The origin, mechanism, and significance of the bile duct proliferation (BDP) associated with cholestasis remain unexplained. This study examined the effect of ...
Digestive Diseases and Sciences, Vol. 38, No. 7 (July 1993), pp. 1291-1296
Effect of Ursodeoxycholic Acid Administration on Bile Duct Proliferation and Cholestasis in Bile Duct Ligated Rat ERMENEGILDO E. FREZZA, GIORGIO E. GERUNDA, MARIO PLEBANI, ALESSANDRA GALLIGIONI, ALDA GIACOMINI, DANIELE NERI, ALVISE MAFFEI FACCIOLI, and CLAUDIO TIRIBELLI*
The origin, mechanism, and significance o f the bile duct proliferation (BDP) associated with cholestasis remain unexplained. This study examined the effect o f oral administration o f ursodeoxycholic acid (UDCA) on both BDP and cholestasis in the rat. After bile duct ligation, male Sprague-Dawley rats were treated for 30 days with either UDCA (5 mg/day) (group A) or saline solution (group B). Animals were sacrificed at day 30. The serum activity o f aminotransferase (ALT, AST), alkaline phosphatase, and ~-glutamyltransferase (GGT) was significantly lower (P < O.01) in the UDCA-treated rats. Total serum bilirubin and total serum bile acids were lower (P < O.001) in group A. Moreover, the control o f BA in bile was reduced also (P < 0.02). Conversely, serum cholesterol levels were not different between the two groups. Histological examination showed that the number o f ductular cells in the portal areas was significantly (P < O.001) reduced in UDCA-treated as compared to saline-treated rats. The replication activity, assessed as the number o f bromodeoxyuridine-positive cells, was also significantly lower in treated animals (33 +_ 11 vs 64 +_22per 1000 cells; P < 0.001). Lobular bile ductules were three times larger in group B, and extrahepatic duct measurements confirmed this increase in size o f the larger biliary ducts (P < 0.001). These findings demonstrate that UDCA reduces BDP in response to BD ligation. Although the mechanism(s) o f this effect is still hypothetical, UDCA may reduce the level o f irritating bile salts such as chenodeoxycholic acid and lithocolate and increase periductular bile acid recirculation. These data support the beneficial effect o f UDCA treatment in chronic cholestatic disease. KEY WORDS: bile duct proliferation; UDCA; liver cholestasis; chenodeoxycholic acid; lithocolate; bile acids; bile duct ligation.
Bile duct proliferation (BDP) was described by Waldayer in 1968 (1) in association with acute liver inflammation due to infection or toxic ingestion (2, 3). BDP has been also observed after administration Manuscript received September 29, 1992; revised manuscript received February i i , 1993; accepted February 22, 1993. From the Istituti di Patologia Chirurgica II, Chimica Clinica, Anatomia Patologica II, Universit~ di Padova, and *Centro Studi Fegato, Dipartimento BBCM, Universith di Trieste, Italy. Address for reprint requests: Dr. Claudio Tiribelli, Dipartimento BBCM, via Giorperi 1 1-34127 Trieste, Italy.
of different hepatotoxic substances (4) as well as during different forms of intra- and extrahepatic cholestasis. While the result of these different conditions is comparable (BDP), the origin of the proliferating cell(s) is not yet known. Based on the histological demonstration of metaplastic (4-6) or rosette formation (7, 8) during chronic extrahepatic cholestasis, portal stem cells have been suggested as the cells of origin for the BDP (8). Other studies have suggested that BDP arises from a proliferation
Digestive Diseases and Sciences, Vol. 38, No. 7 (July 1993) 0163-2116/93/0700-1291507.00/09 1993PlenumPublishingCorporation
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FREZZA ET AL of h e p a t o c y t e s (9), ductule cells (10), or of cholangiocytes (11), the trigger m e c h a n i s m s being cell necrosis (12). Recently, Slott et al (13) suggested that proliferating bile duct cells could arise from preexisting biliary epithelial cells. This conclusion w a s based on the finding that bile ducts of all sizes contain cells with a proliferative capacity. In any case, the changes in bile composition and/or increasing intraluminal pressure appear to be the initiating factors responsible for BDP, while the infiltration of m a c r o p h a g e s and p o l y m o r p h o n u c l e a t e s appears not to be important in initiating BDP. In spite of these findings, it is not clear w h e t h e r bile p e r s e has an initiating m e c h a n i s m in BDP and if a modification in the bile composition can reduce BDP. U r s o d e o x y c h o l i c acid (UDCA) is a bile acid that is used extensively in the treatment of intrahepatic cholestasis (14-16). Clinical studies h a v e p r o v e d it to b e effective in p r i m a r y biliary cirrhosis (17), and in chronic active hepatitis (18, 19). The aim of this study w a s to investigate the effect of U D C A treatment on both cholestatic m a r k e r s and B D P following bile duct ligation (BDL). In particular, the effect of B D L on liver damage, BDP, s e r u m and bile e n z y m e s , and the efficiency of U D C A treatment on BDL-induced liver damage w e r e investigated.
MATERIALS AND METHODS Animals and Chemicals. Adult male Sprague-Dawley rats (60-90 days old) weighing 200 --- 30 g were used. Animals were allowed free access to a standard laboratory chow (RandoinCauseret) and tap water. UDCA as pure compound (purity higher than 98%) was provided by Midi Spa, Milan, Italy. Experimental Design. The common bile duct was ligated as described elsewhere (20). After BDL, the animals were divided at random into two groups and treated for 30 days with either oral UDCA at a dose of 5 mg/kg (group A), or saline solution (group B). Equal volumes of UDCA dissolved in water or saline were given to the animals by oral garage in the morning. After 30 days of treatment, 2 hr before the animals were sacrificed, bromodeoxyuridine (BrdU) (dose 50 mg/kg) was administered intraperitoneally as described by O'Donnel et al (21). Testing. Before sacrifice, plasma samples were collected from 10 randomly selected animals of each group. Plasma (5 ml) was obtained by caval puncture in the presence of 5 units heparin/ml, and the following parameters were immediately determined using an automatized procedure: aminotransferases (ALT and AST), GGT, ALP, and total bilirubin. Plasma bile acids were determined using a kit (Sterognost-3-Flu, Neygaard & Co, Norway), according to the method of Mashige et al (22).
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TABLE 1. SERUM AND BILIARY PARAMETERS IN BILE DUCT-LIGATZD RATS TREATED WITH U D C A (GRouP A) OR WITHOUT TREATMENT (GRouP B)*
Group A A L T (units/liter) AST (units/liter) GGTP (units/liter) Alkaline Phosphatase (units/liter) Total Bilirubin (nmol/liter) Total Bile Acids (nmol/liter) Cholesterol (mg/dl) Biliary Bile Acids (nmol/liter)
185 785 14
• • •
11 81 3
210 2.2 34.7 2.5 196
-x-_ 68 • 0.7 • 13.8 • 0.7 • 175
Group B 336 1357 55 554 54.2 179.0 2.0 684
• 107a • 347a • 7a • 79b • 16.7b • 25.3b • 0.3 • 330c
*Data are reported as mean • 1 SD. Each group consists of 10 animals, a = P < 0.01 v s group A; b = P < 0.001 v s group A; c = P < 0.02 vs group A.
The diameter of the common bile duct was measured three times in each animal with a ruler, and the mean value of the three measures calculated. Bile (2 ml) was collected by puncture of the common bile duct, frozen, and stored at -20~ until a determination of its bile acid content could be accomplished according to Muraca et al (23). All determinations were performed within three weeks. After sacrifice, liver tissue were obtained from the right lobe and processed for histological evaluation after H&E and Van Gieson staining. Histological Examination. Three different sections of each liver stained with both H&E and Van Gieson were evaluated for the number of bile ductules. Forty portal areas were counted in each specimen. Ductular proliferation was graded at a constant magnification (20x) according to the following score: grade 0 = 0-2 ductules; grade 1 = 3-4, grade 2 = 5-7; grade 3 = 8-10, and grade 4 = > 10 ductules. Histological evaluation was performed by a histologist without knowledge of the kind of treatment (UDCA or placebo) the animal was receiving. BrdU Immunoperoxidase Staining. BrdU staining was performed according to O'Donnel (21). Briefly, dewaxed sections were incubated for 60 min with monoclonal antibodies anti-BrdU (Dakko) (diluted 1/30 with PBS), and subsequently with anti-IgG antibody (diluted 1/200 with PBS) for an additional 60 rain. ABC complex (Dakko) was added for 60 min, and diaminobenzidine for 10 min, followed by light counterstaining with hematoxylin. Grading was assessed by counting the number of BrdUpositive cells per 103 cells. Statistical Analysis. Data are reported as mean -- SD and Student's t test was used to establish significant differences.
RESULTS Table 1 shows the results obtained in the two groups of rats b y plasma and bile analysis. A significant reduction in all serum and biliary p a r a m e ters measured, with the exception of cholesterol, w a s o b s e r v e d in the U D C A - t r e a t e d group of animals. In particular, a m a r k e d reduction in the level of both the serum bilirubin and alkaline phosphaDigestive Diseases and Sciences, Vol. 38, No. 7 (July 1993)
UDCA IN BILE DUCT PROLIFERATION 20 G Im
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