The bcl-2 mRNA expression in GCDC-induced obstructive jaundice in rats and its implication in hepatocellular apoptosis / 华中科技大学学报（医学）（英德文版）

The modulatory role of bcl-2 gene in hepatocellular apoptosis of rats with glycochenodeoxycholate (GCDC)-induced obstructive jaundice was investigated. The hepatocytes in normal rats and those with bile duct-ligation for 7 days, 14 days and 21 days were isolated and obtained by in situ collagenase perfusion and primary culture. The expression of bcl-2 mRNA in the hepatocytes was detected by RT-PCR. Primary culture was performed on the hepatocytes from normal rats and those with bile duct-ligation for 14 days. 100 mumol/L GCDC was added to the hepatocytes for incubation for 24 h. The hepatocellular apoptotic ratio was measured by using FCM and hepatocellular apoptosis detected in situ by using TUNEL technique. Results showed that the expression of bcl-2 mRNA was not detectable in the hepatocytes of normal rats by RT-PCR technique, while detectable in the hepatocytes of those with bile duct ligation (BDL) for 7, 14 and 21 days. Hepatocellular apoptosis in the BDL group was obviously decreased as compared with normal control group after addition of 100 mumol/L GCDC to the cells for 24 h. It was concluded that the hepatocytes in the BDL rats expressed bcl-2. During obstructive jaundice, expression of bcl-2 from the hepatocytes can inhibit the bile salt-induced hepatocellular apoptosis.

The role of lipid peroxidation and glutathione on the glycochenodeoxycholic acid-induced cell death in primary cultured rat hepatocytes

Intracellular accumulation of bile acids in the hepatocytes during cholestasis is thought to be pathogenic in cholestatic liver diseases. The objective of this study was to determine the role of lipid peroxidation and glutathione on the bile acid-induced hepatic cell death mechanism in primary cultured rat hepatocytes. To induce hepatic cell death, we incubated primary cultured rat hepatocytes with glycochenodeoxycholic acid (GCDC; 0~400 micrometer) for 3 hours. In electron microscopic examination and agarose gel electrophoresis, low concentration of GCDC treatment mainly induced apoptotic feature. Whereas 400 micrometer GCDC treated cells demonstrated both apoptosis and necrosis. Lipid peroxidation was increased dose-dependently in GCDC treated hepatocyte. And this was also accompanied by decreased glutathione. Therefore, oxygen free radical damage may play a partial role in GCDC-induced hepatic cell death.

Glycochenodeoxycholic acid induces cell death in primary cultured rat hepatocyte: Apoptosis and necrosis

Intracellular accumulation of bile acids in the hepatocytes during cholestasis is thought to be pathogenic in cholestatic liver injury. Due to the detergent-like effect of the hydrophobic bile acids, hepatocellular injury has been attributed to direct membrane damage. However histological findings of cholestatic liver diseases suggest apoptosis can be a mechanism of cell death during cholestatic liver diseases instead of necrosis. To determine the pattern of hepatocellular toxicity induced by bile acid, we incubated primary cultured rat hepatocytes with a hydrophobic bile acid, Glycochenodeoxycholate (GCDC), up to 5 hours. After 5 hours incubation with 400 muM GCDC, lactate dehydrogenase released significantly. Cell viability, quantitated in propidium iodide stained cells concomitant with fluoresceindiacetate was decreased time-and dose-dependently. Most nuclei with condensed chromatin and shrunk cytoplasm were heavily labelled time- and dose-dependently by a positive TUNEL reaction. These findings suggest that both apoptosis and necrosis are involved in hepatocytes injury caused by GCDC.

Optimización del análisis de ácidos biliares séricos por método enzimático fluorométrico / Improvement of an enzymatic fluorometric assay for serum bile acids

Con el objeto de optimizar la determinación fluorométrica de los ácidos biliares séricos (ABS) en cuanto a selectividad y sensibilidad, se desarrolló una metodología de preparación de muestra y preconcentración utilizando columnas de extracción en fase sólida (SPE-C18). Previa desproteinización del suero con acetonitrilo frío, la fase orgánica evaporada y reconstituida con acetonitrilo: agua (3:70), se aplicó a una columna SPE-C18 y los ABS fueron eluidos con metanol. En el extractivo metanólico, evaporado a sequedad y reconstituido con metanol se dosaron los ABS por método enzimático fluorométrico empleando 3Ó-hidroxiesteroide deshidrogenasa, ß-NAD, diaforasa y resazurina. En la validación de la preparación de muestra se utilizó [24-14C] ácido glicocólico. La recuperación fue del 89,0 ñ 1,3 por ciento (SD), con DSR de 1,4 para n=9 (3 días). Se determinaron los ABS en sujetos controles, resultando un valor medio de 2,61 ñ 0,39 µM (SEM) (n=27). La metodología propuesta combina las siguientes ventajas: aumento de la selectividad del método enzimático, eliminación de interferencias y preconcentración de los ABS liberados, previamente, de la unión a proteínas plasmáticas