High concentration of magnolol induces hepatotoxicity under serum-reduced conditions.

Although magnolol is cytoprotective against warm ischemia/reperfusion injury, its effect on cold preservation has not been fully investigated. This study aimed at examining whether magnolol maintains the liver graft integrity after cold preservation and elucidating the underlying mechanisms in terms of apoptotic signaling under both normothermic and hypothermic conditions. After being preserved in Ringer's lactate (RL) at 4 degrees C for 6h ex vivo, the magnolol-treated grafts demonstrated significantly higher AST, ALT, and LDH levels in perfusates than those from negative controls. TUNEL staining showed no difference in the number of apoptotic nuclei in both groups, whereas a more intense apoptotic signal in magnolol-treated grafts was shown as compared with the controls. In vitro data showed no significant difference in viability of RL-preserved clone-9 hepatocytes between the magnolol-treated and control groups, while magnolol pretreatment at 30min before cold preservation prominently induced hepatocyte cell death. RT-PCR and Western blotting analyses revealed a suppression in Bcl-2, but an up-regulation in Bax expression in clone-9 cells after magnolol treatment. Magnolol suppressed the ratios of NF-kappaB to I-kappaBalpha protein contents and I-kappaBalpha phosphorylation induced by TNF-alpha, and potentiated mitochondrial cytochrome c release and subsequent caspase-3 cleavage. Conversely, caspase-3 inhibitor attenuated magnolol-induced hepatotoxicity. We concluded that magnolol could not protect liver grafts from cold ischemia/reperfusion injury. High concentration of magnolol under serum-reduced conditions attenuates NF-kappaB-mediated signaling and induces intrinsic apoptotic pathway, thereby inducing in vitro hepatotoxicity.

Serum factors potentiate hypoxia-induced hepatotoxicity in vitro through increasing transforming growth factor-beta1 activation and release.

BACKGROUND: Although transforming growth factor-beta (TGF-beta), a growth regulator of hepatocytes, induces cell death under pathological conditions, responsiveness of hepatocytes to hypoxic stimulus has not been fully defined. This study aimed at investigating the role of TGF-beta1 in hypoxia-induced hepatotoxicity using cultured clone-9 hepatocytes with or without serum supplementation. METHODS/RESULTS: Presence of serum significantly potentiated hypoxia-induced hepatotoxicity after 72h of exposure, as evidenced by fluorescent viability stain and LDH cytotoxicity assay. Quantitative PCR showed that TGF-beta1 gene expression decreased, while ELISA revealed that latent TGF-beta1 in conditioned media prominently increased in serum-treated groups under hypoxia. Western blotting indicated that both type I and II receptors of TGF-beta were up-regulated in serum-free groups, but down-regulated in serum-treated groups under hypoxia. Smad2 phosphorylation was only detectable in cells supplemented with serum, and hypoxia potentiated the extent of Smad2 phosphorylation, implicating that the activated TGF-beta1 induces hepatotoxicity in an autocrine manner. Addition of exogenous TGF-beta1 deteriorated, while TGF-beta1 blockade by neutralizing antibody ameliorated hypoxia-induced hepatotoxicity with serum supplementation. Gelatine zymography and immunofluorescent stain evidenced that elevated MMP-2 and MMP-9 activity and serum-dependent CD44 expression and its membranous localization may contribute to TGF-beta1 activation. CONCLUSION: The results suggest that the mechanism governing TGF-beta activation plays a crucial role in hypoxia-induced hepatotoxicity. Thus, interventions on TGF-beta1 bioavailability and/or its cognate signaling may be of benefit in preventing hypoxia-related liver injuries.

Propofol pretreatment attenuates LPS-induced granulocyte-macrophage colony-stimulating factor production in cultured hepatocytes by suppressing MAPK/ERK activity and NF-kappaB translocation.

Propofol (PPF), a widely used intravenous anesthetic for induction and maintenance of anesthesia during surgeries, was found to possess suppressive effect on host immunity. This study aimed at investigating whether PPF plays a modulatory role in the lipopolysaccharide (LPS)-induced inflammatory cytokine expression in a cell line of rat hepatocytes. Morphological observation and viability assay showed that PPF exhibits no cytotoxicity at concentrations up to 300 microM after 48 h incubation. Pretreatment with 100 microM PPF for 24 h prior to LPS stimulation was performed to investigate the modulatory effect on LPS-induced inflammatory gene production. The results of semi-quantitative RT-PCR demonstrated that PPF pretreatment significantly suppressed the LPS-induced toll-like receptor (TLR)-4, CD14, tumor necrosis factor (TNF)-alpha, and granulocyte-macrophage colony-stimulating factor (GM-CSF) gene expression. Western blotting analysis showed that PPF pretreatment potentiated the LPS-induced TLR-4 downregulation. Flow cytometrical analysis revealed that PPF pretreatment showed no modulatory effect on the LPS-upregulated CD14 expression on hepatocytes. In addition, PPF pretreatment attenuated the phosphorylation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and IkappaBalpha, as well as the nuclear translocation of NF-kappaB primed by LPS. Moreover, addition of PD98059, a MAPK kinase inhibitor, significantly suppressed the LPS-induced NF-kappaB nuclear translocation and GM-CSF production, suggesting that the PPF-attenuated GM-CSF production in hepatocytes may be attributed to its suppressive effect on MAPK/ERK signaling pathway. In conclusion, PPF as an anesthetic may clinically benefit those patients who are vulnerable to sepsis by alleviating sepsis-related inflammatory response in livers.

Maintenance of normothermia at operation room temperature of 24 degrees C in adult and pediatric patients undergoing liver transplantation.

Hypothermia is common during surgery in regular operating room (OR) temperature. The effect of increasing the OR temperature to 24 degrees C coupled with simple warming measures to maintain normothermia in both pediatric and adult patients during living donor liver transplantation (LDLT) was evaluated. One hundred patients undergoing LDLT were separated into pediatric (GI) and adult (GII) groups. Nasopharyngeal temperature (NT) at each hour for the first 6 h, at the time of anhepatic phase, 5 and 30 min after reperfusion, and each hour for the last 2 h of the operation was recorded, compared and analyzed. A significant difference in core temperature variation was noted between the two groups. GI tended to be hyperthermic, while GII remained mildly hypothermic throughout the procedure. A sudden decrease of NT was observed in both groups during the anhepatic and reperfusion phases. Correlation between liver graft weight over recipient body weight ratio rather than the graft weight itself was found in GI, but no such correlation was found in GII. OR temperature of 24 degrees C, together with simple active and passive warming measures are more effective in maintaining normothermia during liver transplantation in pediatric patients than in adults.

Perioperative normovolemic anemia is safe in pediatric living-donor liver transplantation.

BACKGROUND: Perioperative normovolemic anemia was applied in pediatric living-donor liver transplant (LDLT) recipients with the aim of reducing the use of blood products and decreasing transfusion-related risk. METHODS: The anemic state was allowed to occur by replacing intraoperative blood and transudate loss with colloid solutions and a discriminate use of packed red blood cells. When blood transfusion was required, the amount of blood replacement was calculated to target a hemoglobin level not higher than 8 to 9 g/dL. RESULTS: Forty-eight pediatric patients underwent LDLT. Their mean hemoglobin and hematocrit levels were maintained below 9 g/dL and 27%, respectively, at the end of the operation, at the time of extubation, postoperative days 3, 10, and 20, and at the time of discharge. The mean ventilatory support time was 15.7 hr, and no patient required reintubation. Graft function normalized within the first week posttransplant in all patients, and there was no documented case of acute hepatic artery thrombosis. All the patients were discharged with acceptable liver function, and 98% of them remain alive to date. CONCLUSION: Routine application of perioperative normovolemic anemia in pediatric LDLT has allowed the sparing use of blood products. Approximately half of our patients (42%) did not require intraoperative blood transfusion; 31% of the patients went home without receiving any blood products except 5% albumin. There were no adverse effects with this maneuver, and graft function was good in all patients.