chiang, C.J. Chen, Protective effect of docosahexaenoic acid against brain injury in ischemic rats, J. Nutr. Biochem. 28 (2009) 715–725. [10] A.A. Nanji, K. Jokelainen, G.L. Tipoe, A. Rahemtulla, A.J. Dannenberg, Dietary saturated fatty acids reverse inflammatory and fibrotic changes in rat liver despite continued ethanol administration, J. Pharmacol. Exp. Ther. 299 (2001) 638–644. [11] A.A. Nanji, D. Zakim, A. Rahemtulla, T. Daly, L. Miao, S. Zhao, S. Khwaja, S.R. Tahan, A.J. Dannenberg, Dietary saturated fatty acids down-regulate cyclooxygenase-2 and tumor necrosis factor alpha and reverse fibrosis in alcohol-induced liver disease in the rat, Hepatology 26 (1997) 1538–1545. [12] C.A. Hassel, E.A. Mensing, D.D. Gallaher, Dietary stearic acid reduces plasma and hepatic cholesterol concentrations without increasing bile acid excretion in cholesterol-fed hamsters, J. Nutr. 127 (1997) 1148–1155. [13] R.L. Cowles, J.Y. Lee, D.D. Gallaher, C.L. Stuefer-Powell, T.P. Carr, Dietary stearic acid alters gall bladder bile acid composition in hamsters fed cereal-based diets, J. Nutr. 132 (2002) 3119–3122. [14] Z.J. Wang, C.L. Liang, G.M. Li, C.Y. Yu, M. Yin, Stearic acid protects cultured cortical neurons against oxidative stress, Acta Pharmacol. Sin. 28 (2007) 315– 326. [15] Y. Nishitani, S. Okazaki, K. Imabayashi, R. Katada, K. Umetani, H. Yajima, H. Matsumoto, Saturated and mono-unsaturated fatty acids increase interleukin10 production in rat hepatocytes, Nihon Arukoru Yakubutsu Igakkai Zasshi 42 (2007) 32–35. [16] C.J. Chen, Y.C. Ou, S.L. Liao, W.Y. Chen, S.Y. Chen, C.W. Wu, C.C. Wang, W.Y. Wang, Y.S. Huang, S.H. Hsu, Transplantation of bone marrow stromal cells for peripheral nerve repair, Exp. Neurol. 204 (2007) 443–453. [17] V. Desmet, T. Roskams, P. Van Eyken, Ductular reaction in the liver, Pathol. Res. Pract. 191 (1995) 513–524. [18] M. Bauer, D. Schuppan, TGF-1 in liver fibrosis: time to change paradigms, FEBS Lett. 502 (2001) 1–3. [19] E.S. Fox, J.C. Kim, T.F. Tracy, NF-kappaB activation and modulation in hepatic macrophages during cholestatic injury, J. Surg. Res. 72 (1997) 129–134. [20] S. Demirbilek, E. Tas, K. Gurunluoglu, M. Akin, R.T. Aksoy, M.H. Emre, N.E. Aydin, S. Ay, N. Ozatay, Fluvastatin reduced liver injury in a rat model of extrahepatic cholestasis, Pediatr. Surg. Int. 23 (2007) 155–162. [21] E. Gäbele, M. Froh, G.E. Arteel, T. Uesugi, C. Hellerbrand, J. Schölmerich, D.A. Brenner, R.G. Thurman, R.A. Rippe, TNF is required for cholestasis-induced liver fibrosis in the mouse, Biochem. Biophys. Res. Commun. 378 (2009) 348– 353. [22] A.M. Rutenberg, H. Kim, J.W. Fischbein, J.S. Hanker, H.L. Wasserkrug, A.M. Seligman, Histochemical and ultrastructural demonstration of c-glutamyl transpeptidase activity, J. Histochem. Cytochem. 17 (1969) 517–526. [23] T. Lamireau, M. Zoltowska, E. Levy, I. Yousef, J. Rosenbaum, B. Tuchweber, A. Desmoulière, Effects of bile acids on biliary epithelial cells: proliferation, cytotoxicity, and cytokine secretion, Life Sci. 72 (2003) 1401–1411. [24] X. Xia, H. Francis, S. Glaser, G. Alpini, G. LeSage, Bile acid interactions with cholangiocytes, World J. Gastroenterol. 12 (2006) 3553–3563. [25] E. Olaso, S.L. Friedmann, Molecular regulation of hepatic fibrogenesis, J. Hepatol. 29 (1998) 836–847. [26] S. Dinant, R.L. Veteläinen, S. Florquin, A.K. van Vliet, T.M. van Gulik, IL-10 attenuates hepatic I/R injury and promotes hepatocyte proliferation, J. Surg. Res. 141 (2007) 176–182. [27] C. Mastruzzo, N. Crim, C. Vancheri, Role of oxidative stress in pulmonary fibrosis, Monaldi Arch. Chest Dis. 57 (2002) 173–176. [28] R. Bataller, D.A. Brenner, Liver fibrosis, J. Clin. Invest. 115 (2005) 209–218. [29] Y. Wakabayashi, H. Shimizu, F. Kimura, H. Yoshidome, M. Ohtsuka, A. Kato, M. Miyazaki, Mechanism of neutrophil accumulation in sinusoids after extrahepatic biliary obstruction, Hepatogastroenterology 55 (2008) 1179– 1183. [30] L. Håversen, K.N. Danielsson, L. Fogelstrand, O. Wiklund, Induction of proinflammatory cytokines by long-chain saturated fatty acids in human macrophages, Atherosclerosis 202 (2009) 382–393. [31] C. Shackelford, G. Long, J. Wolf, C. Okerberg, R. Herbert, Qualitative and quantitative analysis of non-neoplastic lesions in toxicology studies, Toxicol. Pathol. 30 (2002) 93–96.
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Stearic acid attenuates cholestasis-induced liver injury Pin-Ho Pan a,1, Shih-Yi Lin b,1, Yen-Chuan Ou c, Wen-Ying Chen d, Yu-Han Chuang b, Yu-Ju Yen d, Su-Lan Liao d, Shue-Ling Raung d, Chun-Jung Chen d,e,f,g,* a
Department of Pediatrics, Tung’s Taichung MetroHarbor Hospital, Taichung, Taiwan Division of Endocrinology and Metabolism, Taichung Veterans General Hospital, Taichung, Taiwan c Division of Urology, Taichung Veterans General Hospital, Taichung, Taiwan d Department of Education and Research, Taichung Veterans General Hospital, Taichung, Taiwan e Center for General Education, Tunghai University, Taichung, Taiwan f Institute of Medical and Molecular Toxicology, Chung-Shan Medical University, Taichung, Taiwan g Institute of Biomedical Sciences, National Chung-Hsing University, Taichung, Taiwan b
a r t i c l e
i n f o
Article history: Received 12 December 2009 Available online 28 December 2009 Keywords: Cholestasis Hepatotoxicity Inflammation Oxidative stress Stearic acid
a b s t r a c t Inflammation is involved in cholestasis-induced hepatic damage. Stearic acid has been shown to possess anti-inflammatory potential. We assessed whether stearic acid has protective effects against cholestasisrelated liver damage. Cholestasis was produced by bile duct ligation (BDL) in male Sprague–Dawley rats for 3 weeks. Daily administration of stearic acid was started 2 weeks before injury and lasted for 5 weeks. In comparison with the control group, the BDL group showed hepatic damage as evidenced by elevation in serum biochemicals, ductular reaction, fibrosis, and inflammation. These pathophysiological changes were attenuated by chronic stearic acid supplementation. The anti-fibrotic effect of stearic acid was accompanied by reductions in a-smooth muscle actin-positive matrix-producing cells and critical fibrogenic cytokine transforming growth factor beta-1 production. Stearic acid also attenuated BDL-induced leukocyte accumulation and NF-jB activation. The data indicate that stearic acid attenuates BDL-induced cholestatic liver injury. The hepatoprotective effect of stearic acid is associated with anti-inflammatory potential. Ó 2009 Elsevier Inc. All rights reserved.
Introduction Cholestasis is characterized by an abnormal accumulation of bile acids, which is caused by defects in the process of bile acid transport. Chronic cholestasis is a key histopathological change that occurs in biliary atresia, primary biliary cirrhosis, and primary sclerosing cholangitis and contributes to the later development of hepatocellular injury, progressive hepatic fibrogenesis, cirrhosis, and death from liver failure [1]. Bile duct proliferation is a hallmark of cholestatic disorders. Histologically, the increase in hepatic bile ductular structures is generally accompanied by hepatocyte death, inflammatory response, and periductular fibrosis [2–5]. Although mechanisms of liver damage in cholestasis are multifactorial, retained bile acids, oxidative stress, and inflammation have been implicated as important factors in cholestatic liver injury [2–8]. Therefore, bile acids, oxidative stress, and inflammation are consid-
* Corresponding author. Address: Department of Education and Research, Taichung Veterans General Hospital, No. 160, Sec. 3, Taichung-Kang Rd., Taichung 407, Taiwan. Fax: +886 4 23592705. E-mail address: [email protected] (C.-J. Chen). 1 These authors contributed equally to this work. 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.12.119
ered to be potential targets for therapeutic intervention in treating cholestatic liver disorders. Polyunsaturated fatty acids have been implicated in the prevention of various human diseases, including inflammation-associated diseases [9]. However, only a few articles have reported the effects of saturated fatty acids on human health. Saturated fatty acids are usually regarded as unhealthy, but nutritionists believe that some types of saturated fatty acids may be beneficial. Several lines of investigation indicate that dietary fat can modulate the severity of liver injury. In experimental animals, 3 weeks of polyunsaturated fatty acid supplementation reduced cholestatic liver injury [2]. Dietary saturated fatty acids have been shown to protect against alcohol-induced liver injury [10,11]. Among the saturated fatty acids commonly found in the food supply, stearic acid (18:0) is unique because, unlike palmitic, myristic, and lauric acid, its hypercholesterolemic effect is negligible [12,13]. A recent study showed that stearic acid may function as a natural ligand of the peroxisome proliferator-activated receptor and protect cells from oxidative damage [14]. Elevated production of anti-inflammatory cytokine interleukin-10 (IL-10) has been detected in stearic acidtreated hepatocytes [15]. Although the consequences of stearic acid supplementation vary, these findings imply that stearic acid is capable of alleviation of oxidative stress and inflammation.
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Cholestasis is a clinically important primary event that contributes to hepatic damage. Currently, little information is available about the effect of stearic acid on chronic cholestatic liver injury. Common bile duct ligation (BDL) and scission in animals represents a classical experimental model for the analysis of cholestasis and consequent hepatic damage. BDL produces cholestasis, triggers free radical generation and inflammation, induces progressive portal fibrosis, causes secondary biliary cirrhosis, and finally leads to liver failure [4]. The aims of the present study were to assay the preventive potential of stearic acid on rat hepatic damage induced by BDL and to elicit the role of the anti-inflammatory effect. Materials and methods Animals. Male Sprague–Dawley rats were randomly divided into six experimental groups. The control (n = 30) and BDL group (n = 30) rats underwent sham and BDL operation, respectively. Intraperitoneal administration of saline and stearic acid (250 and 1000 nmol/kg/day, respectively) was carried out and modified in accordance with the method used in our previous reports [9]. The rats in both groups received saline and stearic acid (10 animals/each group) for 5 weeks starting from 2 weeks before operation. All animals were sacrificed 3 weeks after surgery under pentobarbital anesthesia. The animal study was approved by the Animal Care and Use Committee of Taichung Veterans General Hospital. BDL operation. Rats (200–250 g) were anesthetized with pentobarbital (50 mg/kg) and the common bile duct was exposed and ligated by double ligatures with 3–0 silk. The first ligature was made below the junction of the hepatic ducts and the second ligature was made above the entrance of the pancreatic ducts. The common bile duct was then cut between the double ligatures. Thus, animals had total, permanent biliary obstruction [4]. In sham-operated rats, an abdominal incision was made without a ligation. Biochemical analysis. Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), c-glutamyl transpeptidase (GGT), total bilirubin, and albumin were measured by automated standardized procedures (Roche Hitachi 917/747, Mannheim, Germany). The serum level of transforming growth factor beta-1 (TGF-b1) was measured by enzyme-linked immunosorbent assay (ELISA). Histological and immunohistochemical examination. Excised liver specimens were fixed in 10% formalin and embedded with paraffin. Hematoxylin and eosin (H&E) staining and Sirius Red staining were performed according to standard procedures. For immunohistochemical examination, deparaffinized sections were incubated with a-smooth muscle actin (a-SMA) and CD68 primary antibody and biotinylated secondary antibody, followed by the avidin–biotin–peroxidase complex. The immunoreactive signal was developed by color deposition using diaminobenzidine as substrate. Tissue preparation and Western blot. The resected liver tissues were extracted with lysis buffer (1% Triton X-100; 50 mmol/l Tris–HCl, pH 7.6; 150 mmol/l NaCl; and 1% protease inhibitor cocktail). For Western blot, proteins were separated by SDS–PAGE and electrophoretically transferred to polyvinylidene difluoride membranes. After blocking, the membranes were incubated for 1 h at room temperature with the indicated antibodies including aSMA (Calbiochem Biotechnology), CD68 (BioSource), and GAPDH (Santa Cruz Biotechnology). Then, a 1:10,000 dilution of horseradish peroxidase-labeled IgG was added at room temperature for 1 h. The blots were developed using ECL Western blotting reagents and quantified by optical densitometry. The intensity of each signal was corrected by the values obtained from the immunodetection of GAPDH and the relative protein intensity was expressed as folds of the content in the control group.
Myeloperoxidase activity (MPO) assay. Proteins (10 lg/100 ll) obtained from liver tissues were mixed with 2.9 ml of the assay solution. The optical absorbance was determined at 470 nm for 1 min with a spectrophotometer [9]. MPO activity was calculated from a standard MPO activity curve. The assay solution consisted of: H2O, 26.9 ml; 0.1 M sodium phosphate buffer (pH 7.0), 3.0 ml; 0.1 M H2O2, 0.1 ml; guaiacol, 0.048 ml. Collagen measurement. Proteins (50 lg) extracted from liver tissues were subjected to ELISA with antibody against collagen IV (Sigma Chemical) for the determination of collagen [16]. Preparation of nuclear extracts and electrophoretic mobility shift assay (EMSA). Nuclear proteins were extracted from liver tissues. The isolation of nuclear extract and EMSA was conducted as described previously [9]. The oligonucleotides specific for NF-jB (50 -AGTTGAGGGGACTTTCCCAGGC) were synthesized and labeled with biotin. Nuclear extract (5 lg) was used for EMSA. The binding reaction mixture included 1 lg of poly(dI-dC), 0.1 lg of poly-L-lysine, and 100 fmol of biotin-labeled DNA probe in a 20 ll binding buffer (10 mM HEPES, pH 7.6; 50 mM NaCl; 0.5 mM MgCl2; 0.5 mM EDTA; 1 mM dithiothreitol; 5% glycerol). The DNA/protein complex was analyzed on 6% native polyacrylamide gels. Statistical analysis. All data are presented as mean ± standard deviation. For comparison, the statistical significance between means was determined using one-way analysis of variance (ANOVA) followed by Dunnett’s t-test. A p value of less than 0.05 was considered significant. Results Stearic acid attenuated BDL-induced liver injury Grossly, body mass and the average food intake of BDL rats were lower than those of sham control rats. There were no significant differences in body mass and average food intake between salineand stearic acid-supplemented animals (data not shown). Liver damage was first analyzed by histological examination (Fig. 1). No morphological abnormalities were observed in saline- and stearic acid-supplemented sham-operated control rats showing a regular morphology of liver parenchyma with intact hepatocytes, sinusoids, and portal tract. Diffuse severe/high bile duct hyperplasia, portal edema, and mild portal infiltrates, all features of extrahepatic cholestasis, were present in BDL rats. BDL rats also showed a loss of hepatic structure in periportal areas. Serum levels of AST, ALT, Alk-P, GGT, and total bilirubin, common biochemical indexes of hepatocellular injury, were significantly elevated in BDL rats (Table 1). BDL rats also had decreased serum level of albumin (Table 1). The incidence of BDL-induced histopathological changes was not affected by stearic acid supplementation (Table 2). However, the histopathological changes (Fig. 1) and histopathological scores for bile duct hyperplasia (Table 2) were improved by stearic acid supplementation. In addition, serum biochemical analysis (Table 1) showed moderate reduction in these pathophysiological changes except Alk-P and total bilirubin in BDL rats supplemented with stearic acid. These results indicate that stearic acid supplementation attenuates BDL-induced liver damage. Stearic acid attenuated BDL-induced fibrosis Biliary fibrosis, such as the type observed in conditions of chronic obstruction of the biliary tree, is characterized by accumulation of extracellular matrix at portal tracts, surrounding newly formed bile ducts emerging during the course of ductular reaction and originating from proliferation of pre-existing bile duct epithelial cells [17]. The histopathological scores for bile duct hyperplasia (Table 2) and serum activity measurements of GGT (Table 1) re-
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Fig. 1. Stearic acid attenuated BDL-induced liver injury. The obtained liver sections were subjected to histological examination. Representative photomicrographs of H&E staining are shown. Original magnification: 40.
Table 1 Biochemical parameters. SA (nmol/kg)
AST (U/l) ALT (U/l) Alk-P (U/l) GGT (U/l) Total bilirubin (mg/dl) Albumin (g/dl) TGF-b1 (pg/ml)
Sham 0
BDL 0
Sham 250
BDL 250
Sham 1000
BDL 1000
178 ± 32 44 ± 9 199 ± 88 1.8 ± 1.2 0.6 ± 0.3 3.8 ± 0.2 328 ± 144
924 ± 201** 199 ± 69** 599 ± 86** 53.3 ± 20.1** 8.4 ± 1.9** 2.3 ± 0.4** 1498 ± 395**
193 ± 30 49 ± 5 242 ± 106 1.7 ± 1.0 0.7 ± 0.4 3.8 ± 0.2 415 ± 168
783 ± 233** 168 ± 44** 545 ± 118** 45.2 ± 21.8** 8.1 ± 1.3** 2.8 ± 0.5** 1300 ± 379**
184 ± 32 47 ± 14 225 ± 80 1.5 ± 0.9 0.4 ± 0.3 3.8 ± 0.1 223 ± 122
646 ± 197**,# 134 ± 26**,# 538 ± 154** 32.7 ± 6.1**,# 7.9 ± 2.4** 3.1 ± 0.6*,# 835 ± 357**,#
Data are expressed as mean ± SD *p < 0.05 and **p < 0.01 vs. vehicle sham control group and #p < 0.05 vs. BDL control group, n = 10. AST, aspartate aminotransferase; ALT, alanine aminotransferase; Alk-P, alkaline phosphatase; GGT, c-glutamyl transpeptidase.
Table 2 Scores of bile duct hyperplasia. Histopathological findings
Hyperplasia, bile duct, focal, moderate to severe/higha Histopathology score of bile duct hyperplasiab
Group (SA (nmol/kg)) Sham 0
BDL 0
Sham 250
BDL 250
Sham 1000
BDL 1000
0/10
10/10
0/10
10/10
0/10
10/10
0
4.4 ± 0.5
0
4.0 ± 0.7
0
3.4 ± 0.7*
Data are expressed as mean ± SD. * p < 0.05 vs. BDL control group. a Incidence: affected rats/total examined rats. b Score of bile duct hyperplasia. Severity of lesions was graded according to the methods described by Shackelford et al. [31]. Degree of lesions was graded from one to five depending on severity: 1, minimal (<1%); 2, slight (1–25%); 3, moderate (26– 50%); 4, moderate/severe (51–75%); 5, severe/high (76–100%).
vealed that stearic acid alleviated BDL-induced ductular reaction. The next experiment was conducted to determine whether stearic acid has a role in BDL-induced fibrosis. Fibrosis was assessed by a classical histopathological technique using Sirius Red staining. In BDL rats, significant hepatic collagen deposition/accumulation was present in periportal and portal tracts. The presence of this deposition/accumulation and the bridging fibrosis were reduced in the stearic acid-treated groups (Fig. 2). The extent of liver fibrosis was also measured biochemically as hepatic collagen content.
In parallel to the observed improvement of liver histology, stearic acid reduced BDL-induced elevation in collagen content (Fig. 3A). These results indicate that stearic acid has an attenuating effect against BDL-induced liver fibrosis. Immunohistochemical results showed that BDL caused an elevation in a-SMA immunopositive signals in portal and periportal areas and the immunoreactive signals were reduced by stearic acid treatment (Supplementary Fig. 1). Western blot study showed that stearic acid caused a reduction of a-SMA protein (Fig. 3B). These results indicate that stearic acid decreases the activation of a-SMA-positive matrix-producing cells in hepatic tissues after BDL injury. Numerous studies indicate that TGF-b1 plays an important role in liver fibrogenesis through acting on matrix-producing cells [4,18]. The elevation of serum level of TGF-b1 in BDL rats was reduced by stearic acid in this study (Table 1). These data suggest that the anti-fibrotic effect of stearic acid is associated with reduction of TGF-b1 signaling. Stearic acid attenuated BDL-induced inflammation Prolonged biliary obstruction is associated with inflammatory cell infiltration, NF-jB activation, and cytokine over-production. Inhibition of inflammatory responses prevents cholestatic liver damage [3,5,19–21]. Immunohistochemical staining (Supplementary Fig. 2) and Western blotting (Fig. 4B) revealed an accumulation of monocytes/macrophages in the liver tissues of BDL rats. An increase in neutrophil infiltration in liver tissues assessed by measuring hepatic MPO activity occurred in BDL rats (Fig. 4A). The recruitment/accumulation of inflammatory cells was attenuated by stearic acid. The activation of NF-jB occurs in cholestatic
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Fig. 2. Stearic acid attenuated BDL-induced liver fibrosis. The obtained liver sections were subjected to histological examination. Representative photomicrographs of Sirius Red staining are shown. Original magnification: 40.
Fig. 3. Stearic acid attenuated BDL-induced liver fibrotic changes. (A) Proteins were isolated from livers and were subjected to ELISA for the measurement of collagen. The absorbance was measured at 450 nm using a spectrophotometer. (B) Proteins were isolated from livers and were subjected to Western blot with antibodies against a-SMA and GAPDH. Representative blots and the quantitative data are shown. **p < 0.01 vs. the sham control group and #p < 0.05 vs. the BDL control group.
liver damage and contributes to inflammatory responses [19]. EMSA revealed an increase of NF-jB activity in the liver tissues of BDL rats. BDL-induced NF-jB DNA binding activity was attenuated by stearic acid (Fig. 4C). These data show that stearic acid can attenuate BDL-induced inflammation by suppressing inflammatory cell recruitment/accumulation and NF-jB activation. Discussion Saturated fatty acids such as stearic acid have been proposed to possess several beneficial and detrimental biological activities. Increasing evidence indicates that stearic acid shows antioxidant and anti-inflammatory potential [10,11,14,15]. Previous studies revealed that chronic supplementation with saturated fatty acids prevented alcohol-induced liver damage [10,11]. Using a rat BDL model, we showed that stearic acid supplementation had a benefi-
cial effect against cholestatic liver injury, as evidenced by the results of biochemical analysis and pathohistological examination, decreases in ductular reaction, fibrosis, and inflammatory cell accumulation as well as suppression of NF-jB activity. Our findings suggest that stearic acid could serve as a hepatoprotective agent against cholestasis-related hepatic damage. Obstruction of the biliary tree by BDL causes cholestasis. Cholestasis results in poor bile secretion and, subsequently, impairment in lipid digestion and absorption of fat-soluble nutrients. In addition, the associated bacterial translocation, portal bacteremia, and endotoxemia might alter microflora populations [2,13]. Persistent cholestasis induces bile duct proliferation and dilation, attracts low-grade inflammatory infiltration, activates matrixproducing cells, and then leads to periportal and perineoductular fibrosis [17]. In this study, all these cholestatic changes were moderately attenuated by stearic acid supplementation. In most cases,
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Fig. 4. Stearic acid attenuated BDL-induced inflammation. (A) Proteins were isolated from livers and were subjected to enzymatic assay for the measurement of MPO activity. (B) Proteins were isolated from livers and were subjected to Western blot with antibodies against CD68 and GAPDH. Representative blots and the quantitative data are shown. (C) Nuclear proteins were isolated from livers and were subjected to EMSA for the measurement of NF-jB DNA binding activity. Representative blots and the quantitative data are shown. **p < 0.01 vs. the sham control group and #p < 0.05 vs. the BDL control group.
primary damage to the biliary epithelium leads to the development of cholestasis. Long-standing cholestasis causes an ordered bile duct proliferation or typical ductular reaction [17]. GGT is known to be selectively expressed by bile duct cells [22]. Stearic acid supplementation was accompanied by the alleviation of bile duct proliferation and ductular reaction in this animal model. Since the proliferation of bile ducts is an early event in cholestasis-related changes, the attenuation of hepatic injury and fibrosis in BDL rats by stearic acid might be associated with alleviation of ductular reaction. It is hypothesized that the bile flow, bile acid composition, and bile acid’s effects might be the potential targets for the beneficial effects of stearic acid against ductular reaction and hepatic damage. Hydrophobic bile acids possess biologically active potential. The retained hydrophobic bile acids are able to generate free radicals, trigger inflammatory reaction, cause hepatocyte damage, and stimulate cholangiocyte proliferation [17,23,24]. BDL-induced Alk-P and total bilirubin elevation was not altered by stearic acid, indicating the protective effect of stearic acid was not global. Total bilirubin level is highly reflective of early and acute cholestatic changes [2,5]. Evidence showed that the hepatoprotective effects of omega-3 fatty acids and simvastatin against cholestatic liver damage were independent of reduction of total bilirubin [2,5]. Dietary stearic acid has little effect on bile acid excretion but can alter bile acid composition resulting in lower hydrophobicity [12,13]. Therefore, it is hypothesized that the alteration of bile acid composition might be one of the actions targeted by stearic acid to lower the stimulating effect of bile acid. However, further studies are needed to elicit the action targets of stearic acid. Hepatic fibrosis is the result of the accumulation of matrix proteins in the liver due to the imbalance between fibrogenesis and fibrolysis. Matrix-producing cells play an important role in the positive regulation of fibrogenesis. Among fibrogenic mesenchymal cells in the liver, the most extensively studied populations include the hepatic stellate cells and portal myofibroblasts, in particular the former. In this study, the activated matrix-producing cells proliferated, migrated, and produced collagen. BDL injury caused acti-
vation of a-SMA-positive matrix-producing cells. Stearic acid inhibited the development of hepatic fibrosis and suppressed aSMA-positive cells. These findings indicate that stearic acid exerted a negative regulatory effect on fibrogenesis by suppression of aSMA-positive matrix-producing cells. Evidence indicates that mitogenic and fibrogenic factors are critical for the activation of matrixproducing cells. Tumor necrosis factor alpha, TGF-b1, and interleukin-6 are the most extensively studied mitogenic and fibrogenic factors. Bile duct epithelial cells, Kupffer cells, and other infiltrates are important cell sources for the synthesis of these factors. Among them, TGF-b1 as a key fibrogenic mediator can enhance extracellular matrix deposition by activating stellate cells and inhibiting collagenase activity [18,25]. This study demonstrated that stearic acid down-regulated BDL-induced TGF-b1 production. Taken together, these results indicate that the anti-fibrotic effect of stearic acid is associated with the blockade of TGF-b1-associated mitogenic and/or fibrogenic signaling. The most novel and relevant finding was that stearic acid supplementation was accompanied by the suppression of inflammatory cell recruitment/accumulation and NF-jB activation. Massive hepatocyte cell death and necrosis, inflammation, excessive repair and fibrosis occur in liver diseases induced by different stimuli [4,10,11]. Bile duct epithelial cells, hepatocytes, Kupffer cells, and recruited leukocytes are important cell sources for inflammatory responses. The consequences of inflammatory reaction contribute to regenerative processes and/or augmented liver damage. In these processes, NF-jB plays an important role in cholangiocyte survival/damage, hepatocyte survival/damage, stellate cell and inflammatory cell activation, and inflammatory cytokine production [19]. Chronic stearic acid supplementation attenuated BDL-induced leukocyte infiltration, cytokine production, and NFjB activation in this study. Dietary saturated fatty acids down-regulate inflammatory reactions in the alcohol-injured liver [10,11]. Anti-inflammatory cytokine IL-10 possesses a hepatoprotective effect [26]. Evidence suggests that stearic acid was able to stimulate IL-10 production in hepatocytes [15]. Free radicals are involved in
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the regulation of cell proliferation and death as well as gene expression such as TGF-b1 [27,28]. Stearic acid possesses the potential to neutralize oxidative stress [14]. Thus, the anti-inflammatory effect of stearic acid might be attributable to its antioxidant and/or anti-inflammatory cytokine producing effects. On the other hand, portal endotoxemia also plays an important role in cholestasis-induced inflammation [29]. Stearic acid can alter microflora populations and reduce endotoxemia [13]. Therefore, the effect of stearic acid on the severity of endotoxemia might be another reason to alleviate inflammation. It should be noted that stearic acid also possesses a pro-inflammatory effect by stimulating macrophages to release pro-inflammatory cytokines [30]. However, further study is needed to elucidate the detailed anti-inflammatory mechanisms of stearic acid. In conclusion, our findings suggest that stearic acid could serve as a hepatoprotective agent, and chronic stearic acid supplementation could attenuate cholestasis-related hepatic damage. However, the effect of stearic acid post-treatment against cholestatic liver damage was not investigated in the current study. Acknowledgment This study was supported by Grants (DOH96-TD-F-113-026 and NSC96-2314-B-075A-003-MY3) from the Department of Health and National Science Council, Taiwan. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2009.12.119. References [1] M.E. Guicciardi, G.J. Gores, Bile acid-mediated hepatocyte apoptosis and cholestatic liver disease, Dig. Liver Dis. 34 (2002) 387–392. [2] S. Lee, S. Kim, H.D. Le, J. Meisel, R.A.M. Strijbosch, V. Nose, M. Puder, Reduction of hepatocellular injury after common bile duct ligation using omega-3 fatty acids, J. Pediatr. Surg. 43 (2008) 2010–2015. [3] K. Reyes-Gordillo, J. Segovia, M. Shibayama, V. Tsutsumi, P. Vergara, M.G. Moreno, P. Muriel, Curcumin prevents and reverses cirrhosis induced by bile duct obstruction or CCl4 in rat: role of TGF-b modulation and oxidative stress, Fundam. Clin. Pharmacol. 22 (2008) 417–427. [4] W.Y. Chen, C.J. Chen, J.W. Liao, F.C. Mao, Chromium attenuates hepatic damage in a rat model of chronic cholestasis, Life Sci. 84 (2009) 606–614. [5] S. Dold, M.W. Laschke, S. Lavasani, M.D. Menger, B. Jeppsson, H. Thorlacius, Simvastatin protects against cholestasis-induced liver injury, Br. J. Pharmacol. 156 (2009) 466–474. [6] H. Greim, D. Trulzsch, J. Roboz, K. Dressler, P. Czygan, F. Hutterer, F. Schaffner, H. Popper, Mechanism of cholestasis. Bile acids in normal rat livers and in those after bile duct ligation, Gastroenterology 63 (1972) 837–845. [7] G.S. Baroni, L. D’Ambrosio, G. Ferretti, A. Casini, A.D. Sario, R. Salzano, F. Ridolfi, S. Saccomanno, A.M. Jezequel, A. Benedetti, Fibrogenic effect of oxidative stress on rat hepatic stellate cells, Hepatology 27 (1998) 720–726. [8] T.Z. Liu, K.T. Lee, C.L. Chern, J.T. Cheng, A. Stern, L.Y. Tsai, Free radical-triggered hepatic injury of experimental obstructive jaundice of rats involves overproduction of proinflammatory cytokines and enhanced activation of nuclear factor kappaB, Ann. Clin. Lab. Sci. 31 (2001) 383–390.
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