Bile salts mediate hepatocyte apoptosis by increasing cell surface trafficking of Fas

Toxic bile salts induce hepatocyte apoptosis by a Fas-dependent, Fas ligand-independent mechanism. To account for this observation, we formulated the hypothesis that toxic bile salts induce apoptosis by effecting translocation of cytoplasmic Fas to the cell surface, resulting in transduction of Fas death signals. In McNtcp.24 cells the majority of Fas was cytoplasmic, as assessed by cell fractionation and immunofluorescence studies. However, cell surface Fas increased sixfold after treatment with the toxic bile salt glycochenodeoxycholate (GCDC) in the absence of increased Fas protein expression. Moreover, in cells transfected with Fas-green fluorescence protein, cell surface fluorescence also increased in GCDC-treated cells, directly demonstrating Fas translocation to the plasma membrane. Both brefeldin A, a Golgi-disrupting agent, and nocodazole, a microtubule inhibitor, prevented the GCDC-induced increase in cell surface Fas and apoptosis. In conclusion, toxic bile salts appear to induce apoptosis by promoting cytoplasmic transport of Fas to the cell surface by a Golgi- and microtubule-dependent pathway.     

Bile acids stimulate cFLIP phosphorylation enhancing TRAIL-mediated apoptosis

Bile acids induce hepatocyte injury by enhancing death receptor-mediated apoptosis. In this study, bile acid effects on TRAIL-mediated apoptosis were examined to gain insight into bile acid potentiation of death receptor signaling. TRAIL-induced apoptosis of HuH-7 cells, stably transfected with a bile acid transporter, was enhanced by bile acids. Caspase 8 and 10 activation, bid cleavage, cytosolic cytochrome c, and caspase 3 activation by TRAIL were all increased by the bile acid glycochenodeoxycholate (GCDCA). GCDCA (100 microm) did not alter expression of TRAIL-R1/DR4, TRAIL-R2/DR5, procaspase 8, cFLIP-L, cFLIP-s, Bax, Bcl-xL, or Bax. However, both caspase 8 and caspase 10 recruitment and processing within the TRAIL death-inducing signaling complex (DISC) were greater in GCDCA-treated cells whereas recruitment of cFLIP long and short was reduced. GCDCA stimulated phosphorylation of both cFLIP isoforms, which was associated with decreased binding to GST-FADD. The protein kinase C antagonist chelerythrine prevented bile acid-stimulated cFLIP-L and -s phosphorylation, restored cFLIP binding to GST-FADD, and attenuated bile acid potentiation of TRAIL-induced apoptosis. These results provide new insights into the mechanisms of bile acid cytotoxicity and the proapoptotic effects of cFLIP phosphorylation in TRAIL signaling.     

Protective Effect of Gadolinium Chloride on Early Warm Ischemia/Reperfusion Injury in Rat Bile Duct during Liver Transplantation

Background

Activation of Kupffer cell (KC) is acknowledged as a key event in the initiation and perpetuation of bile duct warm ischemia/reperfusion injury. The inhibitory effect of gadolinium chloride (GdCl₃) on KC activation shows potential as a protective intervention in liver injury, but there is less research with regard to bile duct injury.

Methods

Sixty-five male Sprague-Dawley rats (200–250 g) were randomly divided into three experimental groups: a sham group (n = 15), a control group (n = 25), and a GdCl₃ group (n = 25). Specimen was collected at 0.5, 2, 6, 12 and 24 h after operation. Alanine aminotransferase (ALT), alkaline phosphatase (ALP) and total bilirubin (TBIL) of serum were measured. Tumor necrosis factor-α (TNF-α), Capase-3 activity and soluble Fas (sFas) were detected. The pathologic changes of bile duct were observed. Immunochemistry for bile duct Fas was performed. Apoptosis of bile duct cells was evaluated by the terminal UDP nick end labeling assay.

Results

GdCl₃ significantly decreased the levels of ALT, ALP and TBIL at 2, 6, 12, and 24 h, and increased serum sFas at 2, 6 and 12 h (P<0.05). TNF-α was lower in the GdCl₃ group than in the control group at 2, 6, 12 and 24 h (P<0.05). Preadministration of GdCl₃ significantly reduced the Caspase-3 activity and bile duct cell apoptosis at 2, 6, 12 and 24 h. After operation for 2, 6 and 12 h, the expression of Fas protein was lower in the GdCl₃ group than in the control group (P<0.05).

Conclusions

GdCl₃ plays an important role in suppressing bile duct cell apoptosis, including decreasing ALT, ALP, TBIL and TNF-α; suppressing Fas-FasL-Caspase signal transduction during transplantation.     

Bile acid accumulation in gastric mucosal cells

Bile acids are one of the components of the gastric contents capable of disrupting the mucosal barrier to diffusion. The mechanism by which bile acids can damage the gastric epithelium is not completely understood. Several studies have emphasized mucosal lipid solubilization by bile acids in the pathogenesis of mucosal injury. Bile acid entry into gastric mucosal cells may be a critical and early step in the genesis of mucosal injury, but this possibility has not yet been investigated. The present study was designed to explore the interaction of bile acids with dispersed gastric mucosal cells isolated from the rabbit and guinea pig stomach. Results showed that both glycocholic and deoxycholic acid rapidly associated with the gastric cells and reached a steady state concentration by 30 min. Glycocholic acid accumulated in the cells to a concentration approximately eight times greater than that in the surrounding medium. The amount of bile acid associated with the cells was greater at an acidic than at a neutral pH, and was a function of the concentration of both the cells and the bile acid. The process did not require cellular energy, was nonsaturable, and was not species specific. Experiments with 86Rb, a cytoplasmic marker, revealed that approximately one half of the cellular glycocholic acid was associated with the cytoplasmic compartment and the rest with the membranes. These findings are consistent with a combination of intracellular entrapment of the bile acids due to intracellular ionization and bile acid binding to cellular membrane components being the mechanisms by which bile acids accumulate in cells. Acid-driven bile acid accumulation may explain how relatively low luminal concentrations of bile acid can be damaging to the gastrointestinal mucosa.     

腹腔镜胆囊切除术（LC 术）致胆管损伤的诊治体会

摘要目的：总结腹腔镜胆囊切除术（laparoscopic cholecystectomy,LC 术）中胆管损伤的诊治体会。方法：回顾19 例LC 术致胆管 损伤病例的临床资料,分析其发生的类型及原因,并总结其诊断和治疗要点。结果：19 例患者中,1 例发生在左右肝管汇合以上处 损伤,2 例发生胆总管缺损伤,3 例发生胆总管横断伤,5 例发生胆总管侧面伤,3 例发生胆总管钳夹但未切断,1 例发生右肝管损 伤,4例发生胆囊管残端漏。治疗方法应视胆管损伤类型的不同而不同。采用断端处胆管端端吻合,同时放置T 管引流、单纯胆总 管T 管引流、开腹去除误夹夹子、ERCP检查放置鼻胆管引流及胆肠Rouxeny 吻合术。胆肠Rouxeny吻合术是临床上最常用的修 补胆道损伤的手术方法。随访6 个月～18 年,恢复好,无1 例死亡。结论：胆管损伤是LC 术最常见的并发症之一,规范的操作及 手术适应症的掌握能减少其发生。一旦出现胆管损伤,及时诊断及正确处理能减少其不良后果。     

腹腔镜胆囊切除术（LC术）致胆管损伤的诊治体会

目的：总结腹腔镜胆囊切除术（1aparoscopiccholecystectomy,LC术）中胆管损伤的诊治体会。方法：回顾19例LC术致胆管损伤病例的临床资料,分析其发生的类型及原因,并总结其诊断和治疗要点。结果：19例患者中,1例发生在左右肝管汇合以上处损伤,2例发生胆总管缺损伤,3例发生胆总管横断伤,5例发生胆总管侧面伤,3例发生胆总管钳夹但未切断,1例发生右肝管损伤,4例发生胆囊管残端漏。治疗方法应视胆管损伤类型的不同而不同。采用断端处胆管端端吻合,同时放置T管引流、单纯胆总管T管引流、开腹去除误夹夹子、ERCP检查放置鼻胆管引流及胆肠Rouxeny吻合术。胆肠Rouxeny吻合术是临床上最常用的修补胆道损伤的手术方法。随访6个月～18年,恢复好,无1例死亡。结论：胆管损伤是LC术最常见的并发症之一,规范的操作及手术适应症的掌握能减少其发生。一旦出现胆管损伤,及时诊断及正确处理能减少其不良后果。     

Genetic deletion of Fas receptors or Fas ligands does not reduce infarct size after acute global ischemia-reperfusion in isolated mouse heart

Apoptosis has been shown in cardiac cells under divergent physiological and pathological conditions. However, there has been an ongoing debate upon the relative contribution of cardiomyocyte apoptosis to the myocardial infarct size after ischemia-reperfusion (I-R) injury. We tested the hypothesis that blocking the death receptor pathway of apoptosis through genetic deletion of Fas receptors or Fas ligands would reduce myocardial infarct size caused by acute I-R injury. The hearts isolated from Fas receptor or Fas ligand knockout (KO) mice as well as the C57BL/6J wild-type control mice (N=6–8 per group) were subjected to 20 min of global ischemia and 30 min of reperfusion in Langendorff mode. Our results show that the infarct size, determined with triphenyltetrazolium chloride staining, was not significantly different between the three groups (i.e., 30.2±3.9% for wild-type controls, 30.0±2.1% for Fas ligand KOs, and 23.8±3.6% for Fas receptor KOs; mean±SEM, p>0.05). Postischemic leakage of lactate dehydrogenase, another marker of necrotic cellular injury, also was not significantly different between these groups (p>0.05). In addition, postischemic ventricular contractile function as well as coronary flow were similar for all the experimental groups (p>0.05). In conclusion, contrary to our original hypothesis, the present study in the gene KO mice suggests that the Fas ligand- and Fas receptor-mediated death receptor pathway of apoptosis is not the primary determinant of myocardial infarct size and ventricular dysfunction caused by acute global I-R injury in the isolated perfused mouse heart.     

Bile acids in rat serum as indicators of hepatotoxicity by hornet venom

The effect of hornet venom sac extract (VSE) on hepatic function in rats was evaluated by measurement of bile acid (BA) levels. A significant dose-dependent elevation of BA in rat serum upon repeated VSE administration was found. Rats envenomed for 2 weeks showed a significant rise of serum BA compared with the controls or with rats envenomed for 6 days. In rats envenomed for 1 week and killed after another week without envenomation, an improvement of the biochemical status was observed. These observations demonstrated that BA levels reflect the VSE-induced hepatic injury. They suggest that the cholestatic effects of VSE may be due to an impairment of bile acid excretion, but the elevations could also reflect parenchymal hepatic injury.     

Hormesis in Cholestatic Liver Disease; Preconditioning with Low Bile Acid Concentrations Protects against Bile Acid-Induced Toxicity

Introduction

Cholestasis is characterized by accumulation of bile acids and inflammation, causing hepatocellular damage. Still, liver damage markers are highest in acute cholestasis and drop when this condition becomes chronic, indicating that hepatocytes adapt towards the hostile environment. This may be explained by a hormetic response in hepatocytes that limits cell death during cholestasis.

Aim

To investigate the mechanisms that underlie the hormetic response that protect hepatocytes against experimental cholestatic conditions.

Methods

HepG2.rNtcp cells were preconditioned (24 h) with sub-apoptotic concentrations (0.1–50 μM) of various bile acids, the superoxide donor menadione, TNF-α or the Farsenoid X Receptor agonist GW4064, followed by a challenge with the apoptosis-inducing bile acid glycochenodeoxycholic acid (GCDCA; 200 μM for 4 h), menadione (50 μM, 6 h) or cytokine mixture (CM; 6 h). Levels of apoptotic and necrotic cell death, mRNA expression of the bile salt export pump (ABCB11) and bile acid sensors, as well as intracellular GCDCA levels were analyzed.

Results

Preconditioning with the pro-apoptotic bile acids GCDCA, taurocholic acid, or the protective bile acids (tauro)ursodeoxycholic acid reduced GCDCA-induced caspase-3/7 activity in HepG2.rNtcp cells. Bile acid preconditioning did not induce significant levels of necrosis in GCDCA-challenged HepG2.rNtcp cells. In contrast, preconditioning with cholic acid, menadione or TNF-α potentiated GCDCA-induced apoptosis. GCDCA preconditioning specifically reduced GCDCA-induced cell death and not CM- or menadione-induced apoptosis. The hormetic effect of GCDCA preconditioning was concentration- and time-dependent. GCDCA-, CDCA- and GW4064- preconditioning enhanced ABCB11 mRNA levels, but in contrast to the bile acids, GW4064 did not significantly reduce GCDCA-induced caspase-3/7 activity. The GCDCA challenge strongly increased intracellular levels of this bile acid, which was not lowered by GCDCA-preconditioning.

Conclusions

Sub-toxic concentrations of bile acids in the range that occur under normal physiological conditions protect HepG2.rNtcp cells against GCDCA-induced apoptosis, which is independent of FXR-controlled changes in bile acid transport.     

Bile system morphogenesis defects and liver dysfunction upon targeted deletion of HNF1beta

The inactivation of the Hnf1beta gene identified an essential role in epithelial differentiation of the visceral endoderm and resulted in early embryonic death. In the present study, we have specifically inactivated this gene in hepatocytes and bile duct cells using the Cre/loxP system. Mutant animals exhibited severe jaundice caused by abnormalities of the gallbladder and intrahepatic bile ducts (IHBD). The paucity of small IHBD was linked to a failure in the organization of duct structures during liver organogenesis, suggesting an essential function of Hnf1b in bile duct morphogenesis. Mutant mice also lacked interlobular arteries. As HNF1beta is not expressed in these cells, it further emphasizes the link between arterial and biliary formation. Hepatocyte metabolism was also affected and we identified hepatocyte-specific HNF1beta target genes involved in bile acids sensing and in fatty acid oxidation.     

Acute lung injury and cell death: how many ways can cells die?

Apoptosis has been considered as an underlying mechanism in acute lung injury/acute respiratory distress syndrome and multiorgan dysfunction syndrome. Recently, several alternative pathways for cell death (such as caspase-independent cell death, oncosis, and autophagy) have been discovered. Evidence of these pathways in the pathogenesis of acute lung injury has also come into light. In this article, we briefly introduce cell death pathways and then focus on studies related to lung injury. The different types of cell death that occur and the underlying mechanisms utilized depend on both experimental and clinical conditions. Lipopolysaccharide-induced acute lung injury is associated with apoptosis via Fas/Fas ligand mechanisms. Hyperoxia and ischemia-reperfusion injury generate reactive oxidative species, which induce complex cell death patterns composed of apoptosis, oncosis, and necrosis. Prolonged overexpression of inflammatory mediators results in increased production and activation of proteases, especially cathepsins. Activation and resistance to death of neutrophils also plays an important role in promoting parenchymal cell death. Knowledge of the coexisting multiple cell death pathways and awareness of the pharmacological inhibitors targeting different proteases critical to cell death may lead to the development of novel therapies for acute lung injury.     

Metformin Protects Rat Hepatocytes against Bile Acid-Induced Apoptosis

Background

Metformin is used in the treatment of Diabetes Mellitus type II and improves liver function in patients with non-alcoholic fatty liver disease (NAFLD). Metformin activates AMP-activated protein kinase (AMPK), the cellular energy sensor that is sensitive to changes in the AMP/ATP-ratio. AMPK is an inhibitor of mammalian target of rapamycin (mTOR). Both AMPK and mTOR are able to modulate cell death.

Aim

To evaluate the effects of metformin on hepatocyte cell death.

Methods

Apoptotic cell death was induced in primary rat hepatocytes using either the bile acid glycochenodeoxycholic acid (GCDCA) or TNFα in combination with actinomycin D (actD). AMPK, mTOR and phosphoinositide-3 kinase (PI3K)/Akt were inhibited using pharmacological inhibitors. Apoptosis and necrosis were quantified by caspase activation, acridine orange staining and Sytox green staining respectively.

Results

Metformin dose-dependently reduces GCDCA-induced apoptosis, even when added 2 hours after GCDCA, without increasing necrotic cell death. Metformin does not protect against TNFα/ActD-induced apoptosis. The protective effect of metformin is dependent on an intact PI3-kinase/Akt pathway, but does not require AMPK/mTOR-signaling. Metformin does not inhibit NF-κB activation.

Conclusion

Metformin protects against bile acid-induced apoptosis and could be considered in the treatment of chronic liver diseases accompanied by inflammation.     

Tumor-cell resistance to death receptor--induced apoptosis through mutational inactivation of the proapoptotic Bcl-2 homolog Bax

The importance of Bax for induction of tumor apoptosis through death receptors remains unclear. Here we show that Bax can be essential for death receptor--mediated apoptosis in cancer cells. Bax-deficient human colon carcinoma cells were resistant to death-receptor ligands, whereas Bax-expressing sister clones were sensitive. Bax was dispensable for apical death-receptor signaling events including caspase-8 activation, but crucial for mitochondrial changes and downstream caspase activation. Treatment of colon tumor cells deficient in DNA mismatch repair with the death-receptor ligand apo2 ligand (Apo2L)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selected in vitro or in vivo for refractory subclones with Bax frameshift mutations including deletions at a novel site. Chemotherapeutic agents upregulated expression of the Apo2L/TRAIL receptor DR5 and the Bax homolog Bak in Baxminus sign/minus sign cells, and restored Apo2L/TRAIL sensitivity in vitro and in vivo. Thus, Bax mutation in mismatch repair--deficient tumors can cause resistance to death receptor--targeted therapy, but pre-exposure to chemotherapy rescues tumor sensitivity.     

Bile analysis as a tool for assessing integrity of biliary epithelial cells after cold ischemia--reperfusion of rat livers

Previous morphological studies failed to show appreciable injury of biliary epithelial cells (BEC) after cold ischemia of rat liver, although recent evidence indicated that BEC integrity and function were impaired in this model. We tested the hypothesis that analysis of bile for enzymes, such as lactate dehydrogenase (LDH), alanine transaminase (ALT), and aspartate transaminase (AST), can be used for assessing cold ischemic injury of BEC. Furthermore, we examined whether biliary gamma-glutamyltransferase (GGT) reflects warm ischemic injury of BEC and whether normothermic reperfusion aggravates the negative effect of cold ischemia on BEC integrity and function. Rat livers were reperfused after different periods of cold or warm ischemia using a blood-free perfusion model. Compared with controls, perfusate LDH, ALT, and AST levels and parameters of hepatocyte function, including hepatocyte tight junction permeability, were not significantly altered by 18-h cold ischemia. On the other hand, 9-h cold ischemia markedly increased biliary LDH, ALT, and AST levels. However, only LDH release into the bile was strongly dependent on the time of cold storage. Biliary GGT, LDH, and glucose levels decreased during the reperfusion period following 18-h cold ischemia. The results suggest that biliary LDH can be used for assessing injury of BEC in cold-preserved livers and that normothermic reperfusion does not aggravate preservation-induced injury of BEC after cold ischemic storage.     

Bile secretion in the liver caused by verapamil

Verapamil was studied for its effects on secretory function of liver in rats. In the animals with low initial level of bile secretion, infusion of verapamil resulted in increase of the bile flow conjugated with taurine bile salts and ester of cholesterol, and in reduction of the non-conjugated bile salts secretion, as well as bile salts conjugated with glycine. In the animals with high initial level of the liver secretory function, verapamil decreased the bile flow, the secretion of unconjugated bile salts, and bile salts conjugated with taurine and glycine, phospholipids, cholesterol and its ester. The changes of bile flow and biliary secretion of bile acids and lipids in two groups of animals suggest that verapamil could be influenced in regulation of bile secretion depending on its initial level. Possible mechanisms of the bile secretion regulation by verapamil, are discussed.     

Survival of Rats Undergoing Continuous Bile Drainage Depends on Maintenance of Circadian Rhythm of Bile Secretion

It is very difficult to collect bile secretions from animals for extended periods of time. We compared the use of saline or water as drinking fluids to sustain the animals, which were being continuously drained of bile. Complete bile drainage was performed in 16 male Wistar rats by surgical intervention. After surgery, 8 rats were given tap water, and the other 8 were given normal saline for water. The rats that received water rapidly lost weight after bile drainage, and all died within 8 days after the operation. In contrast, all rats that drank saline maintained their body weight and survived 14 days or longer after surgery. Serum biochemistry of the rats with water intake on the third day after bile drainage revealed hyponatremia, hypochloremia, and acute renal failure resulting in hyperkalemia. In contrast, electrolyte balance and renal function were normal in the rats with saline intake, and bile was secreted continuously with a circadian rhythm. These results clearly demonstrate that saline as drinking water is essential to the replacement of lost fluids and loss of electrolytes due to bile drainage. Further, saline proved efficacious for sustaining experimental animals undergoing continuous bile collection.     

Beyond apoptosis: nonapoptotic cell death in physiology and disease.

Apoptosis is a morphologically defined form of programmed cell death (PCD) that is mediated by the activation of members of the caspase family. Analysis of death-receptor signaling in lymphocytes has revealed that caspase-dependent signaling pathways are also linked to cell death by nonapoptotic mechanisms, indicating that apoptosis is not the only form of PCD. Under physiological and pathological conditions, cells demonstrate a high degree of flexibility in cell-death responses, as is reflected in the existence of a variety of mechanisms, including necrosis-like PCD, autophagy (or type II PCD), and accidental necrosis. In this review, we discuss recent data suggesting that canonical apoptotic pathways, including death-receptor signaling, control caspase-dependent and -independent cell-death pathways.     

Angiotensin II Protects Primary Rat Hepatocytes against Bile Salt-Induced Apoptosis

Angiotensin II (AT-II) is a pro-fibrotic compound that acts via membrane-bound receptors (AT-1R/AT-2R) and thereby activates hepatic stellate cells (HSCs). AT-II receptor blockers (ARBs) are thus important candidates in the treatment of liver fibrosis. However, multiple case reports suggest that AT-1R blockers may induce hepatocyte injury. Therefore, we investigated the effect of AT-II and its receptor blockers on cytokine-, oxidative stress- and bile salt-induced cell death in hepatocytes. Primary rat hepatocytes were exposed to TNF-α/Actinomycin D, the ROS-generating agent menadione or the bile salts: glycochenodeoxycholic acid (GCDCA) and tauro-lithocholic acid-3 sulfate (TLCS), to induce apoptosis. AT-II (100 nmol/L) was added 10 minutes prior to the cell death-inducing agent. AT-1R antagonists (Sartans) and the AT-2R antagonist PD123319 were used at 1 µmol/L. Apoptosis (caspase-3 activity, acridine orange staining) and necrosis (Sytox green staining) were quantified. Expression of CHOP (marker for ER stress) and AT-II receptor mRNAs were quantified by Q-PCR. AT-II dose-dependently reduced GCDCA-induced apoptosis of hepatocytes (−50%, p<0.05) without inducing necrosis. In addition, AT-II reduced TLCS-induced apoptosis of hepatocytes (−50%, p<0.05). However, AT-II did not suppress TNF/Act-D and menadione-induced apoptosis. Only the AT-1R antagonists abolished the protective effect of AT-II against GCDCA-induced apoptosis. AT-II increased phosphorylation of ERK and a significant reversal of the protective effect of AT-II was observed when signaling kinases, including ERK, were inhibited. Moreover, AT-II prevented the GCDCA-induced expression of CHOP (the marker of the ER-mediated apoptosis).

Conclusion

Angiotensin II protects hepatocytes from bile salt-induced apoptosis through a combined activation of PI3-kinase, MAPKs, PKC pathways and inhibition of bile salt-induced ER stress. Our results suggest a mechanism for the observed hepatocyte-toxicity of Sartans (angiotensin receptor blockers, ARBs) in some patients with chronic liver injury.     

Bile acid induces hydrophobicity-dependent membrane alterations

Elevated concentrations of fecal bile acids are a known risk factor for colon cancer, owing to alterations in cellular signaling. In colonic cells, where bile acid uptake is minimal, the hydrophobicity-induced membrane perturbation and alterations have been proposed, but these membrane alterations are largely uncharacterized. In this study, we examined the determinants and characteristics of bile acid-induced membrane alterations, utilizing PKCalpha activation and cholesterol up-regulation as model indicators. We found that bile acid-induced PKCalpha activation is a function of hydrophobicity and correlated with alteration in membrane lipid composition, as evident by the significant up-regulation in membrane cholesterol and phospholipid. We found that bile acid do not cause cell membrane disruption at a concentration sufficient to activate PKCalpha, but do induce drastic alterations in membrane composition. Bile acid also induced the modification and up-regulation of caveolin-1 in a hydrophobicity-dependent manner, implying widespread receptor dysregulation. Similarly, ERK1/2 activation was observed only in response to hydrophobic bile acids, suggesting hydrophobicity-induced caveolar or membrane stress. Experiments with sodium lauryl sarcosine and cholesteryl hemisuccinate showed that bile acid-induced membrane alterations can be mimicked by hydrophobic molecules unrelated to bile acids, strongly implicating hydrophobicity as an important determinant of bile acid signaling.     

Immunohistochemical characterization of Fas (CD95) and Fas Ligand (FasL/CD95L) expression in the injured brain: relationship with neuronal cell death and inflammatory mediators

Traumatic brain injury causes progressive tissue atrophy and consequent neurological dysfunction, resulting from neuronal cell death in both animal models and patients. Fas (CD95) and Fas ligand (FasL/CD95L) are important mediators of apoptosis. However, little is known about the relationship between Fas and FasL and neuronal cell death in mice lacking the genes for inflammatory cytokines. In the present study, double tumor necrosis factor/lymphotoxin-alpha knockout (-/-) and interleukin-6-/- mice were subjected to closed head injury (CHI) and sacrificed at 24 hours or 7 days post-injury. Consecutive brain sections were evaluated for Fas and FasL expression, in situ DNA fragmentation (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling; TUNEL), morphologic characteristics of apoptotic cell death and leukocyte infiltration. A peak incidence of TUNEL positive cells was found in the injured cortex at 24 hours which remained slightly elevated at 7 days and coincided with maximum Fas expression. FasL was only moderately increased at 24 hours and showed maximum expression at 7 days. A few TUNEL positive cells were also found in the ipsilateral hippocampus at 24 hours. Apoptotic, TUNEL positive cells mostly co-localized with neurons and Fas and FasL immunoreactivity. The amount of accumulated polymorphonuclear leukocytes and CD11b positive cells was maximal in the injured hemispheres at 24 hours. We show strong evidence that Fas and FasL might be involved in neuronal apoptosis after CHI. Furthermore, Fas and FasL upregulation seems to be independent of neuroinflammation since no differences were found between cytokine-/- and wild-type mice.