Parenteral nutrition–associated liver disease (PNALD) is a serious complication of PN in infants who do not tolerate enteral feedings, especially those with acquired or congenital intestinal diseases. Yet, the mechanisms underlying PNALD are poorly understood. It has been suggested that a component of soy oil (SO) lipid emulsions in PN solutions, such as plant sterols (phytosterols), may be responsible for PNALD, and that use of fish oil (FO)–based lipid emulsions may be protective. We used a mouse model of PNALD combining PN infusion with intestinal injury to demonstrate that SO-based PN solution causes liver damage and hepatic macrophage activation and that PN solutions that are FO-based or devoid of all lipids prevent these processes. We have furthermore demonstrated that a factor in the SO lipid emulsions, stigmasterol, promotes cholestasis, liver injury, and liver macrophage activation in this model and that this effect may be mediated through suppression of canalicular bile transporter expression (Abcb11/BSEP, Abcc2/MRP2) via antagonism of the nuclear receptors Fxr and Lxr, and failure of up-regulation of the hepatic sterol exporters (Abcg5/g8/ABCG5/8). This study provides experimental evidence that plant sterols in lipid emulsions are a major factor responsible for PNALD and that the absence or reduction of plant sterols is one of the mechanisms for hepatic protection in infants receiving FO-based PN or lipid minimization PN treatment. Modification of lipid constituents in PN solutions is thus a promising strategy to reduce incidence and severity of PNALD.

The accumulation of hydrophobic bile acids plays a role in the induction of apoptosis and necrosis of hepatocytes during cholestasis. The aim of this study was to determine in freshly isolated rat hepatocytes the roles of oxidant stress and the mitochondrial permeability transition (MPT) in bile acid-induced apoptosis. Hepatocytes isolated from adult male Sprague-Dawley rats were incubated for 4 hours in buffer containing the hydrophobic bile acid, glycochenodeoxycholic acid (GCDC, 0-500 mol/L) or the hydrophilic bile acid, glycocholic acid (GCA), and either the antioxidants, alpha tocopherol, ebselen, or idebenone (a coenzyme Q analogue); or the MPT blockers, cyclosporin A, or bongkrekic acid, or a caspase-8 inhibitor.

Infants with intestinal failure who are parenteral nutrition (PN)-dependent may develop cholestatic liver injury and cirrhosis (PN-associated liver injury: PNALI). The pathogenesis of PNALI remains incompletely understood. We hypothesized that intestinal injury with increased intestinal permeability combined with administration of PN promotes LPS-TLR4 signaling dependent Kupffer cell activation as an early event in the pathogenesis of PNALI. We developed a mouse model in which intestinal injury and increased permeability were induced by oral treatment for 4 days with dextran sulphate sodium (DSS) followed by continuous infusion of soy lipid-based PN solution through a central venous catheter for 7 (PN/DSS7d) and 28 (PN/DSS28d) days. Liver injury and cholestasis were evaluated by serum AST, ALT, bile acids, total bilirubin, and by histology. Purified Kupffer cells were probed for transcription of pro-inflammatory cytokines. PN/DSS7d mice showed elevated portal vein LPS levels, evidence of hepatocyte injury and cholestasis, and increased Kupffer cell expression of IL6, TNFα, and TGFβ. Serological markers of liver injury remained elevated in PN/DSS28d mice associated with focal inflammation, hepatocyte apoptosis, peliosis, and Kupffer cell hypertrophy and hyperplasia. PN infusion without DSS pre-treatment or DSS pre-treatment alone did not result in liver injury or Kupffer cell activation. Suppression of the intestinal microbiota with broad spectrum antibiotics or ablation of TLR4 signaling in TLR4 mutant mice resulted in significantly reduced Kupffer cell activation and markedly attenuated liver injury in PN/DSS7d mice. Conclusion These data suggest that intestinal-derived LPS activates Kupffer cells through TLR4 signaling in early stages of PNALI.

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In infants intolerant of enteral feeding because of intestinal disease, parenteral nutrition may be associated with cholestasis, which can progress to end-stage liver disease. Here we show the function of hepatic macrophages and phytosterols in parenteral nutrition-associated cholestasis (PNAC) pathogenesis using a mouse model that recapitulates the human pathophysiology and combines intestinal injury with parenteral nutrition. We combine genetic, molecular, and pharmacological approaches to identify an essential function of hepatic macrophages and IL-1β in PNAC. Pharmacological antagonism of  IL-1 signaling or genetic deficiency in CCR2, caspase-1 and caspase-11, or IL-1 receptor (which binds both IL-1α and IL-1β) prevents PNAC in mice. IL-1β increases hepatocyte NF-κB signaling, which interferes with farnesoid X receptor and liver X receptor bonding to respective promoters of canalicular bile and sterol transporter genes (Abcc2, Abcb11, and Abcg5/8), resulting in transcriptional suppression and subsequent cholestasis. Thus, hepatic macrophages, IL-1β, or NF-κB may be targets for restoring bile and sterol transport to treat PNAC.

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Hydrophobic bile acids may cause hepatocellular necrosis and apoptosis during cholestatic liver diseases. The mechanism for this injury may involve mitochondrial dysfunction and the generation of oxidant stress. The purpose of this study was to determine the relationship of oxidant stress and the mitochondrial membrane permeability transition (MMPT) in hepatocyte necrosis induced by bile acids. The MMPT was measured spectrophotometrically and morphologically in rat liver mitochondria exposed to glycochenodeoxycholic acid (GCDC). Freshly isolated rat hepatocytes were exposed to GCDC and hepatocellular necrosis was assessed by lactate dehydrogenase release, hydroperoxide generation by dichlorofluorescein fluorescence, and the MMPT in cells by JC1 and tetramethylrhodamine methylester fluorescence on flow cytometry. GCDC induced the MMPT in a dose-and Ca 2ϩ -dependent manner. Antioxidants significantly inhibited the GCDC-induced MMPT and the generation of hydroperoxides in isolated mitochondria. Other detergents failed to induce the MMPT and a calpain-like protease inhibitor had no effect on the GCDC-induced MMPT. In isolated rat hepatocytes, GCDC induced the MMPT, which was inhibited by antioxidants. Blocking the MMPT in hepatocytes reduced hepatocyte necrosis and oxidant stress caused by GCDC. Oxidant stress, and not detergent effects or the stimulation of calpain-like proteases, mediates the GCDC-induced MMPT in hepatocytes. We propose that reducing mitochondrial generation of reactive oxygen species or preventing increases in mitochondrial Ca 2ϩ may protect the hepatocyte against bile acid-induced necrosis. 1-3). Although hydrophobic bile acids cause injury to isolated hepatocytes (4), cultured hepatocytes (5), and the intact liver (6), the mechanisms of this toxicity are not fully understood. Both hepatocellular necrosis at higher bile acid concentrations (4) and apoptosis at lower concentrations (7) have been demonstrated and are proposed as playing a role in cholestatic liver injury. Hepatocyte necrosis is characterized by cellular swelling, loss of mitochondrial respiratory function, depleted cellular ATP levels, and formation of plasma membrane blebs that rupture and release cellular contents (8, 9). In cholestatic liver ABSTRACT519