Loss of keratin 19 favours the development of cholestatic liver disease through decreased ductular reaction.

Keratins (K) are cytoprotective proteins and keratin mutations predispose to the development of multiple human diseases. K19 represents the most widely used marker of biliary and hepatic progenitor cells as well as a marker of ductular reaction that constitutes the basic regenerative response to chronic liver injury. In the present study, we investigated the role of K19 in biliary and hepatic progenitor cells and its importance for ductular reaction. K19 wild-type (WT) and knockout (KO) mice were fed: (a) 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC); (b) cholic acid (CA); (c) a choline-deficient, ethionine-supplemented (CDE) diet; or (d) were subjected to common bile duct ligation (CBDL). The bile composition, liver damage, bile duct proliferation, oval cell content and biliary fibrosis were analysed. In untreated animals, loss of K19 led to redistribution of the K network in biliary epithelial cells (BECs) but to no obvious biliary phenotype. After DDC feeding, K19 KO mice exhibited (compared to WTs): (a) increased cholestasis; (b) less pronounced ductular reaction with reduced ductular proliferation and fewer oval cells; (c) impaired Notch 2 signalling in BECs; (d) lower biliary fibrosis score and biliary bicarbonate concentration. An attenuated oval cell proliferation in K19 KOs was also found after feeding with the CDE diet. K19 KOs subjected to CBDL displayed lower BEC proliferation, oval cell content and less prominent Notch 2 signal. K19 deficiency did not change the extent of CA- or CBDL-induced liver injury and fibrosis. Our results demonstrate that K19 plays an important role in the ductular reaction and might be of importance in multiple chronic liver disorders that frequently display a ductular reaction.

Protective effect of the aqueous extract from the root of Platycodon grandiflorum on cholestasis-induced hepatic injury in mice.

CONTEXT: The root of Platycodon grandiflorum (Jacq.) A. DC. (Campanulaceae) has been widely studied for its hepatoprotective effects against various hepatotoxicants. OBJECTIVE: The present study evaluated the protective effect of the standardized aqueous extract of P. grandiflorum (BC703) on cholestasis-induced hepatic injury in mice. MATERIALS AND METHODS: BC703 is a standardized aqueous extract of P. grandiflorum in reference to platycodin D (at least 0.8%). The mice were allocated into five groups as follows: Sham-operated, bile duct ligation (BDL) alone, and BDL with BC703 (1, 5, and 10 mg/kg BW) treated group. BC703 was given for 3 consecutive days before BDL operation. The animals were sacrificed by CO2 anesthesia post-24 h of BDL operations. RESULTS AND DISCUSSION: Serum alanine aminotransferase and serum aspartate aminotransferase increased to 395.2 ± 90.0 and 266.0 ± 45.6 Unit/L in the BDL alone group and decreased with BC703 in a dose-dependent manner. Especially the 10 mg/kg of BC703-treated mice showed a 77% decrease of serum alanine aminotransferase and 56% of aspartate aminotransferase as compared with BDL alone. Decreased antioxidant enzyme levels in BDL alone group were elevated in BC703-treated groups ranging from 7 to 29% for glutathione and from 13 to 25% for superoxide dismutase. BC703 treatment also attenuated malondialdehyde (from 3 to 32%) and nitric oxide levels (from 32 to 50%) as compared with BDL alone. Histopathological studies further confirmed the hepatoprotective effect of BC703 in BDL-induced cholestesis. CONCLUSION: BC703 could attenuate liver injury by BDL in mice, and test results indicate that BC703 might be useful in cholestatic liver injury.

Influence of glutamine on morphological and functional changes of liver in the presence of extrahepatic biliary obstruction in rats.

PURPOSE: To study the influence of glutamine on functional and morphological changes of liver in the extrahepatic biliary obstruction through an experimental model in rats. METHODS: Seventy Wistar rats were divided into four groups: control (group C) fictitious operation, (group FO), submitted to laparotomy with handling of bile ducts, but without hepatic duct ligation, (group EBO) submitted to laparotomy and hepatic duct ligation, one of them submitted to supplementation with glutamine 2% (group G). The control group consisted of 6 animals. The animals from groups FO, EBO and G were divided into three groups consisting of 6 animals each, being sacrificed at 7, 14 and 21 days after operation, respectively. Blood samples were collected for biochemical analysis and a fragment of liver tissue was collected from the middle lobe for histological analysis. RESULTS: Both for biochemical analysis (BT, aspartate and alanine aminotransferase AST, ALT and alkaline phosphatase FAL) and for histopathological changes (fibrosis, portal inflammation, parenchymal inflammation, hepatocytic changes and duct proliferation), no statistical difference between groups submitted to extrahepatic biliary obstruction (EBO) with and without treatment with glutamine (G) was observed. CONCLUSION: Glutamine supplementation did not alter the prognosis of liver enzymes and histopathological changes in animals submitted to extrahepatic biliary obstruction.

Influence of glutamine on morphological and functional changes of liver in the presence of extrahepatic biliary obstruction in rats / Influência da glutamina em alterações funcionais e morfológicas do fígado na vigência de obstrução biliar extra-hepática em ratos

PURPOSE: To study the influence of glutamine on functional and morphological changes of liver in the extrahepatic biliary obstruction through an experimental model in rats. METHODS: Seventy Wistar rats were divided into four groups: control (group C) fictitious operation, (group FO), submitted to laparotomy with handling of bile ducts, but without hepatic duct ligation, (group EBO) submitted to laparotomy and hepatic duct ligation, one of them submitted to supplementation with glutamine 2 percent (group G). The control group consisted of 6 animals. The animals from groups FO, EBO and G were divided into three groups consisting of 6 animals each, being sacrificed at 7, 14 and 21 days after operation, respectively. Blood samples were collected for biochemical analysis and a fragment of liver tissue was collected from the middle lobe for histological analysis. RESULTS: Both for biochemical analysis (BT, aspartate and alanine aminotransferase AST, ALT and alkaline phosphatase FAL) and for histopathological changes (fibrosis, portal inflammation, parenchymal inflammation, hepatocytic changes and duct proliferation), no statistical difference between groups submitted to extrahepatic biliary obstruction (EBO) with and without treatment with glutamine (G) was observed. CONCLUSION: Glutamine supplementation did not alter the prognosis of liver enzymes and histopathological changes in animals submitted to extrahepatic biliary obstruction.

OBJETIVO: Estudar a influência da glutamina em alterações funcionais e morfológicas do fígado na obstrução biliar extra-hepática por meio de um modelo experimental desenvolvido em ratos. MÉTODOS: Setenta ratos Wistar distribuídos em quatro grupos: controle (grupo C); operação fictícia (grupo OF), submetido à laparotomia com manuseio das vias biliares, mas sem ligadura do ducto hepático; (grupo OBE), submetido à laparotomia exploradora e ligadura do ducto hepático, sendo um deles submetido à suplementação com glutamina a 2 por cento (grupo G). O grupo controle era composto por seis animais. Os animais dos grupos OF, OBE e G foram divididos em três grupos compostos por seis animais cada e que foram sacrificados no 7°, 14° e 21° dias após a operação, respectivamente. Foi colhido sangue para análise bioquímica e um fragmento de tecido hepático do lobo médio para estudo histológico. RESULTADOS: Tanto em relação à analise bioquímica (BT, aspartate and alanine aminotransferase AST, ALT e FAL) quanto em relação às alterações histopatológicas (fibrose, inflamação portal, inflamação parenquimatosa, alterações hepatocíticas e proliferação de ducto), não houve diferença estatística entre os grupos submetido a obstrução biliar extra-hepática sem (OBE) e com tratamento com glutamina (G). CONCLUSÃO: A suplementação com glutamina não alterou o prognóstico em relação às enzimas hepáticas e alterações histopatológicas nos animais submetidos à obstrução biliar extra-hepática.

Reduction of hepatocellular injury after common bile duct ligation using omega-3 fatty acids.

PURPOSE: Bile duct obstruction and subsequent cholestasis produces hepatocellular injury and an inflammatory response. Fatty acid constitution of cell membranes plays a major role in the inflammatory cascade. Omega-3 fatty acids are antiinflammatory. We proposed that omega-3 fatty acid supplementation would reduce hepatocellular damage and cell death in a model of murine common bile duct ligation. METHODS: Mice underwent bile duct ligation and were administered either control soy diet (omega-6) or Menhaden diet (omega-3), and parameters of liver injury were measured at postoperative days 1, 4, and 8. Serum was analyzed for liver function tests. Liver tissue was scored for histologic necrosis and inflammation, and apoptosis was qualitatively measured. RESULTS: At day 8, comparing control and Menhaden, liver function tests were not significantly different. The H&E slides were analyzed and scored. At day 4, the mean necrosis scores for the Menhaden-fed group was 0.01 +/- 0.028 and 0.46 +/- 0.108 for the soy-fed group (P = .001) and at day 8, 0.420 +/- 0.107 and 1.22 +/- 0.132 (P < .001). The mean portal inflammation score for day 4 Menhaden-fed and soy-fed mice was 1.40 +/- 0.245 for both groups (P = 1.00) and for day 8, 1.80 +/- 0.200 and 2.80 +/- 0.200 (P = .008). At day 1, the median terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling scores of the Menhaden vs soy group were 6.0 and 0.0 (P < .001); day 4, 24.0 and 3.0 (P < .001); and day 8, 0.0 and 3.0 (P < .001), respectively. CONCLUSION: Although there appears to be a trend toward biochemical protection and a marked reduction of necrosis and inflammation, there was no significant liver function test difference between control and Menhaden groups. Considering our data of blunted histologic hepatotoxicity with omega-3 fatty acid supplementation, we hypothesize that this may be a method of reducing long-term complications of liver injury secondary to diseases of cholestasis such as biliary atresia, namely fibrosis and cirrhosis.

Effect of ATRA on contents of liver retinoids, oxidative stress and hepatic injury in rat model of extrahepatic cholestasis.

The effects of all-trans-retinoic acid (ATRA) administration on the concentration of retinoids (RA and vitamin A) in liver, oxidative stress and the hepatic injury in a rat model of common bile duct ligation (CBDL)-induced liver injury were investigated. Female rats were subjected to a sham (n=5) or CBDL (n=48). Two weeks after operation, rats undergoing CBDL were randomized to receive treatment with either ATRA at three different doses (0.1, 1.5, 7.5 mg/kg) dissolved in bean oil or only bean oil every day over a 4-week experimental period. Rats were killed and blood samples were collected from the heart for determination of the serum transaminase. The contents of retinoids in rat liver were detected by using HPLC. Malondialdehyde (MDA), glutathione (GSH) and superoxide dismutase (SOD) levels in liver were determined by a spectrophotometric method according to the instruction of the kits. Liver pathologic changes were observed under the light microscopy and electron microscopy. The results showed that compared with sham-operated group, the levels of retinoids in the liver tissue were significantly decreased in the CBDL group (P<0.01). ATRA (0.1 mg/kg) administration in CBDL rats partially restored the contents of retinoids (P<0.05). Liver RA and vitamin A contents in CBDL group were significantly increased after ATRA (1.5 and 7.5 mg/kg) supplementation as compared with sham-operated group (P<0.05). However, in ATRA-treated CBDL group, hepatic GSH level and SOD activity, depressed by CBDL, and hepatic MDA level, increased by CBDL were returned to those in sham-operated group (P<0.05). The histologic observation of liver tissues indicated that ATRA treatment notably alleviated hepatocellular swelling, steatosis, the swelling of mitochondria and proliferation of smooth endoplasmic reticulum (SER). Treatment with ATRA could reduce levels of serum transaminase as compared with sham-operated group, more greatly in 1.5 and 7.5 mg/kg ATRA-treated groups than in 0.1 mg/kg ATRA-treated group. It was concluded that ATRA treatment can recover MDA and GSH levels and SOD activity in CBDL rat liver through restoring RA and vitamin A contents, and eventually ameliorate liver injury.

Protective effect of IGF-1 on experimental liver cirrhosis-induced common bile duct ligation.

BACKGROUND/AIMS: The causes of malnutrition in liver cirrhosis are multifactorial. Levels of IGF-1 (insulin like growth factor-1) that is a crucial regulator of intermediary metabolism decreases. The aim of this study was to analyze the effect of IGF-1 supplementation during liver cirrhosis induced by common bile duct ligation. METHODOLOGY: Rats were divided into five different groups: One sham and four experimental groups. Rats in three of four groups were treated with 2 micrograms/day IGF-1 with a different time of experiment in each group. Blood biochemical parameters, tissue malondialdehyde, glutathione levels and the activity of tissue antioxidant enzymes and conventional and immunohistochemical analysis of liver samples were studied for each group. RESULTS: Serum albumin, total protein, fibrinogen levels decreased and prothrombin time was prolonged in the bile duct ligated and transected experimental group but not in the IGF-I treated rats compared with the rats in sham group. Liver malondialdehyde levels significantly increased in control group but not in IGF-1 treated groups. The activities of antioxidant enzymes were decreased compared with the other groups. Histopathology findings of liver biopsy demonstrated intense degree fibrosis and overexpression of fibroblast growth factor and desmin in the control group but a lesser degree of those in the IGF-1 treated groups. CONCLUSIONS: IGF-1 treatment improves liver function and decreases oxidative liver damage and histopathological findings. Further studies are required to delineate the mechanisms of protective effects of IGF-1.