Nonselective inhibition of prostaglandin-endoperoxide synthases by naproxen ameliorates acute or chronic liver injury in animals

The rising prevalence of hepatic injury due to toxins, metabolites, viruses, etc., necessitates development of further mechanisms for protecting the liver and for treating acute or chronic liver diseases. To examine whether inhibition of inflammation is directed by cyclo-oxygenase pathways, we performed animal studies with naproxen, which inhibits prostaglandin-endoperoxide synthases 1 and 2 and is in extensive clinical use. We administered carbon tetrachloride to induce acute liver injury and ligated the common bile duct to induce chronic liver injury in adult rats. These experimental manipulations produced abnormalities in liver tests, tissue necrosis, compensatory hepatocyte or biliary proliferation, and onset of fibrosis, particularly after bile duct ligation. After carbon tetrachloride-induced acute injury, naproxen decreased liver test abnormalities, tissue necrosis and compensatory hepatocellular proliferation. After bile duct ligation-induced chronic injury, naproxen decreased liver test abnormalities, tissue injury and compensatory biliary hyperplasia. Moreover, after bile duct ligation, naproxen-treated rats showed more periductular oval liver cells, which have been classified as hepatic progenitor cells. In naproxen-treated rats, we found greater expression in hepatic stellate cells and mononuclear cells of cytoprotective factors, such as vascular endothelial growth factor. The ability of naproxen to induce expression of vascular endothelial growth factor was verified in cell culture studies with CFSC-8B clone of rat hepatic stellate cells. Whereas assays for carbon tetrachloride toxicity using cultured primary hepatocytes established that naproxen was not directly cytoprotective, we found conditioned medium containing vascular endothelial growth factor from naproxen-treated CFSC-8B cells protected hepatocytes from carbon tetrachloride toxicity. Therefore, naproxen was capable of ameliorating toxic liver injury, which involved naproxen-induced release of physiological cytoprotective factors in nonparenchymal liver cells. Such drug-induced release of endogenous cytoprotectants will advance therapeutic development for hepatic injury.     

Serum alpha-fetoprotein in rats after ligation of the common bile duct: relation to ductular cell (oval cell) proliferation

Serum AFP levels were measured by radio-immunoassay in rats at various periods after ligation and section of the common bile duct and after sham-ligation. Transient elevations of serum AFP reaching statistically significant levels occurred at 3 to 10 days, 17 days and 28 days after ligation of the common bile duct but not in sham-operated rats. The peak elevations of serum AFP followed waves of mitosis in both the ductular cells and the hepatocytes as measured by autoradiography. Proliferation of hepatocytes, however, remained minor when compared to ductular cell multiplication. Ultra-structural and histochemical studies of the liver clearly distinguished ductular (oval) cells from hepatocytes and provided no evidence of transition from ductular cells to hepatocytes. Our findings suggest that ductular cell proliferation in the rat induced by ligation of the common bile duct may produce elevations in the serum AFP.     

Bile acids induce inflammatory genes in hepatocytes: a novel mechanism of inflammation during obstructive cholestasis

Inflammation contributes to liver injury during cholestasis. The mechanism by which cholestasis initiates an inflammatory response in the liver, however, is not known. Two hypotheses were investigated in the present studies. First, activation of Toll-like receptor 4 (TLR4), either by bacterial lipopolysaccharide or by damage-associated molecular pattern molecules released from dead hepatocytes, triggers an inflammatory response. Second, bile acids act as inflammagens, and directly activate signaling pathways in hepatocytes that stimulate production of proinflammatory mediators. Liver inflammation was not affected in lipopolysaccharide-resistant C3H/HeJ mice after bile duct ligation, indicating that Toll-like receptor 4 is not required for initiation of inflammation. Treatment of hepatocytes with bile acids did not directly cause cell toxicity but increased the expression of numerous proinflammatory mediators, including cytokines, chemokines, adhesion molecules, and other proteins that influence immune cell levels and function. Up-regulation of several of these genes in hepatocytes and in the liver after bile duct ligation required early growth response factor-1, but not farnesoid X receptor. In addition, early growth response factor-1 was up-regulated in the livers of patients with cholestasis and correlated with levels of inflammatory mediators. These data demonstrate that Toll-like receptor 4 is not required for the initiation of acute inflammation during cholestasis. In contrast, bile acids directly activate a signaling network in hepatocytes that promotes hepatic inflammation during cholestasis.     

Alterations of hepatocellular intermediate filaments during extrahepatic cholestasis in rat liver

Intermediate filaments (IF) maintain the structural and functional integrity of cells. To investigate whether IF change as a consequence of increased mechanical pressure and what the significance of such alterations is for the integrity of hepatocytes, we investigated alterations of IF in rat liver following common bile duct ligation (CBDL). Immunofluorescence of cytokeratin 18 was performed on extracted cryostat sections which were also used for electron microscopy. Ultrathin sections of mildly extracted liver tissue were applied to reveal the relationship between IF and intercellular junctions and cytoplasmic organelles. Our results showed that hepatocellular IF underwent striking changes during CBDL. The so-called pericanalicular sheath disappeared and IF were rigidly rearranged at the cell periphery, appearing as honeycomb-like structures. Increased amounts of IF were found in close association with increased numbers of desmosomes at the lateral membranes of hepatocytes, and electron-dense desmosome-like bodies were even observed in the ectoplasm at bile canaliculi. Rearrangement of IF in the cytoplasm resulted in segregation of subcellular compartments. The increased density of the IF network and desmosomes are compensatory mechanisms of hepatocytes to resist increased mechanical load and disperse the tension. However, the intracellular rearrangement of IF leading to segregation of subcellular compartments may also have distinct effects on hepatocellular metabolic functions.     

An ultrastructural study of the liver in erythropoietic protoporphyria

An ultrastructural investigation of the liver was performed in two patients with erythropoietic protoporphyria. There were many protoporphyrin crystals in the hepatocytes, Kupffer cells, bile canaliculi, epithelia of bile ducts, and sinusoidal endothelial cells and also free within sinusoids. In hepatocytes, these deposits were composed of granular amorphous materials and numerous slender, straight, or slightly curved needle-like crystals aligned in radial orientation. They were randomly distributed in the cytoplasm and completely replaced other cytoplasmic structures. Some crystals lay free in the cytoplasm and others were surrounded by a single membrane. In the bile canaliculi, severe alterations could be observed. Some of the bile canaliculi were filled with amorphous, noncrystalline pigments, and lumina were enlarged with loss of micro-villi. In addition, despite the absence of protoporphyrin deposits, there were many dilated bile canaliculi. The microfilamentous network around such dilated bile canaliculi was no longer evident, suggesting the depolymerization of actin filaments, which could lead to bile excretory disturbances. The bile duct epithelia showed focal apical membrane bleb formation. The functional or structural alterations of the sinusoidal endothelial cells by the protoporphyrin crystals might lead to the hepatic disturbances. These ultrastructural findings of the liver might contribute to the understanding of the pathogenesis of complicated liver disease in erythropoietic protoporphyria.     

Intrasplenic hepatocyte transplantation in rats with experimental liver injury: morphological and morphometric studies

Isolated rat hepatocytes were transplanted into the splenic parenchyma of syngeneic animals. The effects on the degree of colonization by the transplanted cells of three forms of experimental liver injury in recipient animals were studied. Significant colonization was observed in animals with carbon tetrachloride (CCl4)-induced injury combined with portacaval shunt (PCS) and in animals with common bile duct (CBD) ligation but not in control animals or in animals with CCl4-induced injury alone. Transplanted cells in the CCl4/PCS group resembled normal hepatocytes. In contrast, in the CBD group, the intrasplenic hepatocytes exhibited a pattern of 'ductular metaplasia' similar to that observed in the obstructed liver of the recipients. Transplanted syngeneic hepatocytes can thus proliferate in the spleen in response to liver injury in the recipient. The morphological appearances of the transplanted cells can be modified depending on the nature of the liver injury.     

PSA-NCAM expression in the rat medial prefrontal cortex

Modulation of soluble guanylate cyclase (sGC) by nitric oxide (NO) is altered in brain from cirrhotic patients. The aim of this work was to assess whether an animal model of cirrhosis, bile duct ligation, alone or combined with diet-induced hyperammonemia for 7-10 days reproduces the alterations in NO modulation of sGC found in brains from cirrhotic patients. sGC activity was measured under basal conditions and in the presence of NO in cerebellum and cerebral cortex of the following groups of rats: controls, bile duct ligation without or with hyperammonemia and hyperammonemia without bile duct ligation. In cerebellum activation of sGC by NO was significantly lower in bile duct ligated rats with (12 +/- five-fold) or without (14 +/- six-fold) hyperammonemia than in control rats (23 +/- seven-fold). In cerebral cortex activation of sGC by NO was higher in rats with bile duct ligation with hyperammonemia (124 +/- 30-fold) but not without hyperammonemia (59 +/- 15-fold) than in control rats (66 +/- 11-fold). The combination of bile duct ligation and hyperammonemia reproduces the alterations in the modulation of soluble guanylate cyclase by NO found in cerebral cortex and cerebellum of cirrhotic patients while bile duct ligation or hyperammonemia alone reproduces the effects in cerebellum but not in cerebral cortex.     

Increased Reissner's fiber material in the subcommissural organ and ventricular area in bile duct ligated rats

Hepatic encephalopathy is a common neuropsychiatric complication of acute and chronic liver failure. Whether brain structures with strategic positions in the interface of blood-brain barriers such as the circumventricular organs are involved in hepatic encephalopathy is not yet established. Among the circumventricular organs, the subcommissural organ secretes a glycoprotein known as Reissner's fiber, which condenses and forms an ever-growing thread-like structure into the cerebrospinal fluid. In the present work we describe the Reissner's fiber material within the subcommissural organ and its serotoninergic innervation in an animal model of chronic hepatic encephalopathy following bile duct ligation in experimental rats. The study involved immunohistochemical techniques with antibodies against Reissner's fiber and 5-hydroxytryptamine (5-HT). Four weeks after surgical bile duct ligation, a significant rise of Reissner's fiber immunoreactivity was observed in all subcommissural organ areas compared with controls. Moreover, significant Reissner's fiber immunoreactive materials within the ependyma and inside the parenchyma close to the ventricular borders were also seen in bile duct ligated rats, but not in control rats. Increased Reissner's fiber material in bile duct ligated rats seems to be related to a reduction of 5-HT innervation of the subcommissural organ, the ventricular borders and the nucleus of origin, the dorsal raphe nucleus. Our data describe alterations of the subcommissural organ/Reissner's fiber material and the subcommissural organ 5-HT innervation probably due to a general 5-HT deficit in bile duct ligated rats.     

Induction of prolactin receptors in cholangiocytes of male and female rats after ligation of the common bile duct

Intense expression of prolactin receptors in cholangiocyte nuclei, nuclear membranes, and cytoplasm was demonstrated by the indirect immunoperoxidase method in intact and gonadectomied male and female rats after ligation of the common bile duct. The intensity of staining increased during cholangiocyte proliferative response to the intervention and, in contrast to hepatocytes, did not depend on animal sex and the presence of the gonads. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 125, No. 1, pp. 66–70, January, 1998     

A quantitative assessment of the structural changes the rat's liver following obstruction of the common bile duct.

A study has been made of sequential changes in the rat's liver from 1 to 40 days after obstruction of the common bile duct. The qualitative changes have been described and illustrated. The volume proportions of hepatocytes, bile duct epithelium and biliary stroma have been quantified by histological analysis using a point counting technique. The proliferation of hepatocytes and bile duct cells have been measured by labelling with tritiated thymidine. The absolute quantity of hepatocytes in each liver has been estimated and expressed as a percentage of body weight. Over 40 days there is a relative fall in the volume proportion of hepatocytes and an increase in bile duct cells and biliary stroma. These changes in volume proportions are related directly to the period of jaundice. Biliary stroma increases in support of new bile duct tissue and there is no excessive fibrosis. Hepatocytes proliferate at a greater rate than normal after obstruction of the common bile duct and the degree of proliferation reaches a maximum of 24 times that of normal 4 days after obstruction. Similarly, the proliferation of bile duct epithelium is increased in obstructive jaundice but in this instance it reaches a maximum of 50 times that of normal 24 h after ligation of the common bile duct. The absolute quantity of hepatocytes in the liver probably falls during the period of jaundice. However, the fall is less than anticipated from the volume proportion of hepatocytes because of the overall increase in liver size.     

Gadolinium chloride suppresses hepatic oval cell proliferation in rats with biliary obstruction.

Liver injury due to bile duct ligation (BDL) is histologically characterized by cholestasis, bile ductular proliferation, hepatocellular damage, portal fibrosis, and ultimately biliary cirrhosis. Stem cells within the liver may act as progenitor cells for small epithelial cells termed oval cells that can differentiate into bile duct cells or hepatocytes, whereas myofibroblasts are the principal source of collagen production in fibrosis. The aims of this study were to determine 1) whether BDL induces oval cell proliferation and 2) whether blockade of Kupffer cells affects oval cell proliferation, bile duct proliferation, and myofibroblast transformation in experimental biliary obstruction. Male Sprague-Dawley rats were divided into two groups to receive either a single dose of gadolinium chloride (a selective Kupffer cell blocking agent) or vehicle. One day later, the gadolinium- and vehicle-treated groups were further subdivided to receive either BDL or sham operation. The rats were sacrificed on day 7 postoperatively. Serum was collected for measurement of aspartate aminotransferase, gamma-glutamyl transpeptidase, and bilirubin levels. Liver tissue was taken for evaluation of fibrosis, bile ductular cells, oval cells, and myofibroblasts. BDL resulted in elevated aspartate aminotransferase, gamma-glutamyl transpeptidase, and bilirubin in serum, and gadolinium pretreatment did not modify these effects. BDL induced marked oval cell proliferation, which was completely prevented by gadolinium pretreatment. Gadolinium did not affect the induction of bile duct expansion or myofibroblasts after BDL. We conclude that experimental biliary obstruction induces oval cell proliferation, which can be prevented by gadolinium pretreatment. This suggests that bile ductular proliferation and myofibroblast transformation are not mediated by Kupffer cells and that ductular proliferation can proceed in the absence of oval cells. Alternatively, gadolinium may directly affect oval cell proliferation after BDL.     

Effect of bromocriptine on the expression of prolactin receptors in liver cells after common bile duct ligation

Immunoperoxidase method combined with cytofluorimetry showed that in contrast to hepatocytes, enhanced expression of prolactin receptors on rat cholangiocytes induced by common bile duct ligation cannot be suppressed by the prolactin secretion inhibitor bromocriptine. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 126, No. 7, pp. 52–55, July, 1998     

Vimentin expression of newly formed rat bile duct epithelial cells in secondary biliary fibrosis

Summary The intermediate filament profile and the growth fraction of hepatocytes and bile duct epithelial cells were studied in a rat model of biliary fibrosis secondary to common bile duct ligation and scission. Strong vimentin expression was observed in epithelial cells of newly formed bile ductules, while normal liver contained only few weakly positive bile duct epithelial cells. All epithelial cells reacted with a pan-cytokeratin antibody. A monoclonal antibody specific for human cytokeratin 7 selectively reacted with both normal and newly formed bile duct epithelial cells. The intermediate filament profile of hepatocytes was constant, showing no changes during proliferation or in periportal areas adjacent to excessive bile duct formations. The proliferation-associated antigen detected by the antibody Ki-67 was present in many hepatocytes, homogeneously distributed in the lobules, but was seen only in a small proportion of the epithelial cells of the newly formed bile ducts. We conclude that vimentin may serve as an indicator for cellular reorganization in the bile duct system, and that the epithelial cells of newly formed bile ductules in this particular model of secondary biliary fibrosis were most likely to be derived from an outgrowth of the biliary duct system and recruitment of preductular epithelial cells. No morphological or immunohistological evidence suggesting a derivation from hepatocytes by ductular metaplasia or from oval cells was obtained.     

Role of apoptosis in the remodeling of cholestatic liver injury following release of the mechanical stress

It has been known for a long time that portal fibrosis consecutive to experimental common bile duct ligation is reversible following obstacle removal, but the mechanisms involved remain unknown. We have studied the effect of bilioduodenal anastomosis and of simple biliary decompression on the remodeling of the lesion in bile duct-ligated rats. Rats were subjected to common bile duct ligation for 7 days or 14 days. Bilioduodenal anastomosis was performed after 14 days of bile duct ligation and animals sacrificed at intervals. In other animals, after 7 days or 14 days of ligation, the common bile duct was merely decompressed by bile aspiration and animals sacrificed 24 h later. Collagen deposition, alpha-smooth muscle actin expression and apoptosis were evaluated. Bile was collected and the bile acid profile assessed. After anastomosis, collagen deposition and alpha-smooth muscle actin expression decreased and were back to control values after 7 days. These parameters remained practically unchanged 24 h after biliary decompression. Bile duct ligation by itself induced apoptosis of some fibroblastic and bile ductular cells after 7 days; this was back to normal after 14 days. After anastomosis or decompression, apoptosis of both fibroblastic and bile ductular cells increased greatly and was accompanied by ultrastructural features of extracellular matrix degradation. Total bile acid content decreased after common bile duct ligation, the proportion of dihydroxylated bile acids decreasing and that of trihydroxylated bile acids increasing. Biliary decompression and anastomosis did not modify total concentration and composition of the biliary bile acid pool. In summary, we show that mere biliary decompression, by relieving the mechanical stress, is as effective as bilioduodenal anastomosis to induce apoptosis of portal cells that likely triggers portal fibrosis regression.     

Somatostatin analogue (octreotide) inhibits bile duct epithelial cell proliferation and fibrosis after extrahepatic biliary obstruction.

Extrahepatic biliary obstruction leads to bile duct epithelial cell proliferation. Somatostatin and its analogue, octreotide, have been shown to inhibit DNA synthesis and proliferation in hepatocytes. We investigated the effect of octreotide on the biliary epithelial cell proliferative responses to biliary obstruction. Male Sprague-Dawley rats underwent common bile duct ligation and subcutaneous injection of either saline or octreotide (6 micrograms/kg) twice daily for 7 days. Morphometric analysis of hepatocytes, bile duct epithelial cells, and periportal connective tissue was performed by computerized point counting. Hepatocyte volume was preserved with octreotide treatment, which also significantly decreased bile duct proliferation and periportal extracellular matrix deposition in response to biliary obstruction compared with saline treated, duct-ligated animals. These results indicate that octreotide prevents the morphological changes that accompany extrahepatic biliary obstruction.     

Ultrastructural Changes of Coronary Artery Endothelium Associated with Biliary Obstruction in the Rat

Ultrastructural cellular changes in various target organs have been described in association with experimentally induced biliary obstruction. In the present study, the effect of bile duct ligation on coronary artery endothelium was examined in rats 24 hours after surgery. Electron microscopic examination revealed marked degenerative changes of endothelial cells compared with the lack of such alterations in control animals. Some possible implications of the reported observations for atherogenesis are discussed.     

Effects of Biliary Obstruction on Pulmonary Ultrastructure in the Rat

Hypoxemia and respiratory alkalosis have been described in patients with liver disease. Since no pulmonary parenchymal lesions have been related to the abnormal respiratory function, the effects of common bile duct ligation and division on pulmonary alveolar structure were studied in ninety-two 10- to 14-week-old male Wistar rats. In comparison with controls, the rats with ligated common bile ducts (CBD) developed progressive hepatic and pulmonary changes. The hepatic damage consisted of bile duct proliferation, loss of hepatocytes and generalized cirrhosis. Most pronounced pulmonary histologic differences between the CBD and sham-operated rats were thickening of interalveolar septa and generalized increase in cellularity of alveolar walls. With electron microscopy, frequently observed alterations included swelling and increased vacuolization of endothelial and epithelial (Type I) cells, proliferation of Type II alveolar pneumonocytes, and increase in amounts of collagen fibers and edema of the interstitium.     

Tannic acid inhibits cholangiocyte proliferation after bile duct ligation via a cyclic adenosine 5',3'-monophosphate-dependent pathway

Chronic cholestatic diseases are characterized by morphological changes involving cholangiocyte proliferation and functional alterations of secretory capacity. The plant polyphenol tannic acid inhibits the growth of malignant human cholangiocytes. However, the mechanisms by which tannic acid limits excessive cholangiocyte proliferation are unknown. In this study we assessed the effect of tannic acid on cholangiocyte proliferation after bile duct ligation in rats. Tannic acid feeding decreased cholangiocyte proliferation and ductal mass in vivo after bile duct ligation. These changes were associated with functional changes in bile secretion and with decreases of intracellular cyclic adenosine 5',3'-monophosphate. The anti-proliferative effect of tannic acid was associated with a reduction of ERK1,2 phosphorylation. Additionally, tannic acid feeding decreased protein kinase A phosphorylation and activity. Similar changes were observed in isolated cholangiocytes during in vitro incubation with tannic acid. Furthermore, forskolin abolished the anti-proliferative effect of tannic acid on cholangiocyte proliferation after bile duct ligation. In conclusion, the anti-proliferative effects of tannic acid in cholangiocytes involve modulation of ERK1,2 by a cyclic adenosine 5',3'-monophosphate-protein kinase A-dependent pathway. These data suggest that tannic acid may be useful in limiting excessive cholangiocyte proliferation and modulating secretion during cholestasis.