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Protective effect of polyunsaturated phosphatidylcholine on liver damage induced by biliary obstruction in rats
Protective Effect of Polyunsaturated Phosphatidylcholine on Liver Damage Induced by Biliary Obstruction in Rats By Abdurrahman Karaman, Savas¸ Demirbilek, Nurzen Sezgin, Necla Gu¨rbu¨z, and I˙clal Gu¨rses Malatya, Turkey
Background/Purpose: Persistent inflammatory response secondary to congenital or acquired biliary choleastasis plays an important role in the pathophysiology of hepatic tissue damage. The polyunsaturated fatty acids (PUFA) have been shown to suppress the inflammatory reactions in vivo and in vitro. PUFA has been shown also to protect againts various types of experimental liver damage in animal models and isolated hepatocytes. Therefore, the aim of this study was to investigate the protective effect of PUFA administration on liver damage using the rat chronic biliary obstruction model. Methods: Swiss albino rats of either sex were divided into 4 groups as follows: control group (group 1, 10 rats); rats with sham operation and treated with saline group 2, 10 rats); rats with biliary obstruction (group 3, 15 rats); and polyunsaturated phophatidylcholine (PPC)-treated rats with biliary obstruction (Group 4, 15 rats). Biliary obstruction was induced by double ligation and division of the common bile duct. PUFA treatment was started 2 weeks later from biliary obstruction in doses of 50 mg/d per rat and continued for 2 weeks. All animals were killed after 4 weeks of common bile duct ligation or sham operation. Liver damage and cholesta-
R
EPAIR AFTER cholestatic liver injuries often is complicated by progressive hepatic damage despite adequate technical decompression of the biliary tree.1 This progressive hepatic damage accompanies an increased morbidity and mortality observed in biliary atresia, choledochal cyst, sclerosing cholangitis, and other biliary obstructive pathology.2 The typical histologic characteristics of “failed” or “delayed” repair are persistent biliary hyperplasia, fibrosis, and inflammation despite surgical correction of the outflow obstruction.3 Factors determining the progression of hepatic injury in cholestatic liver disease are not understood fully, but clearly, both cellular and humoral immune responses of the host are involved.2,4-10 Soybean polyunsaturated phophatidylcholine (PPC) is a polyunsaturated fatty acid (PUFA) compound and a 94% to 96% mixture of phosphatidylcholines, about half of which is dilinoleoylphosphatidylcholine.11 At variance with mammalian phospholipids, this plant lipid contains unsaturated fatty acids in both the 1 and 2 positions of the glycerol backbone, which confers a high bioavailability, mainly because of reacylation of the unsaturated 1-acyl-lysophosphotidylcholine with additional unsaturated fatty acids during intestinal absorption.12 The n-3 polyunsaturated fatty acids have been
sis were determined examinations.
by
biochemical
and
histologic
Results: The data showed a decrease in plasma bilirubin level (both conjugated and unconjugated) and liver enzyme levels (AST, ALT, AP, GGT, 5⬘-NT) in group 4, when compared with group 3 (P ⬍ .05). Tissue levels of malondialdehyde (MDA) in group 4 was 20.00 ⫾ 2.93 compared with that in group 3, 27.12 ⫾ 2.96 (P ⬍ .05). Administration of PUFA to the biliary obstructed rats resulted in inhibition of collagen accumulation (P ⬍ .05) and ductal proliferation (P ⬍ .05). Conclusions: PUFA reduced liver damage, ductular proliferation, and fibrosis in biliary obstructed rats. These effects suggest that it might be a useful agent to preserve liver function in patients with biliary obstruction such as biliary atresia. J Pediatr Surg 38:1341-1347. © 2003 Elsevier Inc. All rights reserved. INDEX WORDS: Biliary obstruction, cholestasis, liver damage, polyunsaturated phosphatidycholine.
shown to supress the inflammatory reaction in vivo and to depress the in vitro leukocyte response conducive with this property.13 Long chain polyunsaturated fatty acids have been shown also to significantly inhibit T-lymphocyte proliferation and cytokine production and delayedtype hypersensivity.14 In the light of the above findings, it seems reasonable to expect that administration of PPC might contribute to reduced liver damage in biliary obstruction. Thus, the purpose of our study was to ascertain whether treatment with PPC exerts any beneficial effect on liver histopathology and liver function in 4-week biliary obstructed rats. To mimic what happens in human disease when liver injury is already present, animals were treated in the last 2 weeks of the study.
From the Departments of Pediatric Surgery, Biochemistry and Pathology, I˙no¨nu¨ University School of Medicine, Malatya, Turkey. ¨ niAddress reprint requests to Doc¸. Dr Savas¸ Demirbilek, I˙no¨nu¨ U ¨ zal Tip Merkezi, C¸ocuk Cerrahisi Anabilim Dalı, versitesi, Turgut O 44069, Malatya, Turkey. © 2003 Elsevier Inc. All rights reserved. 0022-3468/03/3809-0013$30.00/0 doi:10.1016/S0022-3468(03)00393-2
Journal of Pediatric Surgery, Vol 38, No 9 (September), 2003: pp 1341-1347
1341
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KARAMAN ET AL
MATERIALS AND METHODS
Materials PPC (Essentiale N) was obtained from Rhono¨ -Poulenc-Rorer (Aventis Pharma, Germany).
Experiments Fifty Swiss albino rats of either sex weighing around 200 g were used. Food was witheld 6 hours before surgery, but free access to water was allowed. Forty of fifty animals were anaesthetised with ketamine HCl (50 mg/kg, intramuscularly). The animals ventilated spontaneously during operation. After a midline incision under sterile conditions, the common bile duct was exposed. Thirty of forty underwent double ligation and division of the common bile duct. Abdominal layers were closed with 4-0 silk sutures. Standart rat chow with free access to tap water was provided at the end of the fourth postoperative hour. All animals were maintained under the same conditions after surgical treatment.16
Experimental Design Four groups of rats were used. The first group was the untreated control (n ⫽ 10). In the second group, animals underwent laparotomy without bile duct ligation and division, and they were treated with saline (sham operated, n ⫽ 10). Rats in the third group underwent common bile duct ligation (n ⫽ 15). The fourth group consisted of bile duct-ligated rats that received PPC (n ⫽ 15). Two weeks after bile duct ligation and division, all rats underwent placement of a jugular venous catheter (Venisystems, Venocath-18, 18 G, Abbott, Ireland). Each animal in this group received one intravenous injection of PPC (Essentiale N) via this catheter at 50 mg/d during the last 2 weeks of the study.17 Sham-operated rats received one intramuscular injection of normal saline (1 mL/d). At the end of the 4-week experimental period, blood samples were taken by intracardiac puncture under ketamine anesthesia, and the animals were killed. To eliminate diurnal effects, all animals were killed at the same time of day. After the animals were killed, the livers were removed immediately, and blood samples were centrifuged at 3,500 rpm for 10 minutes at 4°C to obtain plasma. Serum samples were stored at ⫺40°C until analyzed.
Serum Enzyme Activities and Bilirubins The serum activity of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AP), gamma glutamyl transpeptidase (GGT), and direct and indirect bilirubin were estimated by commercially available kits (Olympus Diagnostica GmbH, Ireland). Levels of 5⬘-nucleotidase (5⬘NT) were determined in sera as described before.18
Liver Collagen Content and Malondialdehyde Levels Malondialdehyde (MDA) was determined in liver homogenates using the thiobarbituric acid (TBA) method according to Uchiyama and Mihara.19 The extent of collagen accumulation was estimated in liver homogenates by measurement of hydroxyproline content according to the method of Woessner.20
Histopathologic Examination Some parts of liver samples were fixed in 10% buffered formalin, embedded in paraffin, and sectioned for H&E and Masson’s trichrome stain. Each slide was reviewed for degree of ductal proliferation, portal inflammation, and collagen deposition.
Fig 1. Liver MDA levels for whole groups. Values are mean ⴞ SEM for rats. *P < .05 significantly from biliary obstructed rats. **P < .05 significantly from control and sham groups.
Statistical Analysis Means and SEMs were calculated for all data. Significant differences between means were evaluated by analysis of variance and, in the case of significance, a Mann-Whitney U test also was applied. All statistical analyses were carried out using SPSS statistical software (SPSS for windows; Chicago, IL). P values less than .05 were considered to be significant.
RESULTS
Lipid peroxidation is thought to be an important mechanism of liver injury, and MDA is one of its end products. Thus, measurements of MDA can be used to assess lipid peroxidation. As shown in Fig 1, MDA levels increased about 2-fold after biliary obstruction. PPC significantly (P ⬍ .05) prevented the increase of hepatic MDA levels. Alkaline phosphatase (AP) is an ectoenzyme of the hepatic plasma membrane; an increase in serum AP activity has been related to damage to the liver cell membrane.21 Gamma-glutamyl transpeptidase (GGT) is an enzyme embedded in the hepatocyte plasma membrane, mainly in the canalicular domain. Increased plasma activity of GGT indicates damage to the cell and thus injury to the liver. It is important to point out that serum GGT activity is considered to be one of the best indicators of cholestasis.22 Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are the cytosolic enzymes of the hepatocyte, and in increase in the serum of these enzymes reflects hepatocyte injury.23 As shown in Fig 2, bile duct ligation produced signifacant increases in these enzymes indicating cholestasis and necrosis. Administration of PPC partly but signifacantly (P ⬍ .05) prevented these increases. Figure 3 shows several-fold increases in serum content of total, conjugated, and unconjugated billirubin. In addition, as shown in this figure, PPC treatment completely prevented the increase in all bilirubins.
EFFECT OF PPL ON LIVER DAMAGE
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Fig 2. Values of serum AP, GGT, ALT, AST, and 5ⴕNT enzyme activities in liver tissue from control (group 1), sham-operated (group 2), biliary-obstructed (group 3) and PPC-treated rats (group 4). Values are mean ⴞ SEM for rats. *P < .05 significantly from biliary obstructed rats (group 3). **P < .05 significantly from control and sham groups. ***P < .6 significantly from control and sham groups. ****P < .2 significantly from control and sham groups.
Liver damage induced by 4 weeks of biliary obstruction is accompanied by a significantly increased collagen content indicating severe hepatic fibrosis in group 3 rats. In contrast, liver samples from bile duct– ligated rats that
received PPC showed significantly (P ⬍ .05) less collagen content and resembled control and sham-operated animals (Fig 4). Figure 5 shows the histologic analysis of liver sec-
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Fig 3. Serum total, conjugate, and unconjugate billuribin levels of groups. Values are mean ⴞ SEM for rats. *P < .05 significantly from biliary obstructed rats (group 3). **P < .05 significantly from control and sham groups.
tions. The following changes were observed in group 3 rats: marked bile duct proliferation in expanded portal tracts (Fig 5A), extension of proliferated bile ducts into lobules (Fig 5A), and dense mononuclear cell infiltration in the widened portal areas (Fig 5B). Findings of choleastasis caused by EBO included pigment accumulations in hepatocytes, bile thrombi in canaliculae and ducts, and phagocytosed pigments by macrophages which were apparent in the portal areas of most sections (Fig 5C). There was less canalicular proliferation (Fig 5D) and mononuclear cell infiltration in portal areas (Fig 5E) and less hepatocyte injury in group 4 rats. DISCUSSION
Biliary cirrhosis caused by congenital or acquired biliary obstruction (eg, biliary atresia) is characterized by impaired hepatic function, fibrosis, and subsequent portal hypertension. Bile duct epithelial hyperplasia and periportal fibrosis are the sterotypic patterns of liver injury induced by biliary obstruction.24 Blockade of hepatofugal bile flow, the accumulation of hydrophobic bile acids in hepatocytes during cholestasis, lipid peroxidation and accumulation of highly toxic products of this process, and the persistent activation of hepatic monocytes have been implicated as pathogenic factors that contribute to hepatic injury.25-29 Failure that occurs despite ongoing repair and biliary decompression suggest a persistent inflammatory state.29 During this persistent inflammatory response, in addition to recruitment of systemic monocytes to the liver, the resident pool of hepatic macrophages or the Kupffer cell mass expands.30,31 Coincident is the activation of these cells that lead to upregulation of porinflammatory cytokines. Recruitment and maintenance of inflammatory cells is dependent on individual cell adhesion molecules
as well as chemotractants. Previous studies have shown increased expression of the cell adhesion molecules ICAM and VCAM in liver specimens from patients with biliary atresia.32,33 Activated hepatic macrophages and upregulation of proinflammatory cytokines have been implicated as those factors responsible to progressive liver injury in cholestatic liver injury.30,31,34 Bile duct ligation in rats for 4 weeks led to severe hepatic fibrosis.35 This obstructive model may prove to be a useful tool for studying human cholestasis. It is also a good model for the evaluation of drugs that are beneficial to the liver. PUFA has been used as an antiinflammatory manipulation in several models of inflammation. PPC has been shown also to protect againts various types of experimental liver damage in animal models and isolated hepatocytes.36,37 PPC also accelerated liver regeneration after partial hepatectomy.38 Considering these effects of PPC, it should be possible to reduce liver damage induced by biliary obstruction in rats. The current study was designed to evaluate the protective effect of PPC in a model of biliary obstruction. In this study, liver cell damage, cholestasis, and collagen deposition in the bileligated rats with or without PPC treatment were analyzed by biochemical and histologic examination. The current results show that PPC administration to the biliary obstructed rats reduced hepatocellular injury (Figs 1 and 2) and prevented collagen deposition (Fig 4). A marked improvement in liver markers of cholestasis was observed after PPC treatment (Fig 3). The mechanisms of action that explain the beneficial effects of PPC on cholestatic liver injury may be complex. Some randomized clinical studies indicated that PPC had beneficial effects in alcoholic steatosis and hepatitis, autoimmune hepatitis, and chronic viral hepatitis. In the baboon, PPC attenuated alcohol-induced liver
Fig 4. Liver collagen contents of groups. Values are mean ⴞ SEM for rats. *P < .05 significantly from biliary obstructed rats. **P < .05 significantly from control and sham groups.
EFFECT OF PPL ON LIVER DAMAGE
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Fig 5. Liver sections from group 3 rats. (A) Prominent bile duct proliferation with extension to the lobules (Masson’s trichrome, original magnification ⴛ200). (B) Dense mononuclear cells infiltration in portal areas (H&E, original magnification ⴛ400). (C) Phagocyted bile pigments by macrophages in the portal areas (H&G, original magnification ⴛ400). (D) Mild bile duct proliferation in portal areas (H&E, original magnification ⴛ200). (E) Mild ductal proliferation and mononuclear cell infiltration in the portal areas (H&E, original magnification ⴛ400).
fibrosis and prevented alcoholic cirrhosis. These studies suggested that PPC might “stabilize” the hepatocyte membrane and thus exert a nonspecific beneficial effect against various types of liver damage.36-41 Animal studies suggest that PPC is incorporated into the membranes of hepatocytes as a substitute for endogenous saturated phosphatidyl-choline molecules. This substitution was shown to result in an increase in membrane fludity and
active transport system activity.38,39 Moreover, PPC selectively prevented the acetaldehyde-induced increase in collagen accumulation in cultured rat hepatic lipocytes, probably by increasing collagenase activity, suggesting that this protective effect of PPC against collagen accumulation may be caused by stimulation of collagen breakdown.42 The beneficial effects of PPC can be explained by its
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antiinflammatuary actions as well as its structural support to the hepatocyte membrane. Dietary PUFA supplementation has been shown to ameliorate a variety of inflammatory conditions such as murine lupus nephritis and collagen-induced arthritis.14,15 Clinical studies have used dietary PUFA as a means to treat inflammatuary disorders such as rheumatoid arthritis.15 In vitro studies have shown that PUFA decreases both leukotriene B4 (LBT4) and PAF production by leukocytes. Additionally, the LT of the B series synthesized from PUFA, which substitute for arachidonate, are a relatively weak agonist compared with LBT4. Thus, a potential common mechanism underlying the protective effect of dietary PUFA
manipulation in inflammation is that the synthesis of proinflammatory lipid mediators is diminished, or the lipid mediators that are produced are relatively inactive.14,15,43 It seems likely that PPC may also act as an anticholestatic drug by an effect on hepatocytes. More investigation is required to establish the antiinflammatuary and anticholestatic mechanisms of PPC on biliary obstructed rats. PPC possesses very important beneficial effects that limit liver damage in rats with biliary obstruction. This suggests that PPC might be a useful drug in preserving liver function in patients with congenital or acquired biliary obstruction.
REFERENCES 1. Okazaki T, Kobayashi H, Yamataka A, et al: Long-term postsurgical outcome of biliary atresia. J Pediatr Surg 34:312-315, 1999 2. Roggin KK, Kim JC, Kurkchubasche AG, et al: Macrophage phenotype during cholestatic injury and repair: The persistent inflammatory response. J Pediatr Surg 36:220-228, 2001 3. Morris J, Gollo G, Scheuer P, et al: Percutaneous liver biopsy in patients with large bile duct obstruction. Gastroenterology 68:750-754, 1975 4. Kobayashi H, Yamataka A, Lane JG, et al: Levels of circulating antiinflammatory cytokine interleukin-1 receptor antagonist and proinflammatory cytokines at different stages of biliary atresia. J Pediatr Surg 37:1038-1041, 2002 5. Davenport M, Gonde C, Redkar R, et al: Immunohistochemistry of the liver and biliary tree in extrahepatic biliary atresia. J Pediatr Surg 36:1017-1025, 2001 6. Kobayashi H, Horikoshi K, Long L, et al: Serum concentration of adhesion molecules in postoperative biliary atresia patients: relationship to disease activity and cirrhosis. J Pediatr Surg 36:1297-1301, 2001 7. Volpes R, van Den Oord JJ, Desmet VJ: Immunohistochemical study of adhesion molecules in liver inflammation. Hepatology 12:5965, 1990 8. Minnick KE, Kreisberg R, Dillon PW: Soluble ICAM-1 (sICAM-1) in biliary atresia and its relationship to disease activity. J Surg Res 76:53-56, 1998 9. Hines J, Johnson S, Burt A: In vivo responses of macrophages and perisinusoidal cells to cholestatic liver injury. Am J Pathol 142: 511-517, 1993 10. Ramm GA, Nair VG, Bridle KR, et al: Contribution of hepatic parenchymal and nonparenchymal cells to hepatic fibrogenesis in biliary atresia. Am J Pathol 153:527-353, 1998 11. Mourella M, Guarner F, Molageloda J: Polyunsaturated phosphatidylcholine prevents stricture formation in a rat model of colitis. Gastroenterology 110:1033-1037, 1996 12. Navder KP, Baraona E, Lieber CS: Polyenphosphatidylcholine decreases alcoholic hyperlipemia without affecting the alcohol-induced rise of HDL-cholesterol. Life Sciences 61:1907-1914, 1997 13. Lefhowith JB, Morrison A, Lee V, et al: Manuplation of the acute inflammatory response by dietary polyunsaturated fatty acid modulation. J Immunol 145:1523-1529, 1990 14. Costabile M, Hii CST, Robinson BS, et al: A novel long chain polyunsaturated fatty acid -Oxa 21:3n-3, inhibits T lymphocyte proliferation, cytokine products, delayed-type hypersensitivity, and carageenan-induced paw reaction and selectively targets intracellulary signals. J Immunol 167:3980-3987, 2001 15. Nielse GLKL, Faarvang BS, Thomsen KL, et al: The effect of
dietary supplementation with n-3 polyunsaturated fatty acids in patients with rheumatoid arthritis: A randomized double blind trial. Eur J Clin Invest 22:687, 1992 16. Cantu¨ rk NZ, Cantu¨ rk Z, Utkan NZ, et al: Cytoprotective effect of alpha tocopherol against liver injury induced by extrahepatic biliary obstruction. East African Med 75:77-80, 1998 17. Ar’Rajab A, Ahren B, Rozga J, et al: Phosphatidylcholine prevents postoperative peritoneal adhesios: An experimental study in the rat. J Surg Res 50:212-215, 1991 18. Burtis CA, Ashwood ER: Tietz Textbook of Clinical Chemistry. Philadelphia, PA, Saunders, 1999 19. Uchiyama M, Mihara M: Determination of malondaldehyde precursor in tissues by thiobarbituric acid test. Anal Biochem 26:271278, 1978 20. Woessner JB: The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid. Arch Biochem Biophsic 99:440, 1961 21. Kaplan MM: Serum alkaline phosphatase, another piece is added to the puzzle. Hepatology 6:226-228, 1986 22. Bulk FP, Mavier P, Zafrani ES, et al: Mechanism of gammaglutamyl transpeptidase release in serum during intrahepatic and extrahepatic cholestasis in the rat. A histochemical, biochemical and molecular approach. Hepatology 11:545-550, 1980 23. Moss DW, Henderson AR: Clinical enzymology, in Burtis CA, Ashwood ER (eds): Tietz Textbook of Clinical Chemistry. Philadelphia, PA, Saunders, 1999, pp 652-653 24. Morris J, Gollo G, Scheuer P, et al: Percutaneous liver biopsy in patients with large bile duct obstruction. Gastroenterology 68:750-754, 1975 25. Slott P, Liu M, Tavaloni N: Origin, pattern, and mechanism of bile duct proliferation following biliary obstruction in the rat. Gastroenterology 99:466-477, 1990 26. Solkol RJ, Winkloher BM, Deverazx MV, et al: Generation of hydroperoxides in isolated rat hepatocytes and hepatic mitochondria exposed to hydrophobic bile acids. Gastroenterology 109:1249-1250, 1995 27. Sigh S, Schakleton G, Ah-Sing E, et al: Antioxidant defenses in the bile duct- ligated rat. Gastroenterology 103:1625-1629, 1992 28. Lumsden A, Handerson J, Kurther M: Endotoxin levels measured by achromogenic assay in portal, hepatic and peripheral vevous blood in patients with cirrhosis. Hepatology 8:232-236, 1988 29. Winwood P, Arther M: Kuppfer cells: Their activation and role in animal models of liver injury and human liver diseases. Semin Liver Dis 13:50-59, 1993 30. Hines J, Johnson S, Burt A: In vivo responses of macrophages
EFFECT OF PPL ON LIVER DAMAGE
and perisinusoidal cells to cholestatic liver injury. Am J Pathol 142: 511-517, 1993 31. Tracy TF, Dillon P, Fox ES, et al: Inflammatory response in pediatric biliary disease macrophage phenotype and distrubition. J Pediatr Surg 31:131-135, 1996 32. Dillon P, Belchis D, Tracy T, et al: Increased expression intracellular adhesion molecules in biliary atresia. Am J Pathol 145:263267, 1994 33. Broome U, Nemeth A, Hultcrantz R, et al: Different expression HLA-DR and ICAM-1 in livers from patients with biliary atresia and Byler’s disease. J Hepatol 26:857-862, 1997 34. Altman RP, Chandra R, Lilly JR: Ongoing cirrhosis after successful portoenterostomy. J Pediatr Surg 10:685-691, 1995 35. Peres W, Tunon MJ, Colldo PS, et al: The flavonoid quercetin ameliorates liver damage in rats with biliary obstruction. Hepatol 33:742-750, 2000 36. Neuberger J, Hegarty JE, Eddleston ALWF, et al: Effect of polyunsaturated phosphatidylcholine on immune mediated hepatocyte damage. Gut 24:751-755, 1983 37. Ma X, Zhao J, Lieber CS: Polyenylphosphatidycholine attenuates non-alcoholic hepatic fibrosis and accelerates its regression. J Hepatol 24:604-613, 1996 38. Holecek M, Mraz J, Koldova P, et al: Effect of polyunsaturated
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phosphatidylcholine on liver regeneration onset after hepatectomy in the rat. Drug Res 42:337-339, 1992 39. Niederau C, Strohmeyer G, Heintges T, et al: Polyunsaturated phosphatidyl-choline Interferon alpha for treatment of chronic hepatitis B and C: A multicenter, randomized, double- blind, placebo-controlled trial. Hepato-Gastroenterology 45:797-804, 1998 40. Gerber GB, Bartsch GG, Deroo J: Influence of phospholipids on liver damage: Carbon tetracholoride poisoning and alterations in amino acid uptake, peroxidation, sialic acid content, and lysosomal enzymes. Acta Hepatogastroenterol 22:175-180, 1975 41. LeKim G, Betzing H: The incorporation of essential phospholipids into the organs of intact and galactosamine intoxicated rats. Arzneimittel forshung 29:1217-1221, 1974 42. Lii J-J, Kim C-I, Leo MA, et al: Polyunsaturated lecithin prevents acetaldehyde-mediated hepatic collagen accumulation by stimulating collagenase activity in cultured lipocytes. Hepatology 15: 373-381, 1992 43. Molvig J, Pociot F, Worsaae H, et al: Dietary supplementation with omega-3-polyunsaturated fatty acids decreases mononuclear cell proliferation and interleukin-1 beta content but not monokine secretion in healthy and insulin-dependent diabetic individuals. Scan J Immunol 34:399-410, 1991
Background/Purpose: Persistent inflammatory response secondary to congenital or acquired biliary choleastasis plays an important role in the pathophysiology of hepatic tissue damage. The polyunsaturated fatty acids (PUFA) have been shown to suppress the inflammatory reactions in vivo and in vitro. PUFA has been shown also to protect againts various types of experimental liver damage in animal models and isolated hepatocytes. Therefore, the aim of this study was to investigate the protective effect of PUFA administration on liver damage using the rat chronic biliary obstruction model. Methods: Swiss albino rats of either sex were divided into 4 groups as follows: control group (group 1, 10 rats); rats with sham operation and treated with saline group 2, 10 rats); rats with biliary obstruction (group 3, 15 rats); and polyunsaturated phophatidylcholine (PPC)-treated rats with biliary obstruction (Group 4, 15 rats). Biliary obstruction was induced by double ligation and division of the common bile duct. PUFA treatment was started 2 weeks later from biliary obstruction in doses of 50 mg/d per rat and continued for 2 weeks. All animals were killed after 4 weeks of common bile duct ligation or sham operation. Liver damage and cholesta-
R
EPAIR AFTER cholestatic liver injuries often is complicated by progressive hepatic damage despite adequate technical decompression of the biliary tree.1 This progressive hepatic damage accompanies an increased morbidity and mortality observed in biliary atresia, choledochal cyst, sclerosing cholangitis, and other biliary obstructive pathology.2 The typical histologic characteristics of “failed” or “delayed” repair are persistent biliary hyperplasia, fibrosis, and inflammation despite surgical correction of the outflow obstruction.3 Factors determining the progression of hepatic injury in cholestatic liver disease are not understood fully, but clearly, both cellular and humoral immune responses of the host are involved.2,4-10 Soybean polyunsaturated phophatidylcholine (PPC) is a polyunsaturated fatty acid (PUFA) compound and a 94% to 96% mixture of phosphatidylcholines, about half of which is dilinoleoylphosphatidylcholine.11 At variance with mammalian phospholipids, this plant lipid contains unsaturated fatty acids in both the 1 and 2 positions of the glycerol backbone, which confers a high bioavailability, mainly because of reacylation of the unsaturated 1-acyl-lysophosphotidylcholine with additional unsaturated fatty acids during intestinal absorption.12 The n-3 polyunsaturated fatty acids have been
sis were determined examinations.
by
biochemical
and
histologic
Results: The data showed a decrease in plasma bilirubin level (both conjugated and unconjugated) and liver enzyme levels (AST, ALT, AP, GGT, 5⬘-NT) in group 4, when compared with group 3 (P ⬍ .05). Tissue levels of malondialdehyde (MDA) in group 4 was 20.00 ⫾ 2.93 compared with that in group 3, 27.12 ⫾ 2.96 (P ⬍ .05). Administration of PUFA to the biliary obstructed rats resulted in inhibition of collagen accumulation (P ⬍ .05) and ductal proliferation (P ⬍ .05). Conclusions: PUFA reduced liver damage, ductular proliferation, and fibrosis in biliary obstructed rats. These effects suggest that it might be a useful agent to preserve liver function in patients with biliary obstruction such as biliary atresia. J Pediatr Surg 38:1341-1347. © 2003 Elsevier Inc. All rights reserved. INDEX WORDS: Biliary obstruction, cholestasis, liver damage, polyunsaturated phosphatidycholine.
shown to supress the inflammatory reaction in vivo and to depress the in vitro leukocyte response conducive with this property.13 Long chain polyunsaturated fatty acids have been shown also to significantly inhibit T-lymphocyte proliferation and cytokine production and delayedtype hypersensivity.14 In the light of the above findings, it seems reasonable to expect that administration of PPC might contribute to reduced liver damage in biliary obstruction. Thus, the purpose of our study was to ascertain whether treatment with PPC exerts any beneficial effect on liver histopathology and liver function in 4-week biliary obstructed rats. To mimic what happens in human disease when liver injury is already present, animals were treated in the last 2 weeks of the study.
From the Departments of Pediatric Surgery, Biochemistry and Pathology, I˙no¨nu¨ University School of Medicine, Malatya, Turkey. ¨ niAddress reprint requests to Doc¸. Dr Savas¸ Demirbilek, I˙no¨nu¨ U ¨ zal Tip Merkezi, C¸ocuk Cerrahisi Anabilim Dalı, versitesi, Turgut O 44069, Malatya, Turkey. © 2003 Elsevier Inc. All rights reserved. 0022-3468/03/3809-0013$30.00/0 doi:10.1016/S0022-3468(03)00393-2
Journal of Pediatric Surgery, Vol 38, No 9 (September), 2003: pp 1341-1347
1341
1342
KARAMAN ET AL
MATERIALS AND METHODS
Materials PPC (Essentiale N) was obtained from Rhono¨ -Poulenc-Rorer (Aventis Pharma, Germany).
Experiments Fifty Swiss albino rats of either sex weighing around 200 g were used. Food was witheld 6 hours before surgery, but free access to water was allowed. Forty of fifty animals were anaesthetised with ketamine HCl (50 mg/kg, intramuscularly). The animals ventilated spontaneously during operation. After a midline incision under sterile conditions, the common bile duct was exposed. Thirty of forty underwent double ligation and division of the common bile duct. Abdominal layers were closed with 4-0 silk sutures. Standart rat chow with free access to tap water was provided at the end of the fourth postoperative hour. All animals were maintained under the same conditions after surgical treatment.16
Experimental Design Four groups of rats were used. The first group was the untreated control (n ⫽ 10). In the second group, animals underwent laparotomy without bile duct ligation and division, and they were treated with saline (sham operated, n ⫽ 10). Rats in the third group underwent common bile duct ligation (n ⫽ 15). The fourth group consisted of bile duct-ligated rats that received PPC (n ⫽ 15). Two weeks after bile duct ligation and division, all rats underwent placement of a jugular venous catheter (Venisystems, Venocath-18, 18 G, Abbott, Ireland). Each animal in this group received one intravenous injection of PPC (Essentiale N) via this catheter at 50 mg/d during the last 2 weeks of the study.17 Sham-operated rats received one intramuscular injection of normal saline (1 mL/d). At the end of the 4-week experimental period, blood samples were taken by intracardiac puncture under ketamine anesthesia, and the animals were killed. To eliminate diurnal effects, all animals were killed at the same time of day. After the animals were killed, the livers were removed immediately, and blood samples were centrifuged at 3,500 rpm for 10 minutes at 4°C to obtain plasma. Serum samples were stored at ⫺40°C until analyzed.
Serum Enzyme Activities and Bilirubins The serum activity of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AP), gamma glutamyl transpeptidase (GGT), and direct and indirect bilirubin were estimated by commercially available kits (Olympus Diagnostica GmbH, Ireland). Levels of 5⬘-nucleotidase (5⬘NT) were determined in sera as described before.18
Liver Collagen Content and Malondialdehyde Levels Malondialdehyde (MDA) was determined in liver homogenates using the thiobarbituric acid (TBA) method according to Uchiyama and Mihara.19 The extent of collagen accumulation was estimated in liver homogenates by measurement of hydroxyproline content according to the method of Woessner.20
Histopathologic Examination Some parts of liver samples were fixed in 10% buffered formalin, embedded in paraffin, and sectioned for H&E and Masson’s trichrome stain. Each slide was reviewed for degree of ductal proliferation, portal inflammation, and collagen deposition.
Fig 1. Liver MDA levels for whole groups. Values are mean ⴞ SEM for rats. *P < .05 significantly from biliary obstructed rats. **P < .05 significantly from control and sham groups.
Statistical Analysis Means and SEMs were calculated for all data. Significant differences between means were evaluated by analysis of variance and, in the case of significance, a Mann-Whitney U test also was applied. All statistical analyses were carried out using SPSS statistical software (SPSS for windows; Chicago, IL). P values less than .05 were considered to be significant.
RESULTS
Lipid peroxidation is thought to be an important mechanism of liver injury, and MDA is one of its end products. Thus, measurements of MDA can be used to assess lipid peroxidation. As shown in Fig 1, MDA levels increased about 2-fold after biliary obstruction. PPC significantly (P ⬍ .05) prevented the increase of hepatic MDA levels. Alkaline phosphatase (AP) is an ectoenzyme of the hepatic plasma membrane; an increase in serum AP activity has been related to damage to the liver cell membrane.21 Gamma-glutamyl transpeptidase (GGT) is an enzyme embedded in the hepatocyte plasma membrane, mainly in the canalicular domain. Increased plasma activity of GGT indicates damage to the cell and thus injury to the liver. It is important to point out that serum GGT activity is considered to be one of the best indicators of cholestasis.22 Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are the cytosolic enzymes of the hepatocyte, and in increase in the serum of these enzymes reflects hepatocyte injury.23 As shown in Fig 2, bile duct ligation produced signifacant increases in these enzymes indicating cholestasis and necrosis. Administration of PPC partly but signifacantly (P ⬍ .05) prevented these increases. Figure 3 shows several-fold increases in serum content of total, conjugated, and unconjugated billirubin. In addition, as shown in this figure, PPC treatment completely prevented the increase in all bilirubins.
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Fig 2. Values of serum AP, GGT, ALT, AST, and 5ⴕNT enzyme activities in liver tissue from control (group 1), sham-operated (group 2), biliary-obstructed (group 3) and PPC-treated rats (group 4). Values are mean ⴞ SEM for rats. *P < .05 significantly from biliary obstructed rats (group 3). **P < .05 significantly from control and sham groups. ***P < .6 significantly from control and sham groups. ****P < .2 significantly from control and sham groups.
Liver damage induced by 4 weeks of biliary obstruction is accompanied by a significantly increased collagen content indicating severe hepatic fibrosis in group 3 rats. In contrast, liver samples from bile duct– ligated rats that
received PPC showed significantly (P ⬍ .05) less collagen content and resembled control and sham-operated animals (Fig 4). Figure 5 shows the histologic analysis of liver sec-
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Fig 3. Serum total, conjugate, and unconjugate billuribin levels of groups. Values are mean ⴞ SEM for rats. *P < .05 significantly from biliary obstructed rats (group 3). **P < .05 significantly from control and sham groups.
tions. The following changes were observed in group 3 rats: marked bile duct proliferation in expanded portal tracts (Fig 5A), extension of proliferated bile ducts into lobules (Fig 5A), and dense mononuclear cell infiltration in the widened portal areas (Fig 5B). Findings of choleastasis caused by EBO included pigment accumulations in hepatocytes, bile thrombi in canaliculae and ducts, and phagocytosed pigments by macrophages which were apparent in the portal areas of most sections (Fig 5C). There was less canalicular proliferation (Fig 5D) and mononuclear cell infiltration in portal areas (Fig 5E) and less hepatocyte injury in group 4 rats. DISCUSSION
Biliary cirrhosis caused by congenital or acquired biliary obstruction (eg, biliary atresia) is characterized by impaired hepatic function, fibrosis, and subsequent portal hypertension. Bile duct epithelial hyperplasia and periportal fibrosis are the sterotypic patterns of liver injury induced by biliary obstruction.24 Blockade of hepatofugal bile flow, the accumulation of hydrophobic bile acids in hepatocytes during cholestasis, lipid peroxidation and accumulation of highly toxic products of this process, and the persistent activation of hepatic monocytes have been implicated as pathogenic factors that contribute to hepatic injury.25-29 Failure that occurs despite ongoing repair and biliary decompression suggest a persistent inflammatory state.29 During this persistent inflammatory response, in addition to recruitment of systemic monocytes to the liver, the resident pool of hepatic macrophages or the Kupffer cell mass expands.30,31 Coincident is the activation of these cells that lead to upregulation of porinflammatory cytokines. Recruitment and maintenance of inflammatory cells is dependent on individual cell adhesion molecules
as well as chemotractants. Previous studies have shown increased expression of the cell adhesion molecules ICAM and VCAM in liver specimens from patients with biliary atresia.32,33 Activated hepatic macrophages and upregulation of proinflammatory cytokines have been implicated as those factors responsible to progressive liver injury in cholestatic liver injury.30,31,34 Bile duct ligation in rats for 4 weeks led to severe hepatic fibrosis.35 This obstructive model may prove to be a useful tool for studying human cholestasis. It is also a good model for the evaluation of drugs that are beneficial to the liver. PUFA has been used as an antiinflammatory manipulation in several models of inflammation. PPC has been shown also to protect againts various types of experimental liver damage in animal models and isolated hepatocytes.36,37 PPC also accelerated liver regeneration after partial hepatectomy.38 Considering these effects of PPC, it should be possible to reduce liver damage induced by biliary obstruction in rats. The current study was designed to evaluate the protective effect of PPC in a model of biliary obstruction. In this study, liver cell damage, cholestasis, and collagen deposition in the bileligated rats with or without PPC treatment were analyzed by biochemical and histologic examination. The current results show that PPC administration to the biliary obstructed rats reduced hepatocellular injury (Figs 1 and 2) and prevented collagen deposition (Fig 4). A marked improvement in liver markers of cholestasis was observed after PPC treatment (Fig 3). The mechanisms of action that explain the beneficial effects of PPC on cholestatic liver injury may be complex. Some randomized clinical studies indicated that PPC had beneficial effects in alcoholic steatosis and hepatitis, autoimmune hepatitis, and chronic viral hepatitis. In the baboon, PPC attenuated alcohol-induced liver
Fig 4. Liver collagen contents of groups. Values are mean ⴞ SEM for rats. *P < .05 significantly from biliary obstructed rats. **P < .05 significantly from control and sham groups.
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Fig 5. Liver sections from group 3 rats. (A) Prominent bile duct proliferation with extension to the lobules (Masson’s trichrome, original magnification ⴛ200). (B) Dense mononuclear cells infiltration in portal areas (H&E, original magnification ⴛ400). (C) Phagocyted bile pigments by macrophages in the portal areas (H&G, original magnification ⴛ400). (D) Mild bile duct proliferation in portal areas (H&E, original magnification ⴛ200). (E) Mild ductal proliferation and mononuclear cell infiltration in the portal areas (H&E, original magnification ⴛ400).
fibrosis and prevented alcoholic cirrhosis. These studies suggested that PPC might “stabilize” the hepatocyte membrane and thus exert a nonspecific beneficial effect against various types of liver damage.36-41 Animal studies suggest that PPC is incorporated into the membranes of hepatocytes as a substitute for endogenous saturated phosphatidyl-choline molecules. This substitution was shown to result in an increase in membrane fludity and
active transport system activity.38,39 Moreover, PPC selectively prevented the acetaldehyde-induced increase in collagen accumulation in cultured rat hepatic lipocytes, probably by increasing collagenase activity, suggesting that this protective effect of PPC against collagen accumulation may be caused by stimulation of collagen breakdown.42 The beneficial effects of PPC can be explained by its
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antiinflammatuary actions as well as its structural support to the hepatocyte membrane. Dietary PUFA supplementation has been shown to ameliorate a variety of inflammatory conditions such as murine lupus nephritis and collagen-induced arthritis.14,15 Clinical studies have used dietary PUFA as a means to treat inflammatuary disorders such as rheumatoid arthritis.15 In vitro studies have shown that PUFA decreases both leukotriene B4 (LBT4) and PAF production by leukocytes. Additionally, the LT of the B series synthesized from PUFA, which substitute for arachidonate, are a relatively weak agonist compared with LBT4. Thus, a potential common mechanism underlying the protective effect of dietary PUFA
manipulation in inflammation is that the synthesis of proinflammatory lipid mediators is diminished, or the lipid mediators that are produced are relatively inactive.14,15,43 It seems likely that PPC may also act as an anticholestatic drug by an effect on hepatocytes. More investigation is required to establish the antiinflammatuary and anticholestatic mechanisms of PPC on biliary obstructed rats. PPC possesses very important beneficial effects that limit liver damage in rats with biliary obstruction. This suggests that PPC might be a useful drug in preserving liver function in patients with congenital or acquired biliary obstruction.
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